Hydro Saline Metabolism: Epidemiology, Genetics, Pathophysiology, Diagnosis and Treatment (Endocrinology) 3031271181, 9783031271182

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Endocrinology Series Editor: Andrea Lenzi Series Co-Editor: Emmanuele A. Jannini

Massimiliano Caprio Fabio Luiz Fernandes-Rosa Editors

Hydro Saline Metabolism Epidemiology, Genetics, Pathophysiology, Diagnosis and Treatment

Endocrinology

Series Editor Andrea Lenzi, Department of Experimental Medicine, Section of Medical Pathophysiology, Food Science and Endocrinology, Sapienza University of Rome, Rome, Italy Series Co-Editor Emmanuele A. Jannini, Department of Systems Medicine, University of Rome Tor Vergata, Rome, Roma, Italy

Within the health sciences, Endocrinology has an unique and pivotal role. This old, but continuously new science is the study of the various hormones and their actions and disorders in the body. The matter of Endocrinology are the glands, i.e. the organs that produce hormones, active on the metabolism, reproduction, food absorption and utilization, growth and development, behavior control, and several other complex functions of the organisms. Since hormones interact, affect, regulate and control virtually all body functions, Endocrinology not only is a very complex science, multidisciplinary in nature, but is one with the highest scientific turnover. Knowledge in the Endocrinological sciences is continuously changing and growing. In fact, the field of Endocrinology and Metabolism is one where the highest number of scientific publications continuously flourishes. The number of scientific journals dealing with hormones and the regulation of body chemistry is dramatically high. Furthermore, Endocrinology is directly related to genetics, neurology, immunology, rheumatology, gastroenterology, nephrology, orthopedics, cardiology, oncology, gland surgery, psychology, psychiatry, internal medicine, and basic sciences. All these fields are interested in updates in Endocrinology. The aim of the MRW in Endocrinology is to update the Endocrinological matter using the knowledge of the best experts in each section of Endocrinology: basic endocrinology, neuroendocrinology, endocrinological oncology, pancreas with diabetes and other metabolic disorders, thyroid, parathyroid and bone metabolism, adrenals and endocrine hypertension, sexuality, reproduction, and behavior.

Massimiliano Caprio • Fabio Luiz Fernandes-Rosa Editors

Hydro Saline Metabolism Epidemiology, Genetics, Pathophysiology, Diagnosis and Treatment

With 46 Figures and 20 Tables

Editors Massimiliano Caprio Department of Human Sciences and Promotion of the Quality of Life San Raffaele Roma Open University Rome, Italy

Fabio Luiz Fernandes-Rosa Université Paris Cité, PARCC Inserm Paris, France

Laboratory of Cardiovascular Endocrinology San Raffaele Research Institute IRCCS San Raffaele Rome, Italy

ISSN 2510-1927 ISSN 2510-1935 (electronic) Endocrinology ISBN 978-3-031-27118-2 ISBN 978-3-031-27119-9 (eBook) https://doi.org/10.1007/978-3-031-27119-9 © Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Paper in this product is recyclable.

Series Preface

Is there an unmet need for a new MRW series in Endocrinology and Metabolism? It might not seem so! The vast number of existing textbooks, monographs, and scientific journals suggest that the field of hormones (from genetic, molecular, biochemical, and translational to physiological, behavioral, and clinical aspects) is one of the largest in biomedicine, producing a simply huge scientific output. However, we are sure that this new series will be of interest to scientists, academics, students, physicians, and specialists alike. The knowledge in endocrinology and metabolism limited to the two main (from an epidemiological perspective) diseases, namely, hypo/hyperthyroidism and diabetes mellitus, now seems outdated and perhaps closer to the practical interests of the general practitioner than to those of the specialist. This has led to endocrinology and metabolism being increasingly considered as a subsection of internal medicine rather than an autonomous specialization. But endocrinology is much more than this. We are proposing this series as the manifest for Endocrinology 2.0, embracing the fields of medicine in which hormones play a major part but which, for various historical and cultural reasons, have thus far been “ignored” by endocrinologists. Hence, this MRW comprises “traditional” (but no less important or investigated) topics: from the molecular actions of hormones to the pathophysiology and management of pituitary, thyroid, adrenal, pancreatic, and gonadal diseases, as well as less usual and common arguments. Endocrinology 2.0 is, in fact, the science of hormones, but it is also the medicine of sexuality and reproduction, the medicine of gender differences, and the medicine of well-being. These aspects of endocrinology have to date been considered of little interest, as they are young and relatively unexplored sciences. But this is no longer the case. The large scientific production in these fields coupled with the impressive social interest of patients in these topics is stimulating a new and fascinating challenge for endocrinology. The aim of the MRW in Endocrinology is thus to update the subject with the knowledge of the best experts in each field: basic endocrinology; neuroendocrinology; endocrinological oncology; pancreatic disorders; diabetes and other metabolic disorders; thyroid, parathyroid, and bone metabolism; adrenal and endocrine

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Series Preface

hypertension; sexuality, reproduction, and behavior. We are sure that this ambitious aim, covering for the first time the whole spectrum of Endocrinology 2.0, will be fulfilled in this vast Springer MRW in Endocrinology Series. Andrea Lenzi Emmanuele A. Jannini

Volume Preface

Different endocrine systems are involved in the maintenance of salt and water homeostasis. Disruptions of these systems are responsible of alterations in sodium and water balance with clinical manifestation ranging from severe salt wasting in the neonatal period to resistant hypertension with increased cardiovascular risk in adulthood. A better understanding of the endocrine mechanisms underlying these alterations may lead to the identification of novel diagnostic and therapeutic tools, improving the standard of care of patients affected by diseases associated with salt and homeostasis imbalance. The aim of the volume Hydro Saline Metabolism is to provide an overview of endocrine diseases associated with alterations of salt metabolism, focusing on basic, translational, and clinical aspects. The volume critically examines the impact of hormone action disruption on renal water and salt reabsorption. The chapters cover different aspects of endocrine control of salt and water homeostasis, including neuroendocrine determinants of water balance, adrenal hormone biosynthesis, hormonal function in target renal cells, and dysfunction of hormonal target effectors in the kidney. A major segment of the volume covers diseases affecting hypothalamic, pituitary, and adrenal diseases and how they affect kidney electrolyte reabsorption, and there are also chapters dedicated to the physiological regulation of hydromineral homeostasis and to the complex relationship between hydrosaline metabolism and metabolic diseases. The addition of these chapters allows a deeper understanding and a wide overview of the field. The chapters cover aspects ranging from bench to bedside, including epidemiology, genetic, pathophysiology, diagnosis, and therapeutics up to date. In this context, an update in the clinical management of endocrine diseases associated with hydrosaline imbalance, with a special focus on novel diagnostic and therapeutic tools, is also addressed. The chapters were written by a combination of outstanding international researchers, with basic and/or clinical expertise in the field of endocrine diseases associated with alterations of hydrosaline metabolism. This volume is intended to serve as a major reference work not only for endocrinologists and nephrologists, but also for pediatricians, neonatologists, cardiologists, and emergency medicine and intensive care specialists, who can be faced with the management of endocrine diseases responsible for salt and water imbalance.

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Volume Preface

The book is part of the SpringerReference program, which provides access to “living editions” that are constantly updated using a dynamic peer-review publishing process. The volume editors wish to sincerely thank and congratulate all of the authors for their commitment and relevant contribution to this major reference work. Rome, Italy Paris, France October 2023

Massimiliano Caprio Fabio Luiz Fernandes-Rosa

Contents

1

Neuroendocrine Regulation of Hydrosaline Metabolism . . . . . . . . Silvia Graciela Ruginsk, Lucila Leico Kagohara Elias, José Antunes-Rodrigues, and André Souza Mecawi

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Physiology of Vasopressin Secretion . . . . . . . . . . . . . . . . . . . . . . . . Giovanna Mantovani, Alessandra Mangone, and Elisa Sala

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3

Tubulopathies and Alterations of the RAAS Marguerite Hureaux and Rosa Vargas-Poussou

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4

Familial Hyperkalemic Hypertension (FHHt) Chloé Rafael and Juliette Hadchouel

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Diabetes Insipidus: Novel Diagnostic Approaches . . . . . . . . . . . . . Marianna Martino, Giulia Giancola, and Giorgio Arnaldi

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Syndrome of Inappropriate Antidiuresis . . . . . . . . . . . . . . . . . . . . Emanuele Ferrante and Júlia Ferreira de Carvalho

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Hydro-saline Alterations in Central Adrenal Insufficiency . . . . . . Rosario Pivonello, Chiara Simeoli, Nicola Di Paola, Rosario Ferrigno, and Annamaria Colao

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Approach to Hyponatremia According to the Clinical Setting . . . . Alessandro Peri, Dario Norello, and Benedetta Fibbi

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9

Electrolyte Disturbance in Congenital Adrenal Hyperplasia due to 21-OH Deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Laetitia Martinerie

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Rare Forms of Congenital Adrenal Hyperplasia Affecting Electrolyte Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sonir Roberto Rauber Antonini and Thais Milioni Luciano

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Apparent Mineralocorticoid Excess . . . . . . . . . . . . . . . . . . . . . . . . Cristian A. Carvajal, Alejandra Tapia-Castillo, Thomas Uslar, and Carlos E. Fardella

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Contents

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Mineralocorticoid Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fabio Luiz Fernandes-Rosa

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Primary Aldosteronism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sheerazed Boulkroun and Maria-Christina Zennaro

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Mineralocorticoid Receptor and Aldosterone: From Hydro-saline Metabolism to Metabolic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . Andrea Armani and Massimiliano Caprio

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Hydrosaline Alterations in Cushing Disease . . . . . . . . . . . . . . . . . . Dario De Alcubierre, Emilia Sbardella, and Andrea M. Isidori

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Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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About the Editors

Massimiliano Caprio San Raffaele Roma Open University Rome, Italy Massimiliano Caprio is full professor of endocrinology at San Raffaele Roma Open University, Rome, Italy, since 2019. He is the head of the Laboratory of Cardiovascular Endocrinology at the Research Center of IRCCS San Raffaele in Rome since 2009 and consultant endocrinologist and diabetologist at CTO Alesini Hospital, University of Rome Tor Vergata, since 2007. He received his MD (1995) and board certification (2001) in endocrinology from Sapienza University of Rome. He obtained a PhD in endocrine sciences from the University of Rome Tor Vergata in 2006. Prof. Caprio was an attending physician in the Units of Nuclear Medicine at Bichat Claude-Bernard Hospital and Pitié-Salpêtrière Hospital in Paris, France (2003–2005). He was a visiting fellow at the National Institute of Child Health and Human Development at the National Institutes of Health, Bethesda, USA (1997–1998), and a postdoctoral fellow in the Unit of Experimental Medicine (U36) at INSERM in Collège de France in Paris, France (2003–2007). He has been adjunct professor of Endocrinology at the Catholic University “Zoja e Këshillit të Mirë” in Tirana, Albania (2013–2016). Prof. Caprio has several national and international committee memberships and editorships. Currently, he is specialty chief editor for Frontiers in Clinical Diabetes and Healthcare (Section on Diabetes Geographical Inequalities) and senior editor for the Journal of Endocrinology and Journal of Molecular Endocrinology. xi

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About the Editors

He is also associate editor for the Journal of Diabetes and Its Complications and Frontiers in Endocrinology (Section on Obesity). He is an international member of the French National Research Agency (Commission 17 – Translational Research in Health) and has served as a member of the Annual Meeting Steering Committee of the Endocrine Society (USA) from 2020. He is also a member of the Executive Committee of the Italian Society of Endocrinology (SIE) and deputy coordinator of the Scientific Committee of SIE. He has been coordinator of the Cardiovascular Endocrinology Club of SIE since 2017. Prof. Caprio has an internationally recognized expertise in the exploration of the roles of mineralocorticoid receptor activation in adipose tissue and vessels. He has contributed a large amount of studies on the pathophysiology of adipose proliferation and endothelial inflammation due to excessive mineralocorticoid activation. His research interests focus on the cardiovascular and metabolic effects of novel nonsteroidal mineralocorticoid receptor antagonists, as well as on the interactions between nutritional status, ketone bodies, and the components of the renin-angiotensin-aldosterone-system. Prof. Caprio is co-inventor of a patent (Mineralocorticoid receptor antagonists for the treatment of corticoidinduced obesity – Code: WO 2012/059594 A1) and has authored more than 150 scientific publications. He has been invited to be a guest speaker and/or visiting scientist by many national and international academic and scientific institutions and has given numerous lectures around the globe (France, Netherlands, Germany, Switzerland, Denmark, Spain, Greece, Poland, Albania, UK, USA, Canada, Australia, Japan, Brazil, etc.). Fabio Luiz Fernandes-Rosa Paris Cardiovascular Research Center (Inserm U970), Université Paris Cité Paris, France Dr. Fabio Luiz Fernandes-Rosa, MD, PhD, is a senior researcher at the Paris Cardiovascular Research Center (PARCC) – Institut national de la santé et de la recherche médicale (Inserm) U970, in the “Genetic Mechanisms of Aldosterone-Related Disorders Towards Integrative Precision Medicine” Team.

About the Editors

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He received his MD at the Federal University of Triangulo Mineiro (Brazil) in 1998 and board certification in Pediatrics and Pediatric Endocrinology (2003) at the School of Medicine of Ribeirao Preto (University of Sao Paulo, Brazil). He received his Master’s Degree (2005) and PhD (2009) in Pediatrics and Pediatric Endocrinology at the University of Sao Paulo on the clinical and genetic characterization of families with renal pseudohypoaldosteronism type 1. He is a former attending physician at the Pediatric Endocrinology Service of the University Hospital of the School of Medicine of Ribeirao Preto (University of Sao Paulo, Brazil) from 2004 to 2009. He completed his postdoctoral studies at Inserm unit 970 (Paris, France) under the direction of Prof. Maria-Christina Zennaro, focusing on the molecular aspects of aldosterone action and the genetic basis of primary aldosteronism and the development of aldosterone-producing adenomas. His research interests have focused for the last 15 years on the clinical and genetic aspects of endocrinology and pediatric endocrinology, in particular translational research in aldosterone-related disorders as pseudohypoaldosteronism type 1 and primary aldosteronism. His actual project concerns genome-wide strategy to explore the genetics and genomics of aldosterone response in target tissues, aiming at the discovery of new mineralocorticoid axis components with potential therapeutic options for hypertension and salt-wasting syndromes. In parallel, he works on the genetics of primary aldosteronism and molecular determinants of adrenal gland pathophysiology. Dr. Fernandes-Rosa is a member of the Endocrine Society and French Society of Endocrinology and participates actively in the scientific dissemination, being invited as a speaker in national and international scientific meetings. He is associate editor of Frontiers Cellular Endocrinology (specialty section of Frontiers in Cell and Developmental Biology and Frontiers in Endocrinology), has been invited editor for Molecular and Cellular Endocrinology, and is a reviewer for many scientific journals focusing on endocrinology and cardiovascular diseases.

Contributors

Sonir Roberto Rauber Antonini Pediatric Endocrinology, Department of Pediatrics, Ribeirao Preto Medical School – University of Sao Paulo, Sao Paulo, Brazil José Antunes-Rodrigues Department of Physiology, School of Medicine of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil Andrea Armani Laboratory of Cardiovascular Endocrinology, IRCCS San Raffaele, Rome, Italy Department of Human Sciences and Promotion of the Quality of Life, San Raffaele Roma Open University, Rome, Italy Giorgio Arnaldi Division of Endocrinology and Metabolic Diseases, Department of Clinical and Molecular Sciences (DISCLIMO), University Hospital of Ancona, Polytechnic University of Marche, Ancona, Italy Sheerazed Boulkroun Université Paris Cité, PARCC, Inserm, Paris, France Massimiliano Caprio Laboratory of Cardiovascular Endocrinology, IRCCS San Raffaele, Rome, Italy Department of Human Sciences and Promotion of the Quality of Life, San Raffaele Roma Open University, Rome, Italy Cristian A. Carvajal Department of Endocrinology, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile Centro Traslacional de Endocrinología UC (CETREN), Santiago, Chile Annamaria Colao Dipartimento di Medicina Clinica e Chirurgia, Sezione di Endocrinologia, Università “Federico II” di Napoli, Naples, Italy UNESCO Chair for Health Education and Sustainable Development, University Federico II, Naples, Italy Dario De Alcubierre Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy Júlia Ferreira de Carvalho Endocrinology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy xv

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Contributors

Nicola Di Paola Dipartimento di Medicina Clinica e Chirurgia, Sezione di Endocrinologia, Università “Federico II” di Napoli, Naples, Italy Lucila Leico Kagohara Elias Department of Physiology, School of Medicine of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil Carlos E. Fardella Department of Endocrinology, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile Centro Traslacional de Endocrinología UC (CETREN), Santiago, Chile Fabio Luiz Fernandes-Rosa Université Paris Cité, PARCC, Inserm, Paris, France Emanuele Ferrante Endocrinology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy Rosario Ferrigno Dipartimento di Medicina Clinica e Chirurgia, Sezione di Endocrinologia, Università “Federico II” di Napoli, Naples, Italy Benedetta Fibbi Pituitary Diseases and Sodium Alterations Unit, Endocrinology, Department of Medicine and Geriatrics, Careggi University Hospital, Florence, Italy Giulia Giancola Division of Endocrinology and Metabolic Diseases, Department of Clinical and Molecular Sciences (DISCLIMO), University Hospital of Ancona, Polytechnic University of Marche, Ancona, Italy Juliette Hadchouel CoRaKID – Inserm UMR_S1155, Paris, France Sorbonne Université, Faculty of Medicine, Paris, France Marguerite Hureaux Department of Genetics, Hôpital Européen Georges-Pompidou, Paris, France Centre de Référence des Maladies Rénales Héréditaires de l’Enfant et de l’Adulte (MARHEA), Paris, France Andrea M. Isidori Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy Thais Milioni Luciano Pediatric Endocrinology, Department of Pediatrics, Ribeirao Preto Medical School – University of Sao Paulo, Sao Paulo, Brazil Alessandra Mangone Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy Endocrinology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy Giovanna Mantovani Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy Endocrinology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy

Contributors

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Laetitia Martinerie Inserm Unit 1185, Paris-Saclay University, Paris, France Pediatric Endocrinology Department, Paris Cite University, Paris, France Marianna Martino Division of Endocrinology and Metabolic Diseases, Department of Clinical and Molecular Sciences (DISCLIMO), University Hospital of Ancona, Polytechnic University of Marche, Ancona, Italy André Souza Mecawi Department of Biophysics, Paulista School of Medicine, Federal University of Sao Paulo, Sao Paulo, Brazil Laboratory of Molecular Neuroendocrinology, Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil Dario Norello Pituitary Diseases and Sodium Alterations Unit, Endocrinology, Department of Medicine and Geriatrics, Careggi University Hospital, Florence, Italy Alessandro Peri Endocrinology, Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy Pituitary Diseases and Sodium Alterations Unit, Endocrinology, Department of Medicine and Geriatrics, Careggi University Hospital, Florence, Italy Rosario Pivonello Dipartimento di Medicina Clinica e Chirurgia, Sezione di Endocrinologia, Università “Federico II” di Napoli, Naples, Italy UNESCO Chair for Health Education and Sustainable Development, University Federico II, Naples, Italy Chloé Rafael CoRaKID – Inserm UMR_S1155, Paris, France Sorbonne Université, Faculty of Medicine, Paris, France Silvia Graciela Ruginsk Department of Physiological Sciences, Biomedical Sciences Institute, Federal University of Alfenas, Alfenas, Brazil Elisa Sala Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy Endocrinology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy Emilia Sbardella Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy Chiara Simeoli Dipartimento di Medicina Clinica e Chirurgia, Sezione di Endocrinologia, Università “Federico II” di Napoli, Naples, Italy Alejandra Tapia-Castillo Department of Endocrinology, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile Centro Traslacional de Endocrinología UC (CETREN), Santiago, Chile Thomas Uslar Department of Endocrinology, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile Centro Traslacional de Endocrinología UC (CETREN), Santiago, Chile

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Contributors

Rosa Vargas-Poussou Department of Genetics, Hôpital Européen Georges-Pompidou, Paris, France Centre de Référence des Maladies Rénales Héréditaires de l’Enfant et de l’Adulte (MARHEA), Paris, France Maria-Christina Zennaro Université Paris Cité, PARCC, Inserm, Paris, France Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Service de Génétique, Paris, France

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Neuroendocrine Regulation of Hydrosaline Metabolism Silvia Graciela Ruginsk, Lucila Leico Kagohara Elias, Jose´ Antunes-Rodrigues, and Andre´ Souza Mecawi

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Historical Background: The Pioneer Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . From Sensors to Effector Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Control of Extracellular Fluid Osmolality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Control of Extracellular Fluid Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Integrating Brain, Heart, and Kidneys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Renin-Angiotensin-Aldosterone System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Natriuretic Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Integrated Autonomic Control of Cardiovascular System and Hydromineral Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Novel Local Players on the Control of Hypothalamic Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gaseous Modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endocannabinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . From Fetal Life to Senescence: The Neuroendocrine Regulation of Hydromineral Balance During the Life Span . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fetal Programming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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S. G. Ruginsk Department of Physiological Sciences, Biomedical Sciences Institute, Federal University of Alfenas, Alfenas, Brazil e-mail: [email protected] L. L. K. Elias · J. Antunes-Rodrigues Department of Physiology, School of Medicine of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil e-mail: [email protected]; [email protected] A. S. Mecawi (*) Department of Biophysics, Paulista School of Medicine, Federal University of Sao Paulo, Sao Paulo, Brazil Laboratory of Molecular Neuroendocrinology, Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil e-mail: [email protected] © Springer Nature Switzerland AG 2023 M. Caprio, F. L. Fernandes-Rosa (eds.), Hydro Saline Metabolism, Endocrinology, https://doi.org/10.1007/978-3-031-27119-9_1

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Sex-Related Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The present chapter will address some evolutionary, historical, and contemporary perspectives concerning the integrative neuroendocrine control of hydrosaline metabolism, focusing on the major neural and endocrine systems implicated in the control of water and sodium balance. This chapter will bring the readers the new advances on the understanding of how our body controls extracellular fluid volume and osmolality, initiating with the sensory afferent mechanisms and advancing on effector responses accomplished by an integrated action of the renin-angiotensin and autonomic systems, atrial natriuretic peptides, and hypothalamic neurohypophysial hormones. We will also discuss the contribution of transcriptomic analyses to the study of magnocellular neurosecretory function, and also how novel regulators (gaseous modulators, endocannabinoids, and glial cells) have increased the complexity of this already robust network. Finally, the chapter will illustrate how the neuroendocrine regulation of hydromineral balance behaves under normal or pathological life span challenges, such as adaptations in response to fetal programing, sex- and age-related management of water/sodium balances, and physical activity. Keywords

Hypothalamic neurohypophysial system · Renin-angiotensin-aldosterone system · Natriuretic peptides · Thirst · Sodium appetite

Introduction The emergence of first cells in earth assumes that the synthesis of organic compounds and their agglomeration due to their physicochemical affinities took place in the pristine ocean. Some theories argue that the first cells would have appeared in a K+-rich environment, which would explain, at least in part, the high intracellular concentration of this ion. Later in the evolutionary process, our first multicellular ancestor most likely originated from a water environment, rich in sodium bicarbonate (NaHCO3), in contrast to the current composition of the ocean, in which NaCl is more abundant. Thus, it is not difficult to understand the higher intracellular K+ levels in relation to the higher extracellular Na+, HCO3
, and Cl
levels in the modern vertebrates. It also explains the high levels of water in both intra- and extracellular medium in all organisms. According to how life appears and evolves on earth, both unicellular and multicellular organisms are surrounded by an extracellular medium rich in Na+ ions, being all biochemical and physiological processes well adapted to occur in an aqueous medium primarily containing constant Na+ concentrations and volume.

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The regulation of cellular volume in unicellular organisms is a complex process, since this ability is the first evolutionary step to individualize a biological system and its biochemical reactions from the universe. In this regard, several adaptive strategies emerged and were selected to guarantee the volume regulation in unicellular species, such as cysts and spores of fungi and bacteria, rigid cellulose walls in bacteria and most algae, and by using contractile vacuoles such as in protozoans. The evolutionary process from single cells to multicellular organisms increases the life ability to regulate and maintain the steady state of internal environment by the emergence of cells, tissues, organs, and systems specialized on the homeostatic balance. The further emerged renal and neuroendocrine systems were crucial for the adaptations needed to the proper control of hydromineral balance, allowing the great evolutionary success and wild range of environments inhabited by vertebrates nowadays. The kidney is the primary organ involved in the control of extracellular volume and composition in humans, but several other osmoregulatory organs are also important, such as bladder and skin. In complex organisms, the intracellular and extracellular compartments, both belonging to the internal environment, establish a constant and intense exchange of water and other substances across the membranes. In order to maintain intracellular/ extracellular volume and composition in a relative steady-state condition, these variables are constantly monitored by integrative control systems, comprised of specific sensory machineries. These systems transduce water or ionic imbalances into the activation of multiple parallel and redundant effector responses, responsible for bringing these physiological variables back to basal levels, thus interrupting input signals. Since life on earth evolved within an aqueous milieu, it is not surprising that water may correspond to the main component in all living beings, despite the well-known discrepancies between intracellular and extracellular ionic compositions. In a single day, most people ingest an average of 2.2 L of water, and normal aerobic metabolism provides an additional volume of 0.3 L, accounting for 2.5 L of input solvent. Under normal conditions, the gastrointestinal tract absorbs virtually all luminal water, excreting only a small amount in feces. The main final route for water excretion is represented by the kidneys: in euhydrated individuals, renal water elimination accounts for approximately 1.8 L, with the remaining output being represented by insensible losses, such as perspiration and air humidification in the respiratory system. On the other hand, sodium chloride (NaCl) is the most abundant electrolyte found in the extracellular fluid (ECF) and drives most of the osmotic-originated movement of water to this compartment. Since ECF NaCl concentrations and osmolality critically regulate cellular volume, this variable should also be maintained within narrow limits of variation. However, increased NaCl consumption has recently become a worldwide concern: most people ingest more than twofold the recommended by the World Health Organization (WHO), which currently advocates a diary consumption of less than 2 g of Na+ or less than 5 g of NaCl. The chronic and excessive consumption of Na+ has been directly associated with the development of arterial hypertension, as well as constitutes an important risk factor for other cardiovascular and renal diseases. As for water, the main final route for sodium excretion is the kidney, with small amounts being eliminated in sweat and in feces (Fig. 1).

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Fig. 1 Pathways for hydrosaline metabolism management in adults. The main route for water and sodium excretion takes place from urinary excretion. Nevertheless, water and sodium can also be lost by defecation, humidification of the inspired air, and sweating. The principal way to acquire water and sodium is through liquids and food intake. (Reproduced with the permission from Ruginsk et al. (2015))

In order to achieve urine concentrations ranging from 50 (hypoosmotic) to 1200 mOsm/kg H2O (hyperosmotic), the kidneys have the ability to decouple water from NaCl reabsorption along some segments of the nephron. This is achieved due to the coordination of a complex structural organization between vascular and tubular renal elements (countercurrent mechanism) and a preserved Na+/urea tubular transport, responsible for the maintenance of a hyperosmotic medullary gradient. However, despite the main role played by the kidneys in hydromineral balance, the activation of thirst and sodium appetite is essential to counterbalance renal output. Thirst and sodium appetite are essential physiological sensations that lead the animal to search for and obtain water and salt from the environment to restore ECF volume and composition. Therefore, most hydromineral imbalances are associated with

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changes in thirst and sodium appetite thresholds in mammals. In a dehydrated animal, for example, the combined ECF volume contraction and increased tonicity generates signals that activate these two motivational behaviors in a temporally dissociated manner: initially, the ingestion of water alone corrects hyperosmolality, allowing a partial restore of volume, then the ingestion of salt is initiated, replenishing, in an isotonic manner, the remaining volume deficit. Although it is hard to know when thirst ends and sodium appetite begins, this double-depletion hypothesis is essential for the overall understanding of dehydration-induced responses and evidences that the increased ECF tonicity is the most critical variable to be regulated, since it is the first to be solved (De Luca et al. 2007). In this context, the participation of the renin-angiotensin system (RAS) should be highlighted, since centrally produced angiotensin II (ANGII) was shown to stimulate both dipsogenic and natriorexigenic responses, in parallel to other important and independent peripheral RAS-mediated actions. Control of water and NaCl balance is triggered by independent afferent sensory pathways that are integrated in the central nervous system (CNS). A negative water balance is associated with enhanced NaCl concentrations and, ultimately, increased ECF osmolality. In specific neuronal groups located around blood-brain barrier windows, hyperosmolality initiates the activation of behavioral and neuroendocrine homeostatic responses that culminate with increased water ingestion and decreased renal water loss. On the other hand, a positive NaCl balance determines an increase in ECF volume, being this parameter monitored by mechanic sensors located particularly in the cardiovascular system. Neural inputs carried by these receptors modulate the activity of brainstem sympathoexcitatory and sympathoinhibitory centers, adjusting cardiac output and vascular resistance. In parallel, renal responses are activated to promote natriuresis and diuresis, thus contributing to the elimination of excessive circulating volume. In the following sections, the readers will be introduced to critical concepts involving body fluid homeostasis, with emphasis on recent advances and new perspectives with clinical relevance in the field of homeostatic systems regulating water and NaCl balances. For review about the evolutionary aspects of hydrosaline metabolism control, please see (Bray 1985; Mecawi et al. 2015a).

Historical Background: The Pioneer Studies The early 1930s through the 1950s were marked by very important morphofunctional advances in neurosciences. Among many achievements, we may highlight the following: (1) anatomic description of brain structures at histological and cellular levels; (2) electrolytic and chemical lesions of brain areas; (3) electrical stimulation of neuronal structures; and (4) the development of the radioimmunoassay, which allowed, by that time, the conduction of studies at the mechanistic level on the involvement of the hypothalamic neurohypophysial system in the regulation of water and salt balance.

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Montemurro and Stevenson reported the involvement of ventromedial hypothalamus in the control of water and food intake and by using electrolytic lesions. At the same period, studies performed in the USA, Sweden, and Brazil also investigated the hypothalamic control of hydromineral balance. Thereafter, the anatomical characterization of the anterior hypothalamus helped to identify the circumventricular organs (CVOs, devoid of blood brain-barrier – BBB), primarily located at the laminae terminalis (LT), as sensory structures involved in monitoring extracellular osmolality and Na+ concentrations. Grossman published the first studies demonstrating that injections of cholinergic agonists into the hypothalamus increased water intake. Additionally, intracerebroventricular and parenchymal injections of carbachol were shown to induce natriuresis, supporting the idea of a stimulatory role for cholinergic neurotransmission on sodium excretion. Also during the 1960s, the development of the radioimmunoassay by Berson and Yalow made it possible to quantify the circulating levels of drugs, peptides, and hormones. By employing this technique, it was possible to uncover the neuroendocrine control of renal sodium and water excretion/reabsorption. In this line, Stricker and Verbalis showed that a decrease in plasma osmolality was able to decrease plasma vasopressin (AVP) and oxytocin (OXT) concentrations. Baldissera et al. demonstrated that carbachol injection into the anteroventral region of the third ventricle (AV3V) releases AVP, OXT, and atrial natriuretic peptide (ANP) into the systemic circulation, and that these effects were associated with antidiuresis and natriuresis. These data linked the CNS cholinergic transmission with a natriuretic effect induced by osmotic stimulation mediated by neuroendocrine changes. After that, Haanwinckel et al. demonstrated that the direct stimulation or lesions of the AV3V region changed ANP circulating levels, as well as the removal of the neural lobe of the pituitary gland completely disrupted ANP release induced by extracellular volume expansion. Taken together, these results indicated that both AV3V region and neural lobe of the pituitary gland were essential for the control of ANP release. Later, it was demonstrated that OXT acts directly on cardiomyocytes to stimulate ANP release. Along the past decades, we experienced a huge methodological advance in biomedical research. Our understanding of how the CNS regulates the hydromineral homeostasis was massively impacted due to the following advances: (1) microscopy/ imaging analyses; (2) hormones, neuropeptides, and neurotransmitter measurements; (3) electrophysiological approaches; (4) protein isolation and characterization; (5) peptide synthesis, immunohistochemistry for neuronal tracing, and activation evaluation; and (7) molecular biology techniques. Using c-Fos imunohistochemical technique, it was demonstrated that both intracerebroventricular and intravenous infusions of ANGII induce the expression of this protein, which was considered as a marker for neuronal activation, in the LT, supraoptic (SON), paraventricular nuclei (PVN), bed nucleus of stria terminalis, and central amygdaloid nucleus. Interestingly, the intravenous ANGII injection was shown to induce more intense increase in c-Fos immunoreactivity in the subfornical organ (SFO) and in the organum vasculosum of the laminae terminalis (OVLT), both located at the LT. In contrast, when ANG II was intracerebroventricularly administered, c-Fos expression

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was observed primarily in the median preoptic nucleus (MnPO). Accordingly, it was recently demonstrated that ANGII is sinaptically released within the MnPO, confirming the central actions of this peptide in activating neurons inside the BBB. Also, still using the c-Fos immunoreactivity mapping, diencephalic, forebrain, and brainstem structures were confirmed as encephalic areas regulating fluid balance. These groups further explored the specific neuronal phenotypes expressed by these structures, confirming, among other findings, the involvement of the dorsal raphe nucleus (DRN) serotonergic neurons in the inhibition of sodium appetite. Furthermore, by manipulating ECF osmolality and/or volume, the studies revealed a complex network integrating PVN and SON (vasopressinergic and oxytocinergic), DRN (serotonergic), A1 (caudal ventral lateral medulla – CVLM), A2 (nucleus of the solitary tract – NTS), and A6 (locus coeruleus – LC), and C1 (rostral ventral lateral medulla – RVLM) catecholaminergic pathways. Finally, Denton, McKinley, and their collaborators made important discoveries on the neuronal circuitry involved in the development of thirst, water intake, and satiety in humans. By employing positron emission tomography, these groups demonstrated a positive correlation between increased plasma sodium levels and the activation of several brain areas such as AV3V region, insula, cerebellum, anterior cingulate region, as well as middle temporal, parahippocampal, inferior, and middle frontal gyri. More recently, through functional magnetic resonance approaches, they have also demonstrated the activation of anterior midcingulate cortex, anterior insula, precentral gyrus, inferior frontal gyrus, middle frontal gyrus, and operculum when fully hydrated participants just imagined an intense thirst sensation or drinking water (Saker et al. 2020), confirming the implication of that brain regions on the genesis of thirst sensation in humans. For review the historical aspects related to the neuroendocrine control of hydrosaline metabolism, please see (Mecawi et al. 2015a).

From Sensors to Effector Systems The Control of Extracellular Fluid Osmolality Osmolality constitutes the measurement of the osmotic strength of a given solution, being expressed as the total osmolyte concentration per kg of solvent (water). The terms osmolality and osmolarity (total osmolyte concentration per liter of solution) are sometimes used interchangeably because for much diluted solutions, such as biological fluids, both osmolarity and osmolality tend to be equivalent. As postulated, the inequality in the concentration of ions and of other molecules between the intra- and extracellular compartments generates chemical gradients, which impel the passive movement of water through the membrane (osmosis). At this point, it is crucial to highlight that water movement is only produced in response to the osmotic gradient generated by solutes that do not cross the membrane (reflection coefficient (σ) ¼ 1). In other words, a hydraulic pressure called osmotic force must be applied to the side of the membrane in contact with a certain solution, in relation to the side in contact with pure water, in order to maintain the water flow between the two

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compartments equal to zero. This hydrostatic pressure then counteracts water’s tendency to move from a region where its chemical potential is higher to another where its chemical potential is lower. Thus, changes in ECF osmolality trend to promote parallel alterations in cellular volume, caused by the osmotically driven movement of water between the two compartments. Since most biological membranes are semipermeable, i.e., allow the passage of solutes in a regulated manner, the osmotic gradient is reduced. Therefore, effective osmolality (or tonicity) is calculated by the product of osmolality by the solute reflection coefficient, which is intrinsically associated to its permeability across the membrane. For example, for a given ion whose σ ¼ 0.5, the tonicity will be only 50% of the measured osmolality. Water movement across the hydrophobic lipid bilayers of cell membranes is strongly facilitated by the presence of specific water channels (aquaporins, AQPs), whose expression could be either ubiquitous or induced. Structurally, AQPs are tetramers, with each monomer acting as an individual pore. Since the 1990s, scientists identified and characterized 13 members of the mammalian AQP family, six of which may be found in the kidneys. In the renal collecting ducts, the expression of AQP2 in the apical membrane of the principal cells mediates AVP-induced increase in water absorption. This process is initiated by AVP binding to type 2 (V2) metabotropic receptors, expressed by the basolateral membrane of these cells. As a result, the serine residue 256 on the C-terminus of AQP2 is phosphorylated by protein kinase A, triggering the translocation of vesicles containing AQP2 to the apical membrane, where they are incorporated. The final outcome is increased water permeability at the distal nephron, where this type of transport normally does not occur in the absence of hormonal stimulation. Mutations in the gene encoding AQP2 are therefore correlated with renal abnormalities, resulting in the production of large volumes of hypotonic urine. More recently, the antagonism to V2 receptors produced by vaptans, with the consequent prevention of AVP-induced increase in AQP2-mediated water permeability, has been also explored in substitution to classical diuretic drugs to treat conditions associated with volume retention. For review about structural and functional aspects of aquaporins, please see (Carbrey and Agre 2009). Sodium is the main determinant of osmotically induced water movement. Its critical role in water homeostasis control can be clearly evidenced by hyponatremia, a severe condition of positive water balance (hemodilution) observed, for example, in response to overhydration with electrolyte-free solutions. In the opposite direction, the excessive water loss in sweat (which is a hypotonic solution) produces a negative water balance, leading to ECF hyperosmolality. Indeed, there is consistent evidence in the literature suggesting that AVP also modulates Na+ transport throughout the distal nephron, which may also contribute to the osmotic drive for water reabsorption. AVP apparently exerts its antinatriuretic effects through actions on diverse molecular targets, including the sodium-potassium-chloride cotransporter (NKCC2), the thiazide-sensitive sodium-chloride cotransporter (NCC), and the epithelial sodium channel (ENaC). Osmo- and Na+ sensors found particularly in CNS areas located in the LT region depolarize in response to increased ECF osmolality or Na+ concentrations. The LT is

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a forebrain structure adjacent to the anterior portion of the cerebral third ventricle. It is comprised of the MnPO and two other CVOs, the SFO and the OVLT. The OVLT and the ventral portion of MnPO, together with both the preoptic periventricular region and the anterior periventricular hypothalamic area, constitute the AV3V region, whose upward continuation contains the SFO. Since the BBB is less restrictive in CVOs, osmo- or Na+-sensitive neurons are in close relationship with the cerebrospinal fluid and, consequently, subject to rapid changes in its ionic composition. Furthermore, the activity of LT neurons is strongly influenced by other circulating factors, whose actions in the control of different aspects of body fluid homeostasis will be further discussed in this chapter. Importantly, osmo- or Na+ receptors are also present in the gastrointestinal tract, particularly in the oropharynx and mesenteric circulation, which enables the ingested volume of water to appropriately match to the need, preventing excessive solvent input even before a detectable correction in peripheral osmolality takes place. Accordingly, water deprivation–induced hyperosmolality was shown to consistently increase neuronal activation in LT structures involved in osmotic signal transduction. It has been also demonstrated that the rate of action potentials in OVLT neurons increases as osmolality is incremented, regardless the presence of an intact synaptic network, indicating that this LT structure would be the one primarily involved in this function. This actually was proven to be a very precise mechanism, since the intracerebroventricular injection of hypertonic sucrose solution was less effective in inducing thirst and antidiuretic responses than hypertonic NaCl infusion, suggesting the existence of a specific Na+-sensor-mediated pathway, as postulated by pioneer studies. The hypothalamus is the main route integrating sensory information carried from the LT and visceral information carried by the NTS, located in the brainstem. The magnocellular neurons of the SON and PVN of the hypothalamus produce AVP (mostly known as antidiuretic hormone), the main regulator of water homeostasis, as introduced before in this section. The terminals of magnocellular neurons are found in the neurohypophysis, where AVP is stored until a proper stimulus reaches the neuronal hypothalamic cell bodies. Importantly, magnocellular neurons also exhibit intrinsic osmosensitive properties, responding with increased cationic membrane conductance and firing rates to the dehydration-induced decrease in cellular volume produced by ECF hypertonicity. Although the molecular identity of these cation channels remains unknown, a growing body of evidence indicates that members of the transient receptor potential vanilloid (TRPV) family of ion channels may underlie these homeostatic responses. In OVLT and hypothalamic neurons, hypertonicity is possibly transduced via the activation of nonselective cation channels, being this effect absent in mice lacking expression of the transient receptor potential vanilloid 1 (Trpv1) but not the Trpv4 gene. On the other hand, recent findings from the same group suggest that Ca2+ influx through Trpv4 channels expressed in OVLT neurons mediate a glia-to-neuron signaling elicited by hypotonicity, ultimately resulting in reduced AVP release from the magnocellular neurosecretory system. This mechanism would be accomplished by the release of taurine from glial cells, which are the only source of this signaling

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amino acid within the SON. Accordingly, taurine was shown to exert a tonic inhibitory action on magnocellular neurons which inversely correlates with ECF tonicity (Prager-Khoutorsky and Bourque 2015; Ciura et al. 2018). Besides hyperosmolality, AVP secretion is also regulated by blood pressure or circulating volume, as shown in Fig. 2. As discussed in the following paragraphs, AVP also interacts with type 1 receptors (V1) to promote vasoconstriction responses, thus regulating peripheral vascular resistance and contributing to adequate tissue perfusion. However, the threshold for AVP secretion is lower in response to hyperosmolality than in response to a volume deficit: a detectable change in AVP secretion is promptly observed in response to osmolality deviations as small as 1–2%, whereas a loss of about 10% in the circulating volume/pressure is required to produce a similar effect in AVP release (Koeppen and Stanton 2013). As discussed before, in the absence of AVP or disrupted V2R-mediated signaling, the kidneys are unable to concentrate urine, culminating in the excretion of large amounts of free water. There are three clinically relevant conditions associated with disrupted AVP secretion or action (Cuzzo et al. 2022). The first is called the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) and is characterized by highplasma AVP concentrations. SIADH may be an outcome in certain malignancies, CNS disturbances (e.g., stroke, hemorrhage, infection, and trauma), and also in response to some surgical or pharmacological interventions, among other causes.

Fig. 2 Vasopressin (AVP) plasma concentrations (arbitrary units, Y axis) in response to volume and osmolality changes (X axis). Under basal conditions, AVP plasma concentrations are very low and hypervolemia can further reduce the threshold for AVP secretion in response to hyperosmolality. Conversely, in response to deficits in plasma volume/pressure greater than 10%, AVP secretion increases. The combined effect of decreased blood volume/pressure and hyperosmolality lowers the set point for AVP secretion, as demonstrated by the increased slope. Max: maximum. (Reproduced with the permission from Koeppen and Stanton (2013))

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Conversely, the two remaining pathologic states related to inappropriate AVP secretion account for diabetes insipidus due to deficiency of AVP secretion or resistance to its effects, resulting in intense polyuria and, consequently, polydipsia. Patients diagnosed with central diabetes insipidus exhibit decreased AVP production, whereas peripheral disruption of AVP action is associated with impaired AQP2- or V2-receptor-mediated signaling. Central diabetes insipidus is the more common form and often observed in response to central damages (hypothalamus or posterior pituitary). Nephrogenic (peripheral) diabetes insipidus, in turn, can be either inherited (mutations in the genes encoding AQP2 or V2 receptor) or appear later in life in response to acquired conditions. A secondary hormone participating in the control of Na+ homeostasis is OXT, a peptide also produced by the hypothalamic neurohypophysial system. OT exhibits a very clear natriuretic action in rodents (but not in some other species), originated from the inhibition of ENaC-mediated Na+ reabsorption in the distal nephron. The main stimulus determining an increase in OT secretion is hypervolemia, although OT can be co-released with AVP in response to ECF hyperosmolality. This pattern is also observed for other natriuretic factors, such as ANP, secreted from atrial cardiomyocytes in response to hypertonicity but primarily regulated by circulating volume. The brainstem provides sensory and motor innervation to the head and neck via the cranial nerves, and also relays most connections originated from the rest of the body. This brain region is situated ventral to the cerebellum, between the diencephalon and the spinal cord and contains the medulla oblongata, pons, and midbrain. By working coordinately with the diencephalon, the brainstem plays a crucial role in the neuroendocrine and autonomic regulation of virtually all vital functions. The peripheral afferents from osmorreceptors, volume receptors, and arterial baro- and chemoreceptors primarily ascend to the NTS through the vagus and/or glossopharyngeal nerves and use glutamate as the main neurotransmitter. Additionally, NTS also receives inputs related to salt taste sensation, integrating this gustatory information to peripheral changes in hydromineral balance. As the main convergent nucleus in the brainstem for peripheral information, the NTS receives afferents not only from the peripheral nervous system, but also from the CVO area postrema (AP). Similarly to SFO and OVLT, the AP is also sensitive to plasma circulating hormones, such as ANGII and ghrelin. The NTS neurons are also under the control of mineralocorticoid hormones, such as aldosterone, that freely cross the BBB and modulate sodium appetite. Therefore, the brainstem neuronal circuitry is crucial for integrating the peripheral cardiovascular and hydromineral information to an appropriate autonomic response that modulates other regulatory systems, such as the RAS. This is possible since the NTS also controls the activity of the inhibitory neurons at the CVLM, which in turn regulates the activity of the RVLM, the most important brainstem nucleus for the control of sympathetic preganglionic neuron activity. Another interesting aspect is the functional connection between the AP and NTS and the lateral parabrachial nucleus (LPBN) and DRN, all essential for the control of the neuroendocrine and behavioral regulation of hydromineral balance. This brainstem circuitry is also

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reciprocally connected with forebrain structures, such as the hypothalamic PVN and SON, modulating AVP and OXT secretion, among other regulatory responses. Thus, the brainstem has a crucial role in the control of body water and sodium balance by modulating sympathetic input to the kidneys, controlling the activity of the neurohypophysial system and regulating thirst and sodium appetite. This neural circuitry is illustrated in Fig. 3. In summary, the control of ECF osmolality is finely regulated by highly specialized sensory systems located mainly in the CNS, but also assisted by peripheral information. Inputs generated by these sensors are then integrated in the hypothalamus and brainstem, so that increased ECF osmolality determines primarily an increment in AVP secretion. As the main regulator of renal water balance, and in the presence of a preserved medullary osmotic gradient, AVP determines an increase in water reabsorption, which ultimately results in the excretion of small volumes of hyperosmotic urine. If the increase in osmolality is accompanied by a parallel

Fig. 3 Sagittal representation of the complex interconnections among hypothalamic and brainstem nuclei is related to the neuroendocrine control of hydrosaline metabolism. The blood-borne signals (ANG) are monitored by the circumventricular organs that are responsible for transmitting these informations to other brain nuclei. Conversely, peripheral afferents (baroreceptors, chemoreceptors, osmoreceptors, volume receptors, and gustatory information) reach the NTS through the vagus and/or glossopharyngeal nerve, which redistributes this information to several brain areas. Finally, the humoral and sensory signals constitute to the multimodal information that are integrated by the central nervous system to control the extracellular volume and osmolality. ANG II angiotensin II, ANP atrial natriuretic peptide, AVP vasopressin, ac anterior commissural nucleus, oc optic chiasm, OXT oxytocin. (organum vasculosum of the lamina terminalis [OVLT], subfornical organ [SFO], and median preoptic nucleus [MnPO]), paraventricular (PVN) and supraoptic (SON) hypothalamic nucleus, midbrain dorsal raphe (DRN) and lateral parabrachial nucleus (LPBN), medullary nucleus of the solitary tract (NTS), area postrema (AP), caudal ventral lateral medulla (CVLM), and rostral ventral lateral medulla (RVLM). (Reproduced with permission from Mecawi et al. (2015a))

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decrease in ECF volume, the threshold for AVP secretion becomes even lower. Finally, if kidneys are unable to restore or maintain normal plasma osmolality, the activation of limbic and cortical areas guarantees the acquisition of water and Na+ through the activation of dipsogenic and natriorexigenic responses, respectively. As further discussed in the following section, these effects also accomplish for the control of ECF volume and are triggered by independent ANGII-mediated central actions. To review the mechanisms related to the extracellular osmolality control, please see (Mecawi et al. 2015a; Bourque 2008; Bichet 2019).

The Control of Extracellular Fluid Volume Water constitutes an average of 60% of total body mass in an adult individual, of which about two-third are located inside the cells. Ions and other molecules are also unevenly distributed between the intra- and extracellular compartments, with Na+ being the major ECF constituent. As discussed in the previous sections, water moves passively between compartments, following the osmotic gradient generated by solutes, particularly Na+. Although the addition of Na+ alone to the ECF transiently increases its osmolality, highly effective and fast effector mechanisms increase water intake and reabsorption, increasing ECF volume proportionally to the amount of Na+ added. In this new state of equilibrium, the addition of Na+ becomes equivalent to the addition of a given volume of isotonic solution, increasing circulating volume. Sodium acquisition occurs mainly through the diet and the kidneys are the main source for its excretion, in a way that only 10% of the total ingested Na+ leaves the body using alternative routes. Therefore, considering the general law of mass balance, if dietary Na+ intake exceeds its excretion (positive balance), the ECF volume increases. On the other hand, if the excretion of Na+ exceeds its intake, the individual undergoes a negative balance and a reduction in ECF volume (Koeppen and Stanton 2013). The response of the kidneys to variations in Na+ consumption is not immediate, taking normally hours to days to be entirely established (Fig. 4). Although the ECF is subdivided between the intravascular and interstitial compartments, plasma volume is the main determinant of cardiac output and blood pressure. Since the primary volume sensors are located in large vessels, changes in intravascular volume, blood pressure, and cardiac output are the main regulators of Na+ excretion by the kidneys. This evidence derives directly from the concept of effective circulatory volume (ECV), which consists of the volume that effectively perfuses tissues in order to allow appropriate nutrient and metabolite exchanges. This is particularly relevant to the understanding of the mechanisms underlying Na+ balance in some clinical situations, such as congestive heart failure, during which renal Na+ excretion may not properly reflect changes in ECV. In advanced stages of this disease, there is a clear reduction in circulating volume, which occurs in parallel with fluid accumulation in the interstitial space (edema). Thus, despite the fact that the total volume of the ECF is increased, the volume sensors located in the vascular system transduce the relative decrease in the ECV volume into the activation of Na+ sparing mechanisms, aggravating rather than solving the original condition.

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Fig. 4 Relationship between changes in NaCl intake/excretion (lower panel) and body weight (upper panel). Na+ excretion by the kidneys (lower panel, dashed line) does not quickly follow abrupt changes in Na+ intake (lower panel, solid line). The change in extracellular fluid volume that occurs in response to positive and negative Na+ balance (black arrows) produces parallel alterations in body weight. (Reproduced with the permission from Koeppen and Stanton (2013))

Considering that the side of the vascular system that deals with low pressures has high compliance, the receptors located in the walls of the atria, right ventricle, and large pulmonary vessels respond mainly to the filling of the vascular system, being referred as volume receptors. The activity of these mechanoreceptors modulates the sympathetic activity and the secretion of AVP. Cardiac atria, particularly the right atrium, exhibit an additional mechanism controlling Na+ excretion: in response to the stretching induced by an increase in circulating volume, atrial cardiomyocytes produce ANP, which inhibits renal Na+ reabsorption through ENaCs expressed by the distal nephron epithelium. In addition to the important natriuretic effect, ANP and also type B natriuretic peptide (BNP), produced by the ventricles, promote vasodilation of afferent arterioles and efferent vasoconstriction, consequently increasing glomerular filtration rate and filtered Na+ load, in parallel with the inhibition of renin, aldosterone, and AVP secretion. On the arterial side, vascular baroreceptors, located in the carotid sinus, aortic arch, and renal afferent arterioles, decrease their firing rates in response to falls in ECV, which ultimately produce parallel effects in blood pressure. Sympathetic nerve endings directly innervate the renal afferent arteriole, promoting an increase in vascular resistance and consequently decreasing the glomerular filtration rate and the filtered Na+ load. In addition, through the activation of beta-adrenergic receptors

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present in the granular cells of the justaglomerular apparatus, the increased sympathetic outflow determines an enhancement of local renin production. Furthermore, the afferent arteriole also responds directly to changes in blood pressure: If the perfusion rate is decreased, granular cells of the justaglomerular apparatus are activated, thus triggering renin release and the consequent activation of peripheral RAS. In parallel with the effects of renal perfusion and sympathetic stimulation, renin synthesis is also activated in response to a decreased filtered Na+ load that reaches the macula densa (tubuloglomerular feedback). Renin is classically responsible for the critical step of RAS activation, i.e., the conversion of the precursor angiotensinogen to angiotensin I (ANGI). ANGI is then converted by endothelial cells into ANGII, which stimulates Na+ reabsorption in the proximal tubule and importantly triggers peripheral vasoconstriction and aldosterone secretion by the adrenal cortex. In addition, ANGII exhibits central natriorexigenic and dipsogenic effects, as previously discussed. As the main RAS regulator of Na+ balance, aldosterone counteracts ANP effects by promoting Na+ reabsorption in the distal segments of the nephron through three main mechanisms: (1) increase in the expression of Na+-K+-ATPase in the basolateral membrane of principal cells; (2) increase in the synthesis and incorporation of ENaCs to the luminal membrane of principal cells; and (3) increase in the Na+/Cl
cotransport in the initial segment of the distal tubule. Opposite neuroendocrine responses are observed following increases in volemia and/or blood pressure, such as in response to an isotonic blood volume expansion. Under this experimental condition, projections from baroreceptor induce a consistent increase in the firing rate of the diagonal band of Broca (DBB) neurons, which are responsible for a selective inhibition of the neurosecretory vasopressinergic neurons of the SON. The signaling initiated by both volume and baroreceptors traffics to the CNS through the vagus and glossopharyngeal nerves, reaching the NTS. From this site, direct or indirect projections achieve the hypothalamus, thus controlling AVP and OXT secretion. As previously discussed in this chapter, hyperosmolality is the main regulator of AVP release; however, ECV/blood pressure contractions greater than 10% can also stimulate AVP secretion, decreasing the endocrine threshold for an additional increase in osmolality. OXT, in turn, has synergistic actions to the other natriuretic peptides, particularly ANP, inhibiting ENaC-mediated Na+ reabsorption in the distal nephron through a cyclic guanosine monophosphate (GMPc)-mediated pathway. In summary, the maintenance of Na+ homeostasis requires a precise balance matching ionic gain and elimination. Since the kidneys take hours to days to respond to variations in Na+ intake, during a transitory period excretion may not properly adjust to ingestion, leading the individual to develop positive or negative Na+ balance, with the respective effects on ECF volume. Under euvolemic conditions, the distal segments of the nephron (distal tubule and collecting duct) are the main sites where Na+ reabsorption is adjusted to maintain excretion at appropriate levels. However, this may only occur if the initial portions of the nephron (proximal tubule and Henle loop) are able to deliver a relatively constant portion of the filtered Na+ load (~8%) to the distal tubule. As the main mechanisms positively regulating sodium balance, we may highlight the sympathetic nervous system and RAS, with important effects on renal and behavioral aspects of the integrated response.

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Secondary to these actions are AVP-mediated water reabsorption, which also accomplishes for partial volume replenishment. Counteracting these volume-sparing mechanisms are the natriuretic peptides, particularly OXT and ANP. Therefore, the kidneys critically participate as the main final route for a coordinated action together with the cardiovascular and neuroendocrine systems to regulate ECF volume. For review of the neuroendocrine control of hydromineral balance, please see (Mecawi et al. 2015a; Abraham and Schrier 1994; Antunes-Rodrigues et al. 2004; Bie 2018).

Integrating Brain, Heart, and Kidneys The Renin-Angiotensin-Aldosterone System The control of renin synthesis and secretion is the primary limiting step for systemic RAS activation. Renin is synthesized as pre-pro-renin, which is cleaved into renin, and is stored in the vacuolar system. Plasma renin is primarily synthesized and secreted by renal juxtaglomerular cells in response to low arterial pressure, reduced Na+ and Cl
ion content in the distal convoluted tubule, and β1-adrenergic stimulation. Once in the plasma, renin cleaves the angiotensinogen, an α-globulin mainly produced and secreted by the liver, generating the ANGI, which is a biological inactive product. One of the metabolic pathways for ANGI is dependent on the action of type 1 angiotensin-converting enzyme (ACE1), which is primarily expressed in the vascular endothelium cells as a transmembrane protein (highly expressed in the lungs). This enzyme converts ANGI into ANGII, which is an octapeptide that is the first biologically active compound of the RAS. ANGII can be further converted to the hexapeptide ANGIII by glutamyl aminopeptidase A or to ANG 1–7 by the monopeptidase type 2 (ACE2). ANG 1–7 can also be formed directly from ANGI by the action of endopeptidases, such as neprilysin. Additionally, membrane alanyl aminopeptidase N cleaves ANGIII to the heptapeptide ANGIV. RAS peptides bind to five receptors subtypes: AT1, AT2, AT3, AT4, and Mas receptors. ANGII actions are mediated by AT1 and AT2 receptors. In contrast, the transmembrane Mas receptor was recently identified as a unique receptor that binds ANG 1–7 (reviewed in Karnik et al. 2015) (Fig. 5). Besides the systemic RAS, several evidences highlight the existence of a brain RAS and its importance for the neuroendocrine control of cardiovascular and hydromineral balance (reviewed in Nakagawa et al. 2020). The main effects of RAS activation are aldosterone secretion, vascular constriction, renal water and sodium reabsorption, thirst/sodium appetite induction, and sympathetic activity stimulation. Central ANGII action is also related to the induction of AVP and OXT neuronal activation, gene expression, and hormone secretion. These effects are primarily mediated by the binding of ANG II on AT1 receptors. Hydromineral challenges, such as sodium deprivation or depletion and intracellular and extracellular dehydration, stimulate renin secretion, activating the RAS and, consequently, inducing thirst and/or sodium appetite. At least under physiological conditions, peripheral ANGII does not cross the BBB, but still can acting in the CNS

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Fig. 5 The cascade of renin-angiotensin system components formation, enzymes involved in these processes, and target receptors to each peptide. (Reproduced with permission from Mecawi et al. (2015a))

through CVOs (AP, SFO, and OVLT), which are devoid of BBB and allow peripheral ANGII to induce its brain effects. The AP is involved in the ANGII pressor response, whereas the SFO and OVLT are related to the drinking, pressor, and AVP secretion responses. Also, the CVO cells can uptake circulating ANGI and convert it into ANGII, which can be then secreted inside the BBB to induce thirst and sodium appetite. It was demonstrated that AT1 receptors in the SON are involved in thirst and sodium appetite induced by ANGII via septal area. ANGII signaling in the CNS was also shown to interact with OXT actions, since the central administration of OXT reduces ANGII-induced sodium appetite in rats, while central administration of an OXT receptor (OXTR) antagonist increases ANG II-induced salt intake. Also, central ANP administration inhibited ANGII- and dehydration-induced thirst. Taking together, these results suggest that OXT and ANP act as counter-regulators of ANGII effects on salt intake (reviewed in Mecawi et al. 2015a; Antunes-Rodrigues et al. 2004; Gutkowska et al. 2014). As the main effector of the RAS effects, several studies have addressed the intracellular signaling pathways recruited by the AT1 receptor activation. As a Gq protein–coupled receptor, ANGII binding to AT1 receptors activates protein kinase C (PKC). Additionally, these receptors also recruit the mitogen-activated protein kinase (MAPK) pathway in Gq-protein-dependent and independent manner. In this context, ANGII-induced thirst, but not sodium appetite, seems to be induced by the generation of intracellular phosphatidylinositol (3,4,5)-trisphosphate (IP3) following AT1 activation, since ANGII effect on water intake is inhibited by PKC inhibitors. On the other hand, ANGII-stimulated sodium appetite, but not thirst, is dependent on the activation of MAPK-mediated pathways, since the inhibition of this pathway in the brain reduces salt but not water intake. The endogenous brain ANGII generation induced by the activation of RAS during furosemide-captopril model was shown to

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activate p44/42 MAPK signaling, which is implicated in the induction of sodium appetite but not thirst or AVP and OXT secretion in rats. Thus, the physiological responses related to the regulation of hydromineral balance induced by the central action of ANGII on its AT1 receptor seems to be mediated by dual intracellular signaling mechanisms (Fig. 6). For review, please see (Daniels 2014). Also of interest for the better understanding of neuroendocrine control of hydromineral balance, it was demonstrated that the AT1 receptors are co-expressed with ENaC (sodium taste receptor) in the tongue epithelial taste cells. This work demonstrates that ANGII-AT1 signaling in the tongue suppresses the Chorda tympani nerve responses to NaCl (Shigemura et al. 2013). These intriguing results suggest that the reduction of amiloride-sensitive response to salt mediated by

Fig. 6 Intracellular pathway activated by AT1 receptors and its effects on thirst, sodium appetite, and neurotransmitter release. The AT1 receptor is coupled to a Gq protein which activates the enzyme phospholipase C (PLC) inducing the cleavage of the plasma membrane phospholipid phosphatidylinositol bisphosphate (PIP2) to produce the inositol 3-phosphate (IP3) and diacilglicerol (DAG). IP3 is responsible for increase intracellular Ca2+ levels, while the DAG activates the protein kinase C (PKC). The AT1 receptors also activate the mitogen-activated protein kinase (MAPKs) pathways which include extracellular regulated kinase types 1 and 2 (ERK 1/2), and its effect can be dependent or independent (mediated by β-arretin) of Gq protein activation. ANG-II-induced thirst is dependent on PKC pathway activation, while ANG-II-induced sodium appetite is dependent on MAPK pathway activation, demonstrating that thirst and sodium appetite induced by ANG II are processed by distinct intracellular pathways (Daniels 2014). Src kinase protein family, Ras GTPase. (Reproduced with permission from Mecawi et al. (2015a))

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ANGII-AT1 signaling may contribute to increased sodium intake in conditions where the RAS is activated, such as during sodium deprivation or depletion. In contrast to the well-defined ANGII-AT1 receptor actions, the physiological role of the other ANG peptides and receptors in the neuroendocrine control of hydromineral balance still needs to be clarified. The central AT2 receptor seems to be important to counter-regulate the AT1-mediated signaling in the brain. In turn, the central blockade of the aminopeptidase A, responsible to generate ANGIII, reduces the thirst, firing rate of vasopressinergic neurons, and pressor response to ANGII. ANG 1–7 are not able to induce thirst or sodium appetite by itself, however it was recently demonstrated that this ANG peptide is able to potentiate the osmotic thirst. Additionally, chronic treatment with an ANG 1–7 antagonist abolishes the hypertensivity and thirst in response to central ANGII. Transgenic mice with increased expression of ACE2 also demonstrated a reduced response to acute or chronic central ANGII-induced hypertensive and thirst responses. These findings suggest that ANG 1–7 can be involved in the thirst responses elicited by central action of ANGII (Fitzsimons 1998). In addition to directly acting in the CNS to induce thirst and sodium appetite, ANGII is also the main secretagogue stimulating mineralocorticoid release, primarily aldosterone, from the adrenal glands. Aldosterone, in turn, plays an essential role in renal sodium handling and in sodium appetite stimulation. Curt Richter was the first to demonstrate that the mineralocorticoids from adrenal glands are crucial for the development of sodium appetite. Different from ANGII, which simultaneously induces thirst and sodium appetite, mineralocorticoids specifically lead to salt intake. The NTS and amygdala seems to be the main sites for the central action of aldosterone to induce sodium appetite. The corticosteroid 11 beta-dehydrogenase isozyme 2 (HSD2) is important for glucocorticoid inactivation, which, at higher plasma concentrations, exhibits affinity for the mineralocorticoid receptor (MR), allowing its predominant action on NTS neurons. So, the sensitivity of this neuronal population to aldosterone is a consequence of the selective co-expression of HSD2 and MR at NTS neurons, which is increased in response to a low sodium diet and mediates the synergistic action of ANGII and aldosterone to induce robust sodium appetite. Conversely, it was also demonstrated that the microinjection of aldosterone into the amygdala stimulates sodium appetite, whereas MR knockdown abolishes such response. Indeed, a bidirectional connection between the NTS HSD2 neurons and the amygdala neurons was demonstrated, which may explain the role of these nuclei in the development of sodium appetite induced by aldosterone. For review, please see (Mecawi et al. 2015a; Geerling and Loewy 2009).

Natriuretic Peptides The family of natriuretic peptides is composed of ANP, BNP, C-type natriuretic peptide (CNP), and Dendroaspis-type natriuretic peptide (DNP). More recently, other members have been added, including urodilatin, guanylin, uroguanylin, and adrenomedullin. The ANP was first identified in the atria, and BNP was first isolated

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from porcine brain tissue, although its major sources are also cardiac cells. CNP is predominantly produced by the CNS but is also found at extremely low concentrations in the peripheral circulation. In addition, DNP was originally isolated from the venom of the Dendroaspis angusticeps or green Mamba snake. Urodilatin is produced at renal level by the alternative cleavage of pro-ANP in the distal tubules. Both guanylin and uroguanylin are predominantly expressed in the intestinal epithelium, where these peptides seem to participate in the transport of sodium and water by the gastrointestinal tract. Finally, adrenomedullin is a ubiquitously expressed peptide that was first isolated from pheochromocytoma, which is a type of adrenal medulla tumor. Both ANP and BNP bind to type A natriuretic peptide receptors (NPRAs), whereas CNP binds to type B natriuretic peptide receptors (NPRBs). NPRAs and NPRBs are associated with type A and B guanylyl cyclase, respectively. Furthermore, all the natriuretic peptides bind with high affinity to type C natriuretic peptide receptors (NPRCs), which do not exhibit the intracellular sequences of the other two receptor subtypes (kinase homology and guanylyl cyclase domains). After binding to NPRCs, natriuretic peptides are internalized and, therefore, removed from the circulation. For this reason, NPRCs are also known as “clearance receptors.” For a review see (Bie 2018; Pandey 2005). In the CNS, the natriuretic peptides and NPRs are predominantly expressed in the hypothalamus and brainstem structures related to the neural control of body fluid homeostasis. When administered into the AV3V region, both ANP and CNP decrease plasma ANP concentrations in volume-expanded rats without changing mean arterial pressure and heart rate. Furthermore, the central administration of ANP was shown to reduce AVP and OXT secretion induced by dehydration or hemorrhage. The effects of ANP on the electrical activity of SON vasopressinergic neurons were characterized by a decrease in the firing rate and membrane hyperpolarization, and are likely to be mediated by the increase in the cytoplasmic cGMP levels. Additionally, the microinjection of ANP into the SON did not change the electric responses induced by local hypertonicity on the magnocellular neurons, but abolished the synaptic excitation of those neurons following hypertonic stimulation at the OVLT, an important brain osmosensitive area that sends glutamatergic inputs to the SON. Interestingly, lesions of the median eminence, AV3V region, and posterior pituitary completely disrupted the extracellular volume expansion–induced increase in ANP plasma concentrations. Later, it was demonstrated that the direct stimulation of the AV3V region increases ANP plasma concentrations. These results indicated that the integrity of these areas of the CNS was essential for ANP release mediated, at least in part, by OXT. In the heart, the identification of OXT and OXT receptors (OTRs) in atrial myocytes supported a functional role for this peptide not only in the autocrine/paracrine regulation of cardiac function but also in the ANP release. In fact, OXT stimulates ANP from the heart in response to both isotonic and hypertonic hypervolemic conditions, increasing the concentration of this peptide in the systemic circulation. These results were also consistent with a hypothetical pathway for the physiological control of ANP release involving: (i) peripheral components (mechanoreceptors in the cardiac atria, carotid and aortic sinuses, and kidneys) and (ii) an afferent input arising from these structures to brainstem

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Fig. 7 Representation of the main actions of atrial natriuretic peptide (ANP) on body fluid homeostasis. Dashed lines represent inhibitory actions of ANP on vasopressin (AVP), renin (Ren), and aldosterone (Aldo) and release, contributing to ANP-induced vasodilation and diuresis/natriuresis effects. (Reproduced with permission from Mecawi et al. (2015a))

noradrenergic neurons and then to the AV3V region, which mediates systemic ANP release via activation of a hypothalamic ANPergic system. The renal effects of natriuretic peptides are induced by three primary mechanisms: (i) reduction in renal afferent arterioles resistance via both cGMP-dependent and independent pathways that control vascular smooth muscle contractility; (ii) decrease in sodium reabsorption in the collecting duct on its medullary portions; and (iii) decrease in AVP release, reducing water permeability in the distal nephron. In addition to contributing to the reduction of ECF volume, natriuretic peptides also exert other effects on the cardiovascular and hydromineral balance: (i) antagonism of the RAS, with inhibition of renin and aldosterone release, consequently decreasing the effects of the related hormones on behavioral, cardiovascular, and renal responses; (ii) negative chronotropic and inotropic effects on the heart; and (iii) inhibition of peripheral sympathetic activity. For review, please see (Mecawi et al. 2015a; Bie 2018; Pandey 2005; Wong et al. 2017) (Figs. 7 and 8).

Integrated Autonomic Control of Cardiovascular System and Hydromineral Balance At the kidney level, sympathetic fibers modulate multiple renal functions, such as blood flow, glomerular filtration rate, solute and water transport, and hormone production and secretion. By releasing norepinephrine, the sympathetic inputs to kidney induce vasoconstriction of glomerular arterioles, increase renin secretion from juxtaglomerular cells, and reduce the urinary output by direct action on the epithelial tubular renal cell. As a consequence of increased renin secretion after sympathetic activation, the RAS is activated. Circulating ANGII then acts on presynaptic sympathetic terminals to stimulate the release of norepinephrine in

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Fig. 8 Schematic representation of atrial natriuretic peptide (ANP) action on kidneys. ANP binds to its type A and C receptors (NPRA and NPRC expressed at the basolateral membrane of tubular cells). NPRA has an intracellular guanylyl cyclase (GS) domain, stimulation the production of cyclic monophosphate of guanosine (cGMP), which is responsible for closing the epithelial Na+ channels (ENaCs) located in the apical membrane of distal tubular cells. The enzyme phosphodiesterase (PD) converts cGMP to GMP, reducing ANP-mediated natriuretic effects. The binding of ANP or other natriuretic peptides to NPRC produces the internalization of the ligand-receptor complex, which is then degraded or recycled. (Reproduced with permission from Mecawi et al. (2015a))

both vessels and tubular epithelial cells. The importance of renal sympathetic and RAS interaction is demonstrated by the reduction of ANGII effects on water and electrolytes tubular reabsorption after kidney denervation. As expected, the RVLM has an important role on the control of renal sympathetic outflow. ANGII, possibly from brain sources, also acts to potentiate the activity of RVLM neurons by acting through the AT1 receptors, which may directly impact on the activity of sympathetic preganglionic neurons that control norepinephrine release in the kidneys. Circulating ANGII may also act to control the sympathetic activity and baroreflex sensitivity, since AP lesions block the ability of peripheral administered ANGII to modulate the maximum renal sympathetic activity. Thus, there is an important positive feedback mechanism between sympathetic and RAS activities at the renal level. For review, please see (Mecawi et al. 2015a; Johns et al. 2011; Ramchandra et al. 2013; Pontes et al. 2015). Changes on AP and NTS neuronal activity are directly involved in the control of renal sympathetic activity and the purinergic neurotransmission looks to be especially important in this process. By modulating the adenosine type 1 receptors (A1) and/or adenosine triphosphate (ATP)-sensitive potassium channels in the AP, adenosine leads to decreases in renal sympathetic nerve activity. On the other hand, by actin on TRPV and/or glutamate N-methyl-D-aspartate (NMDA) receptors, the microinjection of capsaicin into the AP increased renal sympathetic nerve activity. The adenosine type 1 receptors (A1) in the NTS level increase the sympathetic renal outflow, while the adenosine type 2a receptors (A2a) have an inhibitory effect. This signaling pathway seems to be spatially important during the hypotensive phase of

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severe hemorrhage. Noradrenergic NTS neurons seem to be involved in the hypertonicity-induced changes in renal sympathetic nerve activity, since chemical lesions of these neurons prevent the renal sympathoinhibition induced by acute hypernatremia. Also, sinoaortic denervation abolishes the renal vasodilatation in response to blood volume expansion, demonstrating the important role of baroreceptors in the control of renal blood flow. These results indicate that the appropriate control of sympathetic renal activity in response to cardiovascular and hydromineral challenges depend on the baroreflex circuitry (Mecawi et al. 2015a; Johns et al. 2011; Sata et al. 2018). Noradrenergic neurons from the brainstem directly project to PVN and SON in order to modulate oxytocinergic and vasopressinergic neurons in coordination with cardiovascular peripheral information and sympathetic activity. Microinjection of noradrenaline into PVN stimulates AVP secretion, whereas α1-adrenoceptor antagonist and α2-adrenoceptor agonist significantly reduce at basal condition and after intracerebroventricular ANGII administration. The low-pressure receptors in the atrium inhibit AVP release via an NTS-dependent pathway, while the reduction in the activity of this inhibitory circuitry is responsible for the hypovolemia-induced AVP secretion. In general, noradrenergic efferents from NTS and RVLM brainstem nuclei have been shown to facilitate the activity of magnocellular neurons. Stimulation of the cervical vagus induces nuclear c-Fos expression in CVLM noradrenergic neurons and excite AVP cells, while lesions of the LC decrease the hemorrhage-induced AVP and OXT release. Collectively, these data demonstrate the importance of the brainstem noradrenergic system in the integration of peripheral information, such as the baroreflex, in the control of AVP and OXT secretion. As a CVO, the AP is also important to integrate peripheral signals to the neuroendocrine control of neurohypophysial hormone secretion. The lesion of AP decreases hypothalamic AVP mRNA expression, as well as its plasma levels under basal states and after hypovolemic and hyperosmotic stimulation. Additionally, it was demonstrated that both AVP and OXT secretion are disrupted after AP lesions in response to intravenous hypertonic NaCl solution infusion, but not after hypertonic mannitol injection, indicating that this brainstem area is more sensitive to peripheral Na+ concentration than to osmolality. This finding is consistent with the expression of the Na+-sensitive channels Nax in the AP (Iovino et al. 2017; Antunes-Rodrigues et al. 2013; Miyata 2015). It was suggested that the LPBN also sends stimulatory projection to the SON modulating interneurons at its perinuclear zone. In fact, it was already demonstrated that excitatory projections from the LPBN reach the PVN magnocellular and parvocellular AVP and OXT neurons. Supporting these neuroanatomic findings, it was demonstrated that the inhibition of LPBN with a α2-adrenoceptor agonist microinjection impaired the neurohypophysial hormone secretion in response to intragastric hypertonic saline overload. The serotonergic input from the DRN to PVN and SON magnocellular neurons has also been demonstrated. It was also demonstrated that serotonin inhibitors decrease dehydration-induced AVP secretion, while serotonin-releasing agent or selective serotonin reuptake inhibitor potentiates such response. Serotonin seems to act via 5-HT2 and 5-HT4 receptors to modulate

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AVP release, whereas the activation of OXT neurons seems to be more complex and also involves the 5-HT1A and 5-HT3 receptors. For review, please see (Mecawi et al. 2015a; Jørgensen 2007). The control of water and sodium intake according to cardiovascular challenges is essential for proper regulation of the hydromineral balance. In this sense, changes in blood pressure and circulating volume are informed to NTS, which in turn sends projections to brain areas that modulate thirst and sodium appetite via changes of ANGII release inside the BBB. While AP/NTS lesion increases sodium intake in sated rats, the lesion of the NTS has no effect on thirst or sodium appetite in response to extracellular volume depletion, but presents the dipsogenic response to peripheral ANGII administration. Specific lesions of NTS and CVLM catecholaminergic neurons or central microinjection of an adrenergic antagonist inhibit hypertonic saline intake. Furthermore, the microinjection of adrenergic agonists into the third ventricle reduced ANGII-induced thirst. Furthermore, the prior injection of a α2-adrenergic agonist reversed the inhibitory effects of OXT on the hypertonic saline intake induced by sodium depletion (Mecawi et al. 2015a; Fitzsimons 1998; De Luca et al. 2016). Not only cardiovascular information depended on the brainstem integration but also the regulation of water and sodium intake. An inhibitory control of sodium intake mediated by postingestion feedback signaling also takes place in the brainstem. This neuronal pathway is transmitted from the tongue by the vagus and glossopharyngeal nerves via the NTS and LPBN to the forebrain in order to control the ingestive behaviors. The LPBN seems to curtail this regulatory pathway and its reciprocal interaction with the DRN serotonergic neurons is important to transmit sodium balance information to forebrain structures, coordinating adjustments in sodium intake. The use of GABA or opioid agents to inhibit the LPBN neurons leads to a marked increase of salt intake in sodium replete animals, while pharmacological manipulation of serotonin, noradrenaline, cholecystokinin, CRH, glutamate, and ATP potentiates salt intake in water and/or sodium appetite–stimulated rats. The depletion of brain serotonin content or lesions of the DRN were demonstrated to increase salt intake in basal or sodium depletion conditions, demonstrating the important role of the DRN serotonergic neurons in the inhibitory control of sodium appetite. On the other hand, body sodium depletion decreases serotonin synthesis within the DRN neurons. Acute intra-DRN administration of an agonist for the 5-HT1A autoreceptor increased salt intake, whereas repeated injections (intraDRN or peripheral) resulted in a long-lasting reduction in salt intake, demonstrating the possible desensitization of DRN serotonergic neurons. Using specific agonists or antagonists of serotonin receptors it was demonstrated that the 5-HT2C is the main postsynaptic receptor involved in such regulatory mechanism. As for salt intake, the LPBN seems to also inhibit water intake since its pharmacological inhibition or lesions increase water ingestion induced by several challenges in rodents. On the other hand, the role of the serotonergic system in the control of thirst is not clear; electrolytic lesions of DRN lead to an intense dipsogenic response, whereas central administration of 5-HT2C agonist inhibits water intake in rats. These data indicate that 5-HT2C signaling in the DRN suppresses water intake. For review please see (Mecawi et al. 2015a; Reis 2007; Menani et al. 2014).

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Novel Local Players on the Control of Hypothalamic Function Both AVP and OXT are synthesized as pro-hormones in magnocellular neurons of the PVN and SON. The mature AVP and OXT peptides contain nine amino acids and have a molecular weight of 1084 and 1007 kDa, respectively. Once secreted, both hormones are freely transported in the systemic circulation and are rapidly metabolized by endopeptidases, leading to extremely short half-lives (5–10 min). As previously introduced, AVP binds to three different subtypes of G-protein-coupled receptors: (i) V1a, expressed, among other sites, in the vascular smooth muscle, where it determines vasoconstriction; (ii) V1b, expressed in the anterior pituitary corticotrophs, where AVP acts synergistically with corticotrophin releasing hormone (CRH) to stimulate adrenocorticotrophic hormone (ACTH) release; and (iii) V2, expressed in the kidneys and implicated in the antidiuretic effects of this hormone. OXT also binds to receptors belonging to the G-protein-coupled receptors family (OXTRs). Due to the great structural homology, AVP/OXT agonists and antagonists have their selectivity and specificity decreased at high concentrations. However, specific OXTRs are expressed in both female and male reproductive organs, as well as in the cardiovascular system and kidneys. In the macula densa and thin limb of Henle’s loop, OXTRs mediate distinct effects on renal function; however, it remains unclear whether these responses are mediated by different receptor subtypes. More recently, the studies on the magnocellular neurosecretory system function have tremendously advanced with transcriptomic analyses. The transcriptome accounts for the complete set of all RNA transcripts, including coding and noncoding, for an individual cell or a cell group. In the hypothalamus, the responses to diverse physiological stimuli were proved to be underpinned by changes in gene transcription, with up- or downregulation of a series of genes of particular interest (Greenwood et al. 2015a; Dutra et al. 2021). Among these targets are not only AVP and OXT genes, but also other key regulators of cellular function, such as cyclic adenosine monophosphate (cAMP)-responsive element binding protein 3-like 1 (Creb3l1) and gonadotrophin-inducible ovarian transcription factor 1 (Giot1). The relative expression of both Creb3l1 and Giot1 mRNAs is increased in the hypothalamus of water-deprived animals, whereas only Giot1 transcript was upregulated by chronic salt load. Indeed, it has been demonstrated the overexpression of Creb3l1 in the SON and PVN resulted in increased AVP biosynthesis. Furthermore, Creb3l1 expression is differentially affected by cAMP and glucocorticoid in the hypothalamus. As it will be discussed in the following sections, glucocorticoid levels inhibit neurohypophysial hormone secretion, thus supporting an important role for Creb3l1 in the control of AVP production in response to stress (Greenwood et al. 2015b). As the example of Creb3l1, this research group has also described the importance of the newly discovered genes within the hypothalamicneurohypophysial system (Rasd1, Caprin2, Slc12a1, Nr4a1, Ucn, and Azin1) to the control of AVP synthesis and secretion. Besides secreting neurotransmitters and hormones, neurons also produce a great variety of chemical mediators, which are released into the interstitial space and act as paracrine and/or autocrine signals. Since the pioneer studies employing lesions

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performed to AV3V region and to hypothalamic nuclei, a huge body of knowledge has been provided on the participation of classical neurotransmitters (such as acetylcholine, biogenic amines, amino acids, and purines) in the control of hydromineral homeostasis. More recently, gaseous modulators have been included in the increasing list of unusual substances with effects on neuronal activity and renal function. Actually, in the late 1990s, the discovery of the endothelial-mediated vasodilatory properties of nitric oxide (NO) was awarded with the Nobel Prize in Physiology and Medicine, opening a new and intriguing research field in neuroendocrinology. Therefore, in the following paragraphs, we invite the readers to the field of novel regulators of neuroendocrine function, with emphasis on gaseous modulators, endocannabinoids, and the outstanding role played by glial cells in the model of synaptic networking.

Gaseous Modulators Soluble gases are signaling molecules that exhibit a short half-life, which significantly restricts their actions to neighboring target cells. The most well-known gaseous modulator is NO, which is synthesized from L-Arginine by the enzyme nitric oxide synthase (NOS), which exists as two distinct isoforms: (i) the constitutive isoform, consisting of neuronal- and endothelial-derived NOS, whose activity is dependent on intracellular calcium; and (ii) the inducible isoform, which is calcium independent but is stimulated by other molecules, such as cytokines. NO synthesis also requires cofactors, such as nicotinamide adenine dinucleotide phosphate (NADPH), and the presence of oxygen. In the case of constitutive NOS, the formation and binding of a calcium-calmodulin complex is also required. On the other hand, inducible NOS, whose action is calcium independent, exhibits a permanently activated calmodulin subunit. Independent of the enzymatic route, this reaction produces equimolar amounts of NO and L-citrulline. Different from the relatively recent discovery of NO actions, carbon monoxide (CO) effects have been investigated for centuries. Since CO binds with a 200- to 300-fold greater affinity to hemoglobin than oxygen, a great number of studies have focused their attention on CO-induced hypoxia and poisoning. CO can be produced endogenously by the activity of HO isoforms, or by lipid peroxidation of unsaturated fatty acids of cellular membranes. Similarly to NO, there are three distinct isoforms of HO described so far: (i) the inducible isoform, HO-1, which can be activated by numerous stimuli; (ii) the constitutive isoform, HO-2, which is mainly responsive to glucocorticoids, and (iii) HO-3, whose function is not well established yet. The main signaling pathway implicated in the CO-mediated actions as a neuromodulator is the activation of soluble isoform of the enzyme guanylyl cyclase (sGC), followed by the consequent increase in cGMP intracellular concentrations. More recently, H2S appeared as the third gaseous molecule with potential targets in endocrine systems. H2S has proven to be a more potent inhibitor of cytochrome oxidase than cyanide, impairing mitochondrial electron transport, and, in addition, demonstrated the potential to denature protein structure through the reduction of disulfide bridges (Guidotti 2010). Endogenous H2S production was first reported in

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rat liver and kidneys, and consists of a process mediated by three enzymatic pathways: (i) cystathionine γ-lyase (CSE), (ii) cystathionine β-synthase (CBS), and (iii) cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3MST). In the brain, endogenous H2S production was demonstrated only very recently. Not surprisingly, NOS expression is increased in the hypothalamus of experimental animals submitted to hypovolemia, dehydration, and salt load. Although the effects of this neuromodulator are not unanimous in the literature, most reports suggest that the activity of the hypothalamic neurohypophysial system is under tonic inhibition by NO. On the other hand, most studies conducted so far agree that CO may positively regulate AVP and OXT secretion triggered by in vitro and in vivo hydromineral homeostasis imbalances. In the same way, H2S production is apparently upregulated by water deprivation, directly correlating with increased excitability of PVN magnocellular neurons and increased AVP and OXT secretion. Importantly, NO also participates in the regulation of Na+ balance by the kidneys, mediating both ANP- and OXT-induced natriuretic effects through a cGMP-dependent inhibition of ENaCs. Furthermore, NO apparently counteracts H2S pro-inflammatory actions at renal level, suggesting that both mediators could underlie renal/vascular alterations in response to high Na+ dietary exposure. Accordingly, it has been recently demonstrated that the inhibition of endogenous H2S production in rats submitted to a high-salt diet produces anti-inflammatory effects at both vascular and tubular renal structures, further supporting inflammation as a possible mechanism linking increased Na+ intake and the development of systemic chronic diseases. Indeed, a complex interplay between gaseous modulators has been proposed in the regulation of hydromineral balance, with NO apparently playing a central role in this three-way system (Fig. 9). Although most of these insights arouse from studies investigating the integrity of neuroendocrine responses, there is strong evidence suggesting that this interaction may also apply broadly to several homeostatic responses, not only involving the CNS. As reviewed by Ruginsk et al., gaseous modulators may operate locally and in parallel to control organic function, so that when homeostasis is disturbed the individual players would react in a coordinated way to affect each other’s production, thus regulating the final effector response (Ruginsk et al. 2015). However, the intriguing question of which gaseous modulator is altered first in response to a given stimulus remains unanswered so far. It still remains unclear whether each modulator may behave as inhibitory or stimulatory to a given response. Actually, the final outcome depends on a time-dependent and very dynamic process occurring at molecular and enzymatic levels, either through direct interactions between gaseous modulators and the mature proteins, or through induction of altered gene expression. Furthermore, the differential enzymatic expression between neurons and glial cells may also reveal which cell type may be indeed implicated in triggering these responses.

Endocannabinoids Marijuana (Cannabis sativa) is one of the most ancient and widely used drugs in the world. Its effects on the human organism, some of which are clinically explored, are

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Fig. 9 Potential mechanisms by which NOS/NO, HO/CO, and CBS/H2S regulates neurohypophysial hormone secretion in response to hyperosmolality. The osmotic stimulus induces NOS and HO activation, which increases, respectively, NO and CO levels. CO stimulates AVP and OT secretion, whereas NO inhibits hormone release, either directly (via a cGMP-dependent pathway) or indirectly (increasing GABA release). There is also evidence that osmotic stimulation increases H2S production via CBS activation, which increases cyclic AMP (cAMP) intracellular levels. However, the mechanism by which H2S affects AVP and OT remains unknown. A complex interaction among these systems at enzymatic and molecular levels has also been reported. Complete arrows: stimulatory pathways; dashed arrow: inhibitory pathway. AVP arginine vasopressin, CBS cystathionine-β-synthase, cAMP cyclic AMP, cGMP cyclic GMP, CO carbon monoxide, GABA gammaaminobutyric acid, HO heme oxygenase, H2S hydrogen sulfite, NO nitric oxide, NOS nitric oxide synthase, OT oxytocin. (Adapted from Ruginsk et al. (2015))

explained by existence of an endogenous cannabinoid system, which recognizes exogenous synthetic or plant-derived compounds (phytocannabinoids). The endocannabinoid (ECB) system is comprised of two main types of cannabinoid receptors, namely CB1R and CB2R, and also by some orphan receptors, all belonging to GPCR family, endogenous lipophilic ligands (ECBs), and the enzymatic machinery for their synthesis and metabolism. Importantly, some ECBs may behave as promiscuous ligands for other receptor systems, particularly for TRPV channels. Considering that ECBs can be transported bidirectionally across cell membranes, some compounds have been characterized as “endovanilloids,” binding simultaneously to intracellular/extracellular domains of different receptor systems. However, the so-called “promiscuity” of ECBs should not be understood as a lack of specificity but rather as a high degree of flexibility, which allows these lipid mediators to participate in a wide range of homeostatic functions. ECBs are ubiquitous lipophilic agents produced from the hydrolysis of long-chain polyunsaturated fatty acids. Similarly to gaseous modulators, ECBs cannot be stored in vesicles like other conventional neurotransmitters. Therefore, they are produced on demand from the postsynaptic membrane precursors in response to membrane

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depolarization, primarily acting as retrograde messengers at presynaptic CB1Rs/CB2Rs. The two main endogenous ligands for cannabinoid receptors are anandamide (AEA) and 2-arachidonoylglycerol (2-AG), synthesized by N-acylphosphatidylethanolaminephospholipase D (PLD-NAPE) and diacylglycerol lipase (DAG lipase), respectively. After being released to the extracellular space, reuptake of ECBs may occur via diffusion through the cell membrane, which is facilitated by a selective, saturable, and Na+-independent carrier that can be stimulated by NO. Thereafter, AEA and 2-AG are hydrolyzed and metabolized into inactive compounds by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAG), respectively. The main signaling pathways activated in response to CB1R activation by ECBs are (1) inhibition of cAMP and, consequently, protein kinase A (PKA) (Fig. 10); (2) inhibition of voltage-gated calcium channels; and (3) activation of several MAPKs.

Fig. 10 ECBs are lipophilic agents produced from membrane precursors in response to CORT nongenomic actions on mGRs, which are primarily coupled to a stimulatory G protein. ECBs act mainly as retrograde mediators on presynaptic CB1Rs. The AC/cAMP/PKA pathway seem to be implicated not only in ECB production but also in their actions in target cells. In presynaptic terminals, ECBs mediate diverse effects on neurotransmission, such as modulation of ion channels, and regulation of other intracellular signaling pathways. AC adenylyl cyclase, cAMP cyclic adenosine monophosphate, CB1R type 1 cannabinoid receptor, CORT corticosterone/cortisol, ECBs endocannabinoids, mGR membrane-associated glucocorticoid receptor, PKA protein kinase A

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Recently, ECBs have been implicated in a series of physiological and pathological actions on neural activity and synaptic function. Since CB1R is the prominent isoform expressed by neurons, this receptor subtype has drawn great attention as a potential therapeutic avenue in several pathological conditions. Therefore, strong evidence has been provided over the past few decades showing that that ECB-mediated plasticity functionally affects normal brain function and may underlie the development of CNS diseases. Despite the well-described impact on neuroplasticity, the effects of ECBs on the central mechanisms regulating body fluid homeostasis are not yet fully understood. Although fluid and food intakes are intrinsically related, there is evidence suggesting that these motivated homeostatic behaviors are independently affected by ECBs. Indeed, this hypothesis has been recently tested using the salt load experimental model, in which animals lose weight not only because of the decreased ECF volume (dehydration), but also as a result of a well-known hyperosmolality-induced hypophagic effect. In this study, the administration of a CB1R-selective agonist (ACEA) produced hyperphagia but showed no effect in preventing hyperosmolality-induced hypophagia. In addition, the authors demonstrated that ACEA pretreatment potentiated salt load–induced water intake during rehydration, but failed to induce such changes in the pair-fed group. Taken together, these results suggest that ECBs may stimulate both food and fluid intakes through independent regulatory mechanisms. More recently, our group has also demonstrated that ECBs act as intermediates in glucocorticoid-mediated inhibitory actions on AVP and OXT release. These findings corroborated previous in vitro reports in the literature showing that glucocorticoids released during osmotic/volemic challenges activate two independent nongenomic intracellular signaling pathways, culminating with increased production and release of ECBs (Fig. 10). According to these studies, this increased ECB tone would reduce glutamatergic and increase gamma-aminobutyric acid (GABA)ergic inputs onto magnocellular neurons, decreasing the neuroendocrine output. This mechanism was further confirmed by functional studies, which demonstrated that pretreatment with rimonabant, a CB1R antagonist, potentiated OXT secretion induced by extracellular volume expansion and reversed the inhibitory effects of dexamethasone on OXT release. Consistent findings were also obtained in response to the intracerebroventricular administration of AEA and following the inhibition of AEA metabolization. Interestingly, AVP secretion seems to be less affected by glucocorticoids/ECBs than OXT release, which may be related to the more critical role of AVP in maintaining ECF tonicity during osmotic challenges. Taken together, the results indicate that in response to some specific disturbances to body fluid homeostasis, which characterize stressful stimuli, oxytocinergic magnocellular neurons are particularly inhibited by a glucocorticoid induced through ECB-mediated mechanism. Vasopressinergic cells, in turn, may be at some extent protected from glucocorticoid-induced actions possibly through a mechanism involving the control of local ECB availability/metabolization (Ruginsk et al. 2014). As hypothesized before, this may be particularly relevant to maintain AVP plasma concentrations under sustained stress conditions, during which the correction of osmolality deviations should be critically preserved.

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Glial Cells As previously discussed, the direct effect of ECF hyperosmolality on the activity of the hypothalamic neurosecretory system is accomplished by the intrinsic osmosensory properties of magnocellular neurons, which transduce a mechanical signal (hypertonicity-evoked neuronal shrinkage) into depolarizing currents (PragerKhoutorsky and Bourque 2015). However, this dogma has been critically revisited in the past few years due to the evidence that neurons may be not the only cell population responsible for triggering such responses. Glial cells are the most abundant cell type in the human brain and astrocytes represent the majority of these cells in the mammalian CNS. Since the “neuron doctrine” has dominated neuroscience studies until the nineteenth century, glial cells have been neglected over time as mere passive components in brain processes. However, astrocytes were also proved to be chemically excitable cells, with a direct impact on information processing within the CNS. Indeed, fluctuations in ionic conductance across astrocytic membrane may also contribute to an increase in magnocellular neurons excitability during dehydration. ECF hyperosmolality is monitored by astrocytic sodium channels (Nax) and sodium influx through Nax is accompanied by increased Na+/K+-ATPase activity and energy expenditure, culminating with an enhanced glucose anaerobic metabolism and lactate production. This mechanism possibly underlies the regulation of sodium appetite by Nax and may constitute an interface between the control of hydromineral and energy balances (Hiyama and Noda 2016). Increased chloride influx through astrocytic volume-regulated anion channels (VRACs) also reduces taurine release from astrocytes, consequently decreasing glycine inhibitory tone onto magnocellular neurons (Hussy et al. 1997; Choe et al. 2012). In response to ECF hyperosmolality, remarkable citoarchitecture reorganization also takes place in the hypothalamus: astroglial processes undergo retraction, decreasing the coverage of surrounding hypothalamic neurons and increasing postand presynaptic contacts (Fig. 11). This reversible astrocytic remodeling is accomplished by a dehydration-induced decrease in glial fibrillary acid protein (GFAP) expression, which is accompanied by a significant increase in AVP immunoreactivity. Furthermore, astrocytes also play a decisive role in central glutamate-glutamine cycle. Glutamate, the main excitatory neurotransmitter, can be intracellularly stored by astrocytes after being converted to glutamine, a reaction mediated by the enzyme glutamine synthetase, exclusively expressed in the CNS by astrocytes. In the extracellular compartment, glutamate is mostly buffered by astrocytic glutamate transporters, which belong to the family of sodium-dependent excitatory amino acid transporters (EAATs), being glutamate and aspartate transporter (GLAST) and type 1 glutamate transporter (GLT-1) the EAATs are primarily expressed by glial cells. Hence, incubation of hypothalamic cultured astrocytes with hypertonic solution decreases GLAST as well as labeled aspartate uptake by these cells, being this latter effect correlated with increased activation of magnocellular neurons of the SON (Souza et al. 2020). Not surprisingly, many cellular signaling systems known to impact neuronal activity can also strongly modulate astroglial functions. In cultured astrocytes,

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Fig. 11 Schematic diagram summarizing the main effects of hyperosmolality on astrocytes. Under basal conditions, astrocytes are characterized by a very particular morphology, evidenced by abundant and distal processes. The influx of Na+ (through Nax channels) and/or Cl (through volume-regulated anion channels) (left lower panel) is the key electrophysiological event triggering astrocyte activation in response to hyperosmolality. The increased Na+ influx enhances the activity of the Na+/K+-ATPase, increasing energy expenditure and the intracellular lactate production by the anaerobic metabolism. Hyperosmolality also decreases the cellular surface and the EAATsmediated transport of glutamate across the membrane, consequently reducing the intracellular conversion of glutamate to glutamine (catalyzed by GS) and increasing the extracellular availability of this neurotransmitter. Furthermore, hyperosmolality increases the expression of AT1 receptor mRNA in astrocytes, suggesting that ANGII may constitute a downstream mediator in this pathway. ANGII angiotensin II, AT1 type 1 angiotensin II receptor, EAAT excitatory amino acid transporters, GS glutamine synthetase

activation of AT1 receptors by ANGII induces a dose-dependent increase in calcium waves, the main signaling input to these cells. Furthermore, astrocytes constitute the main CNS source of the precursor angiotensinogen and express the two isoforms of angiotensin-converting enzymes (ACE1 and ACE2). Indeed, our group has recently demonstrated in cultured hypothalamic astrocytes that hyperosmolality downregulates the transcript levels of angiotensinogen and both angiotensin-converting enzymes, whereas upregulates AT1 receptor mRNA, indicating that astrocytes may indeed produce and release RAS peptides (Souza et al. 2020). As also recently proposed, an ANGII-induced reduction in astrocytic glutamate uptake may contribute to central ANGII dipsogenic effects and AVP release. Furthermore, Di and coworkers (2013) demonstrated that astrocytic buffering of ECBs may differentially affect glutamatergic and GABAergic synapses opposed to hypothalamic magnocellular neuroendocrine cells (Di et al. 2013). In a series of very elegant experiments, the authors demonstrated that 2-AG release from magnocellular neurons is spatially restricted by astrocytes to

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glutamate synapses, interfering with GABA synapses only when astrocytic buffering is disrupted. The release of AEA, in turn, seems to preferably affect GABA synapses, being insensitive to astrocytic buffering status. In summary, these findings suggest that astrocytes provide very specific control of stimulus-evoked ECB release by neurons, regulating GABAergic synaptic inputs to magnocellular neuroendocrine cells. Taken together, these evidences highlight glial cells as active elements in the dynamic control of endocrine function and suggest that the morphological and spatial rearrangement of astrocytes in response to hydromineral homeostasis challenges may accomplish for a coordinated increase in the excitatory drive onto the hypothalamic neurosecretory system, ultimately culminating with appropriate changes in hormone release. This drive would result from an interdependent production and clearance of transmitters or mediators, including taurine, RAS peptides, and ECBs. Indeed, all these newly described mechanisms are strongly supported by the novel concept of the “tripartite synapse,” in which the classical pre- and postsynaptic neuronal elements are surrounded by active astroglial processes. These findings therefore extend our understanding of the so far mysterious roles of these cell types in the modulation of brain functions, including those related to body fluid homeostasis control.

From Fetal Life to Senescence: The Neuroendocrine Regulation of Hydromineral Balance During the Life Span The addition of sodium without water (high salt intake) to the ECF compartment produces increments in plasma osmolality. In response, several neuroendocrine systems trigger coordinated renal and cardiovascular adaptations to increase the thirst sensation and decrease water excretion proportional to the amount of solute added. Conversely, if excessive amount of water is added to the ECF, a decrease in the plasma osmolality would be expected. So, in this situation, the neuroendocrine systems work in order to retain sodium, excrete excess water, and induce sodium appetite. According to mass balance, in a positive balance situation, when water or sodium intakes are greater than its excretion, several renal mechanisms (regulated by the neuroendocrine systems) are activated in order to eliminate excess ECF. On the contrary, if an excess of water or sodium is lost from the kidneys, skin, respiratory system, and/or the gastrointestinal tract, the negative balance will induce coordinated neuroendocrine changes in order to replenish (through increased ingestion and reabsorption) water and/or sodium to maintain adequate tissue perfusion. Important imbalances of the hydromineral homeostasis are observed during different stages of life, such as the development during the pregnancy, along the adulthood, and aging. Also, gender-related peculiarities on the neuroendocrine control of water and sodium management are responsible for considerable differences in hydromineral balance between females and males. Finally, allostatic conditions, such as during intense exercise or water deprivation, determine the way the neuroendocrine systems will be activated to control the balance between water/salt intake and excretion. In the following paragraphs, we will discuss some

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physiological situations during which such imbalances occur, in order to illustrate the neuroendocrine regulation of the hydromineral balance.

Fetal Programming Recently, an increasing interest has emerged to the understanding of how developmental programming impact adult life, i.e., the developmental origins of health and disease. Several studies indicate that disturbances in critical periods of early life can program the neuroendocrine systems implicated in the control of behavioral and renal responses in juvenile and adult life. In this context, worldwide problems of perinatal malnutrition arouse as responsible for kidney morphofunctional abnormalities implicated in altered sodium taste perception and salt ingestion, probably associated with higher hypertension risk. Furthermore, lifestyle and dietary imbalances during pregnancy and lactation, such as excessive sugar intake, alcohol consumption, and smoking, were shown to induce several changes in the offspring brain, programming thirst, sodium appetite, and AVP secretion in adult life. Accordingly, negative water and sodium ECF imbalances during development were associated with lifelong changes in thirst and sodium appetite. A sodium-rich environment during development, in turn, was demonstrated to disrupt adult kidney morphology and function, water and sodium intake, and c-Fos expression in key neuroendocrine brain areas regulating hydromineral balance. Also, the use of RAS antagonists during pregnancy and lactation leads to renal morphophysiological changes and decreases water and sodium intake in response to dehydration or RAS stimulation at adulthood. These results indicate that several insults, directly or indirectly related to hydromineral balance, performed during development are able to program the adult neuroendocrine systems responsible for the control of water and sodium balance (Fig. 12) (for review, please see Mecawi et al. 2015b).

Sex-Related Differences Sex differences and the action of the reproductive hormones also have great impact on the neuroendocrine control of hydromineral balance. These effects are mainly observed on blood pressure regulation, renal function, neurohypophysial hormone secretion, as well as on thirst and sodium appetite perception. Therefore, men show a greater chance to become hypertensive, whereas women have a greater risk of developing hyponatremia. Accordingly, a sex chromosome modulation of neuronal pathways that control cardiovascular baroreflex and ingestive behaviors in response to peripheral RAS stimulation was demonstrated. Also, estradiol inhibits sodium appetite by modulating the activity of DRN serotonergic neurons related to the sodium appetite-inhibitory circuitry. In this sense, it was also demonstrated that estrogen is able to restrain the sodium appetite by reducing the hedonic orofacial behaviors related to sodium taste. Estrogen was also shown to inhibit brain RAS, which is consistent with its effects regulating hydromineral balance, promoting a reduction of water intake in response to ANGII,

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Fig. 12 Consequences of developmental environmental factors on programing the behavioral component for the neuroendocrine control of hydrosaline metabolism, and the predisposition to pathological conditions. (Reproduced with the permission from Mecawi et al. (2015b))

dehydration, and hypotension. In accordance with the inhibitory effect of estradiol on water intake, several reports have also demonstrated its positive effect on the neurohypophysial hormone secretion under basal or stimulated conditions. Indeed, it was previously demonstrated that estrogen increases the sensitivity for osmotic-induced AVP secretion and thirst. Recently, it was also found that the effect of estrogen by reducing the osmotic threshold and potentiating AVP secretion is mediated by the direct effect of estradiol on type β estrogen (ERβ) receptors expressed in magnocellular neurons, and not via type α estrogen (ERα) receptors expressed in the LT. These findings are in accordance with the fact that ER-β is expressed in the magnocellular hypothalamic and in the serotonergic DRN neurons, whereas ER-α is mainly expressed at the CVOs. These physiological effects may also underlie the important role played by female sexual steroids on the adaptations during healthy pregnancy development. Particularly, estrogens are important for uterine growth, increased blood flow to this organ, and development of the mammary gland and have its plasma concentration elevated progressively throughout pregnancy. The estrogen-induced increases in the

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sensitivity for AVP secretion and thirst development is similarly observed in pregnant rats and women, further highlighting this contribution. For review, please see (Mecawi et al. 2015a; Vivas et al. 2015; Curtis 2015).

Aging Aged people are also at higher risk of developing hydromineral disorders, namely dehydration and hyponatremia common morbidities in elderly people. As discussed above, due to the important role of estrogens on the neuroendocrine control of hydromineral balance, aging-related fluid disturbances are especially important in postmenopause women, in which the risk for the development of hydromineral and cardiovascular disturbances are increased in relation to aged-paired men. Independently of sex, an augmented osmoreceptor sensitivity and disrupted AVP production and secretion are observed in elderly individuals in both animal and human models. Also, humans and rodents studies have demonstrated aging-related reduction in sodium appetite. Such alterations may explain the prevalent hyponatremia observed in elderly people (Cowen et al. 2013).

Exercise During prolonged exercise, water and sodium are lost through sweating. In such condition, the intake of water without electrolytes dilutes the extracellular liquid, decreasing its tonicity and consequently leading to cell swelling. Many neurologic manifestations could be observed as a consequence of brain swelling, such as confusion, loss of motor coordination, intense headache, and even death. The preautonomic oxytocinergic and vasopressinergic neurons of the hypothalamus are involved in the regulation of the sympathetic nervous system to control heart rate and cardiac output during physical exercise. In this context, plasma AVP levels increase according to the intensity of exercise, but are reduced with previous training at absolute submaximal exercise. Recently, it was proposed that the hyperosmolality induced by increased plasma sodium and lactate concentrations may be the primary factors stimulating AVP secretion and thirst in individuals submitted to highintensity exercise. OXT neurons seem to be directly related to the autonomic control during exercise, since OXT released within the solitary-vagal complex increases reflex bradycardia during physical activity, mainly in trained rats. Thus, the neurohypophysial neuropeptides contribute to the regulation of cardiovascular and renal function during exercise (Dampney et al. 2018).

Conclusion During their life span and also during their diary routines, mammals still deal with the primordial task of regulating ECF osmolality and volume. This goal is achieved by the integrated response of several homeostatic systems and consists of very a

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complex reflex triggered, at the initial phase, by the activation of specific sensory systems, followed by stimulation of multiple specialized CNS areas and ended by appropriate effector responses, which ultimately restore basal conditions. In this context, one of the major effector organs for body fluid homeostasis is the kidney. Not surprisingly, most efferent mechanisms (neurohypophysial hormones, natriuretic peptides, RAS, and autonomic systems) act at renal level to control water and sodium output. However, the complete restoration of hydroelectrolytic balance may also require the selective stimulation or inhibition of motivational behaviors (thirst and sodium appetite). In this regard, a complex network of redundant mechanisms (involving multiple local and systemic factors) takes place to allow an accurate and self-limited response. The evolution of single-cell studies, associated with molecular biology techniques, dramatically broadened our understanding of the mechanisms underlying the control of hydroelectrolytic balance since the lesionbased pioneer studies 60 years ago. Now, the main challenge for researches in the area is to equilibrate the advanced understanding of the very specific local mechanisms (cell phenotypes, receptors and ion channels, and signaling cascades) with how the whole-body, intact organism (considering sex- and age-specific mechanisms) deals with novel or repeated challenges to fluid homeostasis.

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pathophysiological angiotensinergic stimuli. Ohlstein EH, editor. Pharmacol Rev [Internet]. 2015;67(4):754–819. Available from: http://pharmrev.aspetjournals.org/lookup/doi/10.1124/pr. 114.010454 Koeppen BM, Stanton BA. Renal physiology|ScienceDirect [Internet]. 2013 [cited 2021 Apr 19]. Available from: https://www.sciencedirect.com/book/9780323086912/renal-physiology Mecawi AS, Ruginsk SG, Elias LLK, Varanda WA, Antunes-Rodrigues J. Neuroendocrine regulation of hydromineral homeostasis. Compr Physiol. 2015a;5:1465–516. Mecawi AS, Macchione AF, Nuñez P, Perillan C, Reis LC, Vivas L, et al. Developmental programing of thirst and sodium appetite. Neurosci Biobehav Rev. 2015b;51:1–14. Menani JV, De Luca LA, Johnson AK. Role of the lateral parabrachial nucleus in the control of sodium appetite. Am J Physiol Regul Integr Comp Physiol. 2014;306:R201–10. Miyata S. New aspects in fenestrated capillary and tissue dynamics in the sensory circumventricular organs of adult brains. Front Neurosci. 2015;9:390. Nakagawa P, Gomez J, Grobe JL, Sigmund CD. The renin-angiotensin system in the central nervous system and its role in blood pressure regulation. Curr Hypertens Rep. 2020;22:7. Pandey KN. Biology of natriuretic peptides and their receptors. Peptides. 2005;26:901–32. Pontes RB, Girardi ACC, Nishi EE, Campos RR, Bergamaschi CT. Crosstalk between the renal sympathetic nerve and intrarenal angiotensin II modulates proximal tubular sodium reabsorption. Exp Physiol [Internet]. 2015 Apr 20 [cited 2021 Apr 13];100(5):502–6. Available from: https://pubmed.ncbi.nlm.nih.gov/25858030/ Prager-Khoutorsky M, Bourque CW. Mechanical basis of osmosensory transduction in magnocellular neurosecretory neurones of the rat supraoptic nucleus. J Neuroendocrinol. 2015;27:507–15. Ramchandra R, Hood SG, Frithiof R, McKinley MJ, May CN. The role of the paraventricular nucleus of the hypothalamus in the regulation of cardiac and renal sympathetic nerve activity in conscious normal and heart failure sheep. J Physiol [Internet]. 2013 Jan [cited 2021 Apr 13];591 (1):93–107. Available from: https://pubmed.ncbi.nlm.nih.gov/22615431/ Reis LC. Role of the serotoninergic system in the sodium appetite control. An Acad Bras Cienc. 2007;79:261–83. Ruginsk SG, Vechiato FMV, Elias LLK, Antunes-Rodrigues J. The endocannabinoid system and the neuroendocrine control of hydromineral balance. J Neuroendocrinol. 2014;26:370–6. Ruginsk SG, de Mecawi AS, da Silva MP, Reis WL, Coletti R, de Lima JBM, et al. Gaseous modulators in the control of the hypothalamic neurohypophyseal system. Physiology. 2015;30: 127–38. Saker P, Carey S, Grohmann M, Farrell MJ, Ryan PJ, Egan GF, et al. Regional brain responses associated with using imagination to evoke and satiate thirst. Proc Natl Acad Sci U S A. 2020;117:13750–6. Sata Y, Head GA, Denton K, May CN, Schlaich MP. Role of the sympathetic nervous system and its modulation in renal hypertension [Internet]. Front Med. 2018 [cited 2021 Apr 13];5:82. Available from: https://pubmed.ncbi.nlm.nih.gov/29651418/ Shigemura N, Iwata S, Yasumatsu K, Ohkuri T, Horio N, Sanematsu K, et al. Angiotensin II modulates salty and sweet taste sensitivities. J Neurosci [Internet]. 2013;33(15):6267–77. Available from: http://www.jneurosci.org/cgi/doi/10.1523/JNEUROSCI.5599-12.2013 Souza MM, Vechiato FMV, Debarba LK, Leao RM, Dias MVS, Pereira AA, et al. Effects of hyperosmolality on hypothalamic astrocytic area, mRNA expression and glutamate balance in vitro. Neuroscience. 2020;442:286–95. Vivas L, Dadam FM, Caeiro XE. Sex differences in body fluid homeostasis: sex chromosome complement influences on bradycardic baroreflex response and sodium depletion induced neural activity. Physiol Behav. 2015;152:416–21. Wong PCY, Guo J, Zhang A. The renal and cardiovascular effects of natriuretic peptides. Adv Physiol Educ. 2017;41:179–85.

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Physiology of Vasopressin Secretion Giovanna Mantovani, Alessandra Mangone, and Elisa Sala

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anatomy of the Neurohypophysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synthesis and Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Circulating Vasopressin and Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Vasopressin Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Osmoregulation and Thirst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Baroregulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vasopressin Release During Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Receptors and Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V1 Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V2 Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V3 Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Vasopressin (AVP) represents the key endocrine regulator of water balance. It is synthesized in the hypothalamic supraoptic and paraventricular nuclei and then transported to the neurohypophysis, where it is released in the bloodstream. Alterations in plasma osmolality represent the main input affecting AVP secretion, yet many other physiological and pathophysiological conditions can intervene in modulating its levels, as hypovolemia and hypotension. AVP primarily controls plasma osmolality and fluid volume by inducing synthesis and insertion of essential water transport proteins in the kidneys, thus reabsorbing water into blood circulation and reducing diuresis. Moreover, AVP exerts vascular and G. Mantovani (*) · A. Mangone · E. Sala Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy Endocrinology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2023 M. Caprio, F. L. Fernandes-Rosa (eds.), Hydro Saline Metabolism, Endocrinology, https://doi.org/10.1007/978-3-031-27119-9_2

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platelet control and intervenes in several metabolic pathways, including glycogenolysis and gluconeogenesis. Furthermore, AVP stimulates the release of adrenocorticotropic hormone (ACTH). AVP exerts its multiple effects by binding to its receptors, classified in different subtypes depending on tissue expression, function, and second messengers. Disorders in AVP synthesis or action can lead to clinical syndromes characterized by water and electrolyte imbalance. Keywords

Vasopressin · Physiology

Introduction Vasopressin (AVP) is secreted by the neurohypophysis in order to maintain body’s homeostasis. It plays a key role in water balance controlling plasma osmolality and extracellular fluid volume. Historically considered two distinct hormones, it is actually also known as antidiuretic hormone (ADH). In fact, its physiologic regulation involves both the osmotic (antidiuretic) and the pressure/volume (vasopressor) system. When vasopressin production or action is altered, different clinical syndromes characterized by disturbances in water and electrolyte balance appear, so that an adequate knowledge of its physiology and pathophysiology are needed in order to understand those disorders.

Anatomy of the Neurohypophysis The neurohypophysis, or posterior pituitary, derives from the forebrain during development and it is composed mainly of neural tissue. The neurohypophysis, laying below the hypothalamus, is connected to the pituitary gland by the pituitary stalk, or infundibular stem, making a structural and functional unit: the hypothalamic-neurohypophyseal system. Taken as a whole, the neurohypophysis is a complex neurohumoral system with a key role in body fluid homeostasis and reproductive function. It consists of three parts: the hypothalamic nuclei containing the cell bodies of the magnocellular, neurosecretory neurons that synthetize and secrete AVP and oxytocin; the supraoctico-hypophyseal tact, which includes the axons of these neurons; and the posterior pituitary, where the axons terminate on capillaries of the inferior hypophyseal artery. Therefore, the posterior pituitary itself does not produce peptides directly, but it stores and secretes oxytocin and vasopressin, synthesized by the hypothalamus. The storage of vasopressin in the posterior pituitary is responsible of its typical MRI feature. In fact, the vasopressin-complex has a macroproteic structure that shortens T1 signal, being responsible of the typical posterior pituitary bright spot (Fig. 1). It is important to note that a posterior pituitary bright spot is not identified in all patients, but rather somewhere between 50% and 100% (Côté et al. 2014).

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Fig. 1 Posterior pituitary bright spot visible in MRI

Vasopressin is synthesized by magnocellular secretory neurons which cell bodies are located in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. Axons of magnocellular neurons terminate on capillaries of the inferior hypophyseal artery in the posterior pituitary. The SON, situated in the proximal section of the optic tract, is constituted mostly by vasopressin-producing magnocellular neurons projecting to the posterior lobe of the hypophysis along the supraoptico-hypophyseal tract. The organization of the PVN, on the contrary, is more complex: it is divided into five subnuclei and contains additional smaller parvocellular neurons. Some of the parvocellular divisions of the PVN, terminating in the anterior pituitary, synthesize vasopressin, while some others regulate anterior pituitary hormones release. Moreover, a small part of vasopressin synthesis takes place in the suprachiasmatic nucleus, which is situated in the midline, at the base of and anterior to the third ventricle (Robinson and Verbalis 2011).

Synthesis and Release The chemical structure of vasopressin was described firstly by Due Vigneaud and colleagues in 1954, synthesizing an identical peptide to the natural hormone isolated in animals’ pituitary, as previously done with oxytocin, from which it differs only by 2 amino acids (du Vigneaud et al. 1954). Vasopressin is an endogenous cyclic nonapeptide consisting of a 6-amino-acid ring, with a cysteine-to-cysteine disulfide bridge between positions 1 and 6, critical for its biological action, and a 3-aminoacid tail in position X (Schally 1972). Due to the presence of an arginine as the eighth amino acid of the peptide, human ADH is called arginine-vasopressin.

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Interestingly, in marsupials and pigs, it is known as lysine-vasopressin since it contains a lysine in place of the arginine (Schally 1972). AVP gene is located on chromosome 20, nearby the oxytocine gene, and it is composed of three exons encoding a 145 amino-acid modular polypeptide (Ball 2007; Szinnai et al. 2007). It firstly synthesizes a precursor molecule called pre-provasopressin (pre-proAVP) consisting of the active nonapeptide, a hormone-specific neurophysin (neurophysin-II) and a carboxyl-terminal peptide that undergoes posttranslational glycosylation, recently named copeptin. The precursor is packed in neurosecretory granules and cleaved to the products during transport to the posterior pituitary (Ball 2007; Szinnai et al. 2007). Neurophysin-II acts as a carrier protein in the transport of AVP from the hypothalamus to the neurohypophysis. Copeptin probably plays a role in the proteolysis of pre-proAVP, thus in the correct structural formation of AVP and may act as a chaperone decreasing the formation of inactive and increasing the formation of active hormones (Barat et al. 2004; Parodi 2000). With respect to AVP, copeptin is more stable in vitro and in human plasma, so, in the last few years, several studies were published, suggesting a possible role as a surrogate biomarker of AVP release (Morgenthaler et al. 2006; Fenske et al. 2018; Garrahy et al. 2019). In particular, from 2006 a novel sandwich immunoassay was developed, starting its use as a biomarker in ADH-dependent diseases, where copeptin is proving an increasingly high diagnostic accuracy (Christ-Crain et al. 2016).

Circulating Vasopressin and Degradation Plasma levels of AVP are very low (0 to 3 pg mL 1 ≈ 3  10–12 M) with a short circulating half-life of about 5–15 min13, being rapidly metabolized by several peptidases (Lauson 1967). The most relevant enzyme is vasopressinase, a type II membrane-spanning protein of the M1 aminopeptidase family that degrades AVP by removing amino acids from its N-terminus (Esposito et al. 2011). Though there is no binding to plasma proteins in the bloodstream, more than 90% of AVP in the circulation is bound to specific receptors on platelets, thus making accurate measurements of its circulating levels very difficult (Dobsa and Edozien 2013; Bichet et al. 1987).

Regulation of Vasopressin Release AVP is secreted and released in response to a wide variety of stimuli (Robinson and Verbalis 2011; Weitzman and Kleeman 1979). Water deprivation, and consequent hyperosmolality, is the strongest stimuli to AVP production directly on the transcription of the hormone in the magnocellular neuron. Moreover, it determines an increase in the length of the poly(A) tail of AVP messenger-RNA (mRNA), influencing mRNA stability and representing a transcription-independent mechanism for modulating AVP synthesis (Carter and Murphy 1991). Hypoosmolality, on the contrary, causes a decrease in the content of AVP mRNA. The transport of the neurosecretory vesicles is subjected to the same system of regulation, as it is

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Ï intracellular water content

Ð plasma osmolality

water excess Ð thirst

Ð AVP release

Ð water intake

Ï water excretion by the kidneys

Ð body water

Fig. 2 Body response during water excess conditions

Ï plasma osmolality

Ð intracellular water content

water deprivation Ï thirst

Ï AVP release

Ï water intake

Ð water excretion by the kidneys

Ï body water

Fig. 3 Body response during water deprivation conditions

upregulated when synthesis is increased and stops when synthesis is turned off, in a complex and coordinated system. In synthesis, the body responds quickly to changes in osmolality, blood pressure, or blood volume by altering, through AVP effect, the amount of water reabsorbed by the kidneys, and the amount of fluid ingested, in both condition of water excess and deprivation (Figs. 2 and 3).

Osmoregulation and Thirst Alterations in plasma osmolality represent the main input affecting AVP secretion, with a tolerance of less than 1%. Osmolality is mainly determined by sodium

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concentration, due to the nature of the pumps and channels regulating intracellular and extracellular ionic concentration. In the human body, the normal reference range is between 280 and 295 mOsm/kg H2O2, which is accurately maintained through the close integration of water excretion, mainly regulated by the antidiuretic action of AVP, and water intake, controlled by the sensation of thirst. The regulation is so accurate that when osmolality exceed a conventional osmotic threshold of 284 mOsm/L, it causes a linear increase in AVP levels (Moses and Miller 1971). Small changes in AVP concentration as of 0.5–4 mU/mL strongly affect urine osmolality and renal water balance. Below the threshold, AVP is suppressed to levels permitting the development of a maximum water diuresis, whereas above it, the hormone rises in direct proportion to plasma osmolarity, quickly reaching levels sufficient to exert a maximum antidiuresis. Substantial inter-individual variations in both the threshold and sensitivity of AVP release have been observed, but they are remarkably reproducible within an individual over time (McKenna and Thompson 1998). Changes in plasma osmolality are detected by osmoreceptors, located in the anteroventral hypothalamus, near the anterior wall of the third cerebral ventricle, as demonstrated in both animals and humans (Johnson and Thunhorst 1997; Baylis and Thompson 1988). They are organized in two circumventricular organs named organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO). These organs are perfused by fenestrated capillaries; therefore osmoreceptors work outside the blood-brain barrier and are in direct contact with the osmolar environment of the systemic circulation (Robinson and Verbalis 2011). However, recent evidences have identified a member of the aquaporins (AQP) water channels family, AQP4, in brain tissue, challenging this traditional vision and suggesting a possible role of aquaporins in the mechanism by which osmoreceptors sense extracellular tonicity (Agre et al. 1995). Independently from the mechanism, increased blood osmolality brings intracellular water into the extracellular space, causing a cell shrinkage in OVLT and SFO that stimulates osmoreceptor discharge. As a result, the transmission of the impulses reaches the supraoptic and paraventricular nuclei, which synthetize and release AVP in response to depolarization (Stout et al. 1999). A surgical destruction or brain damage could results in an alteration of AVP secretion in response to osmolality. Rising levels of AVP activate the renal antidiuretic response through the activation of a specific receptor (V2, explained further): the final effect is the reabsorption of water into blood circulation, with normalization of plasma osmolality. At the same time, increased plasma osmolality causes thirst-generated enhanced water intake, contributing to the mechanism. The reabsorbed water decreases serum osmolality and increases blood volume, hence it inhibits the release of new AVP through a negative feedback on the osmoreceptors (Muhsin and Mount 2016). Maximum diuresis occurs at AVP concentrations of 0.5 pmol/L. As the hormone levels rise, there is a sigmoid relationship between plasma AVP concentration and urine osmolality, with the maximum urine concentration achieved at AVP plasma levels of 3–4 pmol/L. Following persistent AVP secretion, a down-regulation

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of both receptors (V2) and water channels on the kidney (aquaporins family, AQP2) expression occurs, with stabilization of urine concentration. However, different conditions can alter the conventional and linear relationship between plasma osmolality and AVP concentration: for instance, a fast rise in osmolality results in an exaggerated antidiuretic response (Ball 2007). Other examples are represented by pregnancy, when AVP threshold is lower than normal, and luteal phase of the menstrual cycle (Davison et al. 1984; Spruce et al. 1985). Additionally, AVP levels and its response to osmotic stimulation increase with age. Although AVP promotes water reabsorption reducing urine volume to a minimum, it cannot completely eliminate the fluid loss. For this reason, water intake plays an important role in maintaining liquid homeostasis too, hence the role of thirst (Robinson and Verbalis 2011). Thirst is similarly stimulated by a rise in plasma osmolarity, with a slightly higher threshold than the AVP one, as an increase of 2% to 3% of the normal plasma osmolarity is necessary to induce the stimulus (Baylis and Thompson 1988). As for AVP, a linear relationship exists between thirst and plasma osmolality. Nevertheless, most humans typically ingest more fluids than strictly needed, because of social or palatability reasons and through metabolized food, thus plasma osmolality tends to remain below the threshold level, which stimulates thirst. Because of that, AVP is the most important regulator of water balance under physiologic conditions (Robinson and Verbalis 2011).

Baroregulation AVP secretion is also stimulated by hypovolemia and hypotension, as its effects contribute to replace plasma circulating volume, although the main volume regulation system is the Renin-Angiotensin-Aldosterone System (RAAS). The baroregulatory system that regulates AVP production is composed by peripheral high-pressure arterial receptors, located in the aortic arch and carotid sinus, and low-pressure volume receptors, situated in the atria and pulmonary veins. Baroreceptors and volume receptors are stretch-sensitive and normally have a tonic inhibitory effect through the inhibition of the magnocellular neurons. The afferent signals from these receptors are carried via IX and X cranial nerves to the dorsomedial medulla oblongata, finally converging on the supraoptic nucleus (Cunningham et al. 2002). A decrease in the tonic inhibition results in release of AVP and consequent volume expansion. Nevertheless, baroreceptor and volume receptor effect is by all means less sensitive than the one mediated by osmoreceptors, due to the fact that arterial volume and pressure are subjected to alternative and more effective mechanism, as RAAS and sympathetic reflexes (Robinson and Verbalis 2011). Osmolality and plasma volume normally work synergistically in promoting AVP release and its resulting effects. Dehydration causes a decrease in plasma circulating volume and concurrently increases plasma osmolality, thus activating both the pathways resulting in AVP level increase. Similarly, hypervolemia, with increasing volume and osmolality reduction, generates a decrease in AVP secretion. Moreover, an important reduction in blood volume or pressure can lower the osmotic threshold

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for AVP secretion, despite being insufficient to cause direct increase in its levels, hence a greater release of AVP results at any given osmolality (Robinson and Verbalis 2011; Ball 2007). Finally, AVP release can be modulated by additional physiological and pathophysiological stimuli as hypoxia, hypercapnia, hyperthermia, pain, nausea, neuroglycopenia, histamine, infections, drugs, and possibly hyperangiotensinemia (Ball 2007; Weitzman and Kleeman 1979; Robertson 2013) Nausea, for instance, represents a powerful stimulus for AVP secretion, as it typically causes an immediate and important increase in the hormone’s plasma levels even when it is transient and not associated with vomiting or other symptoms. It probably acts via the emetic center in the medulla and it can be blocked completely by treatment with antiemetic drugs such as fluphenazine that acts on dopamine receptors (Robertson 2013).

Vasopressin Release During Pregnancy During pregnancy, there is a physiological expansion of plasma volume, which leads to a decrease in plasma osmolality of about 10 mOsm/kg. This condition causes a reset of the osmoregulatory system, with a consequent lowering in the osmotic threshold needed to release AVP and to stimulate thirst (Lindheimer et al. 1989). This mechanism stimulates increased water intake and consequently dilution of body fluids, without suppressing AVP release at the usual osmotic levels, thus retaining water. This shift of threshold occurs early in pregnancy, at about 5 to 8 weeks of gestation, and is maintained until term, persisting almost after 2 weeks after delivery (Robinson and Verbalis 2011; Lindheimer et al. 1989). Therefore, AVP responds appropriately to decrease and increase of the expanded gestational volume, which is normally increased up to 7–8 l due to vasodilatation. These effects have been demonstrated in murine models to be mediated by the hormone relaxin, a member of the insulin-like growth factor (IGF) family which is normally produced by the corpus luteum during pregnancy (Novak et al. 2001). Finally, the metabolic clearance rate of AVP is also increased, as the placenta produces a type of aminopeptidase, called oxytocinase, which also happens to degrade AVP. Since the production of this enzyme depends on the trophoblastic mass, multi-fetus pregnancies with greater placental masses tends to be associated with higher aminopeptidase activity (Ichaliotis and Lambrinopoulos 1965). For all these mechanisms, human pregnancy can be subjected to several water balance complications.

Receptors and Actions AVP binds to different tissue-specific G-protein-coupled receptors called vasopressin receptors (V-Rs) which are classified in three subtypes: V1, V2, and V3 (Zingg 1996). These subtypes differ in tissue expression, function, and second messenger

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Table 1 Vasopressin receptors: classification and characteristics Receptor V1

Localization Vascular smooth muscle cells, hepatocytes, platelets, brain, and uterus cells

V2

Distal nephron cells, endothelial cells, vascular smooth muscle cells Pituitary corticotroph cells

V3

Signal transduction mechanism Gq/11 phospholipase C activation: Ca2+, inositol triphosphate (IP3), and diacyl glycerol mobilization

Gαs-mediated adenylate cyclase activation: cAMP production and protein kinase A stimulation Gq/11 phospholipase C activation: Ca2+, inositol triphosphate, and diacyl glycerol mobilization

Effects Muscle cell contraction Glycogenolysis Platelet aggregation Neuromodulatory functions Uterine contraction Antidiuretic effect through increased synthesis and insertion of AQP2 ACTH release

systems, so that AVP can exert different actions depending on the receptor bound, as summarized in Table 1.

V1 Receptor V1 receptor is widely expressed in vascular smooth muscle cells, where it promotes their contraction (Ball 2007; Esposito et al. 2011). It also exerts vascular control by modulating levels of nitric oxide (NO), a vasodilator, in particular mitigating the increase of cGMP induced by NO and decreasing the synthesis of inducible NO synthase. V1 receptors are also expressed in liver, where AVP plays a role in different metabolic pathways including glycogenolysis, gluconeogenesis, ureagenesis, glutamine, and proline metabolisms depending on fed or fasted condition and to basal glycemia. These metabolic effects resemble those induced by glucagon, but since they are mediated by different second messengers, such as Ca++ pattern for vasopressin and cAMP for glucagon, they might be additive (Bankir et al. 2017). A secondary important effect of V1 receptor expressed on platelets and endothelial cells is activation of platelet aggregation, production of Von Willebrand factor, and FVIII activating the coagulation cascade.

V2 Receptor V2 receptor is expressed on distal nephron cells, where it promotes the most important effect of AVP: the regulation of water reabsorption. The activation of V2 receptor, through an intracellular cAMP signaling cascade, bring to synthesis and

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insertion of pre-synthesized acquaporin-2 (AQP2) water channels on the luminal membrane of distal nephron cells (Lolait et al. 1992). AQP2, as all aquaporins, is essential to control pure water transport across renal tubular cells (Nielsen et al. 2002). Proximal tubule nephron cells and the thin descending loop of Henle are strongly permeable to water due to their high expression of AQP1 (Nielsen et al. 1993), whereas the terminal part of the nephron, composed by connecting tubules and collecting ducts, do not constitutively express aquaporins in the apical surface, so that their final water permeability is controlled by AVP levels. Vasopressin-induced AQP2 channels presence makes collecting ducts permeable to water, thus allowing water movement along an osmolar gradient from the collecting duct lumen to the hypertonic interstitium of renal pyramids and so resulting in the excretion of a more concentrated urine (Ball 2007). AVP is able to reduce the urinary amount up to 0.4 mL/min with a urine concentration of 1200 mOsm/kg; the absence of AVP could elevate the urinary amount up to 10–20 mL/min with a urine concentration of 30 mOsm/kg. After achieving normal plasma osmolality through water reabsorption, AQP2s are internalized from the plasma membrane, leaving the collecting ducts impermeable to water again (Boone and Deen 2008). AVP also complete its antidiuretic action by helping to maintain hypertonic the medullary interstitium as required for passive water resorption. In fact, it enhances sodium and urea permeability in the collecting duct as well, through stimulation of epithelial sodium channels (ENaC) and urea transporters (UT-A1) expression in the medullary interstitium, ultimately causing an increased interstitial osmolality (Ball 2007; Nielsen and Knepper 1993).

V3 Receptor V3 receptors, also called V1b by some authors, are expressed in the pituitary gland. At this level, the peptide is released not only in posterior pituitary but also by some parvocellular neurons of the paraventricular nuclei that terminate in the anterior pituitary where they co-express vasopressin and corticotropin releasing factor (CRF), thus potentiating ACTH adrenocorticotropic hormone (ACTH) release (Ball 2007). As a result, AVP plays an important role in stimulating the release of ACTH during stress situations, although its relative importance as a secretagogue compared to CRF is not completely understood yet. Recent studies have also shown V3 receptors to be widespread distributed throughout the central nervous system over multiple and functionally distinct neuronal systems, leading to speculations regarding a possible role of AVP in the influence of different neurological functions as cognition, memory, and behavioral regulation (Hernando et al. 2001). Finally, some studies have demonstrated that, due to their similar chemical structure, a cross reaction between AVP and oxytocin receptors is possible. At supraphysiological concentrations, AVP, activating the oxytocin receptors, is responsible of uterine contractions and vasodilatation (Holmes et al. 2004).

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Conclusions Besides being the main responsible of water’s homeostasis by controlling plasma osmolality and extracellular fluid volume, AVP plays an essential role in blood pressure regulation and intervenes in multiple other pathways, some of which are yet to be completely understood. Given its vital role in multiple functions, AVP clearly possesses great clinical significance, hence a good understanding of its physiology and pathophysiology is critical for a proper diagnosis and management of water balance disorders.

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Hernando F, Schoots O, Lolait SJ, Burbach JPH. Immunohistochemical localization of the vasopressin V1b receptor in the rat brain and pituitary gland: anatomical support for its involvement in the central effects of vasopressin. Endocrinology. 2001;142:1659–68. Holmes CL, Landry DW, Granton JT. Science review: vasopressin and the cardiovascular system part 2 – clinical physiology. Crit Care. 2004;8:15–23. Ichaliotis SD, Lambrinopoulos TC. Serum oxytocinase in twin pregnancy. Obstet Gynecol. 1965;25:270–2. Johnson AK, Thunhorst RL. The neuroendocrinology of thirst and salt appetite: visceral sensory signals and mechanisms of central integration. Front Neuroendocrinol. 1997;18:292–353. Lauson HD. Metabolism of antidiuretic hormones. Am J Med. 1967;42:713–44. Lindheimer MD, Barron WM, Davison JM. Osmoregulation of thirst and vasopressin release in pregnancy. Am J Phys. 1989;257:F159–69. Lolait SJ, et al. Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus. Nature. 1992;357:336–9. McKenna K, Thompson C. Osmoregulation in clinical disorders of thirst appreciation. Clin Endocrinol. 1998;49:139–52. Morgenthaler NG, Struck J, Alonso C, Bergmann A. Assay for the measurement of copeptin, a stable peptide derived from the precursor of vasopressin. Clin Chem. 2006;52:112–9. Moses AM, Miller M. Osmotic threshold for vasopressin release as determined by saline infusion and by dehydration. Neuroendocrinology. 1971;7:219–26. Muhsin SA, Mount DB. Diagnosis and treatment of hypernatremia. Best Pract Res Clin Endocrinol Metab. 2016;30:189–203. Nielsen S, Knepper MA. Vasopressin activates collecting duct urea transporters and water channels by distinct physical processes. Am J Phys. 1993;265:F204–13. Nielsen S, Smith BL, Christensen EI, Knepper MA, Agre P. CHIP28 water channels are localized in constitutively water-permeable segments of the nephron. J Cell Biol. 1993;120:371–83. Nielsen S, et al. Aquaporins in the kidney: from molecules to medicine. Physiol Rev. 2002;82:205–44. Novak J, et al. Relaxin is essential for renal vasodilation during pregnancy in conscious rats. J Clin Invest. 2001;107:1469–75. Parodi AJ. Protein glucosylation and its role in protein folding. Annu Rev Biochem. 2000;69:69–93. Robertson GL. Disorders of the neurohypophysis. In: Jameson JL, editor. Harrison’s endocrinology. USA: McGraw-Hill; 2013. p. 50–61. Robinson AG, Verbalis JG. Posterior pituitary. In: Hetherington P, Ryan J, editors. Williams textbook of endocrinology. USA: Elsevier; 2011. p. 291–316. Schally A. Hormones of the neurohypophysis. In: Lock W, Schally AV, editors. The hypothalamus and pituitary in health and disease. Springfield: Thomas; 1972. p. 154–71. Spruce BA, Baylis PH, Burd J, Watson MJ. Variation in osmoregulation of arginine vasopressin during the human menstrual cycle. Clin Endocrinol. 1985;22:37–42. Stout NR, Kenny RA, Baylis PH. A review of water balance in ageing in health and disease. Gerontology. 1999;45:61–6. Szinnai G, et al. Changes in plasma Copeptin, the C-terminal portion of arginine vasopressin during water deprivation and excess in healthy subjects. J Clin Endocrinol Metab. 2007;92:3973–8. Weitzman RE, Kleeman CR. The clinical physiology of water metabolism. Part I: The physiologic regulation of arginine vasopressin secretion and thirst. West J Med. 1979;131:373–400. Zingg HH. Vasopressin and oxytocin receptors. Bailliere Clin Endocrinol Metab. 1996;10:75–96.

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Tubulopathies and Alterations of the RAAS Marguerite Hureaux and Rosa Vargas-Poussou

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of NaCl Reabsorption in the Renal Tubule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of NaCl reabsorption in each Renal Tubule Segment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proximal Tubule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thick Ascending Limb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distal Convoluted Tubule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Collecting Duct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diseases Associated with RAAS Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salt-losing Tubulopathies of Renal Proximal Tubule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salt-losing Tubulopathies of the Thick Ascending Limb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salt-losing Tubulopathies of the Distal Convoluted Tubule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salt-losing Tubulopathies of the Collecting Duct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salt-losing Mixed Tubulopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diseases Associated with RAAS Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liddle Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apparent Mineralocorticoid Excess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

54 55 56 56 58 61 63 65 68 74 81 86 86 89 89 90 92 92

Abstract

The renal tubules have an important role in body homeostasis through reabsorption and secretion of water and solutes. The aldosterone-sensitive distal nephron is crucial for the fine regulation of sodium and potassium reabsorption. Diseases referred to as tubulopathies result from abnormalities of proteins

M. Hureaux · R. Vargas-Poussou (*) Department of Genetics, Hôpital Européen Georges-Pompidou, Paris, France Centre de Référence des Maladies Rénales Héréditaires de l’Enfant et de l’Adulte (MARHEA), Paris, France e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2023 M. Caprio, F. L. Fernandes-Rosa (eds.), Hydro Saline Metabolism, Endocrinology, https://doi.org/10.1007/978-3-031-27119-9_3

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involved directly or indirectly in epithelial transport along the renal tubules. The focus of this chapter is on the inherited tubulopathies associated with chronic activation or inactivation of the renin aldosterone system. The first group are saltlosing tubulopathies, which affect all segments of the nephron: Fanconi renotubular syndrome, Dent disease, and Lowe syndrome in the proximal tubule; Bartter syndromes, HELIX syndrome and the new tubulopathy with cardiomyopathy in the thick ascending limb; Gitelman and Gitelman-like syndromes and EAST syndrome in the distal convoluted tubule; type 1 pseudohypoaldosteronism in the collecting duct; and a new mixed tubulopathy with hypokalemia, salt wasting, disturbed acid-base homeostasis and sensorineural deafness, affecting the proximal and distal convoluted tubule. The second group are tubulopathies characterized by an increase in sodium reabsorption and hypertension. This group includes type 2 pseudohypoaldosteronism or Gordon syndrome, which is caused by an increase of sodium reabsorption in the distal convoluted tubule, and Liddle syndrome and apparent mineralocorticoid excess, which result from an increase of sodium reabsorption in the principal cells of the collecting duct. After a review of the mechanisms of sodium and chloride reabsorption by the different nephron segments, we discuss the physiopathology and main characteristics of diseases of these two groups of tubulopathies, excluding pseudohypoaldosteronism types 1 and 2, which will be discussed in other chapters. Keywords

Aldosterone · Renin · Sodium reabsorption · Salt-losing · Hypertension

Introduction Tubulopathies result from abnormalities of proteins involved directly or indirectly in epithelial transport along the renal tubules. The renal tubules have an important role in body homeostasis through reabsorption and secretion of water and solutes. Inherited tubulopathies are a group of abnormalities with several modes of inheritance, variable presentation (in terms of age and severity), and often clinical and biological overlap. Tubulopathies are more frequently diagnosed in children than in adults, particularly those with autosomal recessive transmission. We will focus here on tubulopathies associated with chronic activation or inactivation of the renin aldosterone system (RAAS). The RAAS is activated in tubulopathies associated with a decrease in NaCl reabsorption and is inactivated in tubulopathies associated with an increase in NaCl reabsorption. In this chapter, we will first describe the mechanisms of NaCl reabsorption in the renal tubules and the common pathophysiological mechanisms that underlie each of these two groups of tubulopathies. Second, we will describe the particularities of these diseases considering the affected tubular segment. We will not discuss pseudohypoaldosteronism type 1 and type 2, because other chapters of this book are dedicated to these two diseases.

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Mechanisms of NaCl Reabsorption in the Renal Tubule The kidneys maintain the extracellular fluid volume by regulating Na+ and its most prevalent anion, Cl . Renal tubules reabsorb about 99.5% of the Na+ filtered by the glomeruli; therefore, small variations in the reabsorption rate can lead to changes in total body Na+ and markedly alter extracellular fluid volume. Figure 1 summarizes the segmental distribution of Na+ reabsorption along the nephron. The proximal tubule reabsorbs the largest fraction (~60–70%) in an isosmotic way (i.e., the Na+ concentration in the lumen at the end of the proximal tubule is the same as that of the plasma). The thick ascending limb (TAL) of the loop of Henle, which is not permeable to water, reabsorbs 20–25%. As a consequence, at the end of this segment the Na+ concentration is approximatively half of the plasma concentration. Finally, the distal convoluted tubule (DCT) and the collecting duct (CD) reabsorb smaller fractions, 5–10% and 2–5%, respectively, but can establish a steep transepithelial concentration gradient and can respond to several hormones including mineralocorticoids. The tubule can reabsorb Na+ and Cl through both transcellular and paracellular pathways. In the transcellular pathway, Na+ and Cl sequentially traverse the apical and basolateral membranes before entering the blood. In the paracellular pathway, these ions move through the tight junctions between cells depending on the electrochemical gradients (generated by ion channels and transporters at the apical and basolateral membranes) and permeability properties of the tight junctions. The

3 S1

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Fig. 1 Schematic representation of different segments of the nephron. Superficial nephrons have glomeruli located near the surface of the kidney and give rise to short-loop nephrons. Juxtamedullary nephrons have glomeruli located deep within the cortex, near the cortico-medullary border, and give rise to long-loop nephrons. The percentages of Na+ reabsorption in each segment are indicated

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mechanism of transcellular Na+ reabsorption is similar in all nephron segments: an apical passive step of entry into the cell is followed by active extrusion out of the cell across the basolateral membrane. All nephron segments express the basolateral Na+K+ ATPase pump, which keeps the intracellular Na+ concentration low and creates driving forces for apical transport. In addition, the cell voltage is negative with respect to the lumen. These two components explain the presence of an electrochemical gradient favourable to passive apical Na+ entry. In epithelial cells, basolaterally located K+-channels also play an important role helping to release potassium taken up by the activity of Na+/K+-ATPase to the extracellular compartment. This K+ release maintains the negative membrane potential across the basolateral membrane, which is a prerequisite for many transport processes. The mechanisms of passive apical Na+ entry differ in the different segments and will be developed hereafter. For further reading on the tubular reabsorption, we suggest the series published by the Clinical Journal of the American Society of Nephrology (Curthoys and Moe 2014; Mount 2014; Subramanya and Ellison 2014; Pearce et al. 2015; Roy et al. 2015; Palmer and Schnermann 2015).

Mechanisms of NaCl reabsorption in each Renal Tubule Segment Proximal Tubule Sodium reabsorption along the proximal tubule is the result of combined action of a multiplicity of processes along of its three heterogeneous segments (S1, S2, and S3, Fig. 1). These transport processes impose a high energy demand, which is sustained by numerous, elongated mitochondria typically observed in proximal tubule cells (Fig. 2). Various cotransporters in the apical membrane couple the uptake of Na+ to the uptake of solutes such as glucose, amino acids, phosphate, sulfate, and lactate. In addition to these cotransporters, Na+ entry is also coupled to the extrusion of H+ through the electroneutral Na+/H+ exchanger (NHE3), which is quantitatively the most important reabsorptive mechanism. Na+ moves from cell to blood via the Na+K+ ATPase pump and, to a lesser extent, via the electrogenic Na+/HCO3 cotransporter (also known as NBCe1). In the basolateral membrane, K+ channels are also expressed; particularly, heteromers of Kir4.2/Kir5.1 are important for the recycling of K+ necessary for the Na+-K+ ATPase pump function and to establish the negative voltage across the basolateral membrane. Cl is reabsorbed by both the transcellular and the paracellular routes; the paracellular route seems to be the dominant route in the early proximal tubule, whereas the transcellular pathway is dominant in the late proximal tubule. The transcellular route involves the apical side, and the exchange of luminal Cl for cellular anions (e.g., formate, oxalate, HCO3 , and OH ), which is mediated by CFEX (also known as SLC26A6). The basolateral exit step for transcellular Cl movement may occur in part through a Cl channel that is analogous in function to the cystic fibrosis transmembrane conductance regulator (CFTR). In addition, the basolateral membrane of the proximal tubule may also have a K+/Cl cotransporter (KCC). The S1 segment initially has no Cl

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Fig. 2 Diagram of proximal tubule cell. Shown are organelles (nucleus, endoplasmic reticulum, peroxisomes, and mitochondria), components of the endocytic pathway and main proteins of transport. Endocitic pathway: the low molecular weight proteins interact with the nonselective megalin/cubilin/amnionless receptor at the apical membrane. After internalization, the complexes formed by receptor and ligands progress along the clathrin-dependent endocytic pathway. The endosomes undergo a progressive increase in chloride concentration and acidification that results in the dissociation of the receptor-ligand complexes, with the receptors being recycled to the apical membrane, whereas the ligand is directed to acidic lysosomes for degradation. Modifications in chloride concentrations and pH in the endosomal vesicles are schematized at the bottom of the figure. Proteins of NaCl transport: apical Na+ reabsorption is mainly accomplished by NHE3, which exchanges Na+ for H+. The H+ luminal secretion participates to the bicarbonate reabsorption. Other apical sodium transporters are: Cl /base exchange (CFEX) and sodium-coupled transporters that use the chemical and electrical gradient of Na+ for the reabsorption of molecules (e.g., glucose, amino acids, phosphate). Basolateral Na+ exit is mediated by the Na+/K+-ATPase but some Na+ also exit with HCO3 via NBCe1. Cl exit is less well defined utilizing a variety of transporters including chloride channel CFTR, K+/Cl cotransporter (KCC). In addition Cl is reabsorbed by the paracellular route. The presence of K+ conductance (heteromers of Kir4.2/Kir5.1) allows the potassium gradient to increase the negative interior potential. The names of proteins defective in diseases described in this chapter are indicated by boxes: in FRTS, the GATM mitochondrial enzyme in the type 1; the NaPi2a cotransporter in the type 2; and the peroxisome enzyme EHHADH in type 3; in Dent-1 disease, the endosomal exchanger ClC-5; in Dent-2 disease and in Lowe syndrome, the phosphatidylinositol 4,5-bisphosphate-5-phosphatase (encode by OCRL); and Kir5.1 in the new mixed tubulopathy with hypokalemia, salt wasting, disturbed acid-base homeostasis and sensorineural deafnessProximal tubule.

concentration gradient between lumen and blood. However, the lumen-negative voltage generated by electrogenic Na+/glucose and Na+/amino acid cotransport establishes a favourable electrical gradient for passive Cl reabsorption. In the S2

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and S3 segments, the Cl concentration in the lumen becomes higher than in the blood due to the preferential HCO3 reabsorption in the earlier portions of the proximal tubule. This chemical gradient for Cl overcomes the electrical gradient so that paracellular movement of Cl in the late proximal tubule proceeds in the reabsorptive direction. An important characteristic of the proximal tubule is that the transepithelial voltage reverses polarity between the S1 and the S2 segments. The early proximal tubule is lumen negative because it reabsorbs Na+ electrogenically, both through electrogenic apical Na+ transporters and the basolateral Na+-K+ pump. The late proximal tubule reabsorbs Na+ at a lower rate. Moreover, because the Cl concentration in the tubular fluid is greater than in the plasma, the paracellular diffusion of Cl from lumen generates a lumen-positive potential that facilitates passive Na+ reabsorption by the paracellular route (Curthoys and Moe 2014; Palmer and Schnermann 2015). In addition, proximal tubular cells reabsorb a significant amount of albumin and low-molecular-weight (LMW) plasma proteins by receptor-mediated endocytosis. Filtered small proteins interact with the nonselective receptor complex (megalin/ cubilin/amnionless). After internalization, the complexes formed by receptor-ligands progress along the endocytic pathway, which involves coated pits and coated vesicles, followed by early endosomes that form recycling endosomes or mature to late endosomes and lysosomes (Fig. 2). In these vesicles the pH drops from 6.0 in early endosomes, 5.5 to 5.0 or below in lysosomes. This vesicular acidification is necessary for dissociation of the ligand-receptor complex, recycling of receptors to the apical membrane, and progression of ligands into lysosomes. In parallel, the Cl concentrations increase from 20–40 mM in early endosomes, to 80 mM or more in lysosomes. The endocytic activity of proximal cells requires the actions of the endosomal Cl /H+ exchanger ClC-5, which facilitates endosomal acidification and trafficking and the activity of the inositol polyphosphate-5-phosphatase (OCRL) which is required for proper vesicular trafficking between intracellular compartments and the plasma membrane. The integrity of this process is important for the normal function of proximal cells (van der Wijst et al. 2019). The names of proteins defective in diseases described in this chapter are indicated by boxes: in FRTS, the GATM mitochondrial enzyme in the type 1; the NaPi2a cotransporter in the type 2; and the peroxisome enzyme EHHADH in type 3; in Dent-1 disease, the endosomal exchanger ClC-5; in Dent-2 disease and in Lowe syndrome, the phosphatidylinositol 4,5-bisphosphate-5-phosphatase (encode by OCRL); and Kir5.1 in the new mixed tubulopathy with hypokalemia, salt wasting, disturbed acid-base homeostasis and sensorineural deafness.

Thick Ascending Limb In the TAL (Fig. 3), the transcellular pathway results from two major mechanisms that move Na+ across the apical membrane: The first is the NKCC2 cotransporter, which is sensitive to furosemide and which couples the entry of one Na+, one K+, and two Cl ions (electroneutral process) driven by the favourable concentration

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Fig. 3 Diagram of thick ascending limb. Shown are the top cells from the cortex and of the outer stripe of the outer medulla (OSOM) and on the bottom cells of the inner stripe of the outer medulla (ISOM). Vertical arrows indicate the tubular flow direction. Na+ is reabsorbed electroneutrally via NKCC2 (inhibited by furosemide). The rate-limiting step for the function of NKCC2 is the availability of K+. It is recycled via the potassium channel Kir1.1 leading to hyperpolarization of the apical membrane. Na+ exits the cell on the basolateral side via the Na+-K+-ATPase, and Cl exits via the chloride channels ClC-Ka and ClC-Kb, which depolarize the basolateral membrane. This process generates a lumen-positive transepithelial potential difference that drives paracellular reabsorption of cations. In the cortex and OSOM, divalent cations, Ca+2 and Mg+2, are reabsorbed via the claudin-16/19 complex, and, in the ISOM, Na+ is reabsorbed via claudin-10b. In the cortex and OSOM, claudins-16/19 and claudin-10b are not expressed in the same tight junctions. As the concentration of NaCl of tubular fluid arriving into the cortical TAL is lower, a paracellular backflow of Na + via the tight junctions expressing claudin-10b occurs (right panel), which contributes to maintenance of the lumen-positive transepithelial potential. The proteins defective in diseases described in this chapter are indicated by boxes: NKCC2 in Bartter type 1; Kir1.1 in Bartter type 2; ClC-Kb in Bartter type 3; barttin (subunit of both chloride channels) in Bartter type 4a; ClC-Ka and ClC-Kb in Bartter 4b; MAGED2 in Bartter type 5 (transient form); and claudin-10b in HELIX syndrome

gradients of Na+ and Cl . The SLC12A1 gene codes for NKCC2, a protein with 12 putative membrane-spanning domains that form a transport pathway after dimerization. Furosemide and other loop diuretics bind to transmembrane domains 11 and 12, whereas portions of domains 2, 4, and 7 are involved in ion transport. NKCC2 has three isoforms (A, B, and F) that arise due to alternative splicing of exon 4. These

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isoforms have different affinities for Cl and different patterns of expression in the TAL: In human kidney, NKCC2A is the dominant isoform and is found along the entire TAL including the macula densa and has the lower affinity for Cl ; NKCC2F is expressed in the outer medulla and cortex cells and has intermediate affinity for Cl and finally NKCC2B expression is limited to the cortical TAL including the macula densa segment and has the highest affinity for Cl . This differential expression is consistent with the presence of lower affinity isoforms in the medullary TAL with a higher tubular Cl concentration and transport rate compared with the presence of a higher affinity isoform in the cortical TAL reabsorbing NaCl from a more dilute tubular fluid. The limiting factor for NKCC2 function is K+ availability, which is provided by the potassium channel Kir1.1 (also known as ROMK), which recycles the K+ across the apical membrane and generates a lumen-positive electrical potential. The Kir1.1 channel is an ATP-sensitive, inwardly rectifying potassium channel encoded by the KCNJ1 gene. Each channel consists of two transmembrane domains flanking a conserved loop that contributes to the pore and selectivity filter, and cytoplasmic NH2 and COOH termini that contain regulatory and oligomerization domains and are expressed at the membrane as an assembly of four subunits (Mount 2014); (Palmer and Schnermann 2015). The second involves the Na+/H+ exchanger NHE3, responsible in a minor proportion of transepithelial Na+ reabsorption in the TAL. It is expressed in the apical membrane of the proximal tubule and TAL and is important for bicarbonate reabsorption. Recently, study of a constitutive tubule specific NHE3 knockout mouse model demonstrated the contribution of this Na+/H+ exchanger in the Na+ homeostasis and urinary concentration mechanism (Fenton et al. 2017). Mice lacking renal NHE3 have significantly elevated fluid intake, urinary flow rate, urinary sodium/creatinine, and pH and significantly lower urine osmolality and GFR than wild-type controls. Interestingly, the mice that lack NHE3 also have a urinary concentration defect after water deprivation that becomes more severe with a low-sodium diet. These data indicate that NHE3 in the medullary TAL contributes to some extent to the generation of the medullary interstitial gradient. As in the proximal tubule, the basolateral Na+-K+ pump keeps intracellular Na+ low and moves Na+ to the blood. The Cl leaves the cell through chloride channels ClC-Ka and ClC-Kb. They require the beta-subunit barttin to facilitate their insertion in the plasma membrane and generate Cl currents. ClC-Kb is also expressed in the basolateral membranes of the DCT cells and intercalated cells of the CD. These two channels are encoded by CLCNKA and CLCNKB, two highly homologous genes. The ClC proteins are functional dimers constituted by two independent permeation pathways called protopores. Each monomer is defined by 18 α-helices (from A to R) with an antiparallel structure, meaning that two structurally related halves (α-helices A to I and J to R, respectively) span the membrane with an opposite orientation. The protopore, localized between the two structurally related halves of the ClC monomer, is formed by an anion-selective filter between two aqueous vestibules comprising three Cl -binding sites. Paracellular transport is a passive process, which means that it is entirely dependent on local concentration gradients and its rate-limiting step is the transport across

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the tight junction. The tight junctions are complexes of structural and anchoring proteins including claudins, a group of integral membrane proteins. Claudins are tetraspan proteins with intracellular N- and C-termini and two extracellular loops; the first loop is important for ion selectivity, and the second for interaction with other claudins. Interactions can occur between claudins of the same plasma membrane (cis interactions) or with claudins of the neighbouring cell across the paracellular cleft (trans interactions); these interactions can happen with the same claudins (homophilic interactions) or with other claudins (heterophilic interactions). The complexes formed by claudins along the cell membranes act primarily as pore or barrier structures, defining permeability and selectivity of the paracellular space and the transepithelial electrical resistance. In the TAL, the paracellular pathway for Na+ reabsorption is favoured by the lumen-positive voltage. Because K+ channels dominate the apical membrane conductance, the voltage of the TAL apical membrane is more negative than that of the basolateral membrane, thereby resulting in lumenpositive transepithelial voltage, which provides the driving force for the diffusion of Na+ and for the passive reabsorption of Ca+2 and Mg+2 through the paracellular pathway. The permeability of the TAL is determined by the presence of several claudins (3, 10b, 14, 16, and 19), which are heterogeneously expressed in different portions of the TAL. Taking as example a long-looped nephron, in the first portion (inner stripe of the outer medulla), the claudin-10b is the only claudin present, whereas in the outer stripe of the outer medulla (OSOM) and in the cortex, claudins have a mosaic pattern of expression with tight junctions expressing either claudin10b or claudins 3, 16, and 19. It has been shown that tight junctions expressing claudin-10b are highly permeable to Na+, making the ISOM the seat of 50% of Na + reabsorption. As the tubular fluid arriving to the cortical TAL has been depleted in NaCl, a backflow of Na + into the lumen along its concentration gradient via the paracellular tight junctions expressing claudin-10b occurs in this segment, which contributes to maintain the lumen-positive transepithelial potential. Claudins 16 and 19 confer selectivity for divalent cations to tight junctions of the cortical and outer stripe of the outer medulla segments of the TAL allowing the reabsorption of 50 to 60% of the filtered Mg+2 and approximately 20% of the filtered Ca+2 (Milatz et al. 2017; Gong and Hou 2017).

Distal Convoluted Tubule In the DCT, Na+ reabsorption occurs almost exclusively by the transcellular route (Fig. 4). The DCT has two segments called DCT1 and DCT2. Cells in DCT1 only express the thiazide-sensitive sodium-chloride cotransporter (NCC), whereas DCT2 cells express both NCC and the epithelial sodium channel (ENaC). In the DCT1, the apical step of Na+ uptake is mediated by NCC, which is sensitive to thiazide diuretics. In the DCT2, in addition to NCC, the apical uptake is also mediated by ENaC, which is sensitive to amiloride. NCC is encoded by the SLC12A3 gene and belongs to the SLC12 family of electroneutral cation-chloride cotransporters that also comprises the NKCC2 protein. NCC contains a central hydrophobic region

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Thiazides

SPAK/OSR1

P

NCC

Cl Na+

2K+

P P

P

WNK4

Ca2+

WNK1

Ð[CL-]

K+

KLHL3 CUL3

Ca2+

TRPV5

RING

E3

Ub Ub Ub

Na+

H+

Na+

Thiazides

Kir4.1

Cl-

ClC-kb

DCT1

Mg2+

Proteasome

ClAmiloride

K+

Ca2+ K+ KCC4 Cl-

WNK1/4

Mg2+

TRPM6

3Na+

P

NCC

Na+

2K+

MT-TF

3Na+

IV IV

K+ MT-TI

ENaC

Na+ +

K

Kir4.1

Cl- ClC-kb K+ Cl-

DCT2

KCC4

Fig. 4 Diagram of the distal convoluted cells. In cells of the early DCT (DCT1), Na+ is reabsorbed at the apical membrane via the electroneutral thiazide-sensitive NaCl cotransporter NCC and exits at the basolateral membrane via the Na+-K+-ATPase. Cl exits via the chloride channel Cl-Ckb and potassium chloride cotransporter 4 (KCC4). In the top part of the figure is shown the regulation of the activity of NCC by the phosphorylation cascade implicating SPAK/OSR/WNKs. WNK4 is active in its phosphorylated form and is autophosphorylated when the intracellular chloride concentration is low. Activated WNK4 phosphorylates SPAK/OSR, which, in turn, phosphorylates NCC increasing the apical NaCl reabsorption. WNK1/4 protein levels are regulated by the complex CUL3-KLHL3E3, which targets WNK1/4 for degradation by promoting their ubiquitylation. Pathogenic variants in the genes encoding for the members of this pathway of NCC regulation (WNK1, WNK4, KLHL3, CUL3) are responsible for pseudohypoaldosteronism type 2 (see Table 5 and the specific chapter dedicated to this disease). In the bottom part of the figure is shown the inhibition of NCC phosphorylation by the dysfunction of oxidative phosphorylation complex IV due to loss of function variants in transfer RNAs for phenylalanine and isoleucine (MT-TF and MT-TI). The heteromer of potassium channel Kir4.1/Kir5.1 recycles the K+ allowing the activity of the Na+-K+-ATPase. In the late DCT (DCT2), both NCC and the amiloride-sensitive epithelial sodium channel ENaC are present. The names of proteins defective in diseases described in this chapter are indicated by boxes: NCC in Gitelman syndrome, Kir4.1 in EAST syndrome and Kir5.1 in the new tubulopathy with hypokalemia, salt wasting, disturbed acid-base homeostasis and sensorineural deafness; as well as the transfer RNAs MT-TI and MT-TF recently implicated in Gitelman-like syndrome. The impaired salt reabsorption in DCT indirectly affects Mg+2 uptake via the magnesium channel TRPM6, explaining the renal magnesium wasting in patients with these diseases

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comprising 12 transmembrane domains with intracellular NH2 and COOH termini, and it functions as a dimer at the plasma membrane. A model suggests that residues involved in Cl affinity are located within the transmembrane 1–7 and for thiazides between transmembrane domain 8 and 12 and that both segments are involved in defining Na+ affinity of the cotransporter. The activity of NCC is regulated by posttranslational modifications such as glycosylation, phosphorylation, and ubiquitination. Two glycosylation sites are found at the connecting loop between transmembrane domains 7 and 8; NCC is functional in a highly glycosylated form. Phosphorylation of NCC is critical for maximal NaCl transport capacity and can alter membrane abundance. Four phosphorylation sites are found in the N-termini. The phosphorylation of NCC is regulated by a variety of hormonal stimuli, which exert several of their effects via activation of the WNK-SPAK kinase cascade. Finally, NCC is regulated by ubiquitination via the aldosterone-SGK1-Nedd4–2 pathway; Nedd4–2 facilitates the degradation of non-phosphorylated NCC. In addition, it has been recently shown that Nedd4–2 inhibits the activity of the basolateral potassium channels Kir4.1/Kir5.1, which are associated with membrane depolarization and reduction of the driving force for Cl exit. The result is an increase of intracellular Cl concentration, which is known to inhibit WNK and SPAK with the consequent inhibition of NCC phosphorylation and activity. The basolateral step of Na+ reabsorption, in both segments and as in other cells, is mediated by the Na+-K+ pump and the Cl leaves the cell by the chloride channel ClC-kb. The transition segment between the DCT is known as the connecting tubule, and its cells resemble the principal cells of the CD and only express ENaC (Subramanya and Ellison 2014; Palmer and Schnermann, 2015).

Collecting Duct Na+ reabsorption in the CD is transcellular and occurs mainly in the principal cells (Fig. 5). Na+ crosses the apical membrane of these cells through ENaC, which is a heteromer of homologous α, β, and γ subunits. Each subunit is encoded by a specific gene (SCNN1A, SCNN1B, and SCNN1G, respectively), which share around 30%– 40% sequence identity. Each ENaC subunit consists of two transmembrane domains (TM1 and TM2), a large extracellular loop, and short cytoplasmic amino and carboxyl termini. Based on one homology model, the three subunits would be arranged around a central ion-pore, lined by the TM2 domains of the subunits. The specificity of ENaC for Na+ depends on the size of the selectivity filter, which allows only the passage of the smallest ions (Na+ and Li+). The activity of ENaC depends on open probability (Po) and on the number (density) of channels expressed at the apical membrane of principal cells. The latter is determined by the balance between the rate of transfer of ENaC from intracellular sites to the apical membrane and the rate of internalization from the apical membrane. One of the mechanisms affecting the ENaC gating and Po is the proteolytic cleavage. The α and ɣ subunits exhibit a high and low molecular weight forms (uncleaved and cleaved, respectively) as result of two cleavages by serine-proteases. ENaC is fully active only in its cleaved state. Aldosterone is the main regulator of ENaC, by interfering with the

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Pendrin Cl-

K+

HCO3-

Intercalated cell β

HCO3Cl-

Na +

ClCl- ClC-Kb H+

HCO3NDCBE Na+

Proton pump H+

HCO3

H+

α Intercalated cell α

Cl-

HCO3AE1

CAII H+

K+

H2CO3

K+

CO2

Amiloride

H2O

(-)

(+) Claudin-4

K+

ENaC Na+ Kir1.1

Na +

ClNa+

Aldosterone

K+

Principal cell

Cortisol

SGK1

H2O

11β -HSD2

MR

Cortisone

H2O

Fig. 5 Diagram of the collecting duct. Shown are the main types of cells of this segment: α and β intercalated cells and principal cells. Na+ is reabsorbed electrogenically in principal cells through the apical amiloride-sensitive sodium channel ENaC and the basolateral Na+-K+-ATPase. This process facilitates K+ secretion through Kir1.1 channel, H+ secretion by the proton pump located in the apical membrane of α intercalated cells, and paracellular Cl reabsorption. Na+ is also reabsorbed electrogenically in β intercalated cells through the apical NDCBE and pendrin and the basolateral Na+-K+-ATPase and ClC-Kb chloride channel. In principal cells, aldosterone binds to nuclear mineralcorticoid receptors (MR), which in turn increase transcription of genes that encode proteins such as the α ENac subunit. Principal cells highly express 11βHSD2, which catalyses the conversion of cortisol to inactive cortisone and allows selective MR activation by aldosterone. The names of proteins defective in diseases described in this chapter are indicated by boxes: activating pathogenic variants in one of the genes encoding for ENaC subunits in Liddle syndrome and lossof-function variants of the gene encoding for the 11βHSD2 in the apparent excess of mineralocorticoid. Inactivating bi allelic pathogenic variants in one of the genes encoding for ENaC subunits and heterozygous loss-of-function variants of the gene encoding for the mineralcorticoid receptors (MR) are responsible for pseudohypoaldosteronism type 1 (a specific chapter is dedicated to this disease)

transcription, the redistribution of ENaC subunits and the Po of the channels. Aldosterone induces SCNN1A transcription with a resulting increase in the expression of α subunit. Aldosterone also induces the expression of other genes involved in regulating posttranslational modifications of ENaC subunits resulting in an increase

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in both the number and the Po of ENaCs expressed at the apical membrane. These include SGK1, which inhibits the removal of ENaC from the apical membrane by phosphorylating the ubiquitin ligase Nedd4–2. SGK1 also directly phosphorylates the carboxyl terminus of the a-subunit of ENaC and plays a role in promoting ENaC trafficking from intracellular compartments to the apical membrane. Levels of arginine vasopressin may also modulate the number of ENaCs that are open in the apical membrane. The abundances of the ß and γ subunits of ENaC are increased by arginine vasopressin. ENaC is specifically blocked by the diuretic amiloride. A mechanism of Na+ and Cl reabsorption is also present in neighbouring ß intercalated cells, which involve two apical exchangers operating in parallel, the Na+ dependent Cl-HCO3 exchanger (NDCBE) and the Cl-HCO3 exchanger pendrin, and two basolateral proteins, the Na+-K+ ATPase and the chloride channel ClC-Kb). As in the other segments, the basolateral step of Na+ reabsorption is mediated by the Na+-K+ pump, which also provides the electrochemical driving force for the apical entry of Na+. These transport properties of the principal cells are also the basis for the lumen-negative transepithelial potential difference in the CD. In addition to ENaC, this segment has both apical and basolateral K+ channels, which play key roles in K+ transport. The lumen-negative potential generated by Na+ entry favours K+ exit from cell to lumen in principal cells, H+ secretion by α intercalated cells, and paracellular Cl reabsorption mediated by claudin-4 (Pearce et al. 2015; Roy et al. 2015; Palmer and Schnermann 2015). The nephron segments in which the expression of sodium ion transporters on the apical side of renal tubular cells is mainly regulated through the action of aldosterone are collectively called the aldosterone-sensitive distal nephron (ASDN). The ASDN is comprised of the DCT2, the connecting tubule, and the cortical CD. The cellular effects of aldosterone are exerted through intracellular mineralocorticoid receptors (MRs) that can bind to both aldosterone and cortisol (Fig. 5). The selective binding of aldosterone to MR in ASDN cells is ensured by the expression of 11ß-hydroxysteroid dehydrogenase type 2, which catalyses the conversion of cortisol to inactive cortisone. This function is critical, because the plasma concentration of aldosterone is much lower than that of cortisol. Upon binding of aldosterone, the MR undergoes a conformational change, translocates to the nucleus, and regulates transcription of target genes including SGK1 and SCNN1A. SGK1 is a Ser/Thr kinase that phosphorylates ubiquitin ligase NEDD4–2, resulting in decreased association between NEDD4–2 and ENaC. This in turn decreases ubiquitination and degradation of ENaC, increasing the number of channels on the plasma membrane. MRs are also expressed in β intercalated cells, and aldosterone upregulates pendrin and the proton pump in these cells, increasing Na+ reabsorption.

Diseases Associated with RAAS Activation The group of diseases associated with RAAS activation include the salt-losing tubulopathies. Any nephron segments can be affected. These diseases and the genes associated are listed in the Table 1.

Thick ascending limb of Henle loop

Nephron segment Proximal tubule

607364 602522

Bartter type 4a

309000

Lowe syndrome

Bartter type 3

300555

Dent disease 2

241200

300009

Dent disease 1

Bartter type 2

615605

Fanconi Renotubular syndrome type 3

601678

613388

Fanconi Renotubular syndrome type 2

Bartter type 1

OMIM number 134600

Disease Fanconi Renotubular syndrome type 1

Table 1 Diseases associated with RAAS activation

AR

AR

AR

AR

X-linked

X-linked

X-linked

AD

AR

Inheritance AD

SLC12A1/ 15q21.1 KCNJ1/ 11q24.3 CLCNKB/ 1p36.13 BSND/1p32.3

OCRL/Xq26.1

CLCN5/ Xp11.23 OCRL/Xq26.1

Gene/ chromosome GATM/ 15q21.1 SLC34A1/ 5q35.3 EHHADH/ 3q27.2

Barttin

ClC-Kb

Kir1.1

Phosphatidylinositol 4,5-bisphosphate-5phosphatase Phosphatidylinositol 4,5-bisphosphate-5phosphatase Na-K-Cl cotransporter NKCC2

Enoyl-CoA-hydratase:3hydroxyacyl-CoA dehydrogenase ClC5 channel

NaPi2a cotransporter

Protein Glycine amidinotransferase

66 M. Hureaux and R. Vargas-Poussou

Mixed (proximal and distal convoluted tubule)

Collecting duct

Distal convoluted tubule

619406

177735

Pseudohypoaldosteronism type 1 (renal)

Tubulopathies with hypokalemia, salt wasting, disturbed acid-base homeostasis and sensorineural deafness

24350

612780

EAST (or SeSAME) syndrome

Pseudohypoaldosteronism type 1 (generalized)

NA

Gitelman-like syndrome

620152 617671

Kidney tubulopathy and cardiomyopathy Helix syndrome 263800

300971

Bartter type 5

Gitelman syndrome

613090

Bartter type 4b

AR

AD

AR

AR

Mitochondrial

AR

AD AR

X-linked

AR

CLCNKACLCNKB/ 1p36.13 MAGED2/ Xp11.21 RRAGD/6q15 CLDN10/ 13q32.1 SLC12A3/ 16q13 MT-TF and MT-TI/ mtDNA KCNJ10/ 1q23.2 SCNN1A/ 12p13.31 SCNN1B/ 16p12.2 SCNN1G/ 16p12.2 NR3C2/ 4q31.23 KCNJ16/ 17q24.3 Kir5.1

Mineralocorticoid receptor

ENaC γ subunit

ENaC β subunit

ENaC α subunit

Transfer RNAs for phenylalanine and isoleucine Kir4.1 channel

NaCl cotransporter NCC

GTPase RagD Claudin-10

MAGE-D2

ClC-Ka/ClC-Kb

3 Tubulopathies and Alterations of the RAAS 67

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It is important to note that some of genetic salt-losing tubulopathies mimic diuretic actions. This is because some transport proteins expressed along the nephron are targets of diuretics (Figs. 2, 3, 4, and 5). For example, Bartter syndrome type 1 mimics furosemide action and Gitelman syndrome mimics thiazide action (Seyberth PN 2015). Other renal tubulopathies can be present with salt loosing and RAAS secondary activation, as is the case of renal tubular acidosis. We will discuss here only diseases affecting directly one of the mechanisms of salt reabsorption previously described. In diseases affecting NaCl reabsorption, salt losing generates a hypovolemic status with a secondary activation of the RAAS and normal or low blood pressure; the compensatory increase of the NaCl reabsorption and K+ and H+ secretion in the aldosterone-sensitive distal tubule are responsible for the hypokalemic metabolic alkalosis often detected in patients. A defect of Na+ reabsorption in one nephron segment is compensated by increased reabsorption rates in the remaining segments (downstream and upstream). If the salt losing starts in the proximal tubule, Na+ reabsorption is increased in TAL and DCT1 and aldosterone-sensitive distal tubule. If the salt losing starts at the TAL, there is upregulation of the DCT and aldosteronesensitive distal tubule as well as of the proximal tubule. If the salt losing starts at the DCT, there is upregulation of aldosterone-sensitive distal tubule as well as of upstream segments (proximal tubule and TAL). The mechanisms involved in these nephron adaptations are complex and involve responses to changes in extracellular fluid volume, responses to increased solute delivery, and structural changes or increase of activity of transporter proteins. These mechanisms are particularly well documented in animal models and in patients with Bartter and Gitelman syndromes. In Bartter syndrome, the impairment of Na+ reabsorption along the TAL increases NaCl delivery to DCT leading to increase reabsorption via NCC and hypertrophy of DCT cells; this hypertrophy further increases the abundance of NCC and NaCl transport capacity. In parallel, the activation of RAAS by the volume depletion contributes to increased DCT reabsorption because NCC is stimulated both by angiotensin II and aldosterone (Cantone et al. 2008). In Gitelman syndrome, the impairment of Na+ reabsorption along the DCT increases NaCl delivery to the aldosterone-sensitive distal tubule leading to increase reabsorption via ENaC in principal cells of the connecting tubule and the CD as well as via pendrin/NDCBE electroneutral NaCl reabsorption in the β intercalated cells. Gitelman syndrome is associated with hypertrophy of connecting tubule and CD principal cells as well as remodelling of the CD with an increase in the number of β intercalated cells. In addition, an increase of solute reabsorption in the proximal tubule and an increase of NaCl reabsorption in the TAL, probably associated with an increase of NKCC2 phosphorylation, has been observed in Gitelman syndrome patients and in animal models (Loffing et al. 2004; Favre et al. 2012; Grimm et al. 2015).

Salt-losing Tubulopathies of Renal Proximal Tubule There is not an isolated form of salt losing that affects strictly the proximal tubule. The defect of salt reabsorption in this segment is observed as part of the Fanconi

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renotubular syndrome (FRTS) also known as de Toni-Debré-Fanconi syndrome, which is a global dysfunction of the proximal cells. Numerous inborn errors of metabolism are associated with FRTS (Wilson disease, tyrosinemia, galactosemia, congenital fructose intolerance, and cystinosis) as well as mitochondrial cytopathies (van der Wijst et al. 2019). Non-metabolic or primary causes of FRTS are listed in Table 1. These diseases are due to anomalies of proteins involved in mitochondrial function (autosomal dominant FRTS type 1 and 3) or in the endolysosomal process (X-linked Dent and Lowe syndromes). Clinically, the complete form of FRTS is diagnosed in children and is characterized by dehydration episodes, rickets, osteomalacia, and growth failure. Further, patients have acidosis and electrolyte imbalances reflecting excessive urinary excretion of salt, potassium, bicarbonate, calcium, and phosphate. The increased excretion of amino acids, glucose, uric acid, and lowmolecular-weight proteins is also present. FRTS type 1 is an autosomal dominant condition caused by gain of function variants in the GATM gene, which encodes a mitochondrial enzyme glycine amidinotransferase; this enzyme is critical for the formation of guanidinoacetate. GATM is fully active as homodimer. Complete GATM deficiency (due to biallelic loss-of-function variants) causes cerebral creatine deficiency syndrome type 3, characterized by mental retardation, language impairment, and behavioural disorders, without muscular or kidney phenotypes, which contrast with the phenotype of FRTS1. This difference is explained by the nature and localization of pathogenic variants in patients with FRTS1. They are clustered in a region important for dimerization leading to creation of new “interaction sites” that allowed the dimeric GATM protein to multimerize and form fibrils that cause mitochondrial toxicity. These aggregates are also responsible for inflammation and renal fibrosis. Accordingly, renal biopsies of affected individuals showed renal fibrosis and proximal tubules showed enlarged mitochondria containing fibril-like structures. Immunogold staining to label GATM showed that these fibrils contained GATM. Patients present with FRTS during childhood, and develop chronic kidney disease during the second decade of life that progresses to end-stage renal disease in adulthood (Reichold et al. 2018). FRTS type 2 has autosomal recessive inheritance and is due to variants in the SLC34A1 gene, which encodes the sodium phosphate co-transporter NaPi-IIa. NaPiIIa is expressed in the apical membrane of proximal tubule cells. The pathogenic variant described result in the intracellular retention of NaPi-IIa, which induces global proximal tubule cell dysfunction; the molecular mechanisms that underlie this retention are not clear. No further patients with SLC34A1 pathogenic variants and FRTS have been described after the first report in 2010 (Magen et al. 2010). In contrast, several patients with bi-allelic pathogenic variants in this gene presenting with infantile hypercalcemia have been described. In this disease, phosphate wasting leads to increases in cholecalciferol, absorptive hypercalciuria, hypercalcemia, and nephrocalcinosis (Schlingmann et al. 2016). FRTS type 3 is an autosomal dominant condition caused by gain-of-function variants in the EHHADH gene. This gene codes for the enzyme enoyl-CoA hydratase-L-3-hydroxyacyl-CoA dehydrogenase, which plays an important role in

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the oxidation of fatty acids in peroxisomes. To date, one family with this disease and one patient with a de novo variant have been described. The missense variant detected in affected patients of that family and in de novo case is the same. This substitution in the third residue of the N-terminal domain was predicted in silico to be responsible of the introduction of a pathological mitochondrial targeting signal. This mechanism was confirmed by in vitro studies, which showed that the abnormal protein is mis-targeted to the mitochondria where it forms aggregates with mitochondrial proteins leading to abnormal production of ATP. Eventually, defects in both fluid and solute transport are the consequence of a reduction of the Na+-K+ATPase activity secondary to the proximal cellular ATP deficiency. Patients present Fanconi syndrome in childhood with rickets. In contrast with type 1, patients with FRTS type 3 do not have chronic kidney disease (Klootwijk et al. 2014). Dent disease, a recessive X-linked tubulopathy, is caused by loss-of-function variants in the CLCN5 gene (Dent-1) or in the OCRL gene (Dent-2). These two genes explain the disease in 50 to 60% and 15% of cases, respectively. Additional genetic heterogeneity should explain the disease in the remaining 25 to 35% of cases. Several gene candidates have been tested but the genetic cause in this group remains unidentified. The CLCN5 gene encodes the exchanger ClC-5, which is expressed in endosomes. It is functionally coupled to H+-ATPase and mediates the acidification and chloride accumulation in these vesicles. CLCN5 pathogenic variants result in loss of antiporter function due either to absence of the protein, or to impaired processing and folding, with endoplasmic reticulum (ER) retention or to loss of Cl conductance. Some pathogenic variants cluster at the dimer interface impairing dimerization and leading to rapid degradation of the mutant protein within the cell. A quiet low number of pathogenic variants are recurrent, which indicates that most families have private variants. Studies in vivo and in vitro in CLCN5 knockout (KO) mice and cells have shown that loss of function of ClC-5 is associated with abnormal endosomal acidification or defective endosomal Cl accumulation or increase of phosphatidylinositol biphosphate in early endosomes resulting in a severe trafficking defect. This defect is responsible for: first, of reduced levels of megalin and cubilin at the brush border and defective receptor-mediated endocytosis with urinary loss of several LMW ligands (including lysosomal enzymes, parathyroid hormone, vitamin carrier proteins, lipoproteins, cell surface antigen and Immunoglobulin light chains). Second, of impaired lysosomal function, which may at least in part be due to defective megalin and finally a defective internalization of NaPi-IIa and Na+/H+ exchanger 3 (NHE3). Although there is a discrepancy in described mouse models regarding the presence of hypercalciuria and their possible mechanisms, the following mechanisms have been suggested. First, an Increase of tubular PTH concentrations, as a result of abnormal endocytosis, which will induce up-regulation of 1-α-hydroxylase, increasing 1.25(0H)2 vitamin-D and intestinal calcium absorption. However, also as consequence of abnormal endocytosis, a great quantity of the 25 (OH)D appears in the final urine, decreasing the substrate. Second, PTH might indirectly favour a renal calcium leak by inhibiting NHE3. As mentioned previously NHE3 expression was reduced in mouse models of Dent-1 disease, and

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PTH-induced endocytosis of NHE3 was also markedly reduced. This decrease could be associated with a decrease of proximal tubular sodium reabsorption and consequently a decrease of paracellular calcium reabsorption. Finally, PTH also stimulates calcium transcellular reabsorption in the distal convoluted tubule, which can counterbalance these effects. It is probably the balance between these features that increase or decrease calcium absorption that will determine the presence or absence of hypercalciuria. Furthermore, the increase of tubular PTH concentrations enhances internalization of sodium/phosphate cotransporter NaPi2a explaining hyperphosphaturia. Studies in animal and proximal cell models as well as urinary proteome analysis in patients with Dent-1 have shown an increase of expression of mRNA or proteins participating to proliferation, oxidative stress and interstitial matrix remodelling. From these studies, the following hypothesis to explain how the abnormal endocytosis could affect the whole proximal cell has emerged: the endolysosomal defect might compromise autophagy, with defective clearance of autophagosomes, which may lead to oxidative stress. It disrupts the integrity of the junctional complex, particularly the zonula occludens protein, which might activate an abnormal signalling cascade involving YBX3 protein, a transcription factor known to promote cell proliferation and represses proximal tubular cells differentiation (van der Wijst et al. 2019; Gianesello et al., 2021). The OCRL gene encodes the inositol polyphosphate 5-phosphatase enzyme (OCRL protein), which is critical for maintenance of low levels of PI(4,5)P2 necessary for proper endolysosomal trafficking. Pathogenic variants of this gene are responsible for the oculocerebrorenal syndrome of Lowe and for the Dent-2 disease. The OCRL protein is a multi-domain protein; the N-terminus of OCRL contains a PH (pleckstrin homology) domain, which is unable to bind phosphoinositides. The PH domain is connected to the central 5-phosphatase catalytic domain by a linker region. The catalytic domain is followed by an ASH domain (ASPM-SPD2-Hydin) and by the C-terminal RhoGAP domain. Although the majority of OCRL pathogenic variants associated with Lowe syndrome are located in exons 8–23 (coding for the catalytic, ASH and Rho-GAP domains) and the majority of pathogenic variants causing Dent-2 are located in exons 1–7 (coding for PH and linker region), some pathogenic variants have been identified as causing both Lowe syndrome and Dent disease 2. A recent analysis of all published cases suggests that the mutation type is also important to explain the phenotype of Lowe syndrome or Dent-2 disease, with truncating variants in the PH and linker domain mainly associated with Dent-2 and missense variants in the 5-phosphatase domain associated with Lowe syndrome (Gianesello et al. 2021). The pathophysiological consequences of loss-of-function of the OCRL protein have been recently confirmed in a humanized mouse model of Dent-2 and Lowe syndrome, which presents with renal (massive urinary losses of LMWP and albumin) and extra renal manifestations (muscular defects with dysfunctional locomotricity). Studies in primary culture of proximal tubule cells derived from these mice kidneys, showed accumulation of phosphatidylinositol 4,5–bisphosphate PI(4,5)P2 in endolysosomes, local hyper-polymerization of F-actin and impaired trafficking of the endocytic megalin receptor. The OCRL deficiency was also associated with a disruption of the lysosomal dynamic affecting their proteolytic activity (Festa et al. 2019).

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Dent disease is frequently diagnosed in children; around 6–11 years in European and North American populations. In Asian population the Dent disease is detected earlier between 2 to 5 years, probably due to the annual school urinary screening program. Low-molecular-weight proteinuria is observed in all Dent disease patients, hypercalciuria and nephrocalcinosis are highly prevalent, nephrolithiasis is present in around 50% of cases. Incomplete FRTS is often observed and a complete FRTS has been described in around 10% of patients. Plasma potassium concentration declined with age and patients can develop a salt-losing tubulopathy with characteristics of Bartter syndrome (salt and potassium with secondary hyperaldosteronism without hypomagnesemia but with polyuria) (Blanchard et al. 2016). Patients with Dent’s disease can have nephrotic range proteinuria without hypoalbuminemia; and the proteinuria can increase with age. Renal biopsies of Dent patients showed either normal histology or a variety of glomerular, tubular and interstitial lesions. The more frequent are glomerulosclerosis, tubular atrophy, interstitial fibrosis and podocytes foot process effacement. To note, there are patients with the same gene mutation and a different histology. In some cases estimated GFR has been shown to correlate with the degree of interstitial fibrosis in renal histopathologic analyses. Progression to end-stage renal failure occurs between the third and the fifth decades of life in 30 to 80% of affected males; no correlation has been observed between the rate of decline of renal function and the presence or absence of nephrocalcinosis. No correlation has been observed between genotype and phenotype in several cohorts of patients and a considerable intra-familial variability in disease severity has also been observed. Heterozygous females are often asymptomatic and exhibit moderate low-molecularweight proteinuria and hypercalciuria (Mansour-Hendili et al. 2015; Blanchard et al. 2016; Gianesello et al. 2021). Although Dent-2 disease has a similar phenotypic presentation as Dent-1, there are some significant differences between these two groups. Glycosuria, hypophosphatemia and hypokalemia are more frequent in Dent-1 and failure to thrive and renal failure in Dent-2. Furthermore, some clinical features have been described only in patients with Dent-2: such as elevated concentrations of muscle enzymes (CPK and LDH), neurologic manifestations as mild intellectual disability and hypotonia (Gianesello et al. 2021). Patients with Lowe syndrome have, in addition to FRTS, specific extra renal manifestations including of bilateral congenital cataracts (100% of cases), glaucoma, and neurological abnormalities including severe neonatal hypotonia and development delay with severe cognitive impairment in the majority of children. Other clinical manifestations described in patients with Low syndrome include: behavioural abnormalities which often develop or worsen with age, febrile convulsions and seizures, undescended testis, chronic constipation, scoliosis, joint dislocation, joint swelling and arthritis, dental abnormalities and a high incidence of bleeding episodes. Patients develop chronic kidney diseases leading to end-stage renal failure in adulthood (around 40 years) (Bokenkamp and Ludwig 2016). The main clinical characteristics of different forms of FRTS are summarized in the Table 2.

Not reported

Rickets or osteomalacia Almost all patients (ESRD between the 4th and 7th decades) –

Nephrocalcinosis

Bone disease

Other manifestations

No

–

–

100% 100% 100% 100% Not reported Not reported 100%

100%

Complete

FRTS3 Children

Yes

Rickets

Only two siblings have been described NA: not available; ESRD: end stage renal disease

a

100% Absent 100% 100% NA

100% 100% 100% 100% 1 case

Chronic kidney disease

Yes 100%

Not reported 100%

Absent

Complete

FRTS2 Infanta

Complete

FRTS1 Children

Incomplete/ complete RTFS Failure to thrive Low molecular weight proteinuria Hypophosphatemia Metabolic acidosis Glycosuria Aminoaciduria Hypokalemia

Age at onset

10–35% (ESRD between the 4th and 5th decades) –

10–28%

22–65%

33% 8–17% 22–45% 47–50% 30–45%

25–30% 100%

Dent-1 Children, adolescent, adults 67–73%/5–11%

Table 2 Main clinical characteristics of Fanconi Renotubular syndrome (FRTS)

Muscle enzymes (CPK and LDH), mild intellectual disability hypotonia

30–50%

15%

10–33%

10% 7–25% 10–20% 22–42% 11–20%

54–65% 100%

55–71%/3–11%

Dent-2 Children, adolescent, adults

Bilateral congenital cataracts (100% of cases), glaucoma, intellectual impairment, severe neonatal hypotonia

74%

NA

45%

15–43% 30–80% 7–15% 40–80% 20%

85–90% 100%

NA

Lowe Infant

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Genetic confirmation of FRTS is important for adapted genetic counselling according to the inheritance mode and for adequate follow-up. Testing relatives is particularly useful to identify heterozygous female carriers in families with X-linked inheritance (Dent disease and Lowe syndrome), to perform early screening of any male children from carrier mother and giving reassurance to noncarrier mothers about the transmission of the disease to their children. To note, for Dent-1 disease the de novo mutation rate has been estimated to about 12%. Considering the genetic heterogeneity and overlapping phenotypes, the simultaneous analysis by highthroughput sequencing using targeted gene panel is recommended. FRTS treatment is based on replacement of the renal solute and water loss and the prevention of bone disease. The normalization of serum phosphate concentrations with oral phosphate supplements is important. Correction of proximal renal tubular acidosis usually requires large doses of alkali.

Salt-losing Tubulopathies of the Thick Ascending Limb Bartter syndromes. Bartter syndromes are caused by loss-of-function variants in proteins that encode transporters or channels participating in NaCl reabsorption in the thick ascending limb. Bartter syndrome is classified into five genetic subtypes: Type 1 is caused by pathogenic variants in the SLC12A1 gene, which encodes the furosemide-sensitive sodium-potassium-chloride cotransporter of the apical membrane of the TAL epithelium. Type 2 results from pathogenic variants in KCNJ1, the gene encoding the apical potassium channel Kir1.1. Type 3 is related to pathogenic variants in the CLCNKB gene, which encodes the basolateral chloride channel ClC-Kb; this channel is expressed in the TAL and DCT and this type is characterized by a phenotypic variability. Pathogenic variants in the BSND gene encoding the protein barttin (an accessory β-subunit of the ClC-Kb and ClC-Ka channels) lead to type 4a. Loss-of-function variants of the two CLCNKA and CLCNKB genes (digenic disease) are responsible for type 4b Bartter syndrome. Finally, type 5 is related to pathogenic variants in MAGED2 gene, which encodes a nuclear protein that affects the expression and function of at least two sodium-chloride cotransporters (Seyberth 2015; Laghmani et al. 2016; Konrad et al. 2021). Locations of the proteins involved in Bartter syndrome are summarized in Fig. 3. Bartter types 1 to 4 are long-life autosomal recessive diseases, but Bartter type 5 is a transient form with X-linked inheritance. Genetic screening and detection of pathogenic variants in genes responsible for Bartter syndrome is crucial to confirm the clinical diagnosis and for genetic counselling. In addition, molecular characterization may help in resolving difficult cases with overlapping phenotypes and is useful for early screening and treatment of deafness in patients with type 4 and for avoiding aggressive treatments in transient type 5. Considering the genetic heterogeneity and the absence of hot spots in these genes, the simultaneous analysis by high-throughput sequencing using targeted gene panel is recommended. Although large rearrangements can be detected by those panels, it is recommended to confirm them by a second independent method (e.g., multiplex ligation-dependent probe

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amplification). Large rearrangements are particularly frequent in the CLCNKB gene but have also been described in KCNJ1, BSND, and MAGED2 genes. Genetic counselling should include cascade screening. Testing relatives is particularly useful to identify heterozygous female carriers in families with an index case carrying a MAGED2 mutation. Prenatal diagnosis and preimplantation genetic diagnosis are technically feasible after genetic counselling and may be considered on an individual basis, according to national ethical and legal standards. Bartter syndrome has two distinct clinical presentations: antenatal and classical. The most severe presentation is antenatal Bartter, which corresponds to genetic types 1, 2, 4, 5 and some cases of type 3. It has common characteristic and some particularities according to the genetic subtype. Common characteristics are: maternal polyhydramnios (within the second trimester of gestation, except type 3 cases, in which it is later), premature birth, intrauterine and postnatal massive polyuria complicated by severe dehydration episodes, recurrent vomiting, failure to thrive, and growth retardation. These symptoms are related to the important role of the TAL in NaCl reabsorption as well as in the generation of the corticopapillary osmolar gradient necessary for urinary concentration. In some patients with antenatal Bartter, the urinary concentrating defect is so severe that it can lead to hypernatremia, resembling nephrogenic diabetes insipidus. In addition, the tubuloglomerular feedback is uncoupled because chloride is not reabsorbed in the macula densa; therefore there is a production of high amounts of prostaglandins (mainly PGE2) irrespective of volume status, with additional stimulation of renin secretion and aldosterone production. This explains why antenatal Bartter is sometimes referred to as Hyperprostaglandin E syndrome and constitutes the rationale for treating patients with prostaglandin synthesis inhibitors. Magnesium wasting is not a common finding in antenatal Bartter, which can be explained by adaptation of the paracellular transport in the TAL or the transcelluar transport in the DCT. Additional clinical manifestations have been described in some cases including: osteopenia and hyperparathyroidism. Hereafter, particularities according to the genetic subtype are described. Type 1 is the most severe form of antenatal Bartter, although some mild forms have been described, including late-onset presentation probably related with particular variants or the affected NKCC2 isoform. Type 2 is characterized by transitory neonatal hyperkalemia, which is observed in about 75% of cases and is attributed to the involvement of the Kir1.1 channel in the secretion of potassium in the CD as well as to the immaturity of the Na-K-ATPase pump and other potassium channels expressed in the CD in the preterm infant. Type 4 antenatal Bartter is associated with deafness, as ClC-Ka, ClC-Kb, and barttin proteins are also expressed in the inner ear where they play important roles in the generation of endo-cochlear potential. As paracellular calcium reabsorption in the TAL is dependent on NaCl reabsorption, hypercalciuria and nephrocalcinosis are frequent in types 1 and 2. In contrast, in patients with types 3 and 4, the calcium excretion can be variable (normal, increased or decreased), which is explained by impairment of salt reabsorption in the DCT, where ClC-Kb and barttin are also expressed. Type 5 or transient antenatal Bartter syndrome has a similar presentation to other antenatal forms in the perinatal period, although in some cases the polyhydramnios is more severe; this type affects mainly

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males and is characterized by a spontaneous resolution, often in the first year of life. Other characteristics of this transient form are harmonious macrosomia, lower bicarbonate values and normal or even high plasma chloride, contrasting with the presence of hypochloremia in the other forms of Bartter syndrome, particularly in Bartter syndrome type 3 (Legrand et al. 2018). In some cases, extra kidney manifestations affecting several systems, including central nervous and cardiovascular, have been described in patients. Loss-of-function variants of MAGE-D2, the affected protein in type 5, which is expressed in the TAL and DCT, have been shown to inhibit expression and activity of NKCC2 and NCC proteins (Laghmani et al. 2016). MAGED2 interacts with the chaperon protein Hsp40 and with the G protein subunit Gs-alpha. Hsp40 protein modulates protein folding and stability especially in conditions associated with stress in the endoplasmic reticulum (ER); the Hsp40/MAGE-D2 interaction protects NKCC2 and NCC from ER-associated degradation. Activation of Gs-alpha enhances AMPc generation as well as phosphorylation and membrane expression of NKCC2 and NCC. It is not clear why this form of Bartter syndrome is transient, but two hypotheses have been put forward. First, the interaction between HSP40 and MAGE-D2 could function to protect the cotransporters from degradation induced by tissue hypoxia that can occur during early pregnancy, a function that is not critical later in development. Second, Gs-alpha activity might be compensated for, resulting in greater sensitivity to vasopressin and reducing the dependence of Gsα on MAGE-D2 activity, as patients age. Future studies are needed to better understand the molecular mechanisms and the role of MAGE-D2 in the regulation of NaCl transport and to decipher possible roles in other kidney tubular functions and/or in other organs explaining the clinical manifestations observed in some cases. Classical Bartter has a milder phenotype with a later onset of symptoms (including polyuria, failure to thrive, and sometimes hypercalciuria and nephrocalcinosis) that begin from infancy to adolescence. Patients with this phenotype have biallelic pathogenic variants in the CLCNKB gene, corresponding to type 3. In these patients a profound hypochloraemia is often present, probably associated with the abnormal presence of the ClC-Kb channel in CD intercalated cells, where it promotes chloride reabsorption. Hypomagnesemia is present in some patients with type 3 Bartter. As previously quoted type 3 presents a variable clinical presentation; the analysis of a large cohort of 115 patients with CLCNKB pathogenic variants showed that 29.5% had a phenotype of antenatal or neonatal Bartter syndrome, 44.5% had classic Bartter syndrome, and 26.0% had Gitelman-like syndrome (Seys et al. 2017). Although there is not a clear correlation between phenotype and genotype, complete loss-of-function variants were associated with younger age at diagnosis. The differential diagnosis during the antenatal period includes other causes of polyhydramnios, mainly congenital chloride diarrhea. This disease of recessive inheritance is often complicated by polyhydramnios with preterm delivery. From the end of the second trimester, dilated intestinal loops are frequently observed by ultrasound. In postnatal period, patients have pronounced metabolic alkalosis secondary to extra renal losses; in consequence biochemical analysis of the urine reveals low chloride concentration. In the neonatal period, type 2 Bartter syndrome can

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mimic pseudohypoaldosteronism type 1; the transient character of the hyperkalemia and the presence of alkalosis support the diagnosis of Bartter type 2. The differential diagnosis in the postnatal period and during the infancy includes: “pseudo-Bartter syndrome” as occasionally observed in patients with cystic fibrosis, in circumstances with increase of extra renal losses; hypokalemic alkalosis may also be present in patients with HNF1B nephropathy; in other salt-losing tubulopathies described in this chapter (Gitelman syndrome particularly when hypocalciuria and/or hypomagnesemia are present, HELIX syndrome, EAST syndrome), and in some patients with autosomal dominant hypocalcaemia due to activating variants in the CASR gene. Finally patients with apparent mineralocorticoid excess (AME) present with polyuria, hypokalemic hypochloremic alkalosis, hypercalciuria and nephrocalcinosis but can be distinguished from classic Bartter syndrome by the presence of volume excess with elevated blood pressure and suppressed renin and aldosterone levels. When the first manifestations are nephrocalcinosis and/or nephrolithiasis, other causes of this phenotype should be considered. These include, distal renal tubular acidosis, which also presents with hypokalemia, but the detection of acidosis helps guide the diagnosis. The differential diagnosis of classic Bartter syndrome includes the extra renal losses due to surreptitious use of loop diuretics (both unusual in children) or laxative abuse and chronic vomiting. Measurement of urinary Cl and urine screening for diuretics are usually useful to diagnose these patients (Konrad et al. 2021). Bartter treatment is supportive: water and electrolyte supplementation are particularly necessary in the antenatal form, and patients may need enteral nutrition to meet high fluid and sodium requirements (150–500 ml/kg/d and 10–45 mmol/kg/d, respectively). Potassium chloride supplementation (1–3 mmol/kg/day) is also often necessary. Indomethacin therapy is initiated after 2 months of age at a dose ranging from 1 to 3 mg/kg/day. This drug allows a decrease of supplementation and dramatically improves growth. However, intolerance or toxicity may develop. Bartter syndrome patients are encouraged to have diets high in sodium and potassium and to increase the supplementation doses during periods of additional losses (warmer environmental conditions or intercurrent diseases associated with vomiting or diarrhea). Patients with antenatal Bartter can have neurological disabilities and cognitive dysfunction linked to prematurity. Early screening for deafness is important to ensure normal speech and general development (Seys et al. 2017; Legrand et al. 2018). End-stage renal failure may occur in patients with all types of Bartter syndrome but seems to occur earlier in patients with Bartter type 4 than other types. The potential mechanisms that could lead to kidney damage include dehydration episodes, hypokalemic nephropathy, nephrocalcinosis and potential nephrotoxicity of indomethacin. Kidney Tubulopathy and cardiomyopathy. In 2021, Schilingmann et al. described a new salt-losing tubulopathy in nine unrelated families with renal magnesium wasting and findings included polyuria, hypokalemia, hypochloremia, a tendency toward metabolic alkalosis, hypercalciuria, and nephrocalcinosis. These clinical manifestations suggest a defect in the TAL; in addition, the prenatal course of two

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individuals was complicated by polyhydramnios, as observed in antenatal Bartter syndrome. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. Using whole exome/genome sequencing, these authors identified heterozygous missense variants in the RRAGD gene that mostly occurred de novo. RRAGD encodes a small Rag guanosine triphosphatase (GTPase), RagD, which is a member of the Ras family of GTP-binding proteins. RagD is an essential component of the nutrient-sensing pathway that activates mTOR signalling. mTOR serves as the main nutrient sensor of the cell, coordinating signals from extracellular growth factors and intracellular nutrient availability, such as amino acids. mTOR forms the catalytic subunit of two distinct protein complexes, known as mTORC1 and mTORC2. Upon amino acid stimulation, Rag GTPases target mTORC1 to the lysosome where its kinase is activated. RagD has a ubiquitous expression pattern with significant transcript levels in heart and kidney. In the kidney, RagD is expressed along the nephron included the thick ascending limb and the distal convoluted tubule. The six different RRAGD missense variants detected in patients of those families affect highly conserved residues involved in the binding of guanosine nucleotides and lead, in vitro, to a constitutively increased activation of mTORC1, as reflected by an increased phosphorylation of its substrate S6K1 and by increased binding to components of the amino acid–sensing, mTORC1-containing complex. Some RRAGD variants results in less S6K1 activation, which may explain the variability of tubular phenotype and the absence of dilated cardiomyopathy in patients harbouring them. However, the variability of tubular phenotype (i.e., absence of hypercalciuria and nephrocalcinosis) could reflect a dysfunction in other tubular segments, particularly in the DCT. Future research will be required to elucidate the exact targets of increased mTOR signalling in distal tubular segments. This study not only has established a novel monogenic disorder of the kidney tubule, but has demonstrated the essential role of mTOR signalling for distal tubular electrolyte handling and cardiac function. In addition, it opens up therapeutic perspectives for a targeted treatment of increased mTOR activity in patients with RRAGD pathogenic variants (Schilingmann et al. 2021a). HELIX syndrome (Hypohidrosis, Electrolyte imbalance, Lacrimal gland dysfunction, Ichthyosis, and Xerostomia. In 2017, three different groups using exome sequencing described patients harbouring biallelic pathogenic variants in the gene that encodes claudin-10b (Bongers et al. 2017; Hadj-Rabia et al. 2018; Klar et al. 2017). These patients were diagnosed with a new tubulopathy clinically characterised by renal hypokalemia, hypermagnesemia, and a trend to hypercalcemia and by extra renal manifestations affecting the exocrine glands. The renal phenotype is similar to the phenotype previously described in mice with cldn10 deletion in the TAL. These mice present with strongly reduced paracellular Na+ permeability that led to a urinary concentration defect accompanied by compensatory increases of K+ and H+ secretion as well as hypermagnesemia (Breiderhoff et al. 2012). Despite these similarities, mice lacking claudin-10b in the TAL have nephrocalcinosis, which was absent in patients. The renal phenotype of HELIX syndrome patients is characterized by electrolyte imbalance: Hypermagnesemia, renal hypokalemia with secondary hyperaldosteronism,

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low blood pressure, and hypocalciuria are the most consistent traits. Hypermagnesemia seems to be most pronounced in children. In contrast, hypokalemia seems to be more frequent in adults. Dynamic studies performed on three patients showed that in basal conditions, patients have higher fractional excretions of Na+ and Cl compared with controls. After furosemide treatment, fractional excretions of Na+ and Cl increased to similar values in patients and controls, but the percentage of increase was significantly lower in patients, consistent with blunted natriuretic and chloruretic responses. Fractional excretions of Ca+2 and Mg+2 were similar in patients and healthy subjects in basal conditions. After furosemide treatment, the fractional excretion of Ca+2 increased significantly, but the increase was not as pronounced in patients as in controls, whereas the fractional excretion of Mg+2 increased to a greater extent in patients than in controls (Hadj-Rabia et al. 2018). The extrarenal phenotype of patients with HELIX syndrome is characterised by manifestations related to exocrine glands. First, there are dermatological manifestations: A hypohidrosis with intolerance to heat has been frequently reported. Although this trait was not noted in the first two patients described by Bongers et al., it was confirmed subsequently (Milatz 2019). In some patients, dry skin with fine white scaling, predominantly on the arms and knees, and plantar keratoderma has been described; histologic examinations were consistent with ichthyosis with an increased number of dilated sweat glands. Skin abnormalities were not detected in patients described by Klar et al. and Meyer et al. (Meyers et al. 2019). Second, an inability to produce tears or alacrima is present in almost all of the cases. Third, a xerostomia with severe reduction of the watery component of saliva was detected in all patients. We analysed saliva from patients and observed a reduction of 98% relative to normal fluid secretion (Hadj-Rabia 2018). Finally, patients can present with severe enamel wear, probably related to the absence of saliva. Two major isoforms are produced from the human CLDN10 gene, isoforms 10a and 10b. They differ in their first extracellular segments and first transmembrane segments. As the first extracellular loop is important for ion selectivity, claudins 10a and 10b exhibit different permeability properties (Milatz and Breiderhoff 2017). Claudin 10a works as an anion channel, and claudin 10b has a strong permeability to all monovalent cations with preference for Na+. The expression of isoform 10a seems to be restricted to the kidney, whereas isoform 10b is expressed in several tissues including kidney, salivary glands, skin, sweat glands, brain, lung, and pancreas. In the kidney, claudin-10a is exclusively expressed in the proximal tubule, and claudin-10b is expressed in the cortical and medullar segments of the TAL. To date, most of pathogenic variants in the 10b isoform of CLDN10 are missense variants. In vitro studies performed showed that these variants cause a partial or complete loss of function of claudin-10b. Due to complete or partial retention inside the cell (Bongers et al. 2017; Hadj-Rabia et al. 2018; Klar et al. 2017), quality and/or quantity of homophilic interactions and assembly are impaired. In normal conditions in the TAL and salivary and sweat glands, the expression of homophilic claudin-10b complex makes tight junctions selectively permeable by Na+ (Fig. 3). The loss of function of claudin-10b reduces Na+ reabsorption in the TAL by the paracellular component and leads to a compensatory increase of

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reabsorption in the more distal nephron segments. This explains the K+ and H+ loss in HELIX syndrome patients as it does in patients with other salt-losing tubulopathies. Hypermagnesemia and hypocalciuria are explained by exaggerated paracellular reabsorption of Mg+2 and Ca+2 in the TAL. In mice lacking claudin-10b in the TAL, increased expression of claudin-16 and -19 is observed in the inner stripe of the outer medulla, and these animals have hypermagnesemia (Breiderhoff et al. 2012, 2018). With the loss of claudin-10b function, the loss of Na+ reabsorption by the inner stripe of the outer medulla segment is compensated for by expression of claudin-16 and -19, and the function of Na+ reabsorption is replaced by increased reabsorption of Mg+2 and Ca+2. In the salivary, sweat, and lacrimal gland epithelia, Na+ and Cl are secreted via the basolateral cotransporter NKCC1 and the apical chloride channel CFTR, creating a lumen-negative transepithelial potential difference that allows the paracellular passive secretion of Na+ via claudin-10b, which acts as a channel (Fig. 6). This is associated with a transcellular water secretion through the aquaporin-5 water channels. The loss of function of claudin-10b in these glands results in loss of Na+ and water secretion in saliva, sweat, and tears explaining the main extrarenal symptoms observed. The characteristics of HELIX syndrome indicate that claudin-10b is essential for Na+ transport in the kidney and exocrine glands. Further studies are

Blood

Lumen

2ClNa+ K+ CFTR

K+

NKCC1

Na+

ClK+ H2O H2O

AQP5

Na+

-

Claudin-10b

+

Fig. 6 Mechanisms of claudin-10b function in salivary and sweat glands. In the secretory part of sweat glands and the acinar cells of salivary and lacrimal glands, the cooperative action of the basolateral NKCC1 co-transporter, the K channel, the Na+-K+ ATPase, and the apical channel CFTR results in Cl secretion into the lumen and Na+ and K+ exit from basolateral side. The secretion of Cl drives Na+ transport via the paracellular pathway due to the function of claudin-10b as a channel and transcellular water transport via aquaporin-5. An abrogation of the claudin-10bmediated Na+ transport results in dysfunction of sweat, saliva, and tear glands

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necessary to better understand the variability of clinical manifestations and their modifications with age in these patients. The long-term prognosis of this new syndrome is unknown; however, patients with stage 3 chronic kidney disease at the time of presentation have been described. No specific treatment is available; high NaCl and fluid intake are recommended, as well as potassium supplements when hypokalemia is present. Artificial tears and saliva can relieve symptoms of eye and mouth dryness. Prolonged intense physical activity should be discouraged, particularly when the outside temperature is high, to prevent the risk of hyperthermia. The main clinical characteristics of salt-losing tubulopathies of the thick ascending limb are summarized in the Table 3.

Salt-losing Tubulopathies of the Distal Convoluted Tubule Gitelman syndrome. Gitelman syndrome is the most frequent salt-losing tubulopathy with prevalence around 1 per 40,000 and potentially higher in Asia. The phenotype is milder compared to patients with Bartter syndrome based on the lower percentage of sodium reabsorption in this segment. Gitelman syndrome is due to biallelic pathogenic variants in the SLC12A3 gene, which encodes NCC, the cotransporter expressed in the apical membrane of the DCT epithelium. In about 3% of cases, patients with Gitelman-like phenotype have pathogenic variants in the CLCNKB gene responsible for Bartter syndrome type 3 (Vargas-Poussou et al. 2011). Most patients with Gitelman syndrome are diagnosed as adolescents or adults. In about 50% of patients the fortuitous discovery of hypokalemia allows the diagnosis. The remaining patients present with fatigue, tetany, paresthesias, cramps, muscle weakness, and salt craving and less frequently with cardiac arrhythmia or failure to thrive. In addition to hypokalemia and metabolic alkalosis, most patients present with hypomagnesemia and hypocalciuria. Symptoms are secondary to hypokalemia and hypomagnesaemia, but there is no correlation between electrolyte imbalance and symptoms severity (Blanchard et al. 2017). By analogy with thiazide-induced hypocalciuria (urinary calcium/creatinine ratio lower than 0.1 mmol/mmol) and hypomagnesemia, it is likely that the mechanisms that underlie these abnormalities are an increase of proximal calcium reabsorption and apical TRPM6 magnesium channel inhibition. Patients with GS have higher bone mineral density, similar to chronic thiazide treatment, which likely arises from increased renal Ca2+ reabsorption and a decreased rate of bone remodelling. Blood pressure is reduced, particularly for patients with severe hypokalemia and hypomagnesemia. However, high blood pressure should not rule out the diagnosis, particularly in older patients. Other clinical manifestations described in patients with Gitelman syndrome include: severe failure to thrive, associated with deficit of growth hormone; autoimmune disease often affecting the thyroid gland; finally vertigo and vestibular syndrome have been recently described in 22 and 8% of patients respectively suggesting a role of NCC in the regulation of endolymphatic composition and/or volume (Alexandru et al. 2020).

Type 3 Type 4 Infant, In utero toddler, preschool or school children. Sometimes in utero Absent-mild Severe

Variable Rare, mild

High

Very frequent

Severe

Type 2 In utero

Type 5 In utero

Rare, mild Deafness risk for chronic kidney disease

Variable

Rare, mild Large for gestational age transient disease

High

Severe (sometimes very severe) Polyuria Polyuria Hypokalemia- Polyuria Polyuria failure to thrivefailure to hypochlorefailure to failure to hypochloremiaathrivehypochlore- miaalkalosis- thrivehypochlore- thrivehypochlorelkalosishypokalemia miaalkalosistran- failure to miaalkalosismiaalkalosissient neonatal thrive hypokalemia hypokalemia hyperkalemia Normal Normal Decreased Decreased Increased

Severe

Type 1 In utero

Plasma Cl/Na ratio Calcium High Excretion Nephrocalcinosis Very frequent Other manifestations

Principal manifestations

Polyhydramnios/ prematurity

Age at onset

Table 3 Main clinical characteristics of salt-losing tubulopathies of the thick ascending limb

No

Very frequent Dilated cardiomyopathy

Variable

No Hipohidrosis, lachrymal dysfunction, ichtyosis, xerostomia

Low

Hypomagnesemia Alkalosishypokalemia polyuria, hypermagnesemia hypokalemia, hypochloremia, metabolic alkalosis, Normal Unavailable

Variable

Kidney Tubulopathy and cardiomyopathy Helix syndrome Children, young Children, adult adult. Sometimes in utero.

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The differential diagnosis of Gitelman syndrome includes other salt-losing tubulopathies such as classic Bartter syndrome and East syndrome (see below), as well as diuretic or laxative abuse, and chronic vomiting. Hypomagnesemia, hypokalemia and hypocalciuria can be present in HNF1B-related disorders. HNF1B has been shown to regulate expression of the γ subunit of the basolateral Na+-K+-ATPase (FXYD2) and the basolateral potassium channel Kir5.1, which in turn regulates the activity of the Na+-K+-ATPase, which is crucial for the transport activity in the DCT. The presence of a family history, dominant mode of inheritance, maturity onset diabetes of the young, chronic kidney disease, and other renal and extrarenal anomalies described in HNF1B-related disorders can help to orientate the diagnosis. Patients with pathogenic variants in the FXYD2 gene were described as a cause of isolated hypomagnesemia; however, subsequent data on newly discovered patients also show a trend to Gitelman-like tubulopathy, which is in agreement with the known pathophysiology of HNF1B transcription factor (Blanchard et al. 2017). Gitelman syndrome treatment is based on ad libitum NaCl intake and magnesium and potassium supplementation. A reasonable target for potassium may be 3.0 mmol/l and for magnesium 0.6 mmol/l. Magnesium deficit is often important and aggravates the hypokalemia; some patients need only magnesium supplementation (magnesium chloride: 4–5 mg/kg/day). Nevertheless potassium chloride (1–3 mmol/kg/day) supplementation is often necessary. These supplements should be divided into four administrations per day to avoid diarrhea and adjusted according to the serum levels of K+ and Mg+2. Some patients may require antialdosterone drugs. Gitelman syndrome patients are encouraged to have diets high in sodium and potassium and to increase the supplementation doses during periods of additional losses (warmer environmental conditions or intercurrent diseases associated with vomiting or diarrhea. Caution should be taken when patients with this syndrome undergo anaesthesia because the effects of local and general anaesthetic agents can be potentiated by hypokalemia and hypomagnesemia (Blanchard et al. 2017). The long-term prognosis of patients with Gitelman syndrome is generally good. Some adult patients present with chondrocalcinosis (calcium pyrophosphate dehydrate crystal deposition in joint cartilage), which is related to chronic hypomagnesaemia. Joint pain and pseudo-gout attacks can be managed with non-steroidal anti-inflammatory drugs. A screen for additional risk factors of cardiac arrhythmia is recommended; indeed prolonged QT interval and ventricular tachycardia or fibrillation have been described in Gitelman syndrome patients. Several studies described abnormal glucose metabolism and insulin secretion, as well as an increased risk for the development of type 2 diabetes in Gitelman syndrome patients, probably due to hypokalemia and hypomagnesemia. In the cross-sectional study published by Blanchard et al., GS patients had resistance to insulin potentiated by high BMI and 14% of patients had prediabetes status. The subgroup of patients with severe hypomagnesemia had higher insulin resistance index and a tendency toward higher BMI irrespective of the presence of severe hypokalemia, suggesting that magnesium depletion is the predominant contributor to insulin resistance (Blanchard et al. 2019). To decrease risk of diabetes in patients with Gitelman syndrome, both low glucose diet and prevention of weight gain are recommended. Pregnancies appear to have a favourable outcome, provided continuous K+ and Mg2+ supplementation.

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Gitelman-like syndrome caused by pathogenic variants in mitochondrial DNA. In a recent study (Viering et al. 2022), the analysis of a cohort of patients with Gitelman-like phenotype without pathogenic variants in the SLC12A3 gene or in the genes phenocopying this syndrome (CLCNKB, HNF1B, KCNJ10, FXYD2) allowed the identification of pathogenic mitochondrial DNA (mtDNA) variants in two genes encoding for the phenylalanine transfer RNA (MT-TF) and isoleucine transfer RNA (MT-TI) in 13 non-related families. First, in three larges families, pedigrees were compatibles with maternal inheritance and the analysis of mtDNA revealed candidate pathogenic variants in the MT-TI or in the MT-TF genes, which segregated with the phenotype. The subsequent screening for variants in additional families and individual patients with an unexplained GS phenotype or unexplained hypomagnesemia allowed the identification of nine more families/individual patients with pathogenic variants in these two genes. In total, nine families carry a variant in MT-TF and 4 families carry a variant in MT-TI. Heteroplasmy levels in blood and fibroblasts ranged from 97% to 100% (homoplasmic) in all tested patients. Ten index patients presented with symptoms related to hypomagnesemia and in two other index patients, hypokalemia or hypomagnesemia was discovered as an incidental finding. Hypomagnesemia and hypokalemia were detected respectively in 86% and 63% of the relatives on the maternal lineage. In most of explored cases the hypomagnesemia was associated with a high fractional magnesium excretion indicating renal magnesium wasting as the cause of the hypomagnesemia. Activation of the RAAS was observed when measured. No clinically overt extrarenal manifestations of mitochondrial dysfunction were observed in patients or relatives of these families. In contrast with Gitelman syndrome, a reduced eGFR was observed in 19 patients from six families harbouring pathogenic variants in the MT-TF gene (6 developed end stage kidney disease). Electron microscopy of kidney biopsy specimens from two individuals with a pathogenic variant in MT-TF and chronic kidney disease demonstrated tubulointerstitial kidney disease and abnormal mitochondria in the distal tubule. Cells in the DCT have the largest number of mitochondria per unit length of the nephron and would therefore be sensitive to mitochondrial dysfunction. Accordingly, measurements of the activity of the mitochondrial oxidative phosphorylation in patient-derived fibroblasts found a reduction of the maximal mitochondrial respiratory capacity, with a significant impairment of complex IV. In HEK293 cells, pharmacologic inhibition of complex IV was shown to result in a reduction in NCC phosphorylation and lower NCC-mediated sodium transport. In addition, mitochondrial dysfunction in DCT might lead to diminished function of the Na+-K+-ATPase, which is critical to maintain basolateral membrane potential, a prerequisite for sodium and magnesium reabsorption. This study points out the importance of screening for mtDNA variants in unexplained Gitelman syndrome patients, which is crucial for adapted genetic counselling and follow-up. Further systematic evaluation of symptoms associated with mitochondrial dysfunction should be performed in patients with variants in mtDNA to definitively exclude the presence of rare or subclinical extrarenal manifestations. EAST syndrome. The genetic basis of a syndrome associated with epilepsy, ataxia, sensorineural deafness, and a tubulopathy with the same characteristics of Gitelman

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syndrome was reported by two groups in 2010 and was named EAST syndrome for Epilepsy, Ataxia, Sensorineural deafness, and Tubulopathy (Bockenhauer et al. 2009) or SeSAME syndrome for Seizures, Sensorineural deafness, Ataxia, Mental retardation and Electrolyte imbalance (Scholl et al. 2009). It is a rare autosomal recessive disease caused by pathogenic variants in the KCNJ10 gene, which codes for the potassium channel Kir4.1. This channel is expressed in glial cells of cerebral and cerebellar cortex and stria vascularis and in the basolateral membrane of the cortical TAL, DCT, and principal cells of the connecting tubule and CD. Kir4.1 may form heterotetramers with other Kir potassium channels. In the DCT, where Kir4.1 is highly expressed, it forms heterotetramers with Kir5.1 but can also form functional homotetramers; their function is to recycle potassium assuring Na-K-ATPase activity and generating a negative membrane potential. It has been shown that the loss of function of Kir4.1 decreases activity of NCC via a complex mechanism. The reduction of the basolateral plasma membrane voltage of DCT cells decreases Cl exit via the chloride channel ClC-Kb, the increased intracellular Cl concentration inhibits the cascade of phosphorylation (WNK4/SPAK/OSR1), and the final consequence is a reduction of the NCC phosphorylation and activity (Wang 2016). NCC inhibition with the subsequent aldosteronedependent activation of Na+ reabsorption and K+ secretion in the CD explains the renal phenotype of patients with this syndrome. Kir4.1 is also expressed in the CD, and a recent mouse model of selective invalidation of this channel in the CD showed that Kir4.1 has a role in the adaptation of ENaC and Kir1.1 to dietary K+ and contributes to the hypokalemia seen in EAST patients (Penton et al. 2020). The first manifestation in EAST syndrome is epilepsy, which presents early in infancy, usually in the first year of life. Most patients also present with generalized tonic-clonic seizures. Ataxia is a prominent feature, evident from when the patient learns to walk. This ataxia limits acquisition of both oral and written communication, challenging intellectual evaluation. Other cerebellar signs reported include intention tremor, dysmetria, and dysdiadochokinesis. Nonprogressive sensorineural hearing loss of variable severity is a consistent feature. Phenotypic variability of motor and mental development within families has also been observed (Scholl et al. 2012; Abdelhadi et al. 2016). Electrolyte imbalances in patient with this syndrome are virtually indistinguishable from those observed in Gitelman syndrome patients, with hypokalemia, metabolic alkalosis, hypomagnesemia, hypocalciuria, and activation of the RAAS. In a group of families studied by Scholl et al. (Scholl et al. 2012), electrolytes were normal in the first years of life but showed significant worsening with age resulting in clinically significant defects after 5 years. In the majority of cases, brain magnetic resonance imaging detected pathological findings, although normal brain images have been reported. The defects reported include abnormalities of the cerebellum such as subtle signal changes in the cerebellar dentate nuclei and cerebellar hypoplasia. Patients with EAST/SeSAME syndrome require a multidisciplinary followup. The treatment is based on antiepileptic drugs, hearing aids, mobility devices, and electrolyte replacement. The main clinical characteristics of salt-losing tubulopathies of the distal convoluted tubule are summarized in the Table 4.

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Table 4 Main clinical characteristics of salting tubulopathies of the distal convoluted tubule

Age at onset Principal manifestations

Calcium excretion Other manifestations

Gitelman Children, adolescent, adult Hypokalemia hypochloremia alkalosis hypomagnesemia Low (sometimes normal) Chondrocalcinosis

East/SeSame Infant Hypokalemia hypochloremia alkalosis Hypomagnesemia Low Deafness, epilepsy, ataxia

Gitelman-like syndrome due to variants in mtDNA Children, adolescent, adult Hypomagnesemia hypokalemia hypochloremia alkalosis Variable Chronic kidney disease

Salt-losing Tubulopathies of the Collecting Duct Salt-losing tubulopathies of the collecting duct correspond to generalized and renal forms of pseudohypoaldosteronism type 1. A chapter of this book is dedicated to this disease.

Salt-losing Mixed Tubulopathy Novel Tubulopathy with Hypokalemia, Salt Wasting, Disturbed Acid-Base Homeostasis, and Sensorineural Deafness. The importance of basolateral inwardly rectifying potassium channels in the regulation of sodium transport has been recently pointed out with the description of a novel disease due to biallelic loss-of-function variants in the KCNJ16 gene coding for the potassium channel Kir5.1 (Schlingmann et al. 2021). In the kidney, this channel forms functional heteromers with Kir4.2 (encoded by KCNJ15) in the proximal tubule and with Kir4.1 (encoded by KCNJ10) in the distal nephron. Patients with pathogenic variants in this gene have a tubulopathy comprising severe hypokalemia, renal salt wasting and variable acidbase homeostasis: either metabolic acidosis reflecting a proximal defect on bicarbonate reabsorption or metabolic alkalosis reflecting a distal tubular defect on salt reabsorption with secondary hyperaldosteronism and potassium wasting. In addition, all patients have sensorineural deafness. Before the identification of this disease, the phenotype observed in animal model with abolished function of one of these channels provided key information about their role in the different nephron segments. First, Paulais et al. in 2011, described the phenotype of Kcnj16 / mice, which presented with hypokalemia, metabolic acidosis, normal blood pressure and increased water intake. Electrophysiologic recordings of DCT basolateral membranes indicated an increased potassium conductance mediated by remaining Kir4.1 homomers with decreased pH sensitivity. Second, homozygous Kcnj10 / mice were not able to survive more than 2 weeks after birth. Accordingly, Zhang et al. studied the consequences of the disruption of Kir4.1 in dissected DCT segment

Collecting duct

Nephron segment Distal convoluted tubule

Apparent mineralcorticoid excess

Disease Pseudohypoaldosteronism type 2C Pseudohypoaldosteronism type 2B Pseudohypoaldosteronism type 2E Pseudohypoaldosteronism type 2D Liddle syndrome

Table 5 Diseases associated with RAAS inactivation OMIM number 614492 614491 614496 614495 618126 177200 611814 218030 Inheritance AD AD AD-AR AD AD AD AD AR

Gene/ chromosome WNK1/12p13.33 WNK4/17q21.2 KLHL3/5q31.2 CUL3/2q36.2 SCNN1A/12p13.31 SCNN1B/16p12.2 SCNN1G/16p12.2 HSD11B2/16q22.1

Protein Wnk1 kinase Wnk4 kinase Kelch-like 3 protein Culin-3 ENaC α subunit ENaC β subunit ENaC γ subunit 11β-hydroxysteroide dehydrogenase type 2

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of homozygous mice of 7 to 10 days old. In this model, they showed: almost complete abolition of the basolateral K conductance, a decrease of the negativity of the cell membrane potential in DCT1, a decrease of the basolateral Cl conductance, an inhibition of the expression of Ste20-related proline–alanine-rich kinase and a decrease of the apical NCC expression. From this model, the authors conclude that in the DCT, the Kir4.1/Kir5.1 heteromers are the sensors of plasma potassium and intracellular pH variations with consequent adjustment of the activity of the apical sodium chloride cotransporter (NCC), modifying sodium delivery to downstream tubular segments (Zhang et al. 2014; Wang 2016). Third, Bignon Y et al. showed that mice deleted for Kcnj15 display metabolic hyperchloremic acidosis with a reduced threshold for bicarbonate reabsorption and impaired ammoniagenesis. This raises the possibility that the acidosis observed in KcnJ16 / mice may reflect a dysfunction of Kir4.2/Kir5 heteromers. Finally, a Gitelman-like phenotype was observed in Dahl salt-sensitive rats with Kcnj16 knockout, contrasting the mouse phenotype and highlighting the spectrum of acid-base abnormalities associated with Kir5.1 dysfunction (Palygin et al. 2017). In some patients with KCNJ16 pathogenic variants presenting with metabolic acidosis phenotype, the characterization of acidification defect after an acid loading showed that they conserved the ability to acidify the urine but failed to increase urinary ammonia excretion, similar to that observed in Kcnj15 / mice model. Altogether, these results suggest that a defect on Kir4.2/Kir5.1 heteromers in the PT cells, could affect membrane depolarization, impairing the basolateral reabsorption of bicarbonate through the Na-HCO3–cotransporter NBCe1 (SLC4A4). This in turn would lead to intracellular alkalinization, potentially inhibiting the ammonia synthesis in this segment. All patients with this new tubulopathy have a profound hypokalemia due to renal potassium wasting, which was also observed in knockout animals (Paulais et al. 2011; Palygin et al. 2017). They exhibit, to a varying degree, signs of renal salt wasting and activation of the RAAS, suggesting the same pathophysiological mechanism observed in Gitelman syndrome. Sodium losses and hypovolemia typically activate the RAAS, promoting increased sodium reabsorption in the CD at the expense of potassium and hydrogen, which results in hypokalemic metabolic alkalosis. However, a contribution of other segments in the development of hypokalemia remains possible. The proximal defect can contribute to hypokalemia in a similar way than in other proximal tubulopathies. In addition, principal cells of the CD also express Kir4.1/Kir5.1 heteromers and a mice model with deletion of Kcnj10 specifically in the collecting system exhibited a higher kaliuresis and lower plasma potassium when treated with thiazide diuretics as well as a higher kaliuresis and renal sodium retention upon dietary potassium restriction. Another constant phenotypic feature observed in all patients with this new tubulopathy was sensorineural hearing impairment diagnosed in childhood or adolescence. Audiograms of demonstrated a moderate hearing loss especially at higher frequencies very similar to the findings in individuals with EAST syndrome. Kir5.1 is also expressed in the inner ear and the pathophysiology might involve a disturbed interaction with additional ion channel subunits in the inner ear.

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The KCNJ16 variants identified in patients are missense or nonsense variants located in the pore-forming domain near the selectivity filter of the ion channel, in the N-terminus near the first transmembrane domain or in the C-terminus. In vitro studies showed that Kir 5.1 mutants decrease both Kir4.1/Kir5.1 and Kir4.2/Kir5.1 currents and surface expression. Electrophysiologic recordings showed differences in their interaction with Kir4.1 and Kir4.2. Some variants exhibited strong effects on both Kir4 channels while other variants showed significant residual activity in vitro, or strong inhibition on Kir4.1 and mild inhibition of Kir4.2/Kir5.1 heteromers. These differences might potentially explain the variability of clinical presentation observed in patients.

Diseases Associated with RAAS Inactivation The group of diseases associated with RAAS inactivation include the tubulopathies associated with increased salt reabsorption in DCT and CD segments, where NaCl reabsorption is highly regulated. We will not discuss pseudohypoaldosteronism type 2, because a specific chapter is dedicated to this disease. The increase of Na+ reabsorption in these diseases shifts fluid into the vasculature. The consequences are high blood pressure and suppression of release of angiotensin II and aldosterone. Patients present with low renin hypertension. These diseases are included in a group of diseases also known as monogenic hypertension.

Liddle Syndrome Liddle syndrome is a rare form of hypertension with autosomal-dominant inheritance that is characterized by severe early onset hypertension associated with decreased plasma levels of potassium, renin, and aldosterone. No more than 100 familial or sporadic cases have been reported to date (Rossi 2019). The prevalence is estimated to be lower than 1 in 10,000,000 (Orphanet). After the initial description by Liddle in 1963, an effective treatment of hypertension and hypokalemia with potassium sparing diuretics that inhibit ENaC (amiloride or triamterene) and sodium restriction was described (Rossi 2019). Liddle syndrome is due to gainof-function variants in the genes encoding the subunits of ENaC; this is the mirror image of the autosomal recessive form of pseudohypoaldosteronism type-1, which is caused by loss-of-function variants of the same genes. ENaC is comprised of three subunits (α, β, and γ), and pathogenic variants occur on the C-terminus of the β and γ subunits, encoded by SCNN1B and SCNN1G, respectively, in a proline-rich region called the PY motif. This motif is highly conserved in the C-termini of all ENaC subunits and serves as a binding site for the Nedd4 family of ubiquitin ligases. Pathogenic variants impair the interaction of the ENaC with Nedd4 and the subsequent degradation of the ENaC by the ubiquitin proteasome system. This results in the constitutive expression of ENaCs in the membrane. Pathogenic variants affecting residues far from the PY motif have been described in SCNN1A, SCNN1B, and

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SCNN1G genes (Salih et al. 2017; Jones et al. 2011; Hiltunen et al. 2002); they increase ENaC activity less than variants that delete or alter the PY motif. The mechanism of pathogenicity of variants far from PY motif is an increase of the open probability of ENaC without modification of its cell expression. The increase of expression and/or activity of ENaC induces sodium reabsorption, secondary potassium and proton secretion, and, ultimately, volume-expanded hypertension. Clinically, a severe hypertension is found in Liddle syndrome patients from infancy to young adulthood (before 35 years of age). Children are usually asymptomatic. Adults can present with symptoms of hypokalemia such as weakness, fatigue, myalgia, constipation, or palpitations. A family history of hypertension across several generations is often present. Diagnosis is suspected by the fortuitous detection of early onset hypertension, especially in the presence of family history. It is then confirmed by blood and urinary electrolyte tests that show hypokalemia, decreased or normal plasma levels of renin and aldosterone, metabolic alkalosis with high sodium plasma levels, and low rates of urinary excretion of sodium and aldosterone, with high rates of urinary potassium excretion. However, it is important to note that a phenotypic variability in clinical and biological parameters has been described including an intrafamilial variability (Rossi 2019). The diagnosis is confirmed by genetic testing. The genetic confirmation allows the identification of the affected parent and the early diagnosis of the offspring of affected patients. The detection of a de novo mutation in a proband allows reassurance of parents for future pregnancies. An early genetic confirmation of Liddle syndrome allows early treatment and surveillance, preventing complications of high blood pressure. Treatment is based on administration of potassium-sparing diuretics, such as amiloride (up to 40 mg/day) or triamterene, which act by blocking ENaC activity. This results in reduction of blood pressure and correction of hypokalemia and metabolic alkalosis. Conventional antihypertensive therapies are not effective (including spironolactone, as it acts via the MR). Patients must also follow a low-sodium diet. With treatment, prognosis is good. Without treatment, cardiovascular (premature stroke, myocardial infarction, or sudden death) and renal complications (end-stage renal disease) usually occur.

Apparent Mineralocorticoid Excess Apparent mineralocorticoid excess (AME) is a rare autosomal recessive disorder, characterized by early-onset severe hypertension, low plasma renin and aldosterone, and hypokalemia. It is due to loss of function variants in the HSD11B2 gene resulting in a deficiency of the renal enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2). Around 100 AME cases with genetic confirmation have been described worldwide and consanguinity is present in most of cases (Yau et al. 2017; Fan et al. 2020). The enzyme 11βHSD2 converts active cortisol into inactive cortisone. Normally, MR has the same affinity for both aldosterone and cortisol. As cortisol circulates at 100 to 1000-fold higher levels than aldosterone, MR is protected from cortisol activation

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by 11βHSD2 activity. In AME syndrome, the functioning of this enzyme is impaired, and, therefore, the MR is occupied and activated by cortisol, resulting in an increase of sodium reabsorption and potassium secretion in the principal cells of the CD. AME can occur in two forms. Type 1 is the more severe form, observed in patients who lack 11βHSD2 activity; clinically, type 1 is diagnosed in infancy. Type 1 AME causes low birth weight, failure to thrive, polyuria and polydipsia, and severe hypertension (Morineau et al. 2006; Yau et al. 2017). In addition to the expression of 11βHSD2 in the collecting duct and in the colonic epithelium, this enzyme is also highly expressed in the placenta, likely explaining the associated intrauterine growth retardation in subjects who lack 11βHSD2 activity. Biological analysis shows hypokalemia, alkalosis, low plasma renin and aldosterone, and a markedly elevated urinary ratio of cortisol to cortisone metabolites. Nephrocalcinosis is frequently observed (75% of cases) (Yau et al. 2017; Fan et al. 2020). It should be noted, however, that the plasma levels of cortisol are not increased in AME, since the reduced conversion of cortisol to cortisone in the kidney leads to a decrease in adrenocorticotropic hormone secretion via negative feedback, which keeps the circulating cortisol level within normal range. Type 2 AME is observed in patients with residual enzyme activity. They have a later age onset and milder hypokalemia and can have normal urinary cortisol-to-cortisone ratio (Ceccato and Mantero 2019). Studies conducted by an international consortium that led to development of a computational model of the 11βHSD2 protein have provided an explanation for the genotype–phenotype of AME: Pathogenic variants that cause severe AME are those that enhance dimerization, disrupt the substrate or coenzyme-binding site, or severely impair structural stability of 11βHSD2. In contrast, pathogenic variants that result in type 2 AME indirectly disrupt substrate binding or cause only slight alterations in protein structure (Yau et al. 2017). The goal of AME treatment is to control blood pressure and correct electrolyte disturbance. Patients respond well to MR antagonists, as the activation of the MR is implicated in the pathogenesis of the disease. Dexamethasone, which reduces cortisol production through the inhibition of adrenocorticotropic hormone secretion, is also effective. In some cases, a calcium channel blocker and amiloride were reported to help to manage hypertension (Ceccato and Mantero 2019). Severe hypertension is associated with end organ damage that can involve the renal, neurological, neuromuscular, cardiovascular, and ocular systems and which may, in some cases, lead to early death in the absence of treatment. In the work of Yau et al., follow-up was available for 16 patients with AME. In this group, cardiovascular complications were prominent. Three patients died from cardiac arrest (two at 16 and one at 17 years of age). Moreover, left ventricular hypertrophy was observed in 12 of the 16 subjects, and hypertensive retinopathy was observed in 10 of the 16. Patients can develop renal insufficiency and need kidney transplant; in the work of Yau et al., four patients developed renal insufficiency more than 10 years after diagnosis, with two progressing to renal failure requiring renal transplantation. In these cases, a reversal of the low-renin hypertension and hypokalemic alkalosis was observed post-transplant, likely reflecting the normal 11βHSD2 activity of the transplanted kidney.

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Conclusion The renin angiotensin aldosterone system plays an important role in electrolyte homeostasis and blood pressure regulation. The molecular genetic studies of diseases associated with increased or decreased NaCl reabsorption demonstrate the consequences of inactivation or over activity of specific transporters in global nephron function. Studies of clinical presentations as well fundamental studies using in vitro and in vivo models have improved our understanding of pathophysiology of these diseases. Pathophysiologies reflect on one hand the physiology of the involved segment and on the other hand common pathways. It is the case of the central role of the aldosterone in the common compensatory mechanism observed in salt-losing tubulopathies. Understanding of the physiopathologies of salt-losing or salt-gain tubulopathies is crucial to adequate follow-up and treatment of patients.

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Familial Hyperkalemic Hypertension (FHHt) Chloe´ Rafael and Juliette Hadchouel

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . From Familial Hyperkalemic Hypertension to the Discovery of New Regulators of Ion Handling by the Distal Nephron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Aspects of Familial Hyperkalemic Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Quick Tour of Na+ Reabsorption and K+ Secretion by the Distal Nephron . . . . . . . . . . . . . At Least Four Genes Implicated in Familial Hyperkalemic Hypertension (FHHt) . . . . . . . . Regulation of NCC Phosphorylation by WNK1 Isoforms and WNK4 . . . . . . . . . . . . . . . . . . . . . . . . The WNK Family of Serine-Threonine Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SPAK and OSR1 – Key Players of Cation-Chloride Cotransporters Activation . . . . . . . . . . . Activation of SPAK and NCC by WNKs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The WNK1-SPAK-NCC Cascade Is Modulated by Extracellular Potassium . . . . . . . . . . . . . . A WNK Network to Modulate NCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The CUL3–KLHL3 Complex Modulates the Abundance of WNK Kinases . . . . . . . . . . . . . . . . . . The CUL3/KLHL3 Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KLHL3 and CUL3 Regulate WNKs Ubiquitination and Degradation . . . . . . . . . . . . . . . . . . . . . Involvement of the CUL3-Ubiquitin Ligase Complex in the Physiological Regulation of the WNK Pathway in the DCT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutations in Any of the Four “FHHt Genes” Result in an Increased Abundance of WNK4 and/or WNK1 Isoforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KLHL3 Mutations Prevent the Recruitment of the Substrate or the Binding to CUL3 . . . CUL3 Mutations Disturb the Activity of the Ubiquitin Ligase Complex . . . . . . . . . . . . . . . . . . WNK4 Mutations Prevent Its Ubiquitination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Two Types of Mutations in WNK1 for Two Different Renal Syndromes . . . . . . . . . . . . . . . . . The Pathophysiology of FHHt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CUL3-Dependent FHHt Is a Renal and Vascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Is FHHt Solely Caused by an Increased Activity of NCC? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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C. Rafael · J. Hadchouel (*) CoRaKID – Inserm UMR_S1155, Paris, France Sorbonne Université, Faculty of Medicine, Paris, France e-mail: [email protected] © Springer Nature Switzerland AG 2023 M. Caprio, F. L. Fernandes-Rosa (eds.), Hydro Saline Metabolism, Endocrinology, https://doi.org/10.1007/978-3-031-27119-9_4

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Are “FHHt Genes” Involved in the Pathogenesis of Essential Hypertension? . . . . . . . . . . . . . . . . Identification of Polymorphisms and Variants Associated with a Higher Risk of Hypertension in the General Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Can the Inhibition of SPAK, OSR1 and/or WNKs Be Considered as a Relevant Anti-Hypertensive Strategy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Familial Hyperkalemic Hypertension (FHHt) syndrome, also known as Gordon syndrome or Pseudohypoaldosteronism type II, is a very rare genetic form of hypertension associated with hyperkalemia and hyperchloremic metabolic acidosis, low renin and a normal GFR. These disorders are all corrected by thiazide diuretics that inhibit the Na+-Cl NCC cotransporter expressed in the distal convoluted tubule of the nephron. The sensitivity of FHHt patients to thiazide diuretics strongly suggested that FHHt is caused by an excessive sodium-chloride reabsorption by NCC. However, no mutations in the SLC12A3 gene, encoding NCC, were discovered in FHHt patients. Mutations were first identified in the genes encoding the serine-threonine kinases WNK1 and WNK4 [With No (K) lysine] and then in genes encoding components of a ubiquitin ligase complex, cullin-3 (CUL3), and Kelch-like family member 3 (KLHL3). Subsequent in vitro and in vivo studies have demonstrated that NCC is stimulated by a signaling pathway activated by WNK1 and WNK4, which are themselves targeted for proteosomal degradation by the Cul3-KLHL3 complex. Therefore, the study of this very rare and easily treatable syndrome has led to the discovery of a new, unsuspected molecular pathway responsible for the regulation of ion handling in the distal nephron. In this chapter, we will describe how thus pathway was discovered and how these studies have allowed to understand how sodium reabsorption by the distal nephron is modulated by both sodium and potassium intakes to maintain blood pressure and plasma potassium balance. Keywords

Hypertension · Distal nephron · WNK · CUL3-KLHL3 · Sodium and water homeostasis · Potassium balance

Introduction Familial Hyperkalemic Hypertension (FHHt), also known as Gordon Syndrome or Pseudohypoaldosteronism type 2 (PHA2), is a very rare autosomal dominant form of hypertension. The French and North American cohorts contain less than a 100 affected families. In addition, and as described below, it is a very easily treated syndrome, as all the clinical abnormalities are corrected with a small dose of thiazide diuretics, a well-known and commonly used anti-hypertensive treatment. So, why has FHHt led to so many publications over the last two decades since the discovery of the first causative mutations?

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It is mainly due to its clinical synopsis (see below). Indeed, it associates signs of both hyperaldosteronism (hypertension) and hypoaldosteronism (hyperkalemia). This suggests that FHHt could be caused by mutations in molecules involved in the coordinated modulation of salt reabsorption and potassium excretion by the distal tubules. It was then hypothesized that the identification of the mutated genes could help solving the so-called “aldosterone paradox,” where aldosterone promotes either NaCl reabsorption or K+ secretion in conditions of hypovolemia or hyperkalemia, respectively. The genetic analysis of FHHt patients has fulfilled its promises and more. Indeed, not one but four mutated genes have been identified in FHHt patients. None of them was known to play a role in the nephron. The characterization of their function unraveled a completely unsuspected regulatory pathway of Na+-Cl reabsorption and K+ secretion in the Distal Convoluted Tubule (DCT).

From Familial Hyperkalemic Hypertension to the Discovery of New Regulators of Ion Handling by the Distal Nephron Clinical Aspects of Familial Hyperkalemic Hypertension FHHt is characterized by low-renin hypertension and metabolic abnormalities, such as hyperkalemia, hyperchloremic metabolic acidosis and hypercalciuria. All these symptoms are completely corrected by the administration of thiazide diuretics (Gordon and Hodsman 1986). If Gordon and collaborators reported their first FHHt patient in 1970 (Gordon et al. 1970), the clinical and biological characteristics of the syndrome were originally described by Paver and Pauline (1964). This concerns a 15-year-old man with a severe hypertension (180/120 mmHg) associated with a major hyperkalemia (8.2 mEq/L), an acidosis with a normal renal function and an altered tubular excretion of potassium. The description of this case was completed a few years later with the report of the correction of all the clinical signs by the administration of chlorothiazide (Arnold and Healy 1969). This sensibility to thiazide diuretic had been reported in other patients (Stokes et al. 1968). The term of “type II pseudohypoaldosteronism” was proposed in 1981 (Schambelan et al. 1981), because of the resistance of hyperkalemia to mineralocorticoids and by opposition to the type 1 pseudohypoaldosteronism, in which hyperkalemia is associated with a loss of salt and a decreased blood pressure. This contested denomination is still widely used now (Gordon 1986). Among the numerous cases described in the literature, there is a huge variability in terms of the age of discovery of the disease (Gordon 1995; Achard et al. 2001). If neonatal cases have been described, a detection late in life is not rare. For example, two girls developed FHHt during the first 2 weeks of their life (Gereda et al. 1996) while another study reported the case of a 52-year-old man suffering from hypertension and hyperkalemia (Brautbar et al. 1978). Despite this variability, the study of all these cases highlighted the sensibility to thiazide diuretics as a characteristic of the FHHt (Lee et al. 1979; Mayan et al. 2002) although the treatment with furosemide, a diuretic of the loop of Henle, has also been reported as efficient in several

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groups of patients (Achard et al. 2001; Lee et al. 1979). There is also a variability in the phenotype of the patients. For example, in a large family from the French cohort (Achard et al. 2001), all affected individuals younger than 20 years of age were detected only because of the systematic familial investigation. Hyperkalemia had been diagnosed in several of them within the previous 10 years, but, since they had normal blood pressure, the diagnosis of FHHt was not made. The studies of other families confirmed this observation, showing that, in contrast to the metabolic abnormalities, hypertension may be absent during childhood and develop during adulthood (Achard et al. 2001). Despite this variability, the study of all these cases highlighted the sensibility to thiazide diuretics as a characteristic of the FHHt (Lee et al. 1979; Mayan et al. 2002) although the treatment with furosemide, a diuretic of the loop of Henle, has also been reported as efficient in several groups of patients (Achard et al. 2001; Lee et al. 1979). Thiazide diuretics act by blocking Na+-Cl reabsorption by the sodiumchloride co-transporter NCC. This co-transporter is expressed in one of the segments of the distal nephron, i.e., the Distal Convoluted Tubule (DCT; Fig. 1). In addition, FHHt is the mirror image of Gitelman’s syndrome, an autosomal recessive pathology resulting from inactivating mutations of the SLC12A3 gene (Simon et al. 1996a), encoding NCC. Taken together, these data strongly suggest that FHHt results from a stimulation of Na-Cl reabsorption in the renal distal tubule, which then leads to a decreased sodium intake in the downstream tubules and thus a decreased potassium excretion, responsible for the hyperkalemia (Fig. 1).

A Quick Tour of Na+ Reabsorption and K+ Secretion by the Distal Nephron As mentioned above, NCC is expressed in the Distal Convoluted Tubule (DCT), where it performs an electro-neutral reabsorption of Na+ and Cl ions (Fig. 1). No K+ is secreted and no water is reabsorbed in this segment. The two cortical downstream segments are the Connecting Tubule (CNT) and Cortical Collecting Duct (CCD). If the DCT contains only one cell type, the CNT and CCD are composed of several cell types: Principal cells and Intercalated Cells. The Principal Cells (PCs) represent about 60% of the CNT/CD cells. They reabsorb Na+ by the apical sodium (Na) Epithelial Channel ENaC. The reabsorption of Na+ through the PCs is electrogenic: it is coupled to the secretion of K+ via the ROMK (Renal Outer Medullary-K) potassium channel (Frindt and Palmer 1989). The functional importance of ROMK is illustrated by the fact that loss-of-function mutations of the channel are responsible for Bartter’s syndrome, characterized by salt loss, hypokalemic alkalosis, hypercalciuria and decreased blood pressure (Simon et al. 1996b). Like NCC, the activity of the ROMK and ENaC channels is directly related to the activity of the Na+/K+-ATPase pump located at the basolateral pole. Gain-of-function mutations in ENaC cause Liddle syndrome, which combines severe hypertension and hypokalemia. These mutations induce an increase in the number of channels present at the apical membrane of PCs, resulting in reabsorption

Familial Hyperkalemic Hypertension (FHHt)

Fig. 1 Na+ and K+ transport in the distal nephron. Schematic representation of the nephron and cells of the distal nephron is shown. The segments of the nephron are each represented by a different color: the proximal tubule is in red, the thin part of the loop of Henle in pink, the thick part of the loop of Henle in yellow, the Distal Convoluted Tubule (DCT) in green, the Connecting Tubule (CNT) in light blue and the Collecting Duct in dark blue. Only the main transporters and channels involved in Na+ reabsorption and K+ secretion are shown. A DCT cell reabsorbs Na+ and Cl through the apical cotransporter NCC in an electroneutral manner. The CNT and Cortical Collecting Duct (CCD) are composed of three cell types: Principal Cell (PC), α-Intercalated Cell (α-IC) and β-Intercalated Cell (β-IC). Both PC and β-ICs reabsorb Na+. PCs do so in an electrogenic fashion via the Epithelial sodium Channel (ENaC), thereby driving K+ secretion by the potassium channel ROMK. β-ICs reabsorb Na+ and Cl through the coordinated activity of the Pendrin exchanger and NDCBE transporter

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of Na+ into the CNT and CCD, coupled with excessive K+ secretion by ROMK (Warnock 2001). Conversely, ENaC loss-of-function results in type I pseudohypoaldosteronism, characterized by urinary loss of Na+, decreased blood pressure and hyperkalaemia. Finally, the PCs reabsorb water through the water channels AQP2 (Aquaporin 2) expressed at the apical pole (Fushimi et al. 1993), and AQP3 and AQP4 present at the basolateral pole (Ecelbarger et al. 1995; Terris et al. 1996). In humans, loss-of-function mutations in the AQP2 gene are responsible for the development of nephrogenic diabetes insipidus (Mulders et al. 1997). Na+ transport in PCs is mainly under the control of aldosterone, which quickly increases the number of ENaC channels at the apical membrane of the cells and the Na+/K+ATPase at the basolateral membrane (Loffing et al. 2001; Mamenko et al. 2012). Aldosterone also regulates the transcription of genes encoding the ENaC channel subunits and Na+/K+-ATPase subunit 1 (Féraille and Doucet 2001). Intercalated Cells (ICs) can be of three types: type A (A-IC or α-IC), type B (B-IC or β-IC) or non-A non-B (Teng-umnuay et al. 1996). Intercalated cells are classically involved in the regulation of the acid-base balance. However, recent results have shown that β-CIs also play a role in the reabsorption of Na+ and Cl . α-ICs secrete protons into the tubular lumen via the apical H+-ATPase proton pump, while bicarbonate ions are transported through the basolateral membrane via the AE1 chloride/bicarbonate exchanger. The H+ and HCO3 ions are generated by a cytoplasmic carbonic anhydrase (CA) from CO2 and water. CAII and IV are predominant in the kidney of humans and rodents (Purkerson and Schwartz 2007). Metabolic acidosis leads to an increase in proton secretion by the H+-ATPase, thus limiting acidosis (Bastani et al. 1991). Mutations in the SLC4A1 gene, which encodes AE1, and inactivation mutations in the H+-ATPase subunits are responsible for renal tubular acidosis characterized by a defect in proton excretion or loss of bicarbonates (Stehberger et al. 2007). β-ICs secrete bicarbonate in response to metabolic alkalosis. This secretion is provided by pendrin, an apical Cl /HCO3 exchanger. The expression of pendrin is regulated by changes in the acid-base state. During metabolic or respiratory acidosis, its expression decreases (Malte et al. 2007). Conversely, an alkaline charge leads to an increase in its expression (Wagner et al. 2002). Leviel et al. showed in 2010 that β-IC also reabsorb sodium and chloride in an electroneutral and sensitive manner to thiazide diuretics. Chloride is reabsorbed by pendrin, while sodium is reabsorbed by NDCBE (also known as SCL4A8), a Na+ dependent Cl /HCO3 co-carrier (Leviel et al. 2010). Like NCC, NDCBE is a target for thiazide diuretics such as hydrochlorothiazide (HCTZ). As a result of this coupling, pendrin plays a role not only in the regulation of acid-base balance but also in the regulation of blood pressure. Overexpression of pendrin in ICs in mice results in hypertension (Jacques et al. 2013), while its genetic ablation in mice leads to a decrease in blood pressure (Trepiccione et al. 2017). The DCT, CNT and CCD act in a coordinated fashion. As we will discuss later, the activities and sizes of the DCT and CNT/CCD are inversely correlated in various pathological situations. As mentioned above, the favored hypothesis for the pathogenesis of FHHt was that an increased Na+ reabsorption by NCC in the DCT would

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decrease the Na+ delivery to the CNT/CCD. Na+ reabsorption by ENaC and the concomitant K+ secretion by ROMK would then be reduced, thereby resulting in hyperkalemia. It was also hypothesized that the genes mutated in FHHt patients would either be gain-of-function mutations of the SLC12A3 gene, encoding NCC, or mutations in gene(s) involved in the regulation of the co-transporter’s activity.

At Least Four Genes Implicated in Familial Hyperkalemic Hypertension (FHHt) No mutation was identified in the SLC12A3 gene, which encodes NCC, in FHHt patients. Three different loci associated with FHHt were initially located on chromosomes 1q31–42, 12p13, and 17p11–q21 (Mansfield et al. 1997; Disse-Nicodème et al. 2000). The study of three new families and the fact that two of them were not linked to any of the three previously identified loci suggested an even greater genetic heterogeneity, with at least a fourth locus (Disse-Nicodeme et al. 2001). The two genes on chromosomes 12 and 17 were respectively identified as WNK1 and WNK4 (Wilson et al. 2001). These genes encode members of a family of serine-threonine kinases, With No lysine (K) kinases, consisting of four members (WNK1–4). However, the mutations found in WNK1 and WNK4 only explained around 10% of the cases in the French and North American cohorts. Several years later, mutations in CULLIN-3 (CUL3) and Kelch-like 3 (KLHL3) were identified as causing FHHt in a higher number of patients (KLHL3 mutations: 46% and 35% in the North American and French cohorts, respectively; CUL3 mutations: 33% and 15%) (Boyden et al. 2012; Louis-Dit-Picard et al. 2012). The proteins encoded by CUL3 and KLHL3 form an E3 ubiquitin ligase complex that mediates ubiquitination and thus proteosomal degradation of cellular proteins (Ferdaus and McCormick 2016). The involvement of WNK1, WNK4, CUL3 and KLHL3 in the regulation of renal ion transport and blood pressure was previously unsuspected. Numerous subsequent in vitro and in vivo studies, sometimes conflicting, have allowed defining the contribution of these four new actors to the regulation of salt reabsorption and potassium secretion in the distal nephron.

Regulation of NCC Phosphorylation by WNK1 Isoforms and WNK4 The WNK Family of Serine-Threonine Kinases The WNK family comprises four members, WNK1–4 (Purkerson and Schwartz 2007). Their name derives from the fact that WNKs lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP (adenosine triphosphate). This lysine is displaced in the first subdomain (Bastani et al. 1991). Despite these changes, WNKs exert kinase activity. WNK1 was identified in 2000 through the search for new members of the mitogen-activated protein (MAP)–extracellular signal-regulated protein kinase

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(ERK) (MEK) family in the rat brain (Xu et al. 2000). Soon after, it was also isolated as a differentially expressed mRNA in human colorectal cancer cell lines (Verissimo and Jordan 2001). The initial identification of WNK1 was very quickly followed by the identification of three additional family members: WNK2, WNK3 and WNK4 (Verissimo and Jordan 2001). WNKs contain a well-described auto-inhibitory domain of kinase activity, located just downstream of the kinase domain, and two coiled-coil domains, classically described as protein-protein interaction domain. It also contains the so-called HQ domain at the carboxy-terminal end of the protein, required for WNK–WNK interaction and, thus, activity (see below) (Stehberger et al. 2007; Malte et al. 2007). WNK1, WNK3 and WNK4, but not WNK2, are expressed in the kidney. We will focus here on WNK1 and WNK4. The WNK1 gene is a complex gene giving rise to two isoforms and several variants (Verissimo and Jordan 2001; Delaloy et al. 2003; O’Reilly et al. 2003; Vidal-Petiot et al. 2012). A proximal promoter, located upstream of the first exon, drives the expression of L-WNK1 (long-WNK1), containing the entire kinase domain, and expressed ubiquitously. A second promoter, located in intron 4, allows for the expression of a shorter isoform called KSWNK1 (kidney specific WNK1), which is expressed only in the distal nephron and is devoid of kinase activity. In addition, the alternative splicing of six exons (8b, HSN2, 11, 12, 26a, and 26b) generates several WNK1 variants expressed in a tissue-specific manner (VidalPetiot et al. 2012). Particularly, the variant containing HSN2 is expressed only in the peripheral nervous system. Mutations located in the HSN2 exon are responsible for another inherited disease, known as hereditary sensory neuropathy type 2 (Shekarabi et al. 2008). The differential functions of the other splicing isoforms are still not known. Within the kidney, L-WNK1 mRNA is detected in all nephron segments (Vidal-Petiot et al. 2012). The highest expression is observed in the glomerulus and the lowest in the DCT. KS-WNK1 is highly expressed in the DCT and to a lesser extent in the connecting tubule (CNT). A low level is also detected in the thick ascending limb (TAL) of Henle’s loop and cortical collecting duct (CCD). WNK4 encodes a 1243-residue protein expressed in several tissues, including the distal nephron. WNK4 transcripts have been detected by in situ hybridization in the TAL (including in the macula densa), the DCT, CCD and the and outermedullary collecting duct (OMCD) (Boyden et al. 2012). Expression is the highest in the DCT. This pattern differs somewhat at the protein level (Louis-Dit-Picard et al. 2012). Immunofluorescence microscopy has confirmed expression in the cortical TAL, DCT, CNT and CCD (principal and intercalated cells), and OMCD. It also revealed that WNK4 is present in podocytes and the inner medullary collecting ducts (Louis-Dit-Picard et al. 2012). There was no signal in the medullary TAL. As mentioned above, the hypothesis generated by the clinical studies of FHHt patients is that the mutated genes regulate the activity of the Na+-Cl cotransporter NCC. Since NCC activity is regulated by phosphorylation (Yang et al. 2013) and that L-WNK1 and WNK4 are kinases, it was postulated that L-WNK1 and WNK4 could phosphorylate directly NCC. However, it was shown that L-WNK1 and WNK4 need an intermediate, i.e., the SPAK and OSR1 kinases.

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SPAK and OSR1 – Key Players of Cation-Chloride Cotransporters Activation Identification of SPAK and OSR1 In the majority of cells, cell volume and homeostasis are maintained through coordinated Cl inputs and outputs by the Na+-K+:2Cl NKCC1 co-transporter and the K+Cl KCCs co-transporters, respectively (Gamba 2005). The decrease in the intracellular concentration of chloride ([Cl ]i) results in the phosphorylation of NKCC1 and KCCs, and their activation and inactivation, respectively. Conversely, an increase in [Cl ]i leads to their dephosphorylation, which inhibits NKCC1 and activates KCCs. These observations suggested that a Cl sensitive kinase was involved in this process. The Sterile20-related proline-alanine-rich kinase (SPAK) and oxidative stress response kinase 1 (OSR1) were identified through the search for the kinase responsible for coordinated phosphorylation of the cation-chloride co-transporters (CCCs). First, a hybrid double screen in yeast demonstrated the interaction between the kinases SPAK and OSR1 and KCC3 as well as with NKCC1 and NKCC2 (Piechotta et al. 2002). Subsequently, SPAK and OSR1 were shown to phosphorylate and thus activate the CCCs (Dowd and Forbush 2003; Richardson and Alessi 2008; Richardson et al. 2011). SPAK and OSR1 phosphorylate NKCC1, NKCC2 and NCC on three threonines located in the N-terminal part, where the sequence is highly conserved between the three co-transporters (residues Thr46, Thr55 and Thr60 for human NCC) (Richardson and Alessi 2008). Thr60 plays a crucial role in the transporter activity. The Alanine mutation of Thr60 inhibits the phosphorylation of the other two threonines and the activation of NCC in osmotically stressed HEK293 cells (Richardson and Alessi 2008). In addition, the human mutation of threonine 60 to methionine is responsible for Gitelman syndrome (Shao et al. 2008). NCC is also phosphorylated at an additional site during a depletion of chloride (Ser91) by a kinase not identified to date. SPAK and OSR1 share a strong sequence homology. The homology rate of their kinase domains is about 89% while it is about 67% in the rest of the protein. The major difference between SPAK and OSR1 is the presence of a region rich in proline and alanine, the PAPA box, in the terminal domain of SPAK, upstream of the catalytic domain. In addition to the catalytic domain, SPAK and OSR1 have a C-terminal domain (CCT), which is unique to them and has a 79% homology rate between the two kinases. The CCT domain is required for the interaction of SPAK and OSR1 with their substrates and activators. It binds to the RFXV/I peptide motif located in the N-terminal end of CCCs and in the C-terminal domain of WNK. A study in mice has shown that this interaction between the C-terminal CCT domain and WNK kinases plays a major role in blood pressure regulation (Zhang et al. 2015). A single mutation in Leu502, present in the SPAK CCT domain, abolishes the strong affinity binding to the RFXI/V motif. The introduction of this mutation in mice (SPAKL502A/L502A mice) leads to decreased abundance and phosphorylation of NCC and NKCC2, resulting in a decrease in blood pressure. SPAK Is Essential for the Phosphorylation and Activity of NCC Two mouse models were used to study the function of SPAK. The first model, the SPAK / mouse, is a global knockout of SPAK (Grimm et al. 2012; Yang et al. 2010).

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The second model, the SPAK243A/243A mouse, was generated by introducing a missense mutation in the T-loop of SPAK (p.Thr243Ala) that prevents the activation of SPAK. Indeed, the activation of SPAK and OSR1 kinase activity requires the phosphorylation of a conserved Thr residue (Thr233 for SPAK, Thr185 for OSR1) in the T-loop catalytic unit of SPAK/OSR1 (Vitari et al. 2005). Ser383 of SPAK and Ser325 of OSR1 are also phosphorylated although the functional consequences are less clear (Richardson and Alessi 2008). Only T-loop phosphorylation is absolutely necessary to activate SPAK and OSR1 in vitro and in vivo (Vitari et al. 2005; Zagórska et al. 2007). SPAK / and SPAK243A/243A mice are viable and fertile. They both show a decrease in baseline blood pressure (Yang et al. 2010). SPAK243A/243A mice display no change in baseline plasma and urinary electrolyte levels except for mild hypomagnesemia and mild hypocalciuria. Na+ restriction results in hypokalemia (Rafiqi et al. 2010). The phenotype of baseline SPAK / mice is more pronounced, with hypokalemia, hypomagnesemia and hypocalciuria (Yang et al. 2010). In both models, NCC expression and phosphorylation are drastically reduced (Yang et al. 2010). This decrease leads to a reduction in the size and surface area of the proximal portion of the distal convoluted tubule (also called DCT1) in SPAK / mice, which is compensated by an increase in the size and activity of the distal part of the DCT (DCT2) and CNT (Grimm et al. 2015). This phenomenon is also observed in mice with NCC inactivation (Schultheis et al. 1998). The analysis of all the mouse models bearing a mutation of SPAK showed that this kinase is essential for maintaining NCC phosphorylation in the distal nephron. This was unexpected since SPAK and OSR1 can both phosphorylate NCC in vitro and are both present in the DCT. The situation is different for the regulation of NKCC2 in the TAL, where SPAK and OSR1 are also both found. The constitutive and ubiquitous inactivation of OSR1 is lethal during embryonic development due to defects in angiogenesis and cardiovascular development (Lin et al. 2011; Xie et al. 2013). Targeted inactivation of OSR1 in the distal nephron (KSP-OSR1 / mice) causes a Bartter syndrome characterized by salt loss, hypokalemic alkalosis, hypercalciuria and decreased blood pressure, all consecutive to a decrease in NKCC2 abundance and phosphorylation (Simon et al. 1996a), showing that OSR1 is the main regulator of this co-transporter. The role of SPAK is more complicated because of the existence of isoforms differentially expressed between the TAL and DCT. Indeed, the expression and phosphorylation of NKCC2 decreases in SPAK243A/243A mice but increases in the SPAK / model. This difference is probably due to inhibition of OSR1 by two shorter SPAK isoforms that are still present in SPAK243A/243A mice but absent in SPAK / mice (McCormick et al. 2011). Indeed, there are three isoforms of SPAK. The “full-length” isoform (FL-SPAK or SPAK), which contains the entire coding sequence, is ubiquitously expressed, with strong expression in the brain, heart and testes (Rafiqi et al. 2010). The other two isoforms lack kinase activity and are expressed specifically in the kidney, whereas FL-SPAK is ubiquitously expressed. Initially identified in the mouse kidney, the existence of these two SPAK isoforms was very recently demonstrated in the human kidney (Mercado et al. 2020). SPAK2, also ubiquitously expressed, lacks the N-terminal

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domain and part of the kinase domain (Rafiqi et al. 2010; McCormick et al. 2011; Piechotta et al. 2003). The third isoform, kidney-specific SPAK (KS-SPAK), also lacks a kinase domain and, as its name implies, is expressed specifically in the kidney. In the kidney, the SPAK isoforms are differentially expressed along the TAL, the site of NKCC2 expression, and the DCT, the site of NCC expression (McCormick et al. 2011). FLSPAK is more strongly expressed in the DCT than in the TAL. SPAK2 and KS-SPAK are the predominant isoforms in the TAL but are only weakly expressed in the DCT (McCormick et al. 2011). In vitro, phosphorylation of NKCC2 is inhibited by KS-SPAK and SPAK2 (McCormick et al. 2011; Park et al. 2013). This suggests that KS-SPAK and SPAK2 would act as inhibitors of FL-SPAK and OSR1 and thus of the phosphorylation of NKCC2. In SPAK / mice (Yang et al. 2010), the absence of KS-SPAK and SPAK2 along the TAL relieves the inhibition of OSR1, thus leading to an increase in NKCC2 phosphorylation at SPAK/ OSR1 sites. In SPAK243A/243A mice, inhibition of OSR1 is maintained since SPAK2 and KS-SPAK are still present (Rafiqi et al. 2010). All these in vivo data demonstrate that SPAK is essential for the phosphorylation of NCC. They also suggest that the absence of SPAK in the TAL can be compensated by OSR1 but that this is not the case in the DCT. Again, these results are surprising since SPAK and OSR1 can similarly activate NKCC2 and NCC in vitro. One possible explanation is that the cellular localization of OSR1 is dependent on SPAK specifically in the DCT. The phosphorylated form of OSR1 is localized predominantly at the apical membrane of the TAL and DCT in control mice (Grimm et al. 2012). Genetic inactivation of SPAK destabilizes this apical localization of OSR1 in the DCT but not in the TAL. Indeed, in the DCT of SPAK / mice, OSR1 is mostly found in cytoplasmic structures distant from the apical membrane, the nature of which has not been clearly determined and which are called “puncta” (Saritas et al. 2013). Abolishing the apical localization of OSR1 would prevent its interaction with NCC.

Activation of SPAK and NCC by WNKs For more than a decade, WNK4 was considered as an inhibitor of NCC and L-WNK1 as an indirect stimulator of NCC, through the inhibition of WNK4. However, these conclusions were drawn from experiments conducted in conditions that were very different from the in vivo one in the DCT. It is now clearly established that L-WNK1 and WNK4 are both capable of activating SPAK and thus NCC (Fig. 2a).

L-WNK1 Activates NCC Independently of WNK4 In vitro studies of the regulation of NCC by L-WNK1 were distorted until 2014 by a mutation in the C-terminal part of the variant used in almost all the studies conducted in Xenopus laevis oocytes (Chávez-Canales et al. 2014). This mutation prevented the activation of NCC (Chávez-Canales et al. 2014). This is why it was believed for a long time that L-WNK1 could not activate NCC on its own. However, these studies

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Fig. 2 L-WNK1 and WNK4 stimulate NCC phosphorylation in a [Cl ]i -dependent manner. (a) In basal conditions, when [K+]e is around 4 mM and [Cl ]i is below 20 mM, L-WNK1 and WNK4 activate SPAK by phosphorylation, which in turn phosphorylates NCC to stimulate its insertion into the apical membrane and activity in DCT cells. At the basolateral membrane, the Kir4.1 potassium channel (as a heterodimer with Kir5.1) allows the recycling of K+ required for Na+ extrusion by the Na+-K+-ATPase and provides the driving force for Cl extrusion for the CLC-Kb channel; (b) When Kir4.1 is genetically activated, Cl accumulates in the cells, thereby inhibiting LWNK1 and WNK4 phosphorylation and activity. In a control, non-genetically modified, mouse, this occurs when [K+]e increases

were in contradiction with those that showed that L-WNK1 can phosphorylate SPAK. A study conducted with a corrected version of the human L-WNK1 cDNA allowed to show that L-WNK1 can indeed activate SPAK and thus stimulate NCC in Xenopus oocyte. This was then confirmed in vivo in a mouse model in which L-WNK1 is overexpressed in the DCT and WNK4 is inactivated. These mice indeed display increased abundance and phosphorylation of SPAK and NCC (ChávezCanales et al. 2014). However, to date, it has not been possible to study the role of L-WNK1 in the adult kidney in physiological conditions since the global inactivation of L-WNK1 leads to embryonic mortality due to severe abnormalities in cardiovascular development (Xie et al. 2013).

WNK4 Is an Activator of SPAK Discordant results between in vitro and in vivo experiments have also complicated the understanding of the role of WNK4 in NaCl transport. Initially, studies conducted in Xenopus oocytes and cell cultures showed an inhibitory effect of WNK4 on NCC activity (Wilson et al. 2003; Yang et al. 2003). However, in vivo studies suggested a crucial role of WNK4 in maintaining NCC expression and activity in the DCT. Indeed, in two independent mouse models, inactivation of WNK4 leads to a strong decrease in NCC expression and phosphorylation, associated with hypokalemia and hypochloremic metabolic alkalosis (Castañeda-Bueno et al. 2012; Takahashi et al. 2014). Taken together, these discordant observations

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suggested that the conditions of the in vitro studies were somewhat different from the physiological in vivo conditions of the DCT. One hypothesis comes from the fact that WNK activity is modulated by changes in the extracellular concentration of sodium, potassium or chloride (Naito et al. 2011). This is consistent with the fact that NKCC1 is activated or inhibited respectively by a decrease or increase in [Cl ]i and that this activation is associated with an increase in its phosphorylation by SPAK and OSR1 on key N-terminal residues (Lytle and Forbush 1996; Pacheco-Alvarez et al. 2006; Ponce-Coria et al. 2008). Accordingly, a study showed that the level of L-WNK1 kinase activity is inversely proportional to the intracellular chloride concentration (Piala et al. 2014). The activation of L-WNK1 requires the phosphorylation of a serine (S382) located in the activation loop of the kinase domain. The binding of chloride to a “pocket” located in the same kinase domain prevents the phosphorylation of this residue by modifying the conformation of this domain. Thus, the higher the chloride concentration is, the lower the activity of L-WNK1 is. The leucine L369 and L371 residues play a crucial role in the formation of the chloride binding site in the kinase domain. Indeed, their mutation into phenylalanine decreases the sensitivity of L-WNK1 to chloride (Piala et al. 2014). The chloride concentration in in vitro cellular models is higher than that observed in DCT cells [40–50 mM (Bazúa-Valenti and Gamba 2015) versus 10–20 mM (Boettger et al. 2002)]. The divergent effects of WNK4 on NCC could therefore result from this difference in intracellular chloride concentration. Indeed, the leucine residues described above are conserved in the WNK4 protein, suggesting that the activity of WNK4 could also be regulated by [Cl ]i. This hypothesis was confirmed by the increased phosphorylation of the WNK4 activation loop (S332) when the intracellular chloride concentration in Xenopus oocytes decreased (Bazúa-Valenti et al. 2015). Under standard conditions, the co-injection of NCC and WNK4 into the Xenopus oocytes results in a decrease in the activity of the co-transporter. However, WNK4 activates NCC when the intracellular chloride concentration is experimentally lowered (Bazúa-Valenti et al. 2015). The sensitivity of WNK4 to chloride concentration was confirmed in vivo, in a mouse model in which the L369 and L371 leucine residues were mutated to phenylalanine (Chen et al. 2019). At baseline, these mice display all the clinical signs of FHHt (hypertension, hyperkalemia and hyperchloremic metabolic acidosis), consecutive to an increase in SPAK and NCC phosphorylation. Taken together, these studies provide an explanation as to why WNK4 inhibits NCC in vitro whereas it activates it in vivo. It also confirms the in vivo studies by showing that WNK4 is indeed able to activate SPAK and thus stimulate NCC, when the correct ionic conditions are met.

The WNK1-SPAK-NCC Cascade Is Modulated by Extracellular Potassium Once the sensitivity of L-WNK1 and WNK4 to intracellular chloride concentration ([Cl ]i) was established, it remained to understand how this sensitivity could

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contribute to the physiological modulation of NCC in the DCT. One hypothesis is that it could be involved in the regulation of NCC by potassium intake. NCC abundance and phosphorylation are indeed increased during potassium depletion and decreased during potassium load in both mice and rats (Castañeda-Bueno et al. 2014; Vallon et al. 2009; Sorensen et al. 2013). During potassium depletion, the increase in NCC activity in the DCT results in a lower delivery of Na+ to the CNT and CCD, thereby reducing Na+/K+ exchange via the ENaC and ROMK channels and thus potassium secretion. Conversely, during potassium load, the reduced NCC activity increases Na+ delivery to the downstream segments, thereby favoring potassium secretion by ROMK. Protein abundance and phosphorylation of WNK4 and SPAK increase in the kidney of potassium-depleted mice (Terker et al. 2015; Wade et al. 2015). But what are the mechanisms responsible for the activation of the WNK-SPAK pathway by potassium depletion? The hypothesis of a modulation of [Cl ]i by extracellular potassium ([K+]e) was proposed and confirmed by in vitro and in vivo experiments (Terker et al. 2015; Su et al. 2020; Argaiz and Gamba 2016). Using a transgenic mouse model expressing an optogenetic kidney-specific Cl-Sensor and measured Cl fluorescent imaging, Su and colleagues demonstrated that [Cl ]i was inversely correlated to [K+]e in isolated mouse DCTs (Su et al. 2020). The modification of [Cl ]i by extracellular potassium concentration in DCT cells involves the basolateral potassium channel Kir4.1 (encoded by the KCNJ10 gene), as demonstrated by the characterization of several mouse models of ubiquitous or kidney-specific KCNJ10 inactivation (for review, Su et al. 2019). This channel ensures potassium recycling and allows the establishment of an electrochemical gradient favoring the efflux of chloride ions through the chloride channel CLC-K2. In isolated DCTs, the pharmacological inhibition of Kir4.1 and CLC-K2 leads to an increase in [Cl ]i (Fig. 4; Su et al. 2020). The involvement of Kir4.1 in the regulation of SPAK and NCC has been demonstrated in vivo: the abundance and phosphorylation of SPAK and NCC are indeed decreased in KCNJ10 / mice, which exhibit Na+ and K+ wasting and hypotension (Zhang et al. 2014). In addition, NCC phosphorylation is also drastically reduced in Clcnk2 / mice (Hennings et al. 2017). Finally, patients with KCNJ10 mutations present with a multi-organs syndrome, whose renal manifestations resemble a Gitelman- syndrome (Scholl et al. 2009; Bockenhauer et al. 2009). The demonstration of the involvement of WNK4 chloride sensitivity in the regulation of NCC activity by extracellular potassium again came from the analysis of the knock-in mice expressing the L319F/L321F double-mutant WNK4 (Chen et al. 2019). As described above, these mice present with FHHt at baseline, with increased SPAK and NCC phosphorylation and activity. When fed a low-potassium diet, these mice fail to upregulate NCC. However, NCC phosphorylation is already stimulated enough to prevent a steep and fatal decrease in plasma potassium, which is only reduced to the level of a wild-type animal. Interestingly, NCC inhibition by a chronic potassium-load is similar in control and mutant mice. However, NCC phosphorylation is not decreased by an acute potassium loading (by oral gavage).

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These observations strongly suggest that WNK4 chloride sensitivity is brought into play during potassium depletion and acute potassium load but that WNK4independent mechanisms, such as phosphatases (Hoorn et al. 2011; Picard et al. 2014; Shoda et al. 2017), are employed during chronic potassium load.

A WNK Network to Modulate NCC Although the chloride-binding site is conserved among all WNK kinases, they do not have the same sensitivity to chloride in vitro. L-WNK1 can activate NCC in the Xenopus oocytes under standard conditions while the intracellular chloride concentration ([Cl ]i) must be lowered for WNK4 (Bazúa-Valenti et al. 2015). Indeed, the kinase activity of WNK4 progressively decreases when the intracellular chloride concentration increases from 10 to 40 mM, whereas that of L-WNK1 is stable at the same concentrations and only begins to decrease when this concentration reaches 60 mM (Terker et al. 2016). The sensitivity of L-WNK1 to chloride is therefore lower than that of WNK4. These in vitro data suggest that WNK4 could be the key physiological regulator of NCC in the DCT (Terker et al. 2015, 2016). This is supported by the following observations: • NCC abundance and phosphorylation are strongly reduced in WNK4 / mice (Castañeda-Bueno et al. 2012; Takahashi et al. 2014) • NCC is not stimulated by a low-potassium diet in WNK4 / mice (Yang et al. 2018) However, it is interesting to note that WNKs are found predominantly as multimers in cells. WNKs interact to form homo- or heteromers by the small HQ motif (HIQEVVSLQT) present in their C-terminal coiled-coil domain (Thastrup et al. 2012). The interaction between WNKs is required for their activation by phosphorylation of the T-loop. When interacting as a complex, WNKs are capable of transphosphorylating (Thastrup et al. 2012). Given that L-WNK1 and WNK4 have different sensitivity to chloride, one can hypothesized that L-WNK1/WNK4 heteromers could have an intermediate sensitivity to chloride. Depending on the intracellular chloride concentration in the DCT, different combinations of L-WNK1 and WNK4 hetero- or homomers could therefore be activated to stimulate NCC at various degrees (Fig. 3).

The CUL3–KLHL3 Complex Modulates the Abundance of WNK Kinases The products encoded by the CUL3 and KLHL3 genes are part of a ubiquitin-ligase complex of which WNK1 and WNK4 are the substrates.

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Fig. 3 Different combinations of WNKs are formed depending on [Cl ]i and [K+]e. At baseline (left panel), when [Cl ]I is low in DCT cells, both L-WNK1 and WNK4 can be activated by phosphorylation. Therefore, all hetero- and homomers of these two kinases can phosphorylate SPAK. When [K+]e increases, [Cl ]I also increases. Only L-WNK1 can be activated by phosphorylation. WNK4 complexes are inactive. L-WNK1/WNK4 complexes could be either less active or inactive. Therefore, only L-WNK1 complexes phosphorylate SPAK, which results in decreased phosphorylation and thus activity of NCC

The CUL3/KLHL3 Complex The CUL3 gene encodes cullin-3 or CUL3, an essential component of a ubiquitin ligase E3 complex allowing the degradation of target proteins by the 26S proteasome after ubiquitination. CUL3 belongs to a seven-member family of proteins (CUL1, -2, -3, -4a, -4b, -5 and -7) (Sarikas et al. 2011). All these cullins are involved in the formation of ubiquitin ligase E3 complex. The N-terminal end of the Cullins interacts with the protein or protein complex responsible for recruiting substrates. The N-terminal end interacts with the RING proteins (Rbx1 or Rbx2) that recruit the E2 enzyme responsible for ubiquitination. This is known as Cullin-RING ligase. Cullin-RING-E3 complexes make up the largest class of ubiquitin ligase E3 in mammals. Most of the seven Cullin proteins interact with their own unique set of substrate adapters. The ability to bind to different substrate adapters allows CullinRING-like ubiquitin ligase complexes to ubiquitinate a multitude of substrates (Harper and Tan 2012). The substrate adapters of CUL3 are BTB proteins (Fig. 4a). They are characterized by a BTB domain that allows interaction with CUL3 (Andérica-Romero et al. 2014). Kelch-like proteins (KLHL) are specific BTB proteins that form the family of BTB-BACK proteins (broad-complex, tramtrack and bric to brac-BTB and C-terminal Kelch) (Pintard et al. 2004). KLHL proteins recruit substrates for the Cullin3-RING ubiquitin ligase via their Kelch domain. It is composed of several kelch patterns (Adams et al. 2000). Each kelch pattern consists

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Fig. 4 WNK4 is ubiquitinated by a CUL3-KLHL3 complex, the mutations of which result in an increased abundance of the kinase. (a) The E3 ubiquitin ligase complex consists of a scaffold protein (CUL3), a specific substrate adapter (KLHL3) and a ubiquitin-ligase RING. The complex is divided into a center for substrate-specific recognition and a catalytic center that ubiquitinates the substrate. The specific ubiquitin ligase complex CUL3/KLHL3 is involved in the FHHt; (b) one of the substrates of the CUL3-KLHL3 complex is the WNK4 kinase, which is itself involves in the stimulation of NCC activity through the phosphorylation of the intermediate kinase SPAK; (c) “FHHt-mutations” in WNK4, KLHL3 or CUL3 result in an increased protein abundance of WNK4. The mutations identified in the acidic motif of WNK4 in FHHt patients prevent the binding

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of four connected β sheets and represents a helix blade. The kelch patterns are linked together by a loop to form a single helix. The helix formed by the kelch domain serves, among other things, as scaffolding for the protein-protein interactions (Gupta and Beggs 2014). The expression of CUL3 is ubiquitous. In the kidney, CUL3 is expressed throughout the nephron, with a slightly higher expression in the proximal tubule (Boyden et al. 2012). KLHL3 is strongly expressed in the brain, specifically in the cortex and hippocampus. Strong expression of KLHL3 is also found in the DCT (Louis-Dit-Picard et al. 2012). KLHL3 is more weakly expressed in the eyes, testes, lungs, heart, liver, stomach and colon (Sasaki et al. 2017). The colocalization of CUL3 and KLHL3 with the NCC co-transporter in the DCT supports the involvement of these genes in the pathology.

KLHL3 and CUL3 Regulate WNKs Ubiquitination and Degradation Patients with CUL3 or KLHL3 mutations display a similar phenotype to those with mutation in WNK1 or WNK4. This observation raises the hypothesis that the products of these four genes could act in the same regulatory pathway for ion transport in the distal nephron. This was confirmed by several in vitro studies that showed that WNK1 and WNK4 are substrates of the ubiquitin ligase E3 CUL3/KLHL3 complex, but not SPAK and NCC (Susa et al. 2014). In vitro, WNK4 is ubiquitinated by the CUL3ubiquitin ligase E3 complex, via recruitment by KLHL3 (Shibata et al. 2013a; Wakabayashi et al. 2013; Wu and Peng 2013). This ubiquitination leads to a reduction in kinase protein abundance. These in vitro results were confirmed by the generation of KLHL3 / mice (Sasaki et al. 2017). The absence of KLHL3 in vivo leads to a degradation defect of WNK4 and an increase in its protein expression level in the kidney. The increased expressions of WNK4 leads to an increase in SPAK and NCC phosphorylation in the kidney and the appearance of a phenotype very close to FHHt, associating hyperkalemia, hyperchloremic metabolic acidosis and a tendency to hypertension during a high-salt diet (Sasaki et al. 2017). Complete inactivation of Cul3 is lethal before 7.5 days of development (Singer et al. 1999). The study of Cul3 / embryos at 6.5 days of development revealed a disorganization of extra-embryonic tissues caused by an accumulation of cyclin E in the ectoderm and trophoblast and a deregulation of the S phase (Singer et al. 1999). To determine the consequences of this inactivation in the adult kidney, McCormick et al. generated a model of Cul3 inactivation specifically in the nephron (KS-Cul3 / mouse) (McCormick et al. 2014). It leads to increased protein levels of WNK4. The authors also observed an increased expression and phosphorylation of NCC, most

ä Fig. 4 (continued) of the kinase to KLHL3. The mutations of the latter prevent its binding to WNK4 or CUL3. Finally, the mutations of CUL3 impair the ubiquitin ligase activity of the complex by a mechanism that still remains to be defined. (Adapted from Andérica-Romero et al. (2014))

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likely due to the increase in WNK4. However, the absence of Cul3 does not lead to FHHt. Mice display renal failure, with tubulo-interstitial inflammation and fibrosis, as well as diabetes, hypochloremic alkalosis and salt-sensitive hypotension (McCormick et al. 2014). This broad phenotype is probably due to the fact that Cul3 deletion in the nephron results in the loss of ubiquitination of all substrates of the Cul3-ubiquitin ligase complex, such as Cyclin E, and not only those recruited by KLHL3. These results also suggest that the CUL3 mutations identified in the patients are not simple loss-of-function mutations (see below). The results obtained for the regulation of WNK1 isoforms by the CUL3-KLHL3 complex are more conflicting. The first studies showed that L-WNK1 is ubiquitinated by this complex in a manner similar to WNK4. A decrease in the protein abundance of L-WNK1 is observed in a cellular model overexpressing KLHL3. The reverse is true, since an increase in L-WNK1 protein abundance is observed when KLHL3 expression is inhibited (Wakabayashi et al. 2013; Ohta et al. 2013). Accordingly, L-WNK1 abundance is increased in KLHL3 / and KS-Cul3 / mice. However, we showed that KS-WNK1 is probably more sensitive to proteosomal degradation following CUL-KLHL3-mediated ubiquitination than LWNK1 (see below).

Involvement of the CUL3-Ubiquitin Ligase Complex in the Physiological Regulation of the WNK Pathway in the DCT While Angiotensin II (angII) stimulates WNK4 kinase activity via the phosphorylation of four serines by PKA and PKC (Castañeda-Bueno et al. 2017), it has also been demonstrated that angII can inhibit the recruitment of WNK4 for ubiquitination by the Cul3 complex (Shibata et al. 2014). Indeed, in response to angII in vitro, PKC phosphorylate KLHL3 on the Ser433 residue. This phosphorylation prevents KLHL3 from binding to WNK4, resulting in its accumulation. It is interesting to note that this residue is mutated in some patients with FHHt (Boyden et al. 2012; Louis-Dit-Picard et al. 2012). The stimulation of the phosphorylation of this KLHL3 residue is observed in the kidneys of mice treated with angII. It is also detected in the DCT of mice fed a low-potassium diet (Ishizawa et al. 2016). Again, it is probably PKC that is responsible for this phosphorylation in this condition.

Mutations in Any of the Four “FHHt Genes” Result in an Increased Abundance of WNK4 and/or WNK1 Isoforms KLHL3 Mutations Prevent the Recruitment of the Substrate or the Binding to CUL3 Most of the KLHL3 mutations are missense mutations, but non-sense mutations, small deletions and splice mutations have also been identified (Boyden et al. 2012; Louis-Dit-Picard et al. 2012). Heterozygous and homozygous mutations were found, which explains the dominant or recessive presentation of the KLHL3-related

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pathology. This in an exception in FHHt genetic, since mutations in WNK1, WNK4 and CUL3 are all heterozygous. KLHL3 mutations are mostly localized in the Kelch domain but some mutations are localized in the BTB-BACK domains. Mutations in the Kelch domain prevent the binding to the substrates (Wakabayashi et al. 2013). Mutations in the BTB or BACK domain lead to a decrease in the interaction between KLHL3 and CUL3 (Mori et al. 2013a). In all in vitro experiments, the direct consequence of these mutations is a decrease in the ubiquitination and subsequent degradation of WNK4 by the proteasome (Wakabayashi et al. 2013; Schumacher et al. 2014). Similar results were obtained in the Xenopus oocytes (Wu and Peng 2013). Since overexpression of KLHL3 leads to decreased expression of L-WNK1 (Wakabayashi et al. 2013), it is likely that KLHL3 mutations also abolish the ubiquitination and degradation of L-WNK1, but this has not been formally demonstrated in vitro (Wu and Peng 2013). In vivo data confirmed the in vitro studies through the characterization of mice carrying a KLHL3 missense mutation, p.Arg528His, located in the Kelch domain of KLHL3 (Susa et al. 2014). Heterozygous KLHL3+/R528H mice present the symptoms of FHHt, as well as increased protein levels of WNK4 and L-WNK1 in the kidney. To test the involvement of WNK4 in FHHt caused by the KLHL3 mutation, Susa et al. genetically inactivated WNK4 in KLHL3+/R528H mice (WNK4 / :KLHL3+/R528H mice). The phosphorylation of SPAK and NCC decreased strongly in the kidney of WNK4 / :KLHL3+/R528H mice. Although the FHHt phenotype of these mice has not been studied, these observations suggest that the presence of WNK4 is essential for the development of FHHt caused by KLHL3 mutations (Castañeda-Bueno et al. 2012).

CUL3 Mutations Disturb the Activity of the Ubiquitin Ligase Complex All CUL3 mutations responsible for FHHt are grouped in the sequences involved in the splicing of exon 9: the acceptor site and potential branching point in intron 8, the donor site in intron 9 and a potential splicing enhancer site (ES enhancer) in exon 9 (Boyden et al. 2012). They all induce a skipping of exon 9, i.e., a deletion of exon 9, corresponding to 57 amino acids (403–459). These amino acids encode the segment between the binding domains to the BTB domain of KLHL and to the RING protein. This skipping of exon 9 was demonstrated in cultured cells through the expression of vectors containing 9 CUL3 mutations identified in patients with FHHt (Boyden et al. 2012). The molecular consequences of the deletion of CUL3 exon 9 have been explored by several groups in vitro and in vivo. The Cullin has an elongated N-terminal domain that is a succession of three repetitions as well as a C-terminal that contains a specific area of the Cullin family. The N-terminal domain recognizes the substrate adapters, while the C-terminal end binds to the RING proteins (Rbx 1 or Rbx2) to which the E2 enzyme and ubiquitin bind (Zimmerman et al. 2010). The structural rigidity of the Cullin, between the elongated N-terminal part and the globular C-terminal part, is important for the ubiquitin ligase function of the complex. Indeed, Cullin flexibility is allosterically regulated, allowing the Cullin-RING E3 ubiquitin

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ligase complexes to increase the distance between the E2 enzyme and the substrate to facilitate substrate polyubiquination. Sequence changes between the N-terminal and C-terminal ends of the Cullins can alter the relative position of the RING with respect to the bound substrate, thus preventing substrate modification (Liu and Nussinov 2011). The efficiency of the Cullin-RING complex also requires the ubiquitination of the C-terminally conserved lysine residue (Lys 721 of CUL3) by the ubiquitin-ligase Nedd8, which binds covalently to CUL3 (Wimuttisuk and Singer 2007). This modification, autocatalyzed by CUL3-RBX1, alters the flexibility of the C-terminal domain of culllins to activate ligase E3 (Duda et al. 2008). While neddylation is necessary for efficient ubiquitination, studies have shown that the cycle between neddylated and de-neddylated states is crucial in a cellular context (Pintard et al. 2004; Cope et al. 2002). Thus, the interaction of Cullins with the COP9-signalosome denedylation complex (CSN) is also necessary for their activity. Another Cullin-RING ligase regulator, CAND1, interacts with the N- and C-terminal ends of the Cullin, only when they are deneddyled, to promote substrate adaptor change (Wu et al. 2013). In the first in vitro analysis, McCormick et al. suggested that CUL3Δ403–459, deleted from the domain encoded by exon 9, degrades KLHL3, which prevents the recruitment and thus ubiquitination of their substrates, including the WNK kinases (McCormick et al. 2014). However, an alternative mechanism has been proposed. The deletion of the domain coded by exon 9 confers greater flexibility to the CUL3Δ403–459 protein (Schumacher et al. 2015). This greater flexibility prevents the ubiquitin ligase complex from directing ubiquitin to the substrates, which is then directed rather to CUL3 and KLHL3. In vitro, the formation of unstable heterodimers Cul3WT:CUL3Δ403–459 indeed leads to a decrease in the activity of Cul3WT. This strongly suggests that Cul3WT is degraded when it is in a complex with CUL3Δ403–459, which decreases the amount of active Cul3WT and leads to a decreased ubiquitination of its substrates (Ibeawuchi et al. 2015). However, this mechanism has not yet been described in vivo. A heterozygous knock-in mouse model for exon 9 deletion of Cul3 (Cul3+/Δ403–459 mouse) was generated (Schumacher et al. 2014). These mice present a typical FHHt phenotype FHHt with hypertension, hyperkalemia and hyperchloremic metabolic acidosis. The authors were unable to obtain mice carrying the mutation in the homozygous state, suggesting that the mutation is lethal in utero. The protein abundance of L-WNK1 and WNK4 are increased in the kidney of CUL3+/Δ403–459 mice, leading to overactivation of SPAK and NCC. The abundance of KLHL3 is unaffected. Conversely, only a small amount of the Cul3Δ403–459 protein is detected in the kidney of these mice. These results are in favor of an instable CUL3WT-CUL3Δ403–459 complex rather than a stimulatory one.

WNK4 Mutations Prevent Its Ubiquitination The mutations found in the WNK4 gene are missense mutations located in two short, highly conserved protein motifs between members of the WNK family and located

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downstream of the two coiled-coil domains. However, the majority of these mutations are found in the acidic motif located downstream of the first coiled-coil domain. These mutations replace negative charges with positive charges (Wilson et al. 2001; Golbang et al. 2005). The consequences of these mutations remained unknown for a long time. Contrary to the majority of mutations, the p.Arg1185Cys mutation is localized in the C-terminal region of WNK4, within a calmodulin (CaM) binding site and close to the phosphorylation sites by SGK1 Ser1190, Ser1201 and Ser1217 (Na et al. 2013). Phosphorylation at the S1201 site is inhibited by CaM. The p.Arg1185Cys mutation reduces the interaction between WNK4 and CaM and thus increases the phosphorylation of Ser1201. In Xenopus oocytes, the p.Arg1185Cys mutation leads to an increase in WNK4 activity (Na et al. 2013). A second study focused on the mutations found in the acidic motif. It showed that the kinase activity of WNK4 could be regulated by Ca2+ ions and that the mutations in the acidic domain could alter this regulation (Na et al. 2012). In vitro, WNK4 activity is positively correlated with Ca2+ concentration. The introduction of acidic domain mutations deregulates the Ca2+ sensor mechanism and increases the kinase activity of WNK4, independently of the Ca2+ concentration. However, this mechanism has not been confirmed in vivo. The discovery of the mutations in CUL3 and KLHL3 finally allowed to understand the consequences of WNK4 mutations. Indeed, the acidic motif is the domain through which WNK4 interacts with the Kelch domain of KLHL3 (Wakabayashi et al. 2013; Ohta et al. 2013). WNK4 proteins carrying a “FHHt-mutation” in the acidic domain no longer interact with KLHL3 and are therefore no longer ubiquitinated and degraded (Shibata et al. 2013a; Wakabayashi et al. 2013; Wu and Peng 2013; Ohta et al. 2013). Several mouse models were then used to confirm this mechanism in vivo. A first model was generated by introducing (knock-in) one of the mutations identified in WNK4 in FHHt patients (p.Asp561Ala) in the mouse WNK4 gene. The protein abundance of WNK4 is increased in the kidney of these mice (Wakabayashi et al. 2013). They show all the characteristic signs of FHHt (Yang et al. 2007), with increased SPAK and NCC phosphorylation. A second model overexpresses the wild-type WNK4 protein, using a transgene containing the entire gene. These mice present all the phenotypes associated with FHHt (Wakabayashi et al. 2013), which proves that the increased abundance of the wild-type form of WNK4 is sufficient to trigger the disease.

Two Types of Mutations in WNK1 for Two Different Renal Syndromes Increased Expression of L-WNK1 Triggers the Development of FHHt The WNK1 mutations responsible for FHHt are not mutations in the coding sequence but 22–41 kbp deletions located within the 60 kbp-long first intron of the gene. These large deletions lead to an increase in L-WNK1 mRNA expression in the leukocytes of human patients (Wilson et al. 2001). Similarly, mice harboring a heterozygous deletion of WNK1 first intron develop a full FHHt phenotype with

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increased NCC abundance and phosphorylation (Vidal-Petiot et al. 2013). All phenotypic alterations are corrected by thiazide treatment in WNK1+/FHHt mice. This study thus confirmed that the intronic deletion is truly responsible for the disease. As a consequence of the intronic deletion on WNK1, a higher L-WNK1 mRNA expression level was observed in the DCT and, to a lesser extent, in the CNT, with no changes in KS-WNK1 level. Therefore, the increase in L-WNK1 expression in the DCT and CNT is responsible for the development of the disease. As mentioned above, the role played by L-WNK1 in the physiological modulation of NCC has not been characterized yet because the knock-out of L-WNK1 is embryonic lethal, following cardiovascular malformations. It is believed that WNK4 is the major physiological regulator of NCC since (1) NCC phosphorylation is greatly reduced (Castañeda-Bueno et al. 2012; Takahashi et al. 2014) and (2) NCC stimulation by potassium depletion is lost in WNK4 / mice (Yang et al. 2018). However, when WNK4 is inactivated in WNK1+/FHHt mice, the phenotype of the WNK1+/FHHt-WNK4 / double-mutant mice was similar to that of the WNK1+/FHHt mice, including the increased activation of NCC (Chávez-Canales et al. 2014). Altogether, these findings provide in vivo evidences that L-WNK1/SPAK pathway is a powerful activator of NCC and that L-WNK1 does not need WNK4 to modulate NCC activity, at least when overexpressed.

An Increased Abundance of KS-WNK1 Results in Hyperkalemic Metabolic Acidosis The French FHHt cohort constituted by the Genetics Department of the European Hospital Georges Pompidou (Paris, France) has about a 100 index cases. 8% of these cases do not carry any of the known mutations of WNK1, WNK4, KLHL3 or CUL3. Interestingly, some of these patients suffer from hyperkalemia and hyperchloremic metabolic acidosis without hypertension. A linkage analysis combined with whole exome sequencing in such a large family revealed a new type of WNK1 mutations. These missense mutations are all located in exon 7, in the acidic “EPEEPEADQH” motif of WNK1 isoforms (Louis-Dit-Picard et al. 2020). This domain, homologous to that in which the WNK4 mutations responsible for FHHt are found, is the domain of interaction with KLHL3 (Ohta et al. 2013). Mutations in the acidic domain of WNK4 prevent the interaction of the kinase with KLHL3. In vitro, in Xenopus laevis oocytes and HEK293 cells, the mutations of the acidic motif of WNK1 lead to a decreased ubiquitination of KS-WNK1 by the KLHL3-CUL3 complex, rather than L-WNK1 (Louis-Dit-Picard et al. 2020). Therefore, the hyperkalemic metabolic acidosis of these patients would be caused by an increased abundance of KS-WNK1, by opposition to the deletions of WNK1 first intron, which result in an increased transcription of L-WNK1. A mouse model bearing the D635E mutation of the WNK1 acidic motif (Wnk1+/delE631 mice) confirms the absence of arterial hypertension. These mice exhibit hyperkalemia and metabolic hyperchloremic acidosis. Despite the absence of arterial hypertension, Wnk1+/delE631 mice display increased activation of the SPAK/OSR1-NCC phosphorylation cascade. The impaired K+ balance was evidenced by a failure to upregulate ROMK in the DCT2/CNT in the presence of

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hyperkalemia. However, ROMK expression is significantly increased in the CCD of Wnk1+/delE631 mice, which could be explained by the effects of hyperkalaemia and by the lack of deregulation of KS-WNK1 expression in this segment, where KLHL3 is only very weakly expressed (Louis-Dit-Picard et al. 2012). This conclusion is consistent with observations in mice that KS-WNK1 gene ablation induces an increase in the apical expression of ROMK in the DCT2 and CNT (Hadchouel et al. 2010). These results highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.

The Pathophysiology of FHHt As described several times in this chapter, the exquisite sensitivity of FHHt patients to low doses of thiazides diuretics strongly suggests that the whole syndrome is solely the consequence of an increased activity of NCC, the target of thiazides. In the following paragraphs, we will discuss the role played by the vasculature in “CUL3dependent FHHt” and the potential roles played by the segments distal to the DCT in all forms of FHHt.

CUL3-Dependent FHHt Is a Renal and Vascular Disease As described above, a mutation in any of the four “FHHt genes” results in an increased abundance of WNK4 and/or L-WNK1. Therefore, the clinical synopsis should be similar in all patients. However, patients with CUL3 mutations have a more severe phenotype in terms of age of onset, degree of hypertension and metabolic disorders (Boyden et al. 2012). Patients with a CUL3 mutation are diagnosed at a younger age: 94% of them are hypertensive before the age of 18 years, while this rate varies from 10% to 17% in other FHHt patients. The kalemia values in these patients are also much higher (above 7 mM in average for “CUL3-patients” vs below 6 mM in average for all others). The majority of patients also have a growth and/or development defect, which is more rarely reported in other patients. Finally, most CUL3 mutations occur de novo, suggesting reproductive impairment for these patients and/or premature death before they are of reproductive age (Boyden et al. 2012). In vitro and in vivo studies have demonstrated that this severity is caused by an impairment of the vascular function in addition to the renal dysfunction. A first series of in vitro studies showed that the Rho1 protein is a substrate of the CUL3-based ubiquitine-ligase complex. RhoA (Ras homolog family member A) is a monomeric G protein of the Rho protein family, itself a member of the Ras superfamily. When stimulated by vasoconstrictor agents, such as angiotensin II, in vascular smooth muscle cells, RhoA activates the ROCK (Rho-associated protein kinase) kinase. The later in turn inhibits myosin light chain phosphatase (MLCP) and myosin dephosphorylation, thereby promoting vascular contraction (Shibata et al. 2015). In vitro and in vivo, the inhibition of CUL3 or the overexpression of the

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CUL3Δ403–459 protein, deleted from exon 9, leads to a decrease in ubiquitination and degradation of RhoA (Schumacher et al. 2015). In mice, the same conditions stimulate vasoconstriction, thus leading to hypertension (Schumacher et al. 2015). These studies allowed to demonstrate that CUL3 mediates RhoA ubiquitination and that the expression of the mutant Cul3 protein results in increased vasoconstriction and hypertension in mice. However, they did not allow to determine the relative contribution of the vasculature and the distal nephron to the FHHt severity caused by CUL3 mutations. Therefore, we generated two mouse models (Abdel Khalek et al. 2019): one with a ubiquitous expression of the mutant Cul3Δ9 protein (pgk-Cul3Δ9 mouse) and one with a vascular-specific expression of this mutant protein (sm22Cul3Δ9 mouse). The pgk-Cul3Δ9 mice present all the signs associated with FHHt, i.e., hyperkalemia, hyperchloremic metabolic acidosis and severe hypertension, associated with activation of the WNK4/NCC signaling pathway. These blood pressure and metabolic disorders are more severe in pgk-Cul3Δ9 mice than in WNK1+/FHHt mice, carrying the intronic WNK1 deletion. Cul3 mutations therefore lead to a more severe phenotype than WNK1 mutations in both mice and humans. The abundance and phosphorylation of NCC are similar in the two groups of mice, demonstrating that the severity of metabolic disorders is not caused by stronger activation of NCC in pgk-Cul3Δ9 mice. In contrast to pgk-Cul3Δ9 mice, sm22Cul3Δ9 mice do not show any metabolic disorders. If both CulΔ9 models are hypertensive, pgk-Cul3Δ9 mice are significantly more hypertensive than sm22Cul3Δ9 mice. As in the overexpression model of Cul3Δ403–459, hypertension in sm22-Cul3Δ9 mice results from an increase in vasorelaxation following an increase in RhoA abundance. This study formally demonstrated for the first time that the severity of hypertension associated with CUL3 mutations in patients results from the deregulation of both renal and vascular functions. It is nevertheless important to note that this study did not allow to explain the severity of metabolic disorders observed in these patients.

Is FHHt Solely Caused by an Increased Activity of NCC? In the distal nephron, WNK1 isoforms and WNK4 are expressed in the DCT but also in the CNT and CCD. However, the expression of SPAK and OSR1 is virtually undetectable in these two segments (Rafiqi et al. 2010), suggesting that WNKs could act independently of these kinases. Several groups have studied the potential involvement of WNK4 and WNK1 isoforms in the regulation of ion transport by principal and intercalated cells. The first observations suggesting that WNK4 and WNK1 isoforms could play a role in these segments came from studies focusing on the regulation of their expression and activity by aldosterone. Indeed, the distal part of the DCT (DCT2), CNT and CCD form the so-called “Aldosterone-Sensitive Distal Nephron” (ASDN), as they express the mineralocorticoid receptor, the nuclear receptor which binds aldosterone and mediates its action. The transcription of KS-WNK1, is stimulated in

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the distal nephron in situations of increased aldosterone secretion, i.e., potassium loading and aldosterone infusion (O’Reilly et al. 2003). While chronic infusion of aldosterone in mice has no effect on the mRNA level of L-WNK1 and WNK4, some studies show that their protein abundance as well as the activity of WNK4 could be modulated by aldosterone. In vitro, the SGK1 kinase phosphorylates WNK4 on residues S1169 and S1196 (Ring et al. 2007a; Rozansky et al. 2009). This phosphorylation could modify the activity of WNK4 since a cDNA encoding a constitutively phosphorylated kinase is no longer able to inhibit NCC and ROMK under standard experimental conditions (high [Cl ]i relative to the DCT) (Ring et al. 2007a). The phosphorylation of WNK4 by SGK1 and its consequences on the activity of WNK4 in vivo have not yet been studied. The regulation of L-WNK1 protein abundance by aldosterone is modulated by the ubiquitin-ligase Nedd4-2 and SGK1 (Roy et al. 2015). In vitro, L-WNK1 is targeted for proteosomal degradation following its ubiquitination by Nedd4-2, a mechanism that is inhibited when Nedd42 is phosphorylated by SGK1. In vivo, the proteic abundance of L-WNK1 increases and is no longer stimulated by aldosterone infusion in a mouse model with Nedd4-2 inactivation specifically in the nephron while it decreases in SGK1 knock-out mice (Roy et al. 2015).

Is ENaC Regulated by L-WNK1 and WNK4? Several groups have studied the regulation of Na+ and K+ by the WNKs in the principal cells of the CNT and CCD. We will focus on ENaC first. As described in Fig. 1 and the corresponding text, the epithelial sodium channel ENaC reabsorbs Na+ in the principal cells of the CNT and CCD. The expression of ENaC is positively controlled by aldosterone by two mechanisms. The first is the stimulation of transcription of the gene encoding the α subunit of ENaC directly by the mineralocorticoid receptor, activated by the binding of aldosterone (Náray-Fejes-Tóth et al. 1999). The second is the inhibition of the degradation of the β and γ subunits of ENaC. These subunits are ubiquitinated by the ubiquitin-ligase Nedd4-2, which is itself inhibited by phosphorylation by SGK1 (Serum and Glucocorticoid activated kinase). SGK1 expression is stimulated by aldosterone (Debonneville et al. 2001; Eaton et al. 2010). The gain-of-function mutations of ENaC identified in patients with Liddle syndrome affect the interaction between Nedd4-2 and ENaC and alter the degradation of the duct, leading to its accumulation at the apical membrane of the principal cells (Pradervand et al. 2003; Schild et al. 1995). In vitro studies have suggested that L-WNK1 and WNK4 could regulate the expression and activity of ENaC by interacting with the SGK1 kinase. A first series of studies has shown that WNK4 reduces the expression of ENaC on the surface of Xenopus oocytes (Ring et al. 2007a; Yu et al. 2013). However, these results should be interpreted with caution in view of the experimental conditions, since, as described above, WNK4 kinase activity is inhibited by the intracellular concentration present in these oocytes. Another series of studies has demonstrated the activation of SGK1 by L-WNK1 and WNK4. The overexpression of L-WNK1 in HEK293 cells stimulates the kinase activity of SGK1 (Xu et al. 2005). The same result was obtained with the overexpression of WNK4 in HEK293 cells (Heise et al. 2010).

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The phosphorylation of SGK1 by WNKs then leads to the inhibitory phosphorylation of Nedd4-2 and thus an increase in ENaC activity. The contradiction with the studies described above is potentially due to differences in the experimental systems, particularly the intracellular and extracellular concentration of ions that influence WNK phosphorylation. The in vivo situation is not less contradictory. WNK4 overexpression models have been used to study the in vivo regulation of ENaC by WNK4. The protein abundance of ENaC subunits is increased in the kidney of mice expressing a mutant “WNK4-FHHt” protein (WNK4D561/+ mouse) (Yang et al. 2007). An overexpression model of mutated WNK4 was also generated by additive transgenesis (TgWNK4PHAII mice; Lalioti et al. 2006). The amiloride-sensitive Na+ flux is increased in the colon of these mice compared to control mice (Ring et al. 2007b), suggesting that WNK4 is an activator of ENaC in vivo in the colon. Finally, in mice which overexpress L-WNK1 in DCT and CNT, the expression and activity of ENaC remain unchanged, suggesting that L-WNK1 does not regulate ENaC activity in these segments (Vidal-Petiot et al. 2013). In conclusion, whether or not L-WNK1 and WNK4 regulate ENaC expression and activity is still not clearly established. The quantitative and molecular analyses of Na+ reabsorption in the CNT and CCD in the mouse models mentioned above are complicated by the fact that Na+ reabsorption by NCC in the DCT is increased. Consequently, Na+ delivery to the downstream segments is decreased. In addition, as we will see below, the different segments of the ASDN can undergo quite extensive remodeling when the activity of one of them is stimulated or inhibited. This is the case in WNK4 / mice, where ENaC activity measured in isolated and perfused CCDs and the natriuretic response to amiloride are strongly increased. However, this is most probably not due to a direct effect of WNK4 inactivation on ENaC, but rather to a compensatory mechanism following the strong decrease in NCC expression and Na+ reabsorption in the DCT (Castañeda-Bueno et al. 2012).

Is ROMK Regulated by L-WNK1 and WNK4? Since FHHt is characterized by a hyperkalemia, it was hypothesized that L-WNK1 and WNK4 could also directly inhibit the ROMK potassium channel in the CNT and CCD. In vitro studies confirmed this hypothesis. In HEK293 cells, showed that L-WNK1 inhibits ROMK by stimulating its endocytosis (Lazrak et al. 2006). The accelerated internalization of ROMK by L-WNK1 is dynamin-dependent and involves clathrincoated vesicles (Cope et al. 2006; He et al. 2007). Regulation of ROMK by L-WNK1 is not direct and two potential partners have been identified: intersectin, a multimodal cytoplasmic scaffold protein (He et al. 2007), and ARH (autosomal recessive hypercholesterolemia) (Fang et al. 2009). In vitro, L-WNK1 and WNK4 interact with intersectin and this interaction is crucial for the internalization of ROMK. One hypothesis is that the interaction between WNKs and intersectin would facilitate the targeting of ROMK to the endocytosis vesicles (He et al. 2007). ARH is a clathrin adaptor molecule that recruits ROMK to the endocytosis vesicles (Fang et al. 2009). Phosphorylation of ARH by L-WNK1 prevents its proteasomal degradation, which stimulates ROMK endocytosis (Fang et al. 2009). It is important to note that a

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decrease in the abundance of ROMK at the apical membrane, as well as diffuse cytoplasmic labelling of ROMK in the DCT2 and CNT of mice overexpressing L-WNK1 (WNK1+/FHHt) is observed (Vidal-Petiot et al. 2013), suggesting that the stimulation of ROMK endocytosis by L-WNK1 could occur in vivo. These changes are observed only in DCT2 and CNT, where the expression of L-WNK1 is increased, and not in CCD, where the expression of L-WNK1 remains unchanged. However, there is currently no evidence for a role for L-WNK1 in the regulation of ROMK expression under physiological conditions. WNK4 is also able to inhibit ROMK in vitro by stimulating clathrin-dependent channel endocytosis, independently of WNK4 kinase activity (Kahle et al. 2003). This inhibition is prevented when WNK4 is phosphorylated by SGK1 (Ring et al. 2007a; Rozansky et al. 2009). This mechanism may contribute to the increase in apical abundance of ROMK in the distal nephron of animals fed a potassium-rich diet, which stimulates aldosterone secretion and hence SGK1 activity. However, inhibition of ROMK by WNK4 has not been demonstrated in vivo.

β-Intercalated Cells: A Partner of DCT Cells to Trigger FHHt? As described in the first part of this chapter, if β-intercalated cells (β-ICs) were originally thought to contribute exclusively to acid-base balance, a study conducted by the group of D. Eladari demonstrated in 2010 that they also play a role in the regulation of water and salt homeostasis, and thus hypertension. Indeed, they reabsorb Na+ and Cl through the coordinated action of pendrin, an apical Cl / HCO3 exchanger and NDCBE (also known as SCL4A8), a Na+ dependent Cl / HCO3 co-carrier (Leviel et al. 2010). The expression of pendrin is stimulated by angiotensin II and aldosterone. However, a potassium-rich diet causes the opposite effect despite a high level of aldosterone. The recent identification of the phosphorylation of the mineralocorticoid receptor on the Ser843 residue preventing aldosterone from binding to its receptor has provided the explanation (Shibata et al. 2013b). This phosphorylation occurs only in β-ICs. In a situation of volume depletion, angiotensin II promotes the dephosphorylation of the receptor, allowing the binding of aldosterone to its receptor and the stimulation of pendrin expression (Shibata et al. 2013b). Conversely, a potassium-rich diet promotes the phosphorylation of the receptor, thus preventing the action of aldosterone. To date, the phosphorylation of MR-Ser843 has only been found in β-ICs and helps explain how aldosterone could trigger two distinct responses to hypovolemia and hyperkalemia in these cells (Shibata et al. 2013b). The authors then investigated the regulation of MR phosphorylation by WNK4, using mice overexpressing a mutant “WNK4-FHHt” protein (TgWNK4PHAII mice; Lalioti et al. 2006), which exhibit the FHHt phenotype. They observed that the phosphorylation of Ser843 of the MR is lower and that pendrin stimulation by aldosterone is stronger in TgWNK4PHAII mice than in controls. WNK4 could therefore be involved in the regulation of MR phosphorylation but the underlying mechanisms remain to be defined. What are the functional consequences of WNK4 increased abundance and lower MR phosphorylation in β-ICs? They were characterized by Lopez-Cayuqueo and

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colleagues in 2018, again using the TgWNK4PHAII mice (López-Cayuqueo et al. 2018). They showed that pendrin activity is increased in isolated perfused CCDs of these mice, leading to a stimulation of thiazide-sensitive Na+-Cl reabsorption in this segment. The number of β-ICs is higher in TgWNK4PHAII mice, whereas that of α-ICs is reduced. This is surprising since the opposite is expected: the number of a-ICs should be induced by the metabolic acidosis. Finally, the genetic inactivation of pendrin in TgWNK4PHAII mice corrects the kalemia and tends to lower the blood pressure. Taken together, these studies suggest that the activation of pendrin in b-ICs could contribute to the pathogenesis of FHHt. Therefore, FHHt could be a disease of not only the DCT but also of the CNT/CCD. Another in vivo study supports this hypothesis.

Activation of SPAK in DCT Cells Is Sufficient to Induce the Development of FHHt To directly test if the hyperkalemia of FHHt patients is the consequence of reduced Na+ delivery to the CNT/CCD, Grimm and colleagues generated a mouse model expressing a constitutively active version of SPAK (SPAK-CA mice), specifically in the proximal part of the DCT (DCT1), and a lack of SPAK expression in all other cells (Grimm et al. 2017). By opposition to the other FHHt mouse models, where the genetic modifications affect not only the DCT but also the other segments of the cortical distal nephron, the FHHt phenotype is here created by a modification that is restricted to the DCT1. These mice display a complete FHHt phenotype (hypertension, hyperkalemia, hyperchloremic metabolic acidosis). NCC expression and phosphorylation are increased only in DCT1, while they are decreased in DCT2 (the distal part of the DCT). All these clinical and molecular manifestations are reversed by thiazide treatment. The authors then tested whether or not the increase in plasma potassium is a direct consequence of increased Na+ delivery by monitoring the kinetic of the changes in Na+ and K+ excretion and plasma K+ over a 3-day thiazide treatment. If that is the case, Na+ and K+ excretion should both increase rapidly when thiazide is administered. If a prompt natriuresis is indeed induced in CA-SPAK mice, plasma and urinary K+ are only slowly restored to the wild-type levels. This suggests that other modifications of the activity of the distal nephron are induced by the activation of SPAK and NCC in DCT1. The authors then explored the remodeling of the CNT and CCD since several studies have shown that it could be induced following NCC activation or inhibition. They showed that the size of DCT1 is increased while that of CNT is decreased, a remodeling that is reversed by thiazides. Consequently, the expression of ENaC subunits is lower in CA-SPAK mice. ROMK expression is also impaired: its abundance at the apical membrane is decreased in the CNT and CCD of CA-SPAK mice but not in the TAL. This is reminiscent of what is observed in the mouse model of “WNK1-FHHt” (WNK1+/FHHt), in which ROMK apical abundance is decreased in the CNT, where L-WNK1 is overexpressed. Taken together, the studies described in the last two paragraphs suggest that FHHt is the consequence of a modified ion transport in the entire distal nephron, which is itself caused by the activation of NCC in the DCT and the remodeling of the downstream segments, i.e., reduced CNT size and increased β-ICs number.

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Are “FHHt Genes” Involved in the Pathogenesis of Essential Hypertension? Identification of Polymorphisms and Variants Associated with a Higher Risk of Hypertension in the General Population Although FHHt is a rare disorder, unravelling the underlying mechanisms contributed to a better understanding of the modulation of the function of the distal part of the nephron. As WNK1, WNK4, CUL3 and KLHL3 are implicated in the regulation of NCC, the inhibition of which is one of the most commonly used anti-hypertensive treatments, several teams hypothesized that genetic variants of the corresponding genes and their signaling pathway could be responsible for the more common forms of hypertension. A study showed an association between increased systolic blood pressure and a polymorphism in an intron of WNK4 in the Japanese population (Kokubo et al. 2004). Another polymorphism located in the tenth intron of WNK4 has been associated with an increased blood pressure in a white American population (Erlich et al. 2003). However, this association was not found in an Australian population (Speirs and Morris 2004). The analysis of the association between WNK1 variants and essential hypertension in the patients of the BRIGHT (British Genetics of Hypertension) cohort showed that the rs1468326 variant is associated with the severity of hypertension (Newhouse et al. 2009). This polymorphism, located closed to the promoter of L-WNK1, is also associated with a greater risk of hypertension in two independent Chinese populations (Han et al. 2011). Others analysis performed in the BRIGHT cohort showed that the polymorphism rs765250, located in the first intron, is associated with an increased systolic blood pressure, to an increased diastolic blood pressure and to hypertension. This variant has been identified in six independent populations (Newhouse et al. 2009). This variant is also strongly associated with the nocturnal systolic blood pressure in a study performed in Caucasian European families (Tobin et al. 2005). Others WNK1 polymorphisms have been associated with the level of response to hydrochlorothiazide treatment in African and non-Hispanic Caucasians populations (Turner et al. 2005), but also with the influence of the diet on blood pressure in a Japanese population of healthy men (Osada et al. 2009). Finally, different variants and haplotypes of WNK1 have been associated with variations in blood pressure, essential hypertension and urinary excretion of potassium (Newhouse et al. 2009). Association tests between 794 SNPs at the KLHL3 locus and blood pressure showed no significant association with systolic or diastolic blood pressure or both combined. Therefore, common variations in KLHL3 do not appear to have an impact on blood pressure regulation in the general population (Louis-Dit-Picard et al. 2012). Only one study suggests that the rs17479770 polymorphism of CUL3 could be a protective factor in the pathogenesis of the essential hypertension. Indeed, the presence of this variant is associated with a decreased risk of essential high blood pressure in a Chinese population (Li et al. 2017).

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While no mutations have been identified in the genes encoding SPAK and OSR1 in patients with FHHt, a study has shown a strong association between increased blood pressure and common variants of the STK39 gene, encoding SPAK, in an Amish population (Wang et al. 2009). These results were confirmed in non-Amish European and East Asian populations, but the association was not found in an African population (Xi et al. 2013). More recently, a whole-genome association study conducted in populations from South Asia, East Asia and Europe identified an association between common variants of OSR1 and changes in blood pressure (Kato et al. 2015). These data obtained in humans confirm the role played by SPAK and OSR1 in the regulation of blood pressure.

Can the Inhibition of SPAK, OSR1 and/or WNKs Be Considered as a Relevant Anti-Hypertensive Strategy? As genetic studies in humans have confirmed the role of the WNK-SPAK/OSR1 pathway in blood pressure control, several strategies have been developed to block the WNK-SPAK/OSR1 signaling pathway. The first strategy was to inhibit the WNK-SPAK interaction. As described previously, the carboxy-terminal conserved domain (CCT) of SPAK plays a key role in the regulation of SPAK activity and function. The SPAK CCT domain binds with high affinity to the RFXV/I motifs of WNK and its substrates (NKCC1/2, NCC, KCC1–4) (Villa et al. 2007). A single mutation in Leu502 in the CCT domain abolishes the binding to the WNKs and mice homozygous for this mutation show a marked decrease in the expression and phosphorylation of NCC and NKCC2 in the kidney, associated with hypotension (Zhang et al. 2015). A molecule targeting the CCT domain of SPAK could therefore be effective against hypertension and could be sufficiently selective to avoid interaction with other signaling pathways. Two components, 1S-50699 and 2S-26016, have been identified as capable of inhibiting the binding of the CCT domain to RFXV/I motifs (Mori et al. 2013b). In vitro, only the 1S-50699 molecule is capable of inhibiting NKCC1 phosphorylation induced by low chloride hypotonic conditions. However, despite encouraging results, the authors point out that pharmacokinetic and pharmaco-dynamic studies are needed to determine whether the 1S-50699 molecule can be used in vivo (Mori et al. 2013b). The identification or generation of an inhibitor of the catalytic activity of SPAK could also be effective in blocking the action of SPAK in the regulation of SCL12 transporters. However, this approach has some limitations. Since this domain is highly conserved between SPAK and OSR1, developing a treatment that blocks the binding between SPAK/OSR1 and SCL12 transporters could affect the function of OSR1 and have unexpected effects such as infertility or reduced gastrointestinal gland secretion (Liu et al. 2016; Yan and Merlin 2008). Due to the competition with OSR1 and the different levels of SPAK expression in tissues, one of the challenges of a SPAK-targeted therapy will be to determine the optimal treatment concentration for blood pressure reduction. The treatment will need to bind to at least 50% of the SPAK content to achieve at least a 20% decrease in NCC phosphorylation in the

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kidney and a 40% decrease in NKCC1 phosphorylation in the vascular system, similar to that observed in SPAK+/243A mice (Rafiqi et al. 2010). Two molecules have been identified as capable of blocking the docking of SPAK to its targets: 1S-14279 and Closantel, a known anti-helmintic. These two molecules cause a significant decrease in the phosphorylation of NCC and NKCC1 in vitro and in vivo in mice (Kikuchi et al. 2015). However, 1S-14279 has been shown to be toxic during a chronic administration. Closantel does not decrease blood pressure (Kikuchi et al. 2015). Another prmoising molecule is Rafoxanide. This anti-parasitic agent has a similar structure and mode of action to Closantel and has been recently identified as an inhibitor of SPAK and OSR1 (AlAmri et al. 2017). Like Closantel, Rafoxanide decreases the phosphorylation of NKCC1 in vitro, but its effects have not been tested in vivo (AlAmri et al. 2017). Such a strategy could also reduce blood pressure, thanks to reduced vasoconstriction via NKCC1 in the blood vessels. More recently, a kinase panel screening showed that Verteporfin, a photosensitizing agent used in photodynamic therapy, inhibits the catalytic activity of SPAK and OSR1 in an ATP-independent manner by binding the kinase domain of SPAK and OSR1 (AlAmri et al. 2018). In vitro, Verteporfin is able to suppress the phosphorylation of NKCC1. In vivo, the decrease in blood pressure has been noted in patients and animals treated with this agent. Although Verteporfin has largely been studied as a modifier of the Hippo signaling pathway, this study indicates that the WNK-SPAK/ OSR1 signaling cascade could also be a target of this clinical agent. Very recently, an original approach led to the development of ZT-1a, a new SPAK inhibitor through the combination of pharmacophores of the 2-(4-amino-2-chloro-5-methylphenyl)-2(4-chlorophenyl)acetonitrile moiety from Closantel and the chloro-substituted 2-hydroxybenzoic acid from 1S-14279 and Rafoxanide (Zhang et al. 2020). In vitro, ZT-1a inhibits the SPAK-dependent phosphorylation of NKCC1. In vivo, in mice, ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral oedema, protects against brain damage and improves outcomes in a model of stroke confirming its efficiency in vivo. Finally, the authors showed that ZT-1a was among the best SPAK inhibitors identified from an ~300-compound chemical library, even better than the inhibitors previously described (Zhang et al. 2020). The second approach involves small molecules capable of binding to the Cab39 chaperone protein necessary for the catalytic activation of SPAK and OSR1. The interest of these molecules is to inhibit the binding of Cab39 to SPAK and OSR1, thus preventing their activation. The HK01 component has been identified from a large library of more than 4000 components through fluorescence interaction screening. HK01 is able to inhibit the activation of SPAK and OSR1 by Cab39 in vitro (Kadri et al. 2017). According to the authors, additional studies are underway to optimize the structure of this molecule and increase the affinity between HK01 and Cab39 (Kadri et al. 2017). A final strategy is the inhibition of WNK kinase activity by targeting the atypical position of the catalytic residue lysine. Through a high-throughput screening of molecules inhibiting L-WNK1 activity, a promising molecule, WNK463, has been identified (Yamada et al. 2016). It can block the kinase activity of all four WNKs in vitro. WNK463 can be administered orally: it leads to a decrease in blood pressure

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in hypertensive rats and mice and to a decrease in the phosphorylation of WNK4 in the mouse kidneys. Despite encouraging results, the development of WNK463 as a therapeutic agent has been suspended due to adverse side effects identified in pre-clinical trials, not described by the authors (Yamada et al. 2016). These adverse effects are likely due to the broad expression of WNKs and their actions other than blood pressure regulation, particularly in the nervous system (Shekarabi et al. 2008; Yamada et al. 2016; McCormick and Ellison 2011). A more recent study compared the selectivity of the WNK463 against the four members of the WNK family by comprehensive molecular modelling and simulations (Jonniya and Kar 2020). They showed that the affinity of the inhibitor decreases in the following order: WNK2 > WNK1 > WNK3 > WNK4. This type of study is essential to elucidated isoform-specific interactions that could be exploited in the design of more potent and selective WNK inhibitors. Despite the current limitations highlighted by all these studies and preventing the development of treatment for the moment, targeting the WNK-SPAK/OSR1 signaling pathway remains a relevant anti-hypertensive strategy. One way to limit these side effects would be to be able to develop molecules capable of specifically targeting the signaling pathway in the kidney and/or the vascular system.

Conclusion The genetic analysis of the rare families affected by Familial Hyperkalemic Hypertension in France and the USA has unraveled a previously unsuspected signaling pathway, which plays a crucial role in Na+ and K+ homeostasis. This pathway, regulated at least in part, by extracellular potassium, stimulates the activity of the Na+-Cl cotransporter NCC in the distal nephron. However, the expression L-WNK1, WNK4, SPAK and OSR1 is far from being confined to the distal nephron. An increasing number of publications over the last 10 years have started to elucidate their roles in several physiological or pathological situations. First, L-WNK1 is expressed in the central and peripheral nervous system. One of its alternatively spliced exons, HSN2, is expressed specifically in the dorsal root ganglia and its mutations result in Hereditary sensory and autonomic neuropathy type 2, a rare human pathology characterized by the early loss of sensory perception (Shekarabi et al. 2008). In neurons of the central nervous system, L-WNK1 inhibits KCC2 activity by phosphorylation (Friedel et al. 2015; Heubl et al. 2017). As this K+-Cl co-transporter KCC2 (SLC12A5) tunes the efficacy of GABAA receptor-mediated transmission by extruding Cl , L-WNK1 signaling could contribute to the homeostasis of synaptic inhibition by rapidly adjusting neuronal [Cl ]i to GABAAR activity (Heubl et al. 2017). Second, L-WNK1 was shown to regulate the migration and adhesion of T lymphocytes (Köchl et al. 2016). Indeed, L-WNK1 reduces integrin-mediated adhesion of T-cells, whereas it stimulates their migration by increasing NKCC1 activity through the phosphorylation of SPAK and OSR1. LWNK1 could therefore be involved in immune and inflammatory responses.

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Finally, there is increasing evidence that L-WNK1 could be implicated in several cancers (for review, see Rodan and Jenny 2017). Indeed, it is part of several pathways that control cell proliferation (such as WNK signaling). Its inhibition impairs the epithelial-mesenchymal proliferation (EMT), suggesting that L-WNK1 activation could favor metastasis. Finally, L-WNK1 can be part of the TGF-ß signaling, which can be either a tumor-suppressor or promote the development of cancers. The precise characterization of the roles played by L-WNK1 is only beginning and requires further studies.

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Diabetes Insipidus: Novel Diagnostic Approaches Marianna Martino

, Giulia Giancola, and Giorgio Arnaldi

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Etiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Confirmation of the Hypotonic Polyuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differential Diagnosis Within the Polyuria-Polydipsia Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . Etiologic Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Diabetes insipidus (DI) is a water balance disorder characterized by the excretion of a large amount of hypotonic urine associated with a compensatory polydipsia. The polyuria-polydipsia syndrome includes four major entities: central DI (CDI), gestational DI (GDI), nephrogenic DI (NDI), and primary polydipsia (PP). It is essential to differentiate accurately CDI from the other forms, because a wrong diagnosis can have fatal consequences. Clinical manifestations do not differ between DI and PP, even though the underlying mechanism is different. The diagnostic process is often challenging, especially in distinguishing partial CDI from PP. Hyponatremia orientates toward PP, whereas sodium levels at the upper end of the normal reference or hypernatremia are suggestive of DI. However, plasma sodium is normal in the majority of cases; therefore, the differential diagnosis of polyuria requires dynamic testing of the arginine-vasopressin (AVP)-renal axis. The classical water deprivation test (WDT) combined with a desmopressin (DDAVP) test has a poor diagnostic accuracy, is stressful, and requires adherence by patients, adequate supervision by M. Martino · G. Giancola · G. Arnaldi (*) Division of Endocrinology and Metabolic Diseases, Department of Clinical and Molecular Sciences (DISCLIMO), University Hospital of Ancona, Polytechnic University of Marche, Ancona, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2023 M. Caprio, F. L. Fernandes-Rosa (eds.), Hydro Saline Metabolism, Endocrinology, https://doi.org/10.1007/978-3-031-27119-9_5

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clinicians, and hospitalization. Copeptin and AVP are secreted in equimolar amounts from the neurohypophysis, but the former is more stable and easier to measure. In recent years, the assessment of circulating copeptin levels, especially after stimulation, has replaced the classical test in clinical practice. Arginine-stimulated copeptin seems to be the most convenient, easy, and accurate tool to diagnose DI, even if a confirmation test using hypertonic saline infusion combined with DDAVP is recommended in doubtful cases. Keywords

Hypotonic polyuria · Water deprivation test · Hypertonic infusion · Vasopressin · Copeptin

Introduction Diabetes insipidus (DI) is a water balance disorder characterized by the excretion of large amounts (>50 ml/kg/24 h) of hypotonic urine and a compensatory polydipsia (>3 L/day). This is a rare disease (prevalence of 1/25,000 case) affecting people of any age without gender differences (Fenske and Allolio 2012; Tomkins et al. 2022; Christ-Crain et al. 2019). The polyuria-polydipsia syndrome includes four major entities: central DI (CDI), gestational DI (GDI), nephrogenic DI (NDI), and primary polydipsia (PP). CDI is caused by absolute or partial deficiency in Arginine Vasopressin (AVP) secretion, whereas NDI expresses the renal resistance to the antidiuretic action of AVP; GDI is a rare complication of pregnancy due to an increased AVP degradation by placental vasopressinases; finally, PP reflects inappropriate and excessive water drinking. Considering the underlying pathogenetic mechanism, a nomenclature change has been recently proposed to avoid misunderstanding between diabetes insipidus and diabetes mellitus, which can cause safety problems when patients with diabetes insipidus are under the care of clinicians other than endocrinologists. Hence, the experts have proposed the name “diabetes insipidus” should be changed into “arginine vasopressin deficiency” (AVP-D) and “arginine vasopressin resistance” (AVP-R) for central and nephrogenic etiologies, respectively (Arima et al. 2022).

Etiology CDI is the most common form of DI. It is caused by either acquired or genetic disorders at the hypothalamic/hypophyseal level leading to loss of AVP secretion (Table 1). The neurohypophysis consists of three parts: (1) the hypothalamic supraoptic and paraventricular nuclei, containing the bodies of vasopressinergic, magnocellular neurons; (2) the supraoptic-hypophyseal tract formed by their axons; and (3) the posterior pituitary, which harbors the axon terminals. The clinical presentation of CDI implies destruction or loss of function of more than 80%

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Table 1 Etiologies of central and nephrogenic diabetes insipidus Types of diabetes insipidus Central DI

Nephrogenic DI

Basic defect Impaired AVP synthesis or secretion

Reduced distal nephron sensitivity to physiological AVP levels

Causes Acquired Trauma/surgery (transsphenoidal pituitary surgery, deceleration injury, and intracranial surgery) Neoplastic (craniopharyngioma, meningioma, germinoma, and metastasis) Vascular (cerebral/ hypothalamic hemorrhage, anterior communicating artery aneurysma infarction, or ligation) Granulomatous or systemic disease (sarcoidosis, granulomatous hypophisitis, Langherans’ cells histiocytosis, and ErdheimChester disease) Infectious (meningitis, encephalitis, tuberculosis, toxoplasmosis, and HIV infection) Inflammatory/autoimmune (lymphocytic infundibuloneurohypophysitis, xanthogranulomatous hypophysitis, IgG4 neurohypophysitis, antivasopressin neuron antibodies, and Guillain-Barré syndrome) Drug/toxin-induced: Temozolomide Immune checkpoint inhibitors Phenytoin Alcohol, snake venom Osmoreceptor dysfunction (adipsic DI) Others (hydrocephalus, ventricular/ suprasellar cyst, and degenerative disease) Idiopathic Congenital (genetic) AVP-neurophysin II gene alterations (autosomal dominant, autosomal recessive, and X-linked recessive inheritance) Septo-optic dysplasia Syndromic: Wolfram “DIDMOAD” (WFS1 gene), Schinzel-Giedion syndrome, CullerJones, Alstrom, Hartsfield, Webb-Dattani syndromes, and central DI in context of malabsorptive diarrhea (PCSK1 gene) Acquired Renal disease/injury (chronic kidney disease, polycystic kidney disease, obstructive uropathy, and acute tubular necrosis) Metabolic (electrolytes disorders): hypokalemia, hypercalcemia (continued)

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Table 1 (continued) Types of diabetes insipidus

Basic defect

Causes Vascular (renal infarction, sickle cell anemia) Neoplastic (multiple myeloma) Granulomatous or systemic disease (amyloidosis, sarcoidosis, and Sjogren’s syndrome) Drug/toxin-induced: Lithium Demeclocycline, methoxyflurane Cisplatin, pemetrexed Aminoglycosides, amphotericin B Congenital (genetic) Autosomal recessive or dominant AQP2channel gene mutations X-linked AVPR2 (V2) gene mutations PMSE syndrome (polyhydramnios, megalencephaly, and symptomatic epilepsy) Type 4b Bartter syndrome

AVP-producing magnocellular neurons. The degree of AVP deficiency varies between a complete and a partial defect in patients with inappropriately low circulating AVP with respect to plasma osmolality. Trauma brain injury (TBI), subarachnoid hemorrhage (SAH), and pituitary-hypothalamic surgery are the most frequent causes of CDI. Both the acute phase of TBI and SAH complicate with CDI in 20% and 15% cases, respectively. Here, CDI is nearly always transient and associated with worse prognosis. Polyuria develops 1–4 days after pituitary surgery at a rate of 0.3–45% and recovers spontaneously only in some cases. A triphasic disease is often observed after surgery: First, an immediate, transient CDI occurs due to axon shock lasting 1–2 days (phase one); this is followed by a second phase of inappropriate antidiuresis (SIAD) and, after 5–10 days, by the development of a permanent CDI (third phase) (Brooks and Inder 2022; Verbalis 2020). If CDI is rarely seen in pituitary adenoma, hypothalamic or pituitary metastases debut with CDI not infrequently (He et al. 2015). Infiltrative forms of CDI include granulomatous diseases (sarcoidosis and tuberculosis) and histiocytosis, where CDI is the typical manifestation of central nervous system involvement (10–50% patients). In children, craniopharyngioma and germinoma are mainly responsible for CDI (Patti et al. 2022). Adipsic CDI is a life-threatening osmoreceptor dysfunction characterized by CDI in the absence of a thirst response to hypernatremia. Patients are thus hypernatremic and dehydrated. In many cases, the brain lesions this severe condition is associated with are the same that cause CDI, but in adipsic CDI they usually grow more rostrally in the hypothalamus, impairing primary osmoreceptor cells. Since osmoreceptors and AVP-producing neurons are very close from an anatomical

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point of view, the risk of adipsic CDI increases following neurosurgical clipping of anterior communicating artery aneurysms, surgery of craniopharyngioma and suprasellar pituitary tumors (Tomkins et al. 2022; Garrahy and Thompson 2020). Despite its lower prevalence in very recent studies, “idiopathic” CDI is still a common diagnosis in both adults and children (25% cases). Indeed, in many cases of idiopathic CDI an autoimmune disease resulting in the destruction of the neurohypophysis has been demonstrated (Tomkins et al. 2022; Patti et al. 2022; Iraqi et al. 2022). One-third patients with idiopathic CDI exhibit circulating antibodies against AVP-producing cells: however, this finding should be considered a surrogate of their immune response rather than a reliable marker of autoimmunity. A lymphocytic infundibulo-neurohypophysitis can be diagnosed presumptively without the need of a biopsy based on the noncontrast T1-weighted magnetic resonance imaging (MRI) finding of a thickened stalk and/or absence of the posterior pituitary bright spot. In patients with Immunoglobulin G4 (IgG4)-related systemic disease, lymphocytic infundibulo-neurohypophysitis is a common presenting feature, occurring in 10–30% patients, mostly women (Langlois et al. 2022). Recently, CDI has been reported as a rare side effect triggered by treatment with immune checkpoint inhibitors (ICI) in patients with cancer (Barnabei et al. 2022). In most cases, CDI was diagnosed in the context of an ipilimumab- and/or nivolumab-induced hypophysitis. Considering pituitary involvement is frequent in patients treated with ICI (about 20%), one can speculate the prevalence of CDI in patients with cancer is underestimated and that the difficulties in assessing the hydration status and the need of fluid supplementation in these patients could account for this discrepancy. A variable degree of AVP deficiency due to increased AVP catabolism operated by placental vasopressinases defines GDI. AVP resistance and a reduced threshold of thirst could also contribute. This rare and transient form of DI (2–6 cases/100,000 pregnancies) typically arises in the third month of pregnancy, peaks in the late second or third trimester, and resolves several weeks after delivery, as vasopressinase activity is undetectable 4–6 weeks after delivery. In normal pregnancy, an approximately fourfold increase in AVP synthesis and secretion is observed. Similarly, the amount of placental vasopressinases increases early and their activity becomes 40- to 50-fold higher by the late second and early third trimester of pregnancy. Despite these physiologic adaptations, most pregnant women are able to maintain stable serum and urine osmolality; therefore, they are not at risk of GDI. Importantly, GDI should be distinguished from a preexisting CDI or NDI in pregnancy (Ananthakrishnan 2020). NDI is characterized by the inability of the kidney to concentrate urine by reabsorbing water in the collecting duct (Bichet 2020; Bockenhauer and Bichet 2015). The resistance to AVP action can derive from either acquired or genetic disorders (Table 1). The clinical presentation is similar to that of CDI, except for basal plasma AVP, which is elevated in NDI. Mild forms of renal AVP resistance are relatively common. Chronic kidney disease, many drugs (mainly lithium), and prolonged electrolyte disorders such as hypokalemia and hypercalcemia are frequently responsible for NDI. The mechanism underlying most cases is a downregulation of aquaporin water channels 2 (AQP2), which are expressed on the luminal surface of the collecting duct epithelium in response to AVP, thus ensuring

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water reabsorption from the lumen into the collecting duct cell. The incidence of NDI in patients on lithium treatment is high (almost 85%). Although the real mechanism of lithium toxicity on the principal cells is still to be elucidated, several data suggest it could be mediated by epithelial sodium channels (ENaCs). PP is characterized by a chronically excessive fluid intake and the subsequent polyuria. In addition, chronic (>3 days) overhydration both inhibits AVP secretion and downregulates the expression of AQP2 at the collecting duct level. PP is common in patients affected by autism, intellectual disabilities and chronic psychotic disorders, such as schizophrenia, schizoaffective disorder, bipolar disorder, and psychotic depression. Up to 30% patients with schizophrenia suffer from this disorder, which is usually associated with abnormal thirst perception (MercierGuidez and Loas 2000). The popularity of lifestyle programs emphasizing fluid intake could explain the increased prevalence of PP in the general population, particularly in women, health-conscious athletes, and runners.

Clinical Presentation Despite a different underlying mechanism, DI and PP share the same clinical manifestations (Fenske and Allolio 2012; Tomkins et al. 2022; Christ-Crain et al. 2019; Angelousi et al. 2023). However, patients with CDI describe more frequently nocturia, a preference for cold drinks and a sudden onset of symptoms, since the ability of concentrating the urine is generally preserved until AVP synthesis falls below 10–15% normal capacity; afterward, urinary output increases suddenly and dramatically in case of complete CDI (Christ-Crain et al. 2019). Symptoms may also vary according to the degree of compensative fluid intake. In patients with PP, symptoms are generally more pronounced during the day, and when sleep is interrupted, nocturia is preceded by drinking. Although psychiatric disorders are more frequent in patients with PP, they are also observed in patients with CDI (27% vs 17%) (Verbalis 2020). Secondary adrenal insufficiency may mask a coexisting CDI, whose symptoms appear as soon as cortisol replacement is started. In children, symptoms of CDI can be accompanied by failure to thrive, growth retardation, fatigue, irritability, headache, emesis, visual field defects, and precocious puberty (Patti et al. 2022). Differentiating PP from DI, as well as among the subtypes of the latter, is of paramount importance to ensure each forms an appropriate treatment and avoids dangerous, sometimes life-threatening, consequences such as severe hyponatremia.

Diagnosis The diagnostic process to differentiate the entities composing the polyuriapolydipsia syndrome is still challenging, not only due to the overlap they show in signs and symptoms, but also because there is no expert agreement in the interpretation of the classically performed diagnostic tests. For this reason, a new diagnostic

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approach has been recently proposed, including three steps (Fenske and Allolio 2012; Tomkins et al. 2022; Christ-Crain et al. 2019; Angelousi et al. 2023; Robertson 2016): 1. Confirmation of the hypotonic polyuria 2. Differential diagnosis within the polyuria-polydipsia syndrome 3. Etiologic characterization

Confirmation of the Hypotonic Polyuria When DI is suspected, collecting a careful medical history is essential in order to exclude other urinary disorders, such as incontinence, urgency, nocturia, or pollakiuria, which may underlie infections of the urogenital tract, prostatic hypertrophy, and other urinary diseases (Garrahy and Thompson 2020). In these cases, no further investigations of osmoregulatory function are required, because the urinary frequency is increased but the urinary output is less than 2.5 L/24 h. Moreover, baseline laboratory tests should be performed to investigate the existence of diabetes mellitus, renal impairment, hypercalcemia, and hypokalemia. It is necessary to assess the water balance by means of a 24-hour urine collection: If the urinary output is >50 mL/kg/24 h (>100–110 mL/kg/24 h for children less than 2 years old, or > 150 mL/Kg/24 h in newborns), there is a real polyuria (Tomkins et al. 2022). Once polyuria is confirmed, urine osmolality should be measured. A random urine osmolality of >800 mOsm/kg excludes DI. Conversely, a low urine osmolality (20.0 pmol/L identified the two patients with NDI included in the study with a 100% sensitivity and specificity. In addition, copeptin levels 3 pmol/L showed the best sensitivity (82%) and specificity (92%) to diagnose PP. This study showed an adjunctive limitation of the classical WDT: An overnight thirsting is able to ensure the increase in serum osmolality over the threshold for AVP secretion in only 50% cases. Despite the 1–2% increase in plasma osmolality following water deprivation is sufficient to stimulate AVP secretion in most patients, the maximal stimulation on AVP-producing cells is achieved after a hypertonic (3–5%) saline infusion. The “osmotic threshold’ for AVP release in healthy subjects is approximately 280 mOsm/kg, with a linear relationship between AVP concentration and plasma osmolality. With this premise, Timper et al. prospectively measured copeptin levels at baseline and after osmotic stimulation in 55 patients with polyuria-polydipsia syndrome (Timper et al. 2015). Serum osmolality increased and reached the osmotic threshold (plasma sodium >147 mmol/L) for AVP release in all patients. A standardized WDT starting at 0800 h without prior fluid restriction was performed until serum sodium exceeded 147 mmol/L, then it was withdrawn. If plasma sodium did not exceed 147 mmol/L following thirsting alone by 1300 h, patients received a 3% saline infusion. Circulating copeptin and AVP levels were measured at baseline and every 30 or 60 min during the combined test. This

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prospective study demonstrated plasma copeptin is a reliable biomarker to discriminate the different forms of polyuria-polydipsia syndrome with superior accuracy than AVP. Without prior thirsting, a single baseline copeptin 21.4 pmol/L showed an excellent diagnostic accuracy (specificity/sensitivity of 100%) in diagnosing correctly the ten patients with NDI, making water restriction unnecessary in this subgroup. Considering the small sample size, this cutoff might need adjustments for patients with partial forms of NDI in the future. Baseline copeptin levels were generally lower in patients with CDI than PP and controls. A baseline copeptin level > 2.9 pmol/L differentiated patients with CDI (complete and partial) from the other forms with good performance (82% sensitivity and 78% specificity); an osmotically stimulated copeptin level of >4.9 pmol/L at sodium levels >147 mmol/L showed excellent diagnostic accuracy (94% sensitivity and 96% specificity) in distinguishing PP from partial and complete CDI (Timper et al. 2015). The osmotically stimulated copeptin cutoff (>4.9 pmol/L) was subsequently validated in a multicenter perspective study including 156 patients, of which 82 were with PP (Fenske et al. 2018). Here, a simplified protocol where only the 3% saline infusion was administered without prior thirsting was performed: After an initial 250 ml bolus, the 3% saline infusion proceeded at a rate of 0.15 ml/kg/min until serum sodium concentration exceeded 150 mmol/L. At this time point, copeptin was measured and normal serum osmolality was restored by means of a glucose infusion and a standardized fluid intake. The overall diagnostic accuracy of this copeptin cutoff in distinguishing patients with PP from CDI was 96.5% (sensitivity 93%, specificity 100%) compared to the 76.5% (sensitivity 86.4%, specificity 69.5%) of the indirect WDT performed in a different day. The limited performance of WDT was not surprising considering that 73% of patients did not achieve hyperosmolality after 16 hours of fluid deprivation, and this finding explains why the addition of copeptin to the standard WDT did not improve its diagnostic accuracy. A morning copeptin 155 mmol/L in 12 patients (6 with PP, 5 with complete CDI, and 1 with partial CDI), as compared to the 2 patients with complete CDI encountering severe hyperntaremia during WDT. All of them were female and had baseline plasma sodium levels ranging from 140 to 144 mmol/L. Although more side effects (nausea, vertigo, headache, and malaise in 50–70% of patients) were reported with the hypertonic saline infusion test than the WDT, the only adverse event reported as serious was a DDAVP-induced hyponatremia during WDT. Despite being less time-consuming than WDT (3 vs 17 h), the widespread use of the hypertonic saline infusion test in clinical practice may be hampered by safety concerns, which include the risk of severe hypernatremia, neurological complications, kidney injury, thrombophlebitis, and thromboembolic events. For these reasons, the test is contraindicated in several conditions (chronic heart failure, liver cirrhosis, and epilepsy seizure disorders), requires close monitoring of sodium levels, and should be performed after adequate hydratation only in specialized

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centers. In order to prevent osmotic overstimulation, as the authors themselves suggest, it may be prudent in clinical practice to establish the osmotic threshold at plasma sodium levels >147 mmol/L instead of 150 mmol/L (Fenske et al. 2018). To overcome these difficulties, Christ-Crain et al. looked for an alternative, rapid, and safe stimulus for AVP secretion. A promising test was then proposed, based on the use of arginine as a nonosmotic stimulus toward the posterior pituitary. Arginine is a precursor for nitric-oxide synthesis currently infused worldwide as a simple and welltolerated stimulus to test growth hormone deficiency, especially in children. In 2019, Winzeler et al. prospectively evaluated the effects of a standardized arginine infusion in 92 healthy volunteers and 96 adult patients with polyuria-polydipsia syndrome (Winzeler et al. 2019). After an overnight fast and fluid restriction for 2 h, 0.5 g/kg arginine diluted in 500 mL of 0.9% saline solution was infused over 30 min starting at 0800 h. Plasma copeptin was measured at baseline and after 30, 45, 60, 90, and 120 min from the infusion start. In healthy adults, median baseline copeptin was 5.2 pmol/L, increasing up to 9.8 pmol/L in the first 60 min following arginine stimulation. A copeptin cutoff of 3.8 pmol/L at 60 min showed a 93% accuracy (93% sensitivity, 92% specificity) in differentiating between DI and PP. This cutoff recognized patients with partial CDI: indeed, patients with complete CDI experienced a negligible copeptin increase, whereas those with partial CDI tended to have higher copeptin concentrations, both at baseline and after stimulation. The diagnostic accuracy of this cutoff in discriminating partial DI and PP was 88% (93% sensitivity, 67% specificity). Seven DI patients had mild hypernatremia (range 146–149 mmol/L) at the end of the test. Nevertheless, the test was safe and well tolerated (mild nausea was the only reported side effect). Therefore, this test is particularly useful in children, provided that copeptin cutoffs need further validation and additional studies are required in this population (Binder et al. 2023; Tuli et al. 2021). Of note, in a recent monocentric, retrospective cohort of 68 children and adolescents without CDI who underwent arginine or growth hormone-releasing hormone (GH-RH) test to assess GH secretory capacity, baseline copeptin levels were highly variable (1.3–44.4 pmol/L) and arginine-stimulated copeptin increased slightly more in children than in adolescents (Binder et al. 2023). If arginine-stimulated copeptin seems to be the most convenient, easy, and accurate tool to diagnose DI, in doubtful cases (e.g., in patients with low copeptin) a confirmation by means of a hypertonic saline infusion combined with DDAVP is recommended. In this purpose, the diagnostic accuracy of argininestimulated copeptin versus the hypertonic infusion test is under direct comparison in a randomized multicentric prospective study (clinical trials.gov NCT03572166, CARGO study (Use of copeptin measurement after arginine infusion for the differential diagnosis of diabetes insipidus – the CARGOx Study (CARGOx))). Recently, a glucagon-stimulated copeptin measurement has been proposed as a way to distinguish among the polyuria-polydipsia syndrome forms (Atila et al. 2022a). As it happens with arginine, glucagon stimulates GH and AVP secretion in a nonosmotic fashion, although the exact underlying mechanism is still unknown. In a double-blind, randomized, placebo-controlled study, 1 mg glucagon was injected subcutaneously at 0800 h, after an overnight fast and fluid restriction for 2 h. Copeptin levels were measured at baseline and 30, 60, 90, 120, 150, and 180 min

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after the injection. The median copeptin increase was 7.56 (range 2.38–28.03) pmol/ L in the 22 healthy subjects and 15.70 (5.99–24.39) pmol/L in PP patients. Conversely, patients with DI experienced no relevant changes in copeptin levels. A glucagon-stimulated copeptin of 4.6 pmol/L at 120 min showed a 100% sensitivity and 90% specificity in discriminating DI from PP. Noteworthy, a correlation between the drop in glucose and the increase in copeptin upon arginine and glucagon stimulation was observed, suggesting a role for hypoglycemia as a trigger for copeptin secretion (Atila et al. 2022b). The glucacon test was safe and well tolerated, but it must be noted all participants received Ondansetron, a selective serotonin receptor antagonist, 10 min before the test to prevent nausea. Due to the small number of patients studied, the usefulness of glucagon-stimulated copeptin for the diagnosis of DI has to be confirmed. The different assays to determine copeptin levels should be taken into account when interpreting cutoffs. Currently, two assays are available and validated: the original manual sandwich immunoluminometric assay and the automated immunofluorescent successor (on the KRYPTOR platform) (Sailer et al. 2021).

Tests to Predict CDI After Pituitary Surgery Postoperative CDI complicates 7–80% surgeries for sellar/parasellar lesions, particularly in case of very large masses, Rathke’s cleft cysts, and craniopharingiomas (Brooks and Inder 2022; de Vries et al. 2021). The incidence of CDI after transphenoidal surgery for pituitary adenomas is 10–26%; the disease is permanent in up to one-third cases. Postoperative CDI may be transient (occurring 24–48 h after surgery and resolving within 1–2 weeks), prolonged (>2 weeks, but 6 months) and has variable severity. The diagnosis and management of postoperative CDI may be difficult for several reasons: the presence of polyuria caused by overload fluid infusion, the triple phase response, and the lack of standardized diagnostic criteria. However, a timely diagnosis and prompt treatment are critical to prevent the neurological consequences of CDI. Recently, postoperative CDI has been defined by the presence of hypotonic polyuria (urine output >300 mL/h for 3 consecutive hours, urine specific gravity 145 mmol/ L or osmolality >300 mOsm/Kg) (Castle-Kirszbaum et al. 2021). Using these criteria, a recent prospective study identified postoperative CDI in 10% of 449 patients operated for anterior skull base pathology. Predictors of CDI on multivariate analysis were suprasellar extension (OR 2.2), age < 50 years (OR 2.8), craniopharyngioma (OR 6.7), and Kelly grade 3 intraoperative cerebrospinal fluid (CSF) Leak (OR 2.1). The SALT score, a simple scoring system based on these parameters, was created. The rates of postoperative CDI were 4%, 6.5%, 15%, 37%, and 86% for SALT scores of zero, one, two, three, and four, respectively. This score seems a good predictor of postoperative CDI, but validation studies are needed (Winzeler et al. 2015). The use of copeptin as a predictor postoperative CDI has been recently evaluated, not without controversies about the best timing of measurement, cutoff values, and the need to start treatment which does not always allow waiting for copeptin results. Of the 205 patients investigated by Winzeler et al., 24% (50) developed postoperative CDI.

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Copeptin values 30 pmol/L had a 95% (sensitivity 94%) negative predictive value (Winzeler et al. 2015). Berton et al. assessed copeptin levels of 66 patients 1 h after extubation, identifiying with this time point the moment of maximal stress: They showed a copeptin peak 12.8 pmol/L was predictive for CDI, while levels >4.2 pmol/L excluded permanent forms (Berton et al. 2020). Recently, another study involving 78 patients demonstrated copeptin levels >3.4 pmol/L on postoperative day 1 helped ruling out DI with a 91% sensitivity and 55% specificity (Rostom et al. 2023). Finally, in a study of 73 patients, copeptin levels 3.4 and 7.5 pmol/L on postoperative day 2 and 7 may have ruled out the occurrence of CDI with a negative predictive value of 100% (Jang et al. 2022). All these studies have limitations, above all the small sample size. For these reasons, the cutoffs they found need validation, but the use of copeptin as a predictive marker of postsurgery CDI offers the possibility to distinguish between transient and permanent forms of CDI, thus optimizing their treatment.

Etiologic Characterization The etiological diagnosis requires a detailed medical history, a thorough physical examination, and biochemical and imaging studies. In cases of family history of or childhood onset DI, genetic counseling is necessary. Most of the acquired etiologies are evident or may be suggested by patients’ history: Headache, visual disturbances, prior TBI, autoimmune, psychiatric disorders, or poor growth and premature puberty in children should be investigated. Finally, a detailed drug history should be recorded. In patients with CDI, pituitary MRI can be useful to identify an underlying pathology, such as pituitary adenoma, craniopharyngioma, germinoma, infiltrative diseases, and metastases. The physiological posterior pituitary “bright spot” in T1-weighted images, which has been attributed to the presence of AVP secretory granules, is absent in complete CDI and is a useful adjunctive element for the differential diagnosis. However, a recent study showed that 20% patients with complete CDI and 40% with partial CDI had persistence of this pituitary bright spot, which was absent in 40% patients with PP (Tomkins et al. 2022). In addition, because of an age-related gradual loss of the posterior pituitary bright spot, up to 25% healthy elders have loss of the bright spot, despite preserved AVP secretion. The thickness of the pituitary stalk can be also useful: an enlargement of more than 2–3 mm is often suggestive but not necessarily diagnostic for idiopathic and autoimmune CDI. Moreover, an isolated pituitary stalk thickening could be a signal of intracranial germ cell tumors (Mootha et al. 1997). Germinomas can occur in the posterior pituitary, more often in the infundibulum or suprasellar region. An initial MRI scan may be normal in these patients and should be repeated within 6–12 months in adults and every 6 months in young patients (Tomkins et al. 2022; Patti et al. 2022).

>3.8 pmol/L

4.9 pmol/L

Hypertonic saline infusion combined with DDAVP (Confirmation test in unclear cases)

21,4 pmol/L

Primary polydipsia

Fig. 1 A new algorithm for the diagnostic approach of the hypotonic polyuria-polydipsia syndrome

Central Diabetes insipidus

147 mEq/L Osmolality > 280 mOsm/Kg

Central Diabetes insipidus

A transversion 11 bp from a splice acceptor site in the human gene for steroidogenic acute regulatory protein causes congenital lipoid adrenal hyperplasia. Hum Mol Genet. 1995;4(12): 2299–305. Valadares LP, et al. Molecular analysis of the CYP11B1 gene in Brazilian patients with 11-betahydroxylase deficiency. Clin Endocrinol. 2018;89(4):432–8. White PC, Curnow KM, Pascoe L. Disorders of steroid 11 beta-hydroxylase isozymes. Endocr Rev. 1994;15(4):421–38. White PC, Dupont J, New MI, Leiberman E, Hochberg Z, Rösler A. A mutation in CYP11B1 (Arg-448----His) associated with steroid 11 beta-hydroxylase deficiency in Jews of Moroccan origin. J Clin Invest. 1991;87(5):1664–7. Zennaro MC, Boulkroun S, Fernandes-Rosa F. Inherited forms of mineralocorticoid hypertension. Best Pract Res Clin Endocrinol Metab. 2015;29(4):633–45. Zhao X, Su Z, Liu X, Song J, Gan Y, Wen P, Li S, Wang L, Pan L. Long-term follow-up in a Chinese child with congenital lipoid adrenal hyperplasia due to a StAR gene mutation. BMC Endocr Disord. 2018;18(1):78.

Apparent Mineralocorticoid Excess

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Cristian A. Carvajal, Alejandra Tapia-Castillo, Thomas Uslar, and Carlos E. Fardella

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mineralocorticoid-Dependent Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apparent Mineralocorticoid Excess Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Etiology of AME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis of AME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nonclassic Apparent Mineralocorticoid Excess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Etiology of the Nonclassic AME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis of NC-AME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetics and Epigenetics Affecting HSD11B2 Gene: Role in NC-AME . . . . . . . . . . . . . . . . . . . . . Epigenetics: DNA Methylation of HSD11B2 Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetics: Non-Coding RNA Affecting HSD11B2 Gene Expression . . . . . . . . . . . . . . . . . . . . Exogenous Inhibitors of 11ΒHSD2 Enzyme: Role in NC-AME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endogenous Inhibitors of 11βHSD2 Enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Novel Combined Phenotype of NC-AME and Primary Aldosteronism . . . . . . . . . . . . . . . . . . Differential Diagnosis from AME and NC-AME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Primary Aldosteronism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypertensive Forms of Congenital Adrenal Hyperplasia (OMIM #202010 and #202110) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucocorticoid Resistance (OMIM #138040) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liddle’s Syndrome (OMIM #177200) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Activating Mutation of Mineralocorticoid Receptor (OMIM #605115) . . . . . . . . . . . . . . . . . . . Gordon Syndrome (OMIM #614495) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment for AME and NC-AME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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C. A. Carvajal · A. Tapia-Castillo · T. Uslar · C. E. Fardella (*) Department of Endocrinology, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile Centro Traslacional de Endocrinología UC (CETREN), Santiago, Chile e-mail: [email protected] © Springer Nature Switzerland AG 2023 M. Caprio, F. L. Fernandes-Rosa (eds.), Hydro Saline Metabolism, Endocrinology, https://doi.org/10.1007/978-3-031-27119-9_11

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Abstract

Context: Arterial hypertension (AHT) is one of the most frequent pathologies in the general population. Subtypes of essential hypertension characterized by low-renin levels allowed the identification of two different clinical entities: aldosterone-mediated mineralocorticoid receptor (MR) activation and cortisolmediated MR activation. The spectrum of cortisol-mediated MR activation includes the classic apparent mineralocorticoid excess (AME) to milder (nonclassic, NC) forms of AME, the latter with a much higher prevalence than classic AME but different phenotype and genotype. AME is a rare autosomal recessive disorder caused by the presence of a severe deficiency of 11βHSD2 activity, mainly due to a multiple pathogenic variant in the HSD11B2 gene. The clinical features are childhood onset hypertension, hypokalemia, and alkalosis with low plasma renin, but unlike primary aldosteronism (PA), AME displays low aldosterone levels in the presence of a high serum or urinary cortisol/cortisone (F/E) ratio. NC-AME is mainly related to partial 11βHSD2 deficiency associated with genetic variations and epigenetic modifications (first hit) and potential additive actions of endogenous or exogenous inhibitors (i.e., glycyrrhetinic acid-like factors (GALFS)) and other factors (i.e., age, high sodium intake) (second hit). Subjects with NC-AME are characterized by high F/E ratio and low E levels, normal and elevated blood pressure, low-renin and increased urinary potassium excretion and microalbuminuria. Subjects with the AME condition should benefit with low-sodium diet, potassium supplementation and monotherapy with MR antagonists. Keywords

AME · Nonclassic AME · Arterial hypertension · Low renin

Introduction Arterial hypertension (AHT) is a multifactorial disease with a complex pathogenesis that occurs from the interplay between genetic and environmental factors that lead to changes in different metabolic pathways associated with the proper control of blood pressure (Padmanabhan et al. 2015; Martinez-Aguayo and Fardella 2009). AHT is also a major risk factor for stroke, myocardial infarction, heart failure and end-stage renal disease. Worldwide, the prevalence of hypertension in adults over 25 years is rather 25% (Collaboration NCDRF 2017), contributing to 45–50% of deaths due to heart disease and stroke (World Health Organization 2011; Kearney et al. 2005), making the AHT a major concern for public health, particularly in western countries (Egan et al. 2010; Danaei et al. 2011; Mills et al. 2016). However, to date, most cases of AHT do not identify a specific etiology and are misclassified as essential hypertension. One-third of patients with hypertension have a low or suppressed renin which suggests a systemic volume expansion and mineralocorticoid receptor (MR) activation as happen in primary

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aldosteronism (PA). However, Adlin et al. (2013) demonstrated in a Framingham-based sample of subjects with low-renin hypertension a bimodal aldosterone distribution, suggesting two different pathophysiological phenotypes: one aldosterone-dependent, and interestingly, a second phenotype that is non-aldosterone-dependent, as has been proposed the apparent mineralocorticoid excess syndrome (AME) (Tapia-Castillo et al. 2019a). Lately, identification of subjects with a combination of both phenotypes has been identified and named as mixed phenotype PA & NCAME.

Mineralocorticoid-Dependent Hypertension Mineralocorticoid-dependent hypertension represents one of the most prevalent causes of secondary hypertension. Pathological conditions associated with aldosterone-mediated MR activation and cortisol-mediated MR activation are responsible for the etiology of AHT in up to 15% of AHT patients. Both conditions are mostly related to an unregulated activation of the MR. The activation of the MR by aldosterone or cortisol is a key step in the pathological process leading to mineralocorticoid hypertension (Funder et al. 2016a), since both can increase sodium transport in renal collecting ducts, increasing water uptake, blood volume, and secondary raising of blood pressure (BP). Aldosterone-MR and cortisol-MR complexes exert important roles in water-electrolyte homeostasis in the epithelial tissues of the kidneys and colon and in nonepithelial tissues (i.e., heart, brain) (Funder 2004; Gomez-Sanchez 2011) through genomic and nongenomic effects.

Apparent Mineralocorticoid Excess Syndrome Apparent mineralocorticoid excess (AME) syndrome (OMIM #218030), also called classic AME, is a rare autosomal recessive disorder caused by the presence of a severe deficiency of the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2), mainly due to multiple pathogenic variants in the HSD11B2 gene (Carvajal et al. 2003, 2018a; Yau et al. 2017). Affected subjects with classic AME display a low birth weight, severe hypertension, nephrocalcinosis, hypokalemia, suppressed renin and low aldosterone (Carvajal et al. 2018a). Biochemically, subjects with AME have a markedly elevated serum cortisol to cortisone ratio (F/E) (Carvajal et al. 2018a; Campino et al. 2010), and urinary 5β-tetrahydrocortisol (THF) plus 5α-THF to tetrahydrocortisone (THE) ratio [(5β-THF + 5α-THF)/THE] (Best and Walker 1997; Palermo et al. 1996).

Etiology of AME HSD11B2 gene is the only gene located in a region of 3 cM (centimorgans) on chromosome 16q22.1 that is associated with blood pressure. Following the description of the first disease-causing pathogenic variation (New and Levine 1977; New et al.

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1977), approximately 40 additional germline mutations have been reported (Yau et al. 2017), mainly in families where consanguineous marriages, endogamy or a founder effect occurred. The recent development of a computational model of the 11βHSD2 protein provide a structural explanation for the genotype-phenotype correlation and the clinical severity of AME syndrome associated to the different pathogenic variants in HSD11B2 gene (Fig. 1) (Carvajal et al. 2003, 2018a; Yau et al. 2017). Yau et al. in 2017 showed in silico changes that are induced by 11βHSD2 mutations. They provide structural explanations for the effect of mutations on 11βHSD2 function involved in enhance dimerization, disrupt the substrate- or coenzyme-binding site, or severely impair structural stability. Of these mutations, those affect multiple salt-bridge interactions, hydrogen bond networks, and hydrophobic patches have a greater impact on disrupting overall structural integrity. In contrast, mutations that only affect a single intramolecular interaction, such as R279C and R359W, cause relatively minor disruptions, and are, therefore, more tolerated. This is because loss of a single interaction is likely to be compensated by interactions formed between other residues. In 2018, Carvajal et al. (2018b) reported subjects with AME syndrome having either the D223N or R213C pathogenic variants in the HSD11B2 gene displayed the classical AME phenotype, including low-renin hypertension, low serum K+, and a high serum cortisol/cortisone ratio. Both mutations show high pathogenic potential as demonstrated previously by the very low activity of mutants in vitro and in silico (Carvajal et al. 2003; Yau et al. 2017; Mune et al. 1995). New in silico models suggest that both mutations induce a structural disruption of the 11βHSD2 protein,

Fig. 1 Gene variants in the human HSD11B2 gene. The diagram shows the human HSD11B2 gene and the location of identified pathogenic variants causing AME. The HSD11B2 gene is located on chromosome 16 and contains five exons, four CpG regions, two main (CA) repeat regions and a promoter with SP1/SP3, NF1, NF-κB and EGR-1 sites. Exons are represented by purple boxes. Gene variants are listed relative to their position in the gene

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impairing its catalytic activity and dimerization (Yau et al. 2017; Mune et al. 1995; Manning et al. 2010). The D223N mutation may alter the plasticity of the binding site or reduce the ability of the charge relay system associated to the Q261 sidechain, reducing the enzyme activity. The R337C inactivates HSD11B2 completely, since is highly conserved across many species, and any change of these residues are unlikely to be tolerated (Atanasov et al. 2007). R213C mutation impairs enzyme stability since affect the side chains forming hydrogen bonds with the backbone atoms of residues A331 and L329, then disrupting the tertiary structure of the protein and impairing the catalytic activity (Mune et al. 1995; Manning et al. 2010). The identification of key residues either in the catalytic site and the NAD+ binding-site facilitate the prediction of AME phenotype associated to known or predicted mutations in HSD11B2 gene.

Pathogenesis of AME This syndrome is characterized by the chronic unregulated activation of MR by cortisol, which induce the same effects as does aldosterone over MR (Arriza et al. 1987, 1988; Whitworth 1987). This is particularly relevant in human physiology, since the concentration of circulating cortisol is 1000–2000 times higher than of aldosterone, the natural ligand of the MR. Under normal condition, the activation of the MR by cortisol does not occur because the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2; NG_016549) efficiently inactivates cortisol (F) to cortisone (E), avoiding the binding of cortisol to MR (Martinez-Aguayo and Fardella 2009; Yau et al. 2017; Ferrari et al. 2000). Impairment of the 11βHSD2 enzyme that results in an inefficient conversion of cortisol to cortisone (F/E) that determines the activation of the mineralocorticoid receptor (MR) by cortisol. The MR has the same affinity for cortisol and aldosterone (Arriza et al. 1987). Activating the renal MR promotes the reabsorption of sodium, water and secondarily high blood pressure. The expression of this enzyme has been reported in the following tissues: the renal distal, collecting tubule, brain, colon, placenta, pancreatic β cells, skin, and human trophoblast. A severe deficiency of 11βHSD2 activity is characterized mainly by severe arterial hypertension associated with an early suppression of plasmatic renin levels (Carvajal et al. 2003, 2018a; Mune et al. 1995; Wilson et al. 1995). At the same time, excessive MR activation can promote endothelial oxidative stress, subclinical inflammation, fibrosis, vascular damage, and heart disease. Mouse models of 11βHSD2 enzyme deficiency have shown increased blood pressure, atherogenesis progression, and proinflammatory changes in the heart and endothelium that are reduced by treatment with MR antagonists such as spironolactone or eplerenone (Deuchar et al. 2011; Savoia et al. 2008; Pitt et al. 1999; Vaclavik et al. 2011). Deuchar et al. (2011) studied Apoe
/
/11βHSD2
/
double-knockout (E/b2) mice and showed that atherogenic effect of 11βHSD2 loss of function leads to remarkable atherogenesis progression and vascular damage associated with a proinflammatory process and further important protective effect seen by treatment with mineralocorticoid receptor antagonists (MRA), as eplerenone

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or spironolactone. Similarly, kidney-specific HSD11B2 knockout mice display high volume low-renin hypertension and renal dysfunction, and this phenotype is reversed by treatment with MRA (Ueda et al. 2017).

Nonclassic Apparent Mineralocorticoid Excess Nonclassic apparent mineralocorticoid excess (NC-AME) is proposed as a novel clinical condition with milder phenotypical spectrum than identified for AME (Tapia-Castillo et al. 2019a; Baudrand and Vaidya 2018a), and similar to the recently spectrum proposed for PA (Tapia-Castillo et al. 2019a; Carvajal et al. 2018a; Yau et al. 2017; Brown et al. 2017) that ranges from normotension to severe arterial hypertension (Fig. 2). The NC-AME condition can be present in subjects without the classic features of disease, but with elevated microalbuminuria, plasminogen

Fig. 2 Phenotypical spectrum of AME and nonclassic AME. Classic AME and nonclassic AME are characterized and shown as a continuous spectrum. We highlight the clinical and biochemical presentation of both conditions associated with first and second hits. NC-AME is a phenotype mainly related to an 11βHSD2 deficiency associated with genetic or epigenetic affecting this gene (first hit) and the potential additive action of endogenous or exogenous inhibitors (i.e., GALFS) (second hit). Treatment for NC-AME and AME is also shown. BP blood pressure, LVH left ventricular hypertrophy, IUGR intrauterine growth restriction, GALFs glycyrrhetinic acid-like factors, NR3C1 human glucocorticoid receptor (GR) gene

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activator inhibitor-1 (PAI-1) and high sensitive c reactive protein (hsCRP), suggesting an association of this phenotype with vascular and renal damage, and also a pro-inflammatory state. Recently, Tapia-Castillo et al. (2019a) identified that 7.1% of a Chilean cohort (including hypertensive and normotensive subjects) fulfilled the criteria of a high F/E ratio (>75th percentile) and low cortisone ( A; rs45483293) and essential hypertension (Brand et al. 1998). Watson and colleagues studied two microsatellites and found an association between one of them: the CA-repeat (D16S301) and hypertension in African Americans with end-stage renal disease (Watson et al. 1996). Campino et al. studied the presence of polymorphisms in the adult Chilean population, especially SNPs in HSD11B2, p.Glu178Glu (c.534G > A; rs45483293) and p.Thr156Thr (c.468C > A; rs5479), detecting an association of c.534G > A with low-renin hypertension (Campino et al. 2012). Moreover, TapiaCastillo et al. (2019a) identified nine subjects with suggestive NC-AME, where two of them have a demonstrated heterozygous pathogenic variant R213C (p. Arg213Cys), which impairs the activity and stability of 11βHSD2 (Carvajal et al. 2018a; Mune et al. 1995; Shackleton et al. 1985). The 11βHSD2 activity and expression can also be modified by different regulators, such as glucocorticoid receptor (GR) (Vitellius et al. 2019), RAC1-GTPase (Lavall et al. 2017), SUMOylation modification (Jimenez-Canino et al. 2017), among others. In 2019, Vitellius et al. describe subjects with pseudohypermineralocorticoidism with a similar phenotype to NC-AME. Authors indicate the heterozygous condition in GR leads to a decrease activity of 11βHSD2 measured as low E/F ratio (or an increased F/E ratio) (Vitellius et al. 2019). Lavall et al. found that Rac1 GTPase regulates 11βHSD2 expression, MR activation and MR-mediated pro-fibrotic signaling (Lavall et al. 2017). Jimenez-Canino et al. (2017) found that SUMOylation of 11βHSD2 at residue K266 modulates cortisol-mediated MR nuclear translocation independently of effects on transactivation (Jimenez-Canino et al. 2017). Zhu et al. found that activation of Hedgehog signaling by a variety of approaches robustly induced 11βHSD2 expression as well as the 11βHSD2 activity, especially in placental tissue (Zhu et al. 2016).

Epigenetics: DNA Methylation of HSD11B2 Gene The gene encoding 11βHSD2 is located in chromosome 16q22 (NG_016549.1; HUGO 5209); its promoter contains to NF1, GRE, and Sp1/Sp3 sites 11, it also includes 5 exons and possesses 2 microsatellite region of cytosine-adenine (CA) repeats; the first CA repeat is located in intron 112 (2174 bp after the start of intron 1 and 1900 bp before the end of intron 1), and the second is located 921 bp after the end of exon 5 (Fig. 1). DNA microsatellites are tandem repeat sequences formed by repeating units of one to six nucleotides. They are located in noncoding

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DNA sequences. The effect of the length of CA repeats may depend on the number of repeats and also on their location, relative to the gene. Microsatellites located near the gene promoter are more likely to influence the expression level of that gene, by promoter methylation. DNA methylation is an epigenetic mechanism potentially associated to cardiovascular diseases, including AHT, and may be linked to environmental-nutritional factors (i.e., salt intake, as a pathognomonic example) (Smolarek et al. 2010; Udali et al. 2013). The HSD11B2 promoter has a highly GC-rich sequence, which contains more than 80% GC, lacks a TATA-like element, and has two typical CpG islands (Fig. 1). This promoter configuration raises the possibility that CpG dinucleotide methylation may play a role in the cell-type-specific and interindividual variable expression of HSD11B2 (Alikhani-Koopaei et al. 2004). In 2008, Friso et al. reported DNA methylation changes in the HSD11B2 promoter in the peripheral leukocytes of essential hypertensive subjects (Friso et al. 2008). In this way, the methylation CpG islands in the promoter region and the first exon of the gene is correlated with reduced gene expression and the demethylation enhances transcription and 11βHSD2 activity in vitro and in vivo. Similarly, the methylation of artificial constructs of the HSD11B2 promoter decreases transcriptional activity by preventing the binding of transcription factors relevant for its expression (Alikhani-Koopaei et al. 2004), which supports CpG methylation as the most relevant epigenetic modification in the HSD11B2 promoter (Alikhani-Koopaei et al. 2004). Here, HSD11B2 gene based in a minigene construct that included the first intron (intron 1), showed that the minigene with 14 CA repeats demonstrated higher messenger RNA levels than the minigene with 23 repeats of CA.14 However, this phenomenon did not occur in the second microsatellite14 after exon 5. Valdivia et al. found 12 alleles with different lengths, according to the number of CA repeats in HSD11B2 gene, ranging from 13 to 24 CA repeats (CA13–CA24), and also reported length of CA-repeat in intron 1 of HSD11B2 did not influence either the serum F/E ratio or the BP in pediatric subject, suggesting that epigenetic modifications, as methylated CpG islands, could be occurring upstream in the HSD11B2 gene promoter.

Epigenetics: Non-Coding RNA Affecting HSD11B2 Gene Expression miRNAs are small noncoding RNA molecules that are approximately 21–23 bp long and they regulate target mRNAs through either translational repression, mRNA destabilization or a combination of both mechanisms. A single miRNA can regulate hundreds of genes, and collectively, miRNAs may regulate approximately 50–60% of the total transcriptome (Baek et al. 2008; Krol et al. 2010). MicroRNAs could play an important role as biomarkers. The expression pattern of miRNAs is subject to change in the onset and progression of the disease, which could be associated to pathophysiological mechanisms related to overt and mild endocrine phenotypes (Shi et al. 2015; Batkai and Thum 2012). MicroRNAs are incorporated within extracellular vesicles, such as exosomes, and appear to be very stable in biofluids, as they are

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extremely resistant to ribonucleases, extreme pH conditions and freeze-thawing (Cheng et al. 2019). The miRNAs influence gene expression both within their parental cells and promote intercellular communication by being transferred to other cells through exosomes, where they regulate cellular processes in the recipient cell (Yu et al. 2016; French et al. 2017; Das and Halushka 2015). Exosomes are small extracellular vesicles (EVs) with a size of 50–150 nm originating from endosomes. They are released from all cell types with a specific cargo (RNA, lipids and protein). Exosome cargo may mirror the physiological state or metabolic change of the cells of origin (Camussi et al. 2011; Michael et al. 2010). Exosomes are isolated from different biofluids by ultracentrifugation (UCF), size exclusion chromatography (SEC), differential centrifugation, ultrafiltration methods, among others technologies and it could be potential biomarkers by itself or by their cargo for a variety of pathophysiological conditions, such as cancer and chronic pathologies as arterial hypertension (Romaine et al. 2016; Butterworth 2015; Bartel 2004). The identification of miRNAs, RNA or proteins within exosomes associated to metabolic changes could be useful for study of cellular physiology and pathophysiology, and also prospective translational studies. Rezaei et al. (2014) provide the first evidence of the association between miRNA, 11βHSD2 and blood pressure. They hypothesized that a differential miRNA expression pattern is linked with a distinct expression of HSD11B2 gene. They compared in Sprague-Dawley with low and Wistar rats with high 11βHSD2 activity and revealed that miRNA rno-miR-20a-5p, rno-miR-19b-3p, and rno-miR-190a-5p were differentially expressed. Shang et al. in 2012 in human placental cell line (BeWo) observed a decrease in the 11βHSD2 activity, associated to a significant increase in miR-498 level. However, they not reported functional analysis using a 30 -UTR construct of HSD11B2 mRNA and they did not report how the miRNA selection was carried out (Shang et al. 2012). In a recent study, Tapia-Castillo et al. identified 355 miRNAs in urinary exosomes, of which only miR-192-5p and miR-204-5p were significant downregulated in NC-AME subjects compared with healthy controls (Tapia-Castillo et al. 2019b). Previous studies indicate that miR-192-5p and miR-204-5p are highly expressed in the kidney (Gracia et al. 2017; Sun et al. 2004). The miR-192 is expressed at higher levels in the renal cortex than in the medulla (Tian et al. 2008) and is 20-fold higher in the proximal tubules than in the glomeruli. miR-192 is involved in regulation of sodium transport in renal epithelial cells (Mladinov et al. 2013). A recent study by Baker et al. showed low expression of miR-192-5p in kidney biopsy specimens from patients with hypertensive nephrosclerosis and hypertension (Baker et al. 2019). Reduced expression of miR-192-5p is associated with an increase in Na/K ATPase function (ATP1B1 gene), which contributes to hypertension and kidney injury (Baker et al. 2019). Similarly, it has been shown that loss of miR-192-5p is associated with fibrogenesis in diabetic nephropathy (Ma et al. 2016). All these examples highlight the role of miR-192-5p in the renal system, which could be useful as a biomarker for some types of kidney diseases, especially in AHT. Bioinformatics studies shown that miR-192-5p could regulate genes related to both small GTPase mediated signal transduction (CUL3, ARHGAP1, ARHGAP36,

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ARHGEF39) and sodium transport (ATP1A2, SCL5A12), which have been previously related to the mineralocorticoid receptor (Nagase and Fujita 2013; Loirand and Pacaud 2014; Tapia-Castillo et al. 2014, 2015) and sodium/potassium exchange (Baker et al. 2019; Kaplan 2005; Dostanic et al. 2005) pathways, suggesting a role in the etiopathogenesis of arterial hypertension. The miR-204-5p is also highly expressed in kidney tissues and has been shown to be downregulated in advanced diabetic nephropathy biopsies (Rudnicki et al. 2016). Other studies have observed a reduction of miR-204-5p expression in epithelial cells associated with reduced expression of claudins 10, 16 and 19, suggesting a critical, albeit indirect, a role of this miRNA in maintaining epithelial cell function (Wang et al. 2010). By the mirWalk tool is possible to find that miR-204-5p could potentially regulate genes downstream MR-activation related to sodium transmembrane transport (NEDD4, ATP1A2, ATP2B4, WNK3 genes), cellular response to hormone stimuli (NEDD4, ATP1A2, NR3C1, NR3C2, YWHAG genes), and genes that regulate of molecular function in the cell (ATP2B4, ATP1A2, NEDD4, WNK3, ARHGEF37, ARHGEF26, ARHGAP30, YWHAG genes), suggesting a potential role for miR-2045p in renal pathways associated with sodium/potassium exchange. Recently, has been reported that miR-204 is a critical regulator of de novo DNA methylation, through affecting the DNA methyltransferase 3-alpha (DNMT3a) (Lin et al. 2018). In this way, we speculate that low expression of miR-204 observed in NC-AME, could be associated with higher expression of DNMT3a and hypermethylation the HSD11B2 promoter (Friso et al. 2008, 2015), decreasing the HSD1B2 expression, and lately affecting the cortisol to cortisone metabolism. These results also show that both miR-192-5p and miR-204-5p could regulate ATP1A2 expression and it has been previously shown that this α2-isoform of the Na/K-ATPase pump mediates ouabaininduced hypertension in mice and increased vascular contractility in vitro (Dostanic et al. 2005). Association studies indicated that miR-192-5p expression is correlated with plasma renin activity (PRA) which suggest being a potential biomarker of MR activation, also can be predicted by aldosterone and sodium urinary excretion, which is in agreement with a previous report by Elvira-Matelot et al. that showed renal miR-192-5p expression is decreased by aldosterone infusion (Elvira-Matelot et al. 2010). Similarly, hsa-miR-204-5p expression was negatively associated with serum cortisol to cortisone and systolic blood pressure (SBP). Authors discussed the possible role of these miRNAs as early biomarkers and regulators of the mineralocorticoid activity in NC-AME subjects, which will be useful to uncover and understand the mechanisms associated to this novel phenotype (Rudnicki et al. 2016; Potus et al. 2014; Courboulin et al. 2011; Yu et al. 2018).

Exogenous Inhibitors of 11ΒHSD2 Enzyme: Role in NC-AME To date, the cause of NC-AME or middle 11βHSD2 deficiencies has not been elucidated. Salt intake, glycyrrhetinic acid-like factors (GALFs) and environmental factors (Carvajal et al. 2020) have been hypothesized to be factors that deregulate the expression and activity of HSD11B2. Previous publications (Latif et al. 1990;

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Kumagai et al. 1957) have addressed the effects of the inhibition of 11βHSD2 by environmental inhibitors, these including licorice components, gossypol, phthalates, organotins, alkylphenols, perfluorinated substances, and carbenoxolone (a succinate derivative of glycyrrhetinic acid) (Latif et al. 1990; Kumagai et al. 1957; Ma et al. 2011; Zhou et al. 2017). The presence of inhibitor substances of the 11βHSD2 enzyme in human urine could affect the adequate metabolism of cortisol (Morris et al. 1992). A large proportion of patients with essential hypertension likely exhibit endogenous GALF-like inhibitors (Morris et al. 1992), particularly endogenous allo3α-5α-reduced corticosterone and 11-dehydrocorticosterone (Morris et al. 2007), derivatives of progesterone and adrenocorticosteroids (Latif et al. 1997; Ma et al. 2011), 11β-OH-testosterone and 11-keto-testosterone, which are potent inhibitors of 11βHSD2 dehydrogenase activity. Chronic ingestion of licorice or licorice-like compounds (such as carbenoxolone) induces a syndrome with findings like those observed in the AME syndrome. The diagnosis is typically based upon the biochemical abnormalities and an elicited history of licorice ingestion. The source of the ingested licorice may not be obvious; it is present, for example, in some forms of flavored chewing gum, chewing tobacco, and tea. Cortisol and cortisone levels may help in the diagnosis, but such testing is not necessary if a history of licorice ingestion has been obtained. Cessation of licorice ingestion (or other source of glycyrrhetinic acid) is usually the only treatment necessary. Potassium supplements or a potassium-sparing diuretic may be initially required to treat hypokalemia but should not be needed once the effect of licorice has worn off (typically less than 1 week). A recent paper by Zhou et al. (2017) describe a group of environmental chemical inhibitors of 11βHSD2 as Dithiocarbamates (DTCs), organotins, Butylated hydroxyanisole (BHA), Perfluoroalkyl substances (PFASs), Bisphenol A (BPA), Zearalenone, 2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane(HPTE), Bortezomib (Velcade, PS-341), Fluoxymesterone, Glycyrrhizin (GL) and its metabolites. Others chemical inhibitors of 11βHSD2 are heavy-metals (i.e., lead) (St-Pierre et al. 2016), fungicides (i.e., poconazole) (Zhou et al. 2017), the silane coupling agent AB110873 (a rubber additive for production of tires and shoe soles), and the antibiotic lasalocid (antibiotic and additive used to improve body weight gain in beef cattle and milk production in dairy cows).

Endogenous Inhibitors of 11βHSD2 Enzyme The 3α5α-tetrahydroderivatives of several adrenal corticosteroid hormones, particularly corticosterone and 11-dehydrocorticosterone (Morris et al. 2007), derivatives of progesterone and adrenocorticosteroids (Latif et al. 1997), 11β-OH-testosterone, 11-keto-testosterone and ACTH, are potent inhibitors of 11βHSD2 -dehydrogenase activity. A large proportion of patients with essential hypertension likely exhibit endogenous inhibitors, particularly allo/3α5α-reduced pathway steroidal products. Earlier studies by Honour (1982) and Bokkenheuser et al. (1979) indicated that a significant proportion of corticosterone and its 5α-ring A-reduced derivatives are

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excreted via the bile (in contrast to cortisol) in both humans and rodents. These steroids can then be 21-deoxygenated by microorganisms in the intestinal flora, yielding 11-oxygenated derivatives of progesterone and its 5α-tetrahydro-derivatives. The 5α-ring A-reduced derivatives of corticosterone and 11β-hydroxy-progesterone are even more potent inhibitors of the dehydrogenase activity of both isoforms of 11βHSD. Metabolomics can be employed to detect global metabolite profiles (Patti et al. 2012), which represent the endpoint of all metabolic activities and help characterize various biological and physiological processes. Previous studies of untargeted metabolomics in resistant hypertension have shown changes in metabolite levels related to fatty acid, lipid, amino acid and purine metabolism (Wawrzyniak et al. 2019), showing that metabolomics helps to elucidate the metabolites that may influence the physiopathology associated with drug-resistant hypertension. Recently TapiaCastillo et al. (2021) published a study based on an untargeted metabolomic analysis of serum samples from NC-AME patients, which identified 36 differentially regulated metabolites, 3 upregulated metabolites and 33 downregulated metabolites. For these 36 metabolites, they evaluated their diagnostic capacity as biomarkers for this phenotype. They observed that L-dopachrome and S-phenylmercapturic acid (SPMA) had the highest sensitivity and specificity to discriminate the NC-AME condition, followed by bilirubin, L-iditol, deoxyribose 1-phosphate, citric acid, gamma-L-glutamyl-L-methionine sulfoxide and 5-sulfoxymethylfurfural (SMF). The combination of these eight metabolites showed a high sensitivity (93%) and specificity (90%) to discriminate for NC-AME from control subjects. The metabolites gamma-L-glutamyl-L-methionine sulfoxide, L-dopachrome and SMF were significantly increased in NC-AME patients. The gamma-L-glutamyl-Lmethionine is an organic compound that belongs to the class of dipeptides and is generated in conditions of oxidative stress (Cabreiro et al. 2006; Picot et al. 2006). Methionine sulfoxide has been proposed as a physiological marker of oxidative stress, which is a key mechanism of endothelial dysfunction, as observed in NC-AME subjects and even more notably in hypertensive patients (Guzik and Touyz 2017). Recently, Zhao and colleagues in a urine metabolomic study revealed the involvement of oxidative stress metabolic pathways and amino acid metabolism in essential hypertension (Zhao et al. 2018). Additional evidence suggested that essential hypertensives (EH) may be a disorder of inherited amino acid metabolism (Min et al. 2004). However, the increased levels of these two metabolites have never been explored in NC-AME. Similarly, L-dopachrome belongs to the class of organic compounds known as l-alpha-amino acids, and elevated levels of this metabolite indicate an increase in tyrosine metabolism, which includes the biosynthesis of melanin (Tsukamoto et al. 1992; Leonard et al. 1988). In this way, gamma-Lglutamyl-L-methionine, L-dopachrome or metabolites associated with this metabolic pathway should be also evaluated as endogenous inhibitors of 11βHSD2, and further research is required to reveal such effects. Along with the findings described above, subjects with NC-AME have high levels of the organic compound SMF, which is also negatively associated with serum cortisone. SMF is generated from the metabolism of 5-hydroxymethylfurfural, a

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reactive metabolite that can bind to DNA and cause mutagenic effects (Monien et al. 2012). SMF is toxic, since it accumulates in kidney proximal tubules by improper excretion due to renal reabsorption processes (Bakhiya et al. 2009), which leads to the above-mentioned damage to DNA and proteins. In addition, we showed that SPMA had a good diagnostic ability to identify this phenotype of NC-AME. SPMA belongs to the family of N-acyl-alpha amino acids and derivatives and is a benzene metabolite that is catalyzed by glutathione S-transferases and has been considered a biomarker of oxidative damage. These findings may support the complementary use of biomarkers associated with oxidative stress and renal damage, such as microalbuminuria, in NC-AME subjects. Bilirubin also has a good discriminatory capacity to identify the NC-AME phenotype and is decreased in these subjects. Bilirubin has previously been characterized as an antioxidative and anti-inflammatory protective factor with respect to peripheral vascular diseases (Djousse et al. 2001; Kunutsor et al. 2015), suggesting that NC-AME subjects with lower bilirubin levels may have lower protective antioxidant effects (Chin et al. 2009). Other metabolites that are decreased in NC-AME, such as L-iditol and deoxyribose 1-phosphate, also have a good discriminatory capacity to identify the NC-AME phenotype. L-Iditol is a sugar alcohol and is part of various metabolic reactions in organisms that include fructose and mannose metabolism. Similarly, Tapia-Castillo et al. (2021) found low levels of citric acid and S-adenosyl-L-homocysteine (SAH) in NC-AME subjects. The citric acid cycle (CAC) provides precursors of certain amino acids, as well as the reducing agent NADH, that are used in numerous reactions. On 11βHSD2 enzymatic activity, it is known that this enzyme is highly dependent on cofactor NAD+ (Carvajal et al. 2003), which is essential for proper cortisol catabolic activity in the kidney and other nonepithelial tissues. Thus, a decrease in the activity of the citric acid cycle should affect the synthesis of NAD+, which is a critical cofactor for the 11βHSD2 enzymatic activity. S-adenosyl-L-homocysteine (SAH) is the metabolic precursor of homocysteine and is a negative regulator of most cell methyltransferases associated with DNA hypermethylation (James et al. 2002). Low SAH levels observed in NC-AME subjects are associated with higher expression of DNA-methyltransferase and hypermethylation of the HSD11B2 promoter (Pizzolo et al. 2015), which is expected to decrease HSD1B2 expression and subsequently affect cortisol to cortisone metabolism in these subjects. In this respect, Lana et al. found in a similar untargeted metabolomics assay in essential hypertensives (Lana et al. 2019), a dysregulation in the urine levels of sulfur-containing metabolites (thiocysteine and homomethionine), purines (SAH, AMP, allantoate, and hydroxyisourate) and pyrimidines (dihydrothymine, uracil, and UDP), among others (Lana et al. 2019), suggesting that NC-AME may be associated with impaired sulfur-containing metabolites, such as SAH. Other articles have addressed the effects of the inhibition of 11βHSD2 by endogenous inhibitors, such as cholic acid derivatives, and exogenous inhibitors, such as perfluorohexane sulfonic acid, perfluorooctanesulfonic acid, perfluorooctanoic acid, diethyl-phthalic acid, and monoethylphthalate (Ma et al. 2011; Zhou et al. 2017). These metabolites and their respective metabolic pathways should help identify the pathophysiological mechanism governing this condition.

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The Novel Combined Phenotype of NC-AME and Primary Aldosteronism Mineralocorticoid-dependent hypertension represents one of the most prevalent causes of secondary hypertension. One-third of patients with hypertension have low or suppressed renin, which suggests systemic volume expansion and unregulated activation of mineralocorticoid receptor (MR) by either excess aldosterone or cortisol, affecting not only blood pressure (BP) control and urinary potassium excretion but also causing cardiac and renal damage, endothelial dysfunction and subclinical inflammation (Funder 2017a; Kosicka et al. 2013; Funder et al. 2016b). Primary aldosteronism (PA) is the most prevalent form of endocrine hypertension, with an estimated prevalence of 5–10% in the general hypertensive population, characterized by increased aldosterone secretion, independent of renin, angiotensin II or sodium status (Carvajal et al. 2020; Vaidya et al. 2018). The deleterious effects of PA are mediated by MR activation in various tissues. At the renal level, activation of MR increases sodium and water reabsortion, leading to an increase in blood pressure (BP). At the cardiovascular level, its effects include myocardial fibrosis, reduction of fibrinolysis and endothelial dysfunction (Milliez et al. 2005). Additionally, aldosterone has an impact on the immune system, promoting an inflammatory state characterized by vascular infiltration of immune cells, reactive oxidative stress and proinflammatory cytokine production (Herrada et al. 2010; Carvajal et al. 2009; Leroy et al. 2009). On the other hand, AME (Carvajal et al. 2018a) or non-classic AME (NC-AME), which is already mentioned in this chapter, as a mild form of classic AME without the classic clinical symptoms, characterized by normal or elevated blood pressure, a high serum cortisol to cortisone (F/E) ratio and concomitant low cortisone (E) levels, normal-low aldosterone, low renin and increased urinary potassium excretion (Tapia-Castillo et al. 2019a). Emerging evidence suggests that beside the general etiologic categorizations of these diseases, such as primary aldosteronism (PA) and apparent mineralocorticoid excess (AME), among other conditions, there may exist a broad phenotypic spectrum that extends from overt or “classic” presentations to milder or “non-classic” phenotypes, suggesting that the prevalence of these phenotypes may be more common and recognized earlier using novel biomarkers (Milliez et al. 2005; Gilbert and Brown 2010; Connell et al. 2008; Rossi et al. 2006). Moreover, it has been suggested the coexistence of both phenotypes, NCAME and PA, associated with endocrine hypertension, generate a more severe phenotype with higher BP, higher NGAL levels (e.g., a surrogate MR biomarker), urinary electrolyte abnormalities (e.g., higher FEK) and higher albumin excretion. In this combinate phenotype (NCAME&PA), the prevalence of hypertension increases with the severity of the phenotype, reaching 93% in NCAME&PA subjects, which is even higher than EH patients. This finding suggests that excessive activation of the mineralocorticoid receptor by both cortisol (glucocorticoid) and aldosterone (mineralocorticoid) could lead to an increased sodium transport in renal collecting ducts, increased water uptake and blood volume, and a secondary increase in the blood pressure. Moreover, NCAME&PA group had higher potassium excretion (FEK), which is suggested to be

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associated to an increase in mineralocorticoid activity and recruitment of basolateral Na+/K+-adenosine triphosphatase pumps in renal principal cells (Feraille et al. 2003). Subjects with the NCAME&PA condition are characterized by having lower renin compared to those in PA or NCAME patients. Low-renin hypertension (LRH) has been associated to a high aldosterone and recently to low cortisone, which is secondary to 11βHSD2 deficiency (Baudrand and Vaidya 2018b). Thus, seems reasonable to include cortisone in the diagnostic algorithm of low-renin hypertension (LRH). A high albuminuria excretion has been observed in NCAME&PA patients. Microalbuminuria was considered not only a renal risk factor, but also a marker of cardiovascular risk and target organ damage in patients with hypertension, chronic kidney disease and diabetes, and its recognition is important for identifying patients at higher risk for cardiovascular mortality (Kolkhof et al. 2014). In this respect, mineralocorticoid receptor antagonists (MRAs) were shown to reduce albuminuria and end organ damage (Marquez et al. 2019). Other finding in NCAME&PA combinate phenotype showed elevated MMP9 and NGAL levels, which is consistent with reported increase in MMP-9 levels in plasma from PA and hypertensive patients (Berk et al. 2007; Martinez et al. 2006; Flamant et al. 2007). NGAL has been found increased in primary aldosteronism and familial hyperaldosteronism (Krug and Ehrhart-Bornstein 2008; Schinner et al. 2007; Engeli et al. 2005). Also in mice models, NGAL was indicated as a MR target gene associated to fibrosis of heart and cardiovascular system (Martinez-Martinez et al. 2017). Recent reports have shown that EV and EV-cargo assays could be a novel approach in the diagnosis of endocrine hypertension (Friso et al. 2015; Barros and Carvajal 2017; Romero et al. 2008). Similarly, our group found differences in EV size in the NCAME&PA group, suggesting that different sizes may reflect variations in the biogenesis or content of individual vesicles, compared with normotensives, PA or NCAME subjects. In this respect, they found four miRNAs (miRNA-192, miRNA-133a, miRNA-21 and let-7i-5p) downregulated in the uEVs of NCAME&PA subjects, and their decreasing expression corresponded with the severity of the phenotype, which suggest these miRNAs are potential biomarkers of this novel phenotype. miR-192-5p showed a correlation with low plasma renin activity, consistent with previous publications (Tapia-Castillo et al. 2019b). Additionally, miR-192-5p was associated with aldosterone, which could be involved in the regulation of sodium transport in renal epithelial cells, by affecting ATP1B1 gene expression (Na/K-ATPase activity) (Baker et al. 2019). miR-133a expression was negatively associated with DBP and positively associated with plasma renin activity. This is consistent with previous studies that have shown decreased miR-133a levels in plasma in patients with arterial hypertension, heart disease and left ventricular diastolic dysfunction (Koval et al. 2020). In fact, a protective role for miR-133a against myocardial fibrosis has been suggested (Abdellatif 2010; Matkovich et al. 2010) and its downregulation has been associated with Col1A1 and CTGF overexpression and myocardial fibrosis (Castoldi et al. 2012; Angelini et al. 2015). Downregulation of miR-21-5p in NCAME&PA was associated negatively with blood pressure, aldosterone and cortisol, and positively with PRA. miR-21 has been

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identified as an aldosterone-regulated miRNA mediating RAAS effects in the adrenal gland and heart (Ball et al. 2017; Syed et al. 2018) causing inflammation (Sheedy 2015) and fibrosis (Chen et al. 2017). Moreover, the low expression of miR-21-5p promotes an inflammatory state characterized by vascular infiltration of immune cells by regulating target genes, such as IL1B and IL12A. In addition, like miR-133, which also is target of COL1A1, miR-21 has a role in the reorganization of extracellular matrix scaffolding. Similarly, let-7i-5p also has a role in fibrosis and inflammation regulating target genes, such as IL-6, TGFBR1, COL1A1, COL1A2, COL3A1 and ORM1. Previous studies have shown that Ang II stimulation activates fibroblast transforming growth factor-β and IL-6 signaling pathways (Ma et al. 2012), which are the major profibrogenic mediators of cardiac fibrosis. In fact, overexpression of let-7i significantly reduces Ang II-induced inflammation and cardiac fibrosis (Wang et al. 2015). Inhibition of the expression of IL-6 and collagen by let-7i could be one potential mechanism for the reduced cardiac inflammation and fibrosis. Altogether, these results represent the first attempt to link this novel phenotype with EV-miRNAs, which could be add to a novel diagnostic algorithm as complementary biomarkers.

Differential Diagnosis from AME and NC-AME Primary Aldosteronism Primary aldosteronism (PA) is a condition in which the biosynthesis of aldosterone is increased, independent of renin and sodium status, leading to AHT, cardiovascular damage, heart diseases (Funder 2015, 2017b; Stowasser 2015), and renal and immune system alterations (Muñoz-Durango et al. 2013; Stehr et al. 2008, 2010; Zhu et al. 2011; Carvajal et al. 2009). PA is considered an uncommon disease that is suspected in patients with severe hypertension and hypokalemia. However, several studies have shown that PA is a prevalent condition, showing a prevalence around 5% in general hypertensive population and around 10% in reference centers (Hannemann and Wallaschofski 2012; Monticone et al. 2017; Mosso et al. 2003), which is detected by screening through a high serum aldosterone-to-renin ratio (ARR) or a high urinary aldosterone in a low-renin state, and further salt or fludrocortisone confirmatory tests (Mosso et al. 1999, 2003; Fardella et al. 2000; Cortes et al. 2000; Mulatero et al. 2004). Currently, the prevalence of PA is dependent on the population being targeted, the ARR threshold for positive screen test, and the confirmatory test performed (Vaidya et al. 2018; Baudrand et al. 2017). PA can be commonly caused by an adrenal adenoma, unilateral or bilateral adrenal hyperplasia (BAH), or in rare cases adrenal carcinoma. The majority of cases are sporadic, but PA can be transmitted as an inherited condition of familial hyperaldosteronism (Funder et al. 2016a). According to the Endocrine Society guidelines, in presence of family history of PA or early-onset hypertension further genetics studies are recommended (Funder et al. 2016a). Here, at least four forms of familial hyperaldosteronism have been reported: FH-I (glucocorticoid-remediable aldosteronism, GRA), which displays a severe phenotype with

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early-onset hypertension, and the estimated prevalence is only 1% of PA subjects and is secondary to a chimeric gene (CYP11B1/CYP11B2) (Mosso et al. 2003); FH-II, which has been described in 6% of the familial form of PA associated with gain-offunction mutations in the CLCN2 gene coding for the CLC-2 chloride channel (Stowasser et al. 1992; Scholl et al. 2018; Fernandes-Rosa et al. 2018); FH-III, which was described secondary to a gain-of-function germline mutation in the KCNJ5 gene (Mulatero et al. 2013); and FH-IV, which is caused by a heterozygous mutation in the CACNA1H gene (Scholl et al. 2015) (OMIM #607904) on chromosome 16p13. Currently, the importance of diagnosing PA has become clear, not only for the deleterious consequences of high blood pressure but also because of the direct deleterious effects of MR activation, independent of the blood pressure in several organs and tissues, such as those of the cardiovascular, renal, and immune system as well as adipose tissue. The deleterious effects of PA on the cardiovascular system include myocardial fibrosis, a reduction in fibrinolysis, and endothelial dysfunction (Milliez et al. 2005; McCurley and Jaffe 2012). Similarly, cell signaling by the aldosterone/MR complex is a key mediator of kidney injury and renal dysfunction (Rossi et al. 2006) identified by high levels of urinary microalbuminuria (MAC) and creatinine. And lately aldosterone also has been associated to changes in the immune system (Herrada et al. 2010) and adipose tissue metabolism (Schutten et al. 2017), promoting an inflammatory state characterized by a vascular infiltration of immune cells, reactive oxidative stress, and proinflammatory cytokine production (Herrada et al. 2010). Recently, Brown et al. (2017) performed a longitudinal cohort study using physiological phenotypes of autonomous aldosterone secretion and MR activity. In this study, they proposed that a broader spectrum of clinically relevant reninindependent aldosteronism (PA) may extend even to persons with no apparent clinical syndrome of excessive MR activation by aldosterone and normotensives. Moreover, they suggest that a proportion of cases of hypertension may not be “essential” (or idiopathic) and could be associated with MR-mediated hypertension as a pathogenic mechanism, potentially treated with MR antagonists. Identifying PA is relevant since it has an increased prevalence and is associated to higher rate of cardiovascular morbidity and mortality when compared with age- and sex-matched patients with essential hypertensives (Stowasser et al. 2000). In patients diagnosed with PA, treatment of the mineralocorticoid excess with antagonist of MR (e.g., spironolactone, eplerenone) improvement of the hypertension and the cardiovascular risk.

Hypertensive Forms of Congenital Adrenal Hyperplasia (OMIM #202010 and #202110) The most common hypertensive congenital adrenal hyperplasia (CAH) is due to a deficiency in 11β-hydroxylase (OMIM #202010). It is present in 5–8% of all CAH cases, occurring in approximately 1–200,000 live births (White et al. 1994).

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This type of CAH is an autosomal recessive disorder of corticosteroid biosynthesis resulting in androgen excess, virilization, and hypertension. It is caused by a mutation in the CYP11B1 gene, which encodes the 11β-hydroxylase enzyme, which disrupts the conversion of 11-deoxycortisol to cortisol (White et al. 1994). Because of deficient cortisol production, the pituitary secretes large amounts of ACTH, stimulating adrenal steroidogenesis and increasing the plasma levels of 11-deoxycortisol, deoxycorticosterone and androgen precursors. Because deoxycorticosterone is an effective mineralocorticoid, excess causes retention of salt and water and suppression of the PRA and aldosterone, leading to hypertension. The disorder responds to suppressive doses of dexamethasone. Another hypertensive form of CAH is caused by a deficiency in 17α-hydroxylase (OMIM #202110). This rare condition is caused by defects in cytochrome P450c17, the single enzyme that has 17α-hydroxylase and 17,20-lyase activities. This disorder is characterized by moderate arterial hypertension, suppression of the renin-angiotensin system, absence sex steroid synthesis, resulting in female external genitalia in 46XY patients, and by impaired production of cortisol with a compensatory hypersecretion of ACTH. ACTH stimulates the synthesis of large amounts of deoxycorticosterone and corticosterone, leading to sodium reabsorption and suppression of PRA and aldosterone. Affected patients may be treated with suppressive doses of dexamethasone or physiologic doses of hydrocortisone during childhood.

Glucocorticoid Resistance (OMIM #138040) Glucocorticoid resistance is a rare condition characterized by generalized, partial, target-tissue insensitivity to glucocorticoids. Compensatory elevations in circulating adrenocorticotropic hormone (ACTH) concentrations lead to increased secretion of cortisol and adrenal steroids with mineralocorticoid and/or androgenic activity, but without any clinical evidence of hypercortisolism. The clinical spectrum of the condition is broad, ranging from asymptomatic to severe cases of hyperandrogenism, fatigue and/or mineralocorticoid excess. The molecular basis of glucocorticoid resistance has been ascribed to mutations in the human glucocorticoid receptor gene (NR3C1), which impair glucocorticoid signal transduction, thereby altering tissue sensitivity to glucocorticoids (Charmandari and Kino 2007). Genetic studies have documented that mutations and polymorphisms at the GR gene might be associated with different metabolic syndromes, such as glucocorticoid resistance, glucocorticoid sensitivity, obesity, and hypertension.

Liddle’s Syndrome (OMIM #177200) Liddle’s syndrome is caused by mutations in the subunits of the renal epithelial sodium channel (ENaC) (Shimkets et al. 1994). The amiloride-sensitive ENaC is considered the rate-limiting step for sodium absorption in the distal nephron, and it is composed of three subunits named α, β and γ (Canessa et al. 1994). In Liddle’s

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syndrome, mutations have been found in the β and γ subunits. Because of these mutations, a constitutive activation of the epithelial channel leads to increased sodium absorption and volume expansion. This syndrome is inherited as an autosomal dominant disorder in which affected patients have hypertension, suppressed plasma renin activity, and low aldosterone levels. The defect in the sodium channels of the distal nephron results in an excessive salt absorption and potassium wasting. ENaC also has been implicated as a candidate gene for the development of essential hypertension. A genetic analysis of the amiloridesensitive ENaC is recommended when assessing patients with low-renin, saltsensitive hypertension whose blood pressure is not responsive to spironolactone treatment. This disorder responds to inhibitors of epithelial sodium transport, e.g., triamterene (Botero-Velez et al. 1994). Affected patients also respond to renal transplantation, which results in normalization of blood pressure and of electrolyte abnormalities.

Activating Mutation of Mineralocorticoid Receptor (OMIM #605115) This disorder can be caused by mutation in the MR gene in the locus 4q31.1 (OMIM #600983). A gain of function mutation resulting in the substitution of leucine for serine at codon 810 (S810L) in the human MR is responsible for early-onset hypertension that is exacerbated in pregnancy. All steroids, including progesterone, that display antagonist properties when bound to the wild-type MR are able to activate the mutant receptor (MR(L810)). These findings suggest that progesterone may contribute to the dramatic aggravation of hypertension in MR(L810) carriers during pregnancy.

Gordon Syndrome (OMIM #614495) Familial hyperkalemic hypertension, also known as Gordon syndrome or type II pseudohypoaldosteronism (PHA-II), is a rare autosomal dominant disease that causes altered renal salt absorption. It was first described in the 1960s and consists of a pathological net positive sodium ion balance associated with renal potassium ion retention, resulting in hypertension, hyperkalemia, and hyperchloremic metabolic acidosis (Athimulam et al. 2019). Other manifestations include low renin with low-normal aldosterone concentrations and hypercalciuria. In the last decades, it was elucidated that this condition occurs secondary to pathogenic variants in WNK1, WNK4, CUL3, or KLHL3 genes. Those genes regulate the expression of the Na/Cl cotransporter in the distal nephron, while CUL3 and KLHL3 genes are involved in the ubiquitination and proteasomal degradation of the WNK kinases (O’Shaughnessy 2015). The consequence of these alterations is upregulation of the Na/Cl cotransporter resulting in increased Na reabsorption and a reduced expression of renal outer medullar K channel, which results in hyperkalemia. Affected patients respond to thiazide diuretics that act inhibiting the Na/Cl cotransporter.

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Treatment for AME and NC-AME Treatment for AME must be started at diagnosis to control the blood pressure and BP-independent damage occurring in other tissues, such as the heart, blood vessels, among others by unregulated activation of MR (Fig. 3). MR antagonists are safe and cardioprotective in essential hypertension, even in diabetics and heart failure patients, and at low doses, they also specifically lower blood pressure in patients with mineralocorticoid-dependent hypertension. Spironolactone is a progesterone derivative, categorized as a potassium-sparing diuretic. Because of its origin, it retains progestational activity, and therefore, it is not a selective MR antagonist. Its main adverse consequence is hyperkalemia, however, rather rare in patients with normal renal function in the presence of overactivation of MR receptor. In men, side effects are dose-related and include erectile dysfunction and gynecomastia. In recent years came up eplerenone as a highly selective MR antagonist to complement and perhaps as a replacement to spironolactone. However, eplerenone has only 2–3% of the affinity for MR of spironolactone in vitro but is much less tightly bound to plasma protein. Therefore, it has approximately 60% of the potency of spironolactone. Its half-life is 4–6 h rather than the 18 h of spironolactone, mainly because it is much more rapidly metabolized by P450 cytochrome and the metabolites of eplerenone are inactive. It has the marked advantage of MR selectivity and safety with less marked effect in terms of hyperkalemia (Funder 2013). Lastly, this has led to the development of nonsteroidal dihydropyridine-based third generation MRA. Finerenone is a bulky passive antagonist highly selective for

Fig. 3 Renal and extrarenal deleterious effects by aldosterone and cortisol-mediated MR activation. Aldosterone and cortisol can activate MR in epithelial and nonepithelial tissues. The condition of 11βHSD2 deficiency leads to cortisol binds MR in epithelial tissues, and induce MR activation, similar as does aldosterone. The unregulated MR activation by cortisol can induce also deleterious effects in kidney and other tissues, such as the heart, blood vessels, the immune system, among others

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MR, its potency in vitro is like spironolactone, and it is more potent than eplerenone. Finerenone is uniformly distributed between cardiac and renal tissues, whereas spironolactone and eplerenone reach higher concentrations in the kidneys than in the heart (Azizi et al. 2019). In patients with chronic kidney disease (CKD) and type 2 diabetes, treatment with finerenone resulted in lower risks of CKD progression and cardiovascular events than placebo (Bakris et al. 2020). MR antagonists at age-appropriate doses are mandatory: relatively low doses are sufficient, given the side effects (spironolactone) or cost (eplerenone), with amiloride and calcium channel blockers occasionally required to normalize blood pressure. In addition, patients can also rarely be treated with glucocorticoids that have a long half-life, such as dexamethasone or betamethasone, decreasing ACTH levels and endogenous cortisol. According to Funder in 2017, treatment of mild AME or NC-AME (Tapia-Castillo et al. 2017) with elevated blood pressure should be straightforward with low-dose spironolactone (12.5–25 mg/day) (Williams et al. 2018) or eplerenone (25–50 mg/day) with the intention to correct BP and improve renin levels. Despite there is no prospective data in NC-AME, inference from PA studies support the concept of improve renin levels as a proxy of MR inactivation (Hundemer et al. 2018). Hundemer et al. showed that MR antagonist treatment addressed to normalize renin levels leads to better cardiovascular outcomes (Hundemer et al. 2018). As shown in the PATHWAY-2 clinical trial, MR antagonists are indisputably the most robust add-on therapy in individuals with low renin hypertension, even after “secondary” causes of hypertension were supposedly excluded among participants. NC-AME and NCAME&PA may respond appropriately to MR antagonists, and therefore, future investigation is needed to corroborate the efficiency of personalized therapy in these conditions.

Conclusion Classic AME is a relatively rare disorder characterized by a severe low-renin hypertensive phenotype in childhood. The diagnosis of AME requires a demonstration of a biallelic deficiency of HSD11B2 and a very low 11βHSD2 enzyme activity. The treatment of AME includes MR antagonists, glucocorticoid and potassium supplementation and additional antihypertensive therapy. NC-AME differs from classic AME syndrome because it has a milder phenotype, is diagnosed later in life in adolescents and adults, and can be observed even in normotensive subjects. NC-AME phenotype is mainly associated to epigenetic modifications that along with a second hit are able impair the proper cortisol metabolism. Since NC-AME is associated to low-renin phenotype and to an increased cardiovascular risk due to MR activation, these subjects are candidates to be treated with MR antagonists to improve blood pressure, end-organ damage and modulate renin levels. Acknowledgements This study was supported partially by grants ANID-FONDECYT 1160695 (CEF), 1212006 (CAC) and 3200646 (ATC); CONICYT-FONDEQUIP EQM150023 (CAC); SOCHED 2019-09 (CAC) and CETREN-UC.

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Mineralocorticoid Resistance

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aldosterone Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adrenal Cortex Steroidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aldosterone Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of Action of Aldosterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aldosterone Action in Distal Nephron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mineralocorticoid Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transcriptional Regulation by the MR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ENaC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Actions of Aldosterone in Epithelial Target Tissues: Genomic Effects . . . . . . . . . . . . . . . . . . . . Renal PHA1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MR Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal Models of Renal PHA1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Generalized PHA1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ENaC Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal Models of ENaC Subunits Invalidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Secondary PHA1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differential Diagnosis of PHA1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PHA1 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Aldosterone and the mineralocorticoid receptor are major regulators of fluid and electrolyte homeostasis as well as regulation of blood pressure by stimulating sodium reabsorption in the distal nephron. Pseudohypoaldosteronism type F. L. Fernandes-Rosa (*) Université Paris Cité, PARCC, Inserm, Paris, France e-mail: [email protected] © Springer Nature Switzerland AG 2023 M. Caprio, F. L. Fernandes-Rosa (eds.), Hydro Saline Metabolism, Endocrinology, https://doi.org/10.1007/978-3-031-27119-9_12

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1 (PHA1) is a rare disease characterized by salt loss in the neonatal period secondary to a target organ resistance to mineralocorticoid action. Patients exhibit salt wasting in the neonatal period with failure to thrive, vomiting, and dehydration, associated with hyponatremia, hyperkalemia, and metabolic acidosis despite high levels of aldosterone and renin. The renal form of PHA1 occurs in most cases by loss of function mutations in the mineralocorticoid receptor (MR) gene, and the generalized PHA1 is due to inactivating mutations in the genes coding for the subunits of the epithelial amiloride-sensitive sodium channel ENaC. In addition, transitory forms of PHA1 are associated with malformations in the urinary tract and/or urinary tract infections. PHA1 is a major differential diagnosis for salt wasting syndromes occurring in the first weeks after birth. The knowledge of its genetic background and pathophysiology may improve the early recognition of the disease and patients care, resulting in decreased morbidity and mortality. Keywords

Pseudohypoaldosteronism type 1 · Mineralocorticoid receptor · EnaC · Aldosterone · Salt wasting

Introduction Pseudohypoaldosteronism type 1 (PHA1) is a rare pediatric disorder characterized by resistance to mineralocorticoid hormones. PHA1 is due to defects in the mineralocorticoid receptor (MR) and the amiloride-sensitive epithelial sodium channel ENaC, molecules that play a crucial role in aldosterone-mediated regulation of water and electrolyte balance. In neonates, proper action of the mineralocorticoid axis is necessary because immaturity of tubular function at this time may alter the regulation of water and electrolyte balance by the kidneys. In addition, prematurity, infections, and also physiological partial resistance to aldosterone in the newborn may contribute to altered aldosterone-dependent regulation of electrolyte balance. The original case was reported by Cheek and Perry in 1958 (Cheek and Perry 1958), who described an infant with severe salt loss and growth retardation without any renal or adrenal abnormalities. Typically, patients present with severe salt loss during the neonatal period, with hyponatremia, hyperkalemia, and metabolic acidosis despite extremely high plasma renin and aldosterone levels (Zennaro and FernandesRosa 2017). Classically, two different forms of PHA1 have been described, a mild renal form and a severe generalized form (Hanukoglu 1991) with different genetic alterations described. In addition, a secondary form of PHA1 associated with renal abnormalities has also been described. Furthermore, some studies have shown PHA1 forms with an intermediate phenotype between the renal and generalized forms. In this chapter, we will describe the biosynthesis and action of the hormone aldosterone, the genetics and molecular mechanisms of the mineralocorticoid receptor in epithelial tissues, particularly the distal nephron, and clinical and genetic aspects of PHA1.

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Aldosterone Biosynthesis Adrenal Cortex Steroidogenesis Aldosterone is the main mineralocorticoid hormone in humans. It is synthesized in the zona glomerulosa (ZG) of the adrenal cortex following a series of enzymatic reactions at specific subcellular loci. The human adrenal gland is composed of two distinct parts, the outer cortex and the inner medulla, which have different functions. The cortex, itself subdivided into three zones, is responsible for the production of corticosteroids while the medulla releases catecholamines (adrenaline and noradrenaline). Each area of the cortex is specialized in the synthesis and secretion of a particular type of steroid. Thus, the subcapsular ZG produces aldosterone, the zona fasciculate (ZF) produces cortisol, and the zona reticularis (ZR) produces androgens. In steroidogenic cells, cholesterol has different origins. It can be synthetized de novo from acetate or derived from the mobilization of cholesterol esters stored in lipid droplets upon cellular uptake of HDL cholesterol via the scavenger receptor, class B type 1 (SR-B1) or from lipoprotein-derived cholesterol esters. In the cells of the ZG, the cholesterol must cross the outer mitochondrial membrane to the inner mitochondrial membrane where the first enzyme of the steroidogenic pathway is located. However, a pathway involving the StAR (steroidogenic acute regulatory protein) protein expressed in all steroidogenic tissues (Stocco 2001) has been demonstrated in a study of patients with congenital adrenal lipoid hyperplasia, where mutations in the StAR gene cause an inability to synthesize steroids and an accumulation of cholesterol in the adrenal gland (Lin et al. 1995). The StAR gene expression is induced by most agents known to stimulate steroid biosynthesis and the cholesterol translocation is considered the limiting step in steroidogenesis (Stocco 2001). StAR facilitates the transport of cholesterol to the outer mitochondrial membrane where TSPO acts in concert with other proteins to bind cholesterol and participate in its transport from the outer mitochondrial membrane to the inner membrane, which is the initial step for the conversion of cholesterol to pregnenolone. For aldosterone synthesis, pregnenolone is converted by a series of enzymatic reactions catalyzed by endoplasmic reticulum dehydrogenases and mitochondrial oxidases, most of which belong to the cytochrome P450 (CYP) superfamily of heme enzymes. These require coupling with coenzymes (adrenodoxin/adrenodoxin reductase) that transfer an electron to the P450 enzyme for the hydroxylation reaction (Lambeth et al. 1982). The first reaction in steroidogenesis is the conversion of cholesterol to pregnenolone, catalyzed by the P450 enzyme encoded by the CYP11A1 gene located on chromosome 15 (Lieberman and Lin 2001), which cleaves the side chain in three reactions (20α-hydroxylation, following by 22-hydroxylation, and the cleavage of the bond between C-20 and C-22). Pregnenolone is then released into the cytosol, where it is converted to progesterone by dehydrogenation of the 3β-hydroxyl group and isomerization of the C-5 double bond by the enzyme 3β-dehydrogenase (3β-HSD II). Progesterone is then 21-hydroxylated by the enzyme 21-hydroxylase, encoded by the CYP21A gene, located on the surface of the smooth endoplasmic reticulum to produce 11-deoxycorticosterone (DOC) (Shinzawa et al. 1988). The

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conversion of DOC to aldosterone then proceeds through three consecutive enzymatic reactions: 11β-hydroxylation of DOC to form corticosterone, 18-hydroxylation leading to 18-hydroxycorticosterone (18-OH-B), and finally 18-methyloxidation for the production of aldosterone. These enzymatic reactions are catalyzed by aldosterone synthase, encoded by the CYP11B2 gene. It is expressed exclusively in the zona glomerulosa, in islands of cells specifically involved in aldosterone synthesis (Boulkroun et al. 2010). Aldosterone synthase is highly homologous to the enzyme 11β-hydroxylase (encoded by CYP11B1), which catalyzes the conversion of 11-deoxycortisol to cortisol in the ZF of the adrenal cortex. These two genes are located in tandem on chromosome 8 in humans (Chua et al. 1987; Mornet et al. 1989).

Aldosterone Biosynthesis A large number of factors have been shown to stimulate or inhibit aldosterone production such as adrenaline, serotonin, ouabain, atrial natriuretic peptide, dopamine, heparin, and adrenomedullin. More recently, new factors secreted by adipose tissue have also been shown to stimulate aldosterone synthesis in vitro. However, the major regulators of aldosterone production remain angiotensin II, extracellular potassium concentration, and ACTH (Williams 2005). Aldosterone biosynthesis is primarily regulated by the renin-angiotensin system (RAS) (Fig. 1). In the kidney, angiotensin II mainly affects hemodynamics through Increased Na+ reabsorption Increase in blood pressure Aldosterone

Angiotensin II Angiotensin-converting enzyme

Increased [K+]

Angiotensin I Renin Angiotensinogen Decrease in Blood Flow, dehydration...

Fig. 1 Regulation of aldosterone biosynthesis. Renin is secreted by the juxtaglomerular apparatus of the kidney in response to a decrease in blood volume, dehydration, or hyponatremia. It cleaves the angiotensinogen produced in the liver to produce Angiotensin I. Angiotensin I is converted by the angiotensin-converting enzyme to Angiotensin II in the lungs. Angiotensin II acts via the AT1 receptor in ZG cells, leading to the activation of calcium signaling and aldosterone production. Aldosterone increases sodium reabsorption and blood pressure, decreasing, in turn, the renin production and consequently the aldosterone biosynthesis. The figure has been created in Biorender.com

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afferent and efferent arteriolar vasoconstriction and tubular function by increasing water reabsorption through stimulation of aldosterone synthesis by activation of the angiotensin II type 1 receptor (AT1R). Angiotensin II is formed from angiotensinogen by a series of proteolytic cleavages. Angiotensinogen is synthesized in the liver and secreted into the bloodstream. It is cleaved to angiotensin I, by renin, a circulating enzyme whose renal synthesis and secretion is the limiting step for angiotensin II synthesis. Angiotensin I is converted to angiotensin II by the angiotensin-converting enzyme (ACE). Angiotensin II exerts its physiological effects on a large number of tissues including the adrenal cortex, kidney, brain, and vascular smooth muscle cells. The secretion of renin is the limiting factor for angiotensin II formation. The juxtaglomerular apparatus is an endocrine structure that regulates the function of each nephron. It is located between the vascular pole of the glomerulus and the distal convoluted tube of the nephron. This location is essential for the regulation of renal blood flow and glomerular filtration rate. Renin is synthesized and released by juxtaglomerular cells located in the media of the terminal part of the afferent arteriole, in the intravascular space, and in the interstitium. Renin secretion is positively regulated by the second messenger cyclic adenosine monophosphate (cAMP). Renin is released upon a decrease in renal perfusion pressure detected directly by the juxtaglomerular cells in the afferent arteriole, perceived by their intrarenal baroreceptors. Its decrease causes a decrease in the elasticity of the blood vessel walls, a decrease in the intracellular calcium concentration, and an increase in renin secretion. Renin secretion is also stimulated by decreased sodium uptake in the macula densa, activation of sympathetic renal nerves, and stimulation of β-adrenergic receptors. Renin secretion is inhibited by an increase in renal perfusion pressure. Angiotensin II exerts rapid negative feedback on renin secretion and positive feedback on hepatic angiotensinogen synthesis (Bie and Damkjaer 2010). Angiotensin II receptor type 1 (AT1R) is AT1R is a G-protein-coupled receptor (GPCR) associated with Gαq/11 protein alpha subunit. Angiotensin II binding to AT1R leads to the activation of phospholipase C (PLC), which in turn stimulates intracellular production of inositol 1, 4, 5-triphosphate (IP3) and 1, 2-diacylglycerol (DAG). The generation of IP3 induces a transient increase of cytosolic calcium concentration via calcium release from the endoplasmic reticulum (ER). The increase of cytosolic free calcium results in the activation of specific calciumbinding proteins, especially the calcium/calmodulin-dependent kinases (CaMKs) such as CaMKI, CaMKII, and CaMKIV. CamK phosphorylate and activate transcription factors (Nurr1; ATF-1 and ATF-2) and cAMP-response element binding protein (CREB) (Spat and Hunyady 2004). These different transcription factors bind CRE and other cis-acting elements in the regulatory region of the CYP11B2 gene, leading to increased CYP11B2 expression and aldosterone production. DAG is an activator of protein kinase C (PKC). The PLC-β/DAG/PKC pathway leads to the activation of the protein kinase D (PKD) which phosphorylates CREB who will increase the expression of steroidogenic genes involved in aldosterone production such as STAR and CYP11B2. DAG is also known to inhibit TASK-1 and TASK-3 channels in ZG cells, leading to cell membrane depolarization which induces the opening of voltage-gate dependent calcium channels, which also contribute to the

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increase in cytosolic calcium concentration. This results in increased CYP11B2 expression and aldosterone biosynthesis. The second main activator of aldosterone production is the extracellular concentration of potassium. Increase in potassium concentration stimulates aldosterone secretion in order to maintain potassium homeostasis, and aldosterone secretion is stimulated by changes in potassium concentration as small as a few tenths of a millimolar. This highly sensitive response to changes in kalemia is related to the potassium conductance of the ZG cells, which is controlled by potassium channels responsible for a very negative membrane potential under resting conditions. K+ sets the negative resting membrane potential in ZG cells, around -80 mV, through the leak/background channels TWIK-related acid-sensitive K+ (TASK) and TWIKrelated K+ (TREK). A small rise in serum potassium concentrations of 1 mmol/L is sufficient to depolarize cells by reducing the reversal potential for potassium, leading to the increase of intracellular calcium concentration through the activation of L- and T-type calcium channels and the release of intracellular calcium stores. The increase in intracellular calcium concentration induces calcium binding to calmodulin and thus activation of CaM kinases that phosphorylate transcription factors responsible for CYP11B2 gene transcription and aldosterone biosynthesis (Spat 2004). The importance of the cell membrane potential, intracellular calcium concentration, and calcium signaling on aldosterone biosynthesis was confirmed by the identification of mutations in genes coding for potassium channels, L- and T- type calcium channels, and ATPase pumps as responsible for primary aldosteronism (Zennaro et al. 2020). Other regulators of aldosterone biosynthesis include ACTH, serotonin, interleukin 6, epidermal growth factor, and leptin. It is also regulated by autocrine/paracrine mechanisms by bioactive signals released by endothelial cells, chromaffin cells, immune system cells, adipocytes, nerve endings, and autonomic nervous system (Wils et al. 2020).

Mechanism of Action of Aldosterone Aldosterone Action in Distal Nephron The knowledge of the physiological role of aldosterone in the distal nephron, regulating volume and electrolyte homeostasis, is fundamental to understand the clinical consequences of the mineralocorticoid resistance. Aldosterone acts in a region of the nephron that includes the late distal convoluted tubule (DCT), the connecting tubule (CNT), and the collecting duct (CD), named aldosterone-sensitive distal nephron (ASDN) (Fig. 2). The ASDN expresses the mineralocorticoid receptor (MR, encoded by the NR3C2 gene), the final effector of aldosterone in epithelial tissues (the amiloride-sensitive epithelial sodium channel ENaC), and the enzyme 11-β hydroxysteroid dehydrogenase type 2 (11β-HSD2), responsible for mineralocorticoid selectivity (Farman and Rafestin-Oblin 2001). Two-thirds of the total sodium load filtered by the glomerulus is reabsorbed in the proximal tubule and

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Aldosterone sensitive distal nephron proximal tubule (60-70%)

distal convoluted tubule and connecting tubule (5-10%)

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glomerulus Cortisol

cortical colecting duct (1-5%)

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Cortisone 11β-HSD2

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Aldosterone target genes Co-R

Descending limb of loop of Henle

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Thick ascending limb of loop of Henle (20-25%) Thin ascending limb of loop of Henle

Fig. 2 Renal sodium reabsorption and aldosterone action in distal nephron. (a) Segmental distribution of Na+ reabsorption along the nephron. Approximately 60–70% of sodium is reabsorbed in the proximal tubule, 20–25% in the thick ascending branch of Henlé’s loop, 5–10% in the distal convoluted tubule and connector tubule, and 1–5% in the cortical collecting duct. The regulated zone of Na+ reabsorption corresponds to the aldosterone-sensitive distal nephron (ADSN), comprising the distal convoluted tubule, connector tubule, and cortical collecting duct. (b) ASDN cells express the mineralocorticoid receptor (MR), the epithelial sodium channel (EnaC), and the enzyme 11-β hydroxysteroid dehydrogenase type 2 (11-HSD2), responsible for mineralocorticoid selectivity. In the ASDN, aldosterone binding to MR leads to the nuclear translocation of the hormone-receptor complex, binding to specific DNA regulatory regions in aldosterone-target genes and recruitment of co-regulators (Co-R). The DNA binding of MR will induct the expression of specific target genes related to the stimulation of Na+ reabsorption, including genes coding for ENaC subunits and the Na+/K+ ATPase pump. These two proteins ensure a vectorial transport of sodium from the lumen of the epithelium to the internal environment and a secretion of potassium to the external environment. The figure has been created in Biorender.com

20–25% in the loop of Henle; about 5–10% is finely regulated by the ASDN (Fig. 2). Activity along this segment is controlled by dietary sodium intake but also by hormones, mainly aldosterone (Loffing et al. 2001a). Therefore, the ASDN is responsible for the fine-tuning of the renal reabsorption of Na+ (Loffing et al. 2001b). Aldosterone stimulates sodium reabsorption and potassium excretion by involving two membrane transporters in particular: ENaC, located at the apical membrane of the epithelial cells, and the Na+/K+ ATPase pump, located at the basolateral membrane. These two proteins ensure a vectorial transport of sodium from the lumen of the epithelium to the internal environment and a secretion of potassium to the external environment. Sodium entry, which is the limiting step in the process, is via ENaC, and then sodium is extruded at the basolateral pole of the cell through the action of Na+/K+ ATPase, which provides the driving force of the system. In addition to its direct effects on transporter expression, aldosterone also stimulates the activation of signaling cascades modulating the activity of these transporters. The

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action of aldosterone is mediated by its receptor, the MR, which acts as a transcriptional factor, regulating the expression of genes encoding proteins involved in sodium transport in the tight junctional epithelia (Fig. 2). MR was also identified in various non-epithelial tissues with moderate (heart) or high (hippocampus) levels of expression (Pearce and Funder 1987b). It was shown that the MR has an equivalent affinity for aldosterone and cortisol, and a higher affinity for corticosterone and deoxycorticosterone (DOC) (Arriza et al. 1987). The level of freely circulating glucocorticoids is approximately 100–1000 times higher than that of aldosterone; therefore, without mechanisms of mineralocorticoid selectivity, the MR could be always occupied by glucocorticoids, avoiding the occupancy by aldosterone in its epithelial target tissues in order to regulate water balance. The enzyme 11β-HSD2 has a pivotal role of conferring binding specificity to the MR (Funder et al. 1988). This enzyme is expressed at high levels in aldosterone-targeted epithelial tissues, converting active glucocorticoids (cortisol in humans and corticosterone in rodents) to inactive 11-keto-analogues (cortisone and 11-dehydrocorticosterone, respectively) and preventing illicit occupancy of the MR by glucocorticoids. The response to aldosterone follows a particular kinetics, which occurs in three phases. First a latent phase lasting approximately 1 h, during which the MR-dependent transcriptional program is established, in the absence of measurable electrophysiological changes. Second, an early phase, 1–2 h after aldosterone treatment that lasts for 3 h, characterized by increased sodium transport and decreased transepithelial electrical resistance. This phase probably involves proteins induced early by aldosterone and/or mechanisms of activation of preexisting proteins (phosphorylation/dephosphorylation, methylation, insertion of new proteins at the membrane or at tight junctions, and modifications of protein/cytoskeleton interactions) (Verrey 1999). Finally, a late phase characterized by the persistence of high short circuit current and a low transepithelial electrical resistance, reflecting ionic transport through the epithelium. During this phase, there is neosynthesis of ENaC subunits and the Na+/K+ ATPase pump, through increased transcription and translation of some of their subunits (the α-subunit of ENaC and the α1-subunit of the Na+/K+ ATPase in the cortical collecting duct in mammals) (Escoubet et al. 1997). This late effect of aldosterone probably involves the interaction between several transcriptional activation pathways that would act in a coordinated manner. In ASDN, Na+/K+ ATPase activity at the basolateral membrane of the cell creates the electrochemical gradient for sodium reabsorption via ENaC. Basolateral potassium channels Kir4.1 and Kir5.1 are involved in the creation of the membrane potential and K+ recycling, while apical potassium channels, mainly ROMK1 (or Kir1.1) whose activity increases with high dietary potassium intake, are responsible for potassium secretion (Wang et al. 2010). It is by rendering the interior of the cell electronegative to the exterior that the Na+/K+ ATPase pump contributes to the generation of a large concentration gradient that leads to the secretion of potassium from the apical side (Reilly and Ellison 2000). Aldosterone also regulates acid-base transport, which occurs in the intercalary cells (IC). Type A ICs secrete protons into the urine via the vacuolar H+-ATPase channel expressed on the apical side of the cells. This channel is coupled on the

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basolateral side to the Cl-/HCO3- exchanger, EA1, which releases bicarbonate ions into the blood. Type B ICs express the apical Cl-/HCO3- exchanger which excretes bicarbonate into the urine, while protons are secreted into the blood by the basolateral H+-ATPase. Proton secretion through the vacuolar H+-ATPase pump is thought to be indirectly coupled to sodium reabsorption through the ENaC channel, which creates a negative luminal potential that favors H+ secretion (Kovacikova et al. 2006).

Mineralocorticoid Receptor The NR3C2 gene, encoding the MR, which spans 450 kb, is located on chromosome 4 in humans and on chromosome 8 in mice. Its structure was first described in 1995 by experiments of hybridization with a human MR cDNA probe on cosmid and lambda phage libraries obtained from human placenta and cosmid libraries obtained from human leukocytes, allowing to identify the organization of the gene and the transcription initiation site (Zennaro et al. 1995). The NR3C2 gene is composed of 10 exons of variable size. The first two exons are untranslated, undergoing alternative transcription from two separate P1 and P2 promoters. Exons 1α and 1β are alternatively transcribed to generate two mRNA isoforms, MRα and MRβ. The translation starting site is located in the exon 2, which encodes the N-Term portion of the protein. Exons 3 and 4 code for the entire DNA-binding domain (DBD) and exons 5–9 code for the hinge region and the ligand-binding site (LBD). NR3C2 expression is under control of two alternative promoters, P1 and P2, responsible for the synthesis of two isoforms of 1α and 1β mRNA encoding a single 984 AA protein (Zennaro et al. 1995). P1 promoter has about tenfold higher basal activity than P2. In contrast, P2 promoter is sensitive to aldosterone even though it lacks a consensus GRE site (Zennaro et al. 1996). The alternative use of the P1 and P2 promoters leads in humans to the transcription of two mRNA isoforms, MRα and MRβ, encoding a single protein. In situ hybridization studies in classical aldosterone target tissues such as distal tubule, heart, skin, colon, or sweat glands showed equivalent expression of α and β mRNA isoforms. No tissue-specific expression of these isoforms was described in the kidney glomerulus or proximal tubule. However, MRβ expression is decreased in the sweat glands of patients with functional hypermineralocorticism (primary aldosteronism and Liddle’s syndrome) independently of aldosterone levels (Zennaro et al. 1997). Different isoforms of messenger RNA can be observed from the same coding sequence, called alternative transcripts, which can either result in the same protein or result in a different protein. In the steroid receptor family, the many different messenger RNA isoforms most often result in the expression of the same protein. Another possibility to obtain different protein isoforms from the same transcript is the use of different translation initiation sites or preceded by a Kozak sequence (Kozak 1987). Some MR isoforms have been described, in particular an MRΔ5,6 isoform resulting from alternative splicing of exons 5 and 6 in the NR3C2 coding sequence and resulting in the change of the reading frame. This results in the

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appearance of a premature stop codon 35 residues downstream of the amino acid p.671Gly (Zennaro et al. 2001). This variant, expressed in many tissues and unable to bind aldosterone, has a constitutive transcriptional activity in the range of 30–40% of that of wild-type MR, which may have an important effect in modulating aldosterone target genes. It has also been shown that NR3C2 encodes two different sized receptors called MR A and MR B. MR A (107 kDa) corresponds to the wildtype MR while MR B is smaller (105.4 kDa). MR B is translated from a second translation starting site corresponding to a Kozak sequence located 15 amino acids downstream of the first ATG site (Pascual-Le Tallec et al. 2004). Both receptors have the same ED50 as wild-type MR (ED50 ¼ 10–11 M), but MR A is a more potent transactivator than MR B. The structure of MR resembles that adopted by most members of the nuclear receptors superfamily: a modular structure with an N-terminal domain, a DNA-binding domain, a hinge region, and a ligand-binding domain. The N-terminal domain (NTD) is the least conserved domain among the different members of the NR superfamily (Agarwal and Mirshahi 1999). In contrast, this domain is highly conserved across species with up to 85% homology in mammals, suggesting that this domain has a crucial role in the function of the protein. Exon 2 encodes the entire N-terminal domain of the MR, which is the longest of the steroidal receptors. It has two ligand-independent activating functional (AF-1a and AF-1b) sites capable of transactivating reporter genes highlighting the key role of the N-Term in the recruitment of coregulators (Pascual-Le Tallec and Lombes 2005). The different regions of the N-Term domain are responsible for modulating the transcriptional activity of the MR in a complex transactivation system composed of distinct structures and functional domains (Fischer et al. 2010). The DNA-binding domain of the MR, between residues 603 and 670, is encoded by exons 3 and 4 and is the most conserved domain between different nuclear receptors. The DBD has two zinc fingers positioned in a perpendicular way, each composed of four cysteine residues containing a central zinc atom. The first zinc finger interacts directly with the large groove of the DNA helix via a P-box specific to the receptor. The P-box consists of three residues that are essential for the recognition of a hemisite of an ERH. As with most nuclear receptors, the second zinc finger is involved in dimerization between two receptor (Dahlman-Wright et al. 1991). Thus, the DBD is central to MR function as it is involved in both dimerization and recognition of DNA-binding sites at HREs. The hinge region that extends between residues 671 and 734 is poorly conserved between the different receptors. It contains a repeat of proline residues and would allow conformational change or torsion upon binding of the DBD to DNA or when the LBD needs to be in a position accessible for interaction with coactivators and the transcriptional machinery (Tsai and O’malley 1994). The ligand-binding domain or LBD of the MR is relatively well-conserved across receptors and is encoded by exons 5–9. The LBD, which extends from residues 735 to 984, is involved in ligand binding and binding to the heat shock protein HSP90, receptor dimerization, nuclear localization, and interaction with cofactors. The crystallographic structure of the MR LBD was determined taking into account its homology with other members of the nuclear receptor superfamily, in particular

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date from the LBD of RARγ bound to all-trans retinoic acid (Fagart et al. 1998). The MR LBD consists of a β-sheet and 11 α-helices composing a sandwich of three antiparallel helix layers (denoted H1 to H12) forming a hydrophobic ligand-hosting pocket. The positioning of aldosterone in made possible by the existence of two polar sites located on either side of the hydrophobic ligand-hosting pocket, interacting with specific sites of the ligand. These interaction sites would then allow a good stabilization of the ligand in the binding pocket and allow the creation of interaction surfaces between the AF-2 transactivation domain, present on helix H12, and transcriptional cofactors (Farman and Rafestin-Oblin 2001). Classical target tissues of aldosterone are essentially the epithelia with high transepithelial electrical resistance. They are the site of transepithelial sodium transport: the ASDN in the kidney, the colonic epithelium, and the excretory ducts of the sweat and salivary glands. For all these tissues, co-expression of MR with 11β-HSD2 ensures the specificity of aldosterone binding to its receptor (Hirasawa et al. 1997). In the kidney, MR mRNA and protein have been also detected in glomeruli, particularly in mesangial cells (Miyata et al. 2005) and podocytes (Shibata et al. 2007), where aldosterone has been reported to modulate podocyte function (Shibata et al. 2007 #9236). In the lung, hormone-binding sites have been demonstrated in the airways from the bronchiolar epithelium to the trachea (Krozowski and Funder 1981 #8286). MR expression (mRNA and/or protein) has been clearly revealed in the salivary glands, sweat glands, and in the inner ear. MR was also detected in non-epithelial tissues in which 11β-HSD2 expression was absent or very low. Specific aldosterone-binding sites have been identified in mononuclear leukocytes and in the heart (Armanini et al. 1985; Pearce and Funder 1987a). MR transcripts have been detected in specific structures of the hippocampus and in the hypothalamus (Han et al. 2005). MR was localized at the cellular level in cardiomyocytes, endothelial cells, and large vessels (Lombes et al. 1992). The expression of MR was identified in the skin, not only in sweat or sebaceous glands but also in the keratinocytes constituting the stratified epithelium (Farman et al. 2010). Other studies have shown that MR is transcribed and translated in both brown and white adipose tissue (Caprio et al. 2007; Zennaro et al. 1998). The MR protein is also expressed in the eye, notably in the retina and the ciliary body of the iris (Zhao et al. 2010). MR is also expressed in the placenta and at the messenger level in the uterus, ovaries, and testes (Le Menuet et al. 2000). Like most proteins, the MR is subject to posttranslational modifications that modulate its activity depending on the tissue in which it is expressed. The best known are phosphorylation, ubiquitinylation, sumoylation, and acetylation. These modifications can influence its dimerization, DNA binding, or the recruitment of transcriptional co-regulators (Faresse 2014). It has been shown that MR can be phosphorylated by the CDK5 kinase on serine and threonine residues of the N-terminal region, resulting in a decrease in MR transcriptional activity (Kino et al. 2010). Ubiquitination is the addition of a small protein of 76 amino acids that allows the protein to be addressed by the proteasome for degradation. MR is monoubiquitinated in the basal state and poly-ubiquitinated by ERK1/2 kinases after stimulation by aldosterone in a negative feedback loop (Faresse 2014). Sumoylation is the addition of small ubiquitin-like

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SUMO proteins (SUMOs for small ubiquitin-related modifiers) on lysine residues preferentially in the NTD domain. Sumoylation could decrease MR transcriptional activity or instead promote the recruitment of MR coactivators (Tirard et al. 2007). Finally, the acetylation level of the MR is one of the factors regulating its activity. Other posttranslational modifications have also been described, such as O-glycosylation, which stimulates its transcriptional activity, or O-GlcNAc, which has been described in diabetes mellitus (Faresse 2014; Jo et al. 2023 #80).

Transcriptional Regulation by the MR When the MR is not bound to its ligand, it interacts with a large number of proteins, especially in the cytoplasmic compartment, forming a hetero-oligomer. MR interacts with chaperone proteins, such as HSP90 (heat shock protein 90) (Binart et al. 1995), and interacts indirectly with HSP70, P23 and P48 proteins, immunophilin FKBP-59, and cyclophilin CYP40. These chaperones maintain the MR in a suitable conformation for ligand binding. Upon hormone binding, the MR dissociates from a number of proteins and is translocated into the nucleus interacting with numerous molecular partners in a coordinated and sequential manner to ensure proper transcriptional regulation. The MR is a member of the nuclear receptor superfamily, the largest family of transcription factors in eukaryotes. Prior to any interaction with DNA, the MR dimerizes, either as MR-MR homodimers or MR-GR heterodimers (Tsai and O’malley 1994), especially in certain tissues such as the brain, in which these two receptors cooperate to regulate transcription. After transfer to the nucleus, the receptor interacts with specific DNA sequences to induce transcription of aldosterone target genes. Glucocorticoid receptor (GR), MR, progesterone receptor (PR), and the androgen receptor (AR) bind to the same 15-nucleotide consensus sequence, called glucocorticoid response element (GRE), classically consisting of the palindrome AGAAGAnnnTGTTCT, a motif that varies between promoters (Lombes et al. 1993). Understanding of the mechanisms of action of nuclear receptors has shown that these receptors make contacts primarily with transcriptional coregulators and not directly with the basal transcription machinery. Coregulators act as molecular bridges that are in direct contact with general transcription factors. The cellular response to aldosterone is mediated by MR-dependent transcriptional regulation, which involves the recruitment of specific transcriptional coregulators to the promoters of target genes involved in the hormone response.

ENaC The ENaC channel has an exclusive selectivity for sodium, a low conductance, and a high opening probability (Rossier et al. 2002). ENaC consists of three subunits called α, β, and γ encoded by three distinct genes SCNN1A, SCNN1B, and SCNN1G, respectively. The α subunit is absolutely necessary for channel activity, whereas the β and γ subunits participate in maximal channel expression at the cell surface and in

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the regulation of channel activity. The gene encoding the α subunit is located on chromosome 12 at locus 12p13, whereas the genes encoding the β and γ subunits are located on chromosome 16 at locus 16p12–13. Despite low sequence homology (35%) (Canessa et al. 1994), the three subunits show the same structural organization: two transmembrane domains separated by a large extracellular loop. The extracellular loop is involved in the interaction with amiloride and has putative glycosylation sites and two conserved cysteine-rich regions involved in addressing the channel to the membrane (Firsov et al. 1999). The second transmembrane domain participates in the formation of the pore and the ion selectivity filter (Fyfe et al. 1999). The N- and C-Term ends are cytoplasmic and have specific domains involved in posttranslational modifications. Indeed, the N-terminal domain contains potential phosphorylation and ubiquitination sites while the C-terminal domain has two proline-rich regions named PY (motif: xPPxY) that can be recognized by SH3 and WW domains. Moreover, the interaction of the PY sequences of the ENaC channel subunits with the SH3 and WW domains is involved in membrane addressing and channel regulation (Staub et al. 1996). ENaC is a low unit conductance channel, 4–5 pS, with very high selectivity for sodium and lithium and long opening times, up to 1–5 sec, and displaying a short half-life (less than 1 h)(Alvarez De La Rosa et al. 2002). Its activity can be inhibited by pharmacological agents such as amiloride and its derivatives (phenamil or benzamil) that act on the extracellular side by binding to a common site at transmembrane segment 2 near the channel pore (Kellenberger et al. 2003). Investigation of the localization of the mRNAs encoding the different subunits of the ENaC channel showed a nearly ubiquitous expression. The three ENaC subunits have been detected in the surface epithelium of the distal colon but absent from the crypts and in the respiratory system (epithelium of the nasal, tracheal, and bronchial surfaces and in the alveolar epithelium) (Farman et al. 1997). It is also expressed in the excretory ducts of the salivary and sweat glands, in the taste buds of the tongue, and in some epithelia of the eye and inner ear (Duc et al. 1994; Couloigner et al. 2001). In the kidney, ENaC is expressed in the ASDN (Bachmann et al. 1999). The subcellular localization and expression level of ENaC channel subunits change dramatically with increasing plasma aldosterone levels in response to a low sodium diet (Masilamani et al. 1999; Loffing et al. 2000). In rodents under normal sodium supply, the different subunits are detectable by immunohistochemistry in the cytosol of the principal cells, with weak localization in the apical membrane for the distal DCT and CNT segments. Under low sodium supply, which causes an increase in plasma aldosterone level, the subunits are localized in the apical membrane of DCT, CNT, and CCD principal cells, with decreasing expression from early to late ASDN. This axial distribution of ENaC along the ASDN was corroborated by patch-clamp experiments on microdissected segments of the ASDN from rats fed different diets: under normal diet conditions, there is no detectable amiloride-sensitive cell current in the principal cells of the CNT or CCD. In contrast, under low dietary sodium intake, a very strong significant amiloride-sensitive current in the CNT is recorded compared with the CCD (Frindt and Palmer 2004). Expression of the β and γ subunits is constant in the ASDN, whereas expression of the α subunit of ENaC is

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under the tight transcriptional control of aldosterone. Indeed, it is the availability of this subunit that dictates the level of channel activity in the distal nephron. Expression of the α-subunit doubles in microdissected CCD from rat kidneys 2 h after aldosterone treatment (Loffing et al. 2001c). The increased expression of the α-subunit of ENaC is part of the direct genomic response to aldosterone. The activity of the channel is also subject to indirect genomic regulation, as aldosterone induces its expression by a factor of 2 to 3, but further stimulates its activity as will be discussed in the next paragraph. In the apical membrane of major cells, ENaC is the major pathway for Na+ ions from the tubule lumen to the cell cytoplasm (Palmer and Frindt 1996). Because ENaC is a constitutively open channel, all factors that control the number of active channels at the cell surface have a major effect on sodium uptake in the ASDN (Palmer and Andersen 2008).

Actions of Aldosterone in Epithelial Target Tissues: Genomic Effects Aldosterone stimulates the activation of signaling cascades modulating the activity of salt reabsorption. Some of these aldosterone-activated signaling cascades are dependent on the effects of the MR on transcription, while other, more rapid events are independent of the effects of aldosterone on transcription (Thomas et al. 2007). Determining the interaction between these genomic and non-genomic events is crucial in understanding the full range of aldosterone effects. In general terms, aldosterone promotes Na+ uptake and K+ secretion in epithelial target tissues. In addition, as water follows sodium by osmosis, the net effect of aldosterone action is to increase blood volume and regulate electrolyte balance, leading to an increase in blood pressure through both genomic and non-genomic effects. In the late DCT and CD, aldosterone stimulates the expression and activity of several proteins involved in transepithelial sodium transport, including ENaC and Na+/K+ ATPase (Stockand 2002). Furthermore, aldosterone regulates the expression of genes encoding proteins capable of regulating the activity of preexisting ENaC channels, thus allowing a rapid physiological response, well before the accumulation of newly synthesized channels and pumps. In the CD, when aldosterone is low, no ENaC channel activity is observed (Garty and Palmer 1997). When plasma aldosterone levels increase, there is an increase in transepithelial sodium transport within a few hours, due to the neosynthesis of Na+/K+ ATPase subunits and ENaC (Frindt et al. 2001; Frindt et al. 2002). In the kidney, aldosterone induces expression of the ENaC α-subunit and the α1-subunit of the Na+/K+ ATPase; there is also a redistribution of cytoplasmic pools of channels at the membrane (Escoubet et al. 1997). This corresponds to the late phase of the effects of aldosterone on sodium transport in addition to the latent and early phases which do not involve protein neosynthesis but rather activation of preexisting channels as described earlier. ENaC ubiquitination by the Nedd4-2 ubiquitin ligase plays a major role in the regulation of its activity. ENaC PY motifs act as binding sites for the protein-protein interaction with the Nedd4-2 domain. The ubiquitination of the channel results in a decrease in its expression at the cell surface and also interferes with cAMP-

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dependent translocation from the subapical pool to the membrane (Abriel et al. 1999). ENaC ubiquitination via Nedd4-2 is regulated by various physiological mechanisms, including different sodium diets, which can influence Nedd4-2 expression in ASDN (Loffing-Cueni et al. 2006). Aldosterone acts by antagonizing ENaC ubiquitination. Aldosterone rapidly increases the mRNA levels of the serine/threonine-protein kinase sgk1, which stimulates ENaC activity by binding to and phosphorylating Nedd4-2. This prevents the binding of Nedd4-2 to ENaC, thus causing a reduction in ubiquitinated ENaC channels and leading to an accumulation of channels at the cell surface (Bhalla and Hallows 2008). In parallel, transcriptional regulation of the deubiquitinating enzyme USP2-45, leads to increased ENaC expression on the surface, which indirectly increases the channel activity (Verrey et al. 2008). Furthermore, aldosterone increases the expression of the glucocorticoidinduced protein “leucine zipper” (Gilz) which acts, in parallel to sgk1, to increase the localization of ENaC in the plasma membrane through inhibition of the extracellular signal-regulated kinase ERK pathway (Soundararajan et al. 2005). Thus, expression of ENaC on the cell surface is controlled via channel ubiquitination, which in turn is regulated by aldosterone target proteins (Verrey et al. 2008). Some studies suggest that sgk1, via its induction by aldosterone, may also have a role in renal potassium secretion by increasing the export of ROMK channels from the endoplasmic reticulum to the membrane, and also by suppressing the inhibitory effect of serine/ threonine kinase WNK4 on ROMK channels (Vallon et al. 2005).

Renal PHA1 Clinical Features Renal PHA1 (MIM#177735), also called autosomal dominant PHA1, is a mild and the most frequent form of PHA1, with a prevalence of ~1 per 80,000 newborns (Zennaro and Fernandes-Rosa 2017). Familial cases with an autosomal dominant inheritance and sporadic cases were described. The phenotypic expression is restricted to the kidney, with patients exhibiting a salt-losing syndrome enduring the neonatal period with different degrees of weight loss, failure to thrive, vomiting, and dehydration. Biological analysis is characterized by hyponatremia, hyperkalemia, metabolic acidosis, and inappropriately high urinary sodium excretion. In contrast, urinary potassium excretion is low, with a reduced excretion fraction and transtubular potassium gradient, and low urinary potassium excretion. The diagnosis is confirmed by the presence of elevated plasma aldosterone and renin levels and an elevated urinary aldosterone concentration. Despite the clinical and biological findings compatible with decreased aldosterone, affected neonates exhibit high plasma and urinary aldosterone and high plasma renin levels, confirming the mineralocorticoid resistance. The symptoms of renal PHA1 improve with age as the mineralocorticoid axis becomes more active to better compensate for sodium loss. In adulthood, patients are usually asymptomatic but have elevated plasma aldosterone levels while plasma renin returns to normal levels (Zennaro et al. 2012). The

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mechanisms underlying this compensation are not known: renal maturation, access to saltier food, and tubulo-glomerular feedback regulation are the most likely hypotheses to explain compensation for sodium loss. Phenotypic variability is observed within the PHA1 families, ranging from subjects with severe salt loos in the neonatal period to asymptomatic subjects. Interestingly, adult patients with renal PHA1 have no adverse cardiovascular outcome despite lifelong increase in plasma aldosterone and renin levels but rather an improved diastolic left ventricular function, suggesting that the cardiovascular consequences of aldosterone excess require full MR signaling (Escoubet et al. 2013).

MR Mutations The genetic defect underlying the development of renal PHA1 was elucidated in 1998 by Geller et al. The authors identified heterozygous mutations in the NR3C2 gene in four dominant and one sporadic cases of PHA1 (Geller et al. 1998). Since this description, more than 70 heterozygous NR3C2 mutations were described in patients with renal PHA1 (Zennaro and Fernandes-Rosa 2017). Interestingly, in about 30% of cases, no MR mutations were identified. Mutations leading to renal PHA1 are always heterozygous and were identified in all coding exons of the NR3C2 gene. To date, no missense mutations were reported in exon 2, suggesting that missense mutations located in the N-terminal domain do not sufficiently affect MR function. NR3C2 mutations occur at high frequency in patients with familial autosomal dominant and in sporadic PHA1 (Pujo et al. 2007). The pathogenic mechanism of PHA1 in patients with heterozygous MR mutations is dependent on the type of mutation, which may be due to MR haploinsufficiency but also dominant negative effects on the wild-type receptor (Riepe et al. 2006; Sartorato et al. 2003; Sartorato et al. 2004b). MR mutations may differentially affect individual gene expression in a promoter-dependent manner (Fernandes-Rosa et al. 2011; Sartorato et al. 2004a). In this case, the same MR mutation may induce complete functional loss of transcriptional activity on one target promoter while retaining a partial transcriptional activity on another gene. Thus, the phenotype of PHA1 may be modulated, in terms of target gene expression, not only by the quantitative extent of functional reduction but also by the specific qualitative impact of MR function over target genes (Zennaro et al. 2012). The clinical severity of renal PHA1, however, is not correlated with a particular MR genotype (Riepe 2009). This could be explained by adaptive and compensatory mechanisms occurring in the distal part of the nephron. Adult PHA1 patients carrying NR3C2 mutations had higher morning plasma cortisol and higher 24 h urinary cortisol, associated with higher plasma renin, lower HDL cholesterol, and higher waist circumference. These findings were not correlated with blood pressure, carotid intima-media thickness, or echocardiographic parameters, suggesting that the glucocorticoid excess is mediated by GR on liver lipid metabolism and adipose tissue distribution, without adversely affecting cardiac and vascular remodeling in the absence of normal signaling through the MR (Walker et al. 2014).

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Interestingly, one patient with a severe PHA1 phenotype was shown to carry two NR3C2 mutations, each inherited from one parent. The gravity of the salt wasting syndrome was similar to the generalized PHA1 despite the MR mutation which is associated to the mild renal PHA1. Independent segregation of the mutations occurred in the family, with p.Ser166X being transmitted from the affected father and p.Trp806X from the asymptomatic mother. Whereas the truncated MR(166X) protein was degraded, MR(806X) was expressed both at the mRNA and protein level. Functional studies demonstrated that despite its inability to bind aldosterone, MR(806X) had partial ligand-independent transcriptional activity, with minimal residual MR activity. This case suggests that hypomorphic NR3C2 alleles may be more common than expected from the prevalence of detected PHA1 cases (Hubert et al. 2011). In addition to loss of function mutations, one activating mutation of the MR (p. Ser810Leu) was described in on family presenting a rare autosomal dominant form of hypertension (Geller et al. 2000). The p.Ser810Leu variant segregates with patients presenting early and severe hypertension, and in female patients, the hypertension is exacerbated during pregnancy. This variant leads to abnormal structure and binding affinities of the mutant MR resulting in hypersensitivity to steroids that are normally antagonists of the wild-type MR, including progesterone and spironolactone. The first MR LBD X-ray crystal structure to be reported was that of the mutant MR carrying the p.Ser810Leu mutation (Fagart et al. 2005) complexed with deoxycorticosterone (DOC) and progesterone, which both act as agonists on the mutated receptor. The Leu810 residue (in helix 5) establishes hydrophobic contacts with the Gln776 residue (helix H3) and with the 19-methyl group of both DOC and progesterone. The binding of mutant MR to cortisone and 11-dehydrocorticosterone is responsible for the early onset hypertension in nonpregnant patients the hypertension. Those steroids are the main metabolites of cortisol and corticosterone produced by the action of the 11HSD2. In contrast with their low affinity for the wild-type MR, cortisone and 11-dehydrocorticosterone bind MRS810L with high affinity, leading to its activation and induction of MR-dependent transcriptional activation (Rafestin-Oblin et al. 2003). Frequent NR3C2 polymorphisms (SNP) have been shown to exert quantitative effects on MR function and to modulate salt balance, blood pressure, stress, and the hypothalamic–pituitary–adrenal (HPA) axis. The NR3C2 c.-2G > C (rs2070951, MAF ≈ 0.45) is a frequent SNP located in two nucleotides upstream of the first translation start site, in the Kozak region. In vitro characterization has revealed that the G allele is associated, in the presence of aldosterone or cortisol, with decreased MR protein levels and reduced transcriptional activation compared to the C allele. The G allele of the c.-2G > C SNP is associated with increased activation of the renin–angiotensin–aldosterone axis and with increased blood pressure, probably related to decreased MR expression. Homozygous G carriers had significant higher plasma renin levels in a mild hypertensive group subjected to a salt sensitivity test and in a healthy normotensive group receiving high and low Na/K diets. The GG genotype was also correlated with higher plasma aldosterone levels in healthy subjects. In mild hypertensive subjects, male GG carriers exhibited higher systolic blood pressure. These studies provide evidence that frequent MR SNP may modulate the vulnerability for hypertension (Van Leeuwen et al. 2010). The functional effects

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of the common NR3C2 SNP c.538A > G/p.Ile180Val (rs5522, MAF 0.12) was also studied in a cohort of healthy patients subjected to psychosocial challenges and mild hypertensive patients submitted to different sodium intake. Carriers of the MR180Val allele had higher plasma and saliva cortisol levels and higher heart rate responses to the psychosocial challenges than noncarriers, but no difference in salt sensitivity according to genotypes was observed in the group of mild hypertensives (Derijk et al. 2006). The differences in central and renal MR effects were explained by the functional consequences of the p.Ile180Val variant on receptor function. On the other hand, the association of the frequent MR SNP p.I180V with hypertension and markers of cardiovascular risk was previously analyzed in a Brazilian cohort of young hypertensive patients. The MR p.Ile180Val polymorphism was associated with cardiovascular risk factors including BMI and LDL-cholesterol levels, reinforcing the role of MR in adipocyte biology and in cardiovascular disease in the general population (Fernandes-Rosa et al. 2010).

Animal Models of Renal PHA1 Invalidation of the NR3C2 gene in mice by homologous recombination was performed by the group of G. Schütz (Berger et al. 1998). Inactivation of the NR3C2 gene in mice is responsible for early death of the mice as early as the second week of life due to severe dehydration caused by massive hydrosodic loss but does not result in embryonic lethality. From the first week, these mice show a PHA1 picture characterized by dehydration, hyperkalemia, and hyponatremia associated with extremely high plasma levels of aldosterone and renin. In homozygous transgenic animals, rapid weight loss is observed due to dehydration and salt loss. Hypovolemia and hyponatremia result in major RAAS activation in these animals at 8 days of age but also in heterozygotes of the same age: plasma renin levels increase 440-fold, angiotensin II 50-fold, and aldosterone 65-fold compared with levels in control mice (Hubert et al. 1999). In this model, the activity of the ENaC channel is strongly reduced but not totally abolished: the effects of amiloride on the fraction of sodium excretion by the kidney and on the transepithelial voltage of the colon are measured to be 24% and 16% in the kidney and colon, respectively, in homozygous transgenic mice aged 8 days compared with measurements in control mice of the same age (Berger et al. 1998). However, the compensatory mechanism is not sufficient to maintain blood volume and sodium in these animals, which die between days 8 and 13 of life. Death of homozygous mice can be prevented by subcutaneous injections of an isotonic saline solution from day 5 after birth until weaning and then by sodium supplementation in the diet. Breeding of homozygous adult mice showed that water and sodium loss persists in the kidney and colon as well as hyperkalemia: adult MR/ mice supplemented with NaCl have a fourfold increase in sodium excretion fraction. The RAAS of these mice is highly activated with a tenfold increase in plasma renin and aldosterone levels compared to control mice subjected to the same treatments (Bleich et al. 1999).

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These experiments demonstrated that the MR is not a critical component during prenatal development but is required for adaptation to limited sodium intake after birth. A second model of MR invalidation allowing to avoid lethality due to a complete KO of the NR3C2 gene was then performed in rats by additive transgenesis of a lentiviral interfering RNA. The challenge in this model was to have a sufficient degree of NR3C2 gene silencing to obtain a PHA1 phenotype. The authors demonstrated that the vector used specifically reduced the expression of the NR3C2 gene but that it is impossible to control the expression mosaic in a noninvasive way. Furthermore, the degree of NR3C2 gene silencing was animal and tissue-specific depending on the site of provirus integration and copy number. Although this complicates the analysis, this technique reflects the phenotypic heterogeneity observed in many genetic diseases and would mimic human pathophysiology: shMR rats are a useful model for PHA1 and the study of mental retardation, but also to explain altered hormone levels and disruption of electrolyte homeostasis (Lim et al. 2008). In order to study the role of the MR in the kidney, a transgenic mouse in which MR expression is specifically silenced in the major cells in the CCD by the CRE-loxP system under the control of the aquaporin 2 (AQP2) promoter was created. Surprisingly, mice on a normal diet develop without any alteration of urinary sodium excretion, despite very high plasma aldosterone levels. On a salt-deficient diet, AQP2-MR/ mice show increased urinary sodium and water excretion, responsible for continued weight loss. Thus, these data suggest that MR inactivation in CD can be compensated for under normal conditions but more so when salt intake is severely restricted (Ronzaud et al. 2007). Interestingly, these data also suggest that other segments of the ASDN, such as the late DCT, play a key role in the regulation of aldosterone-dependent sodium reabsorption.

Generalized PHA1 Clinical Findings Generalized PHA1 (MIM #264350) is a severe, autosomal recessive, form of PHA1. It is characterized by salt wasting from multiple organs, including kidney, distal colon, and the salivary and sweat glands (Savage et al. 1982). Patients exhibit with severe dehydration, vomiting, and failure to thrive in the neonatal period, and the clinical course may be complicated by cardiac dysrhythmias, collapse, shock, or cardiac arrest. Biological analysis is characterized by severe hyperkalemia, high plasma aldosterone, and high plasma renin levels, confirming the mineralocorticoid resistance. The presence of elevated sweat and salivary Na+ and Cl- and absent nasal amiloride-sensitive Na+ transport corroborate the systemic mineralocorticoid unresponsiveness (Hanukoglu 1991). Signs of systemic salt loss may include a respiratory syndrome characterized by persistent rhinorrhea of clear liquid, congestion, tachypnea, wheezing, fever, and recurrent pulmonary infections, due to their reduced capacity to absorb liquid from airway surfaces (Kerem et al. 1999). Cutaneous lesions similar to those appearing in miliaria rubra, associated to inflammation

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and damage in the eccrine structures, are due to high concentration of sweat salt (Hanukoglu et al. 2017). Cholelithiasis and salt loss from the Meibomian glands were also reported in children with PHA1 (Kuhnle 1997). Early diagnosis is critical to survival in generalized PHA1 (Belot et al. 2008). In contrast to renal PHA1, patients with generalized PHA1 suffer from recurrent lifethreatening episodes of salt loss in childhood (Hanukoglu et al. 2008). Interestingly, the evaluation of patients with systemic PHA1 throughout adolescence and early adulthood showed gradual normalization of urinary Na/K ratios with age (Hanukoglu et al. 2008). In the same way, the patients with near normal lives on a lifelong high salt diet and even PHA1 improvement with cessation of salt supplementation were described (Adachi et al. 2010).

ENaC Mutations Systemic PHA1 is caused by mutations in SCNN1A, SCNN1B, and SCNN1G genes coding for the ENaC subunits α, β, and γ, respectively. SCNN1A is located on chromosome 12p13.3, whereas SCNN1B and SCNN1G are located within 400 kb on chromosome 16p12.2-p13.11. The three genes are comprised of 13 exons spanning over 17 kb. Parts of exons 2 and 13 encode the transmembrane regions and exons 3–13 code for the extracellular loop (Saxena et al. 1998; Thomas et al. 1996; Ludwig et al. 1998). After the association of the locus of ENaC subunits on chromosomes 12 and 16 with generalized PHA1 (Strautnieks et al. 1996b), Chang et al. identified mutations in SCNN1A and SCNN1B in PHA1 patients from Arabian and Iranian Jewish ethnicity (Chang et al. 1996). A mutation affecting SCNN1G was described in three families originating from the Indian subcontinent showing linkage to chromosome 16p (Strautnieks et al. 1996a). Different homozygous or compound heterozygous inactivating mutations of ENaC subunits, as well as a large deletion in the promoter region of βENaC, were subsequently reported (Adachi et al. 2001; Bonny et al. 2002; Edelheit et al. 2005; Kerem et al. 1999; Riepe et al. 2009; Saxena et al. 2002; Schaedel et al. 1999). ENaC mutations are in majority homozygous, mostly in consanguineous families, although compound heterozygous mutations can occur in non-consanguineous pedigrees. In contrast to the renal PHA1, ENaC mutations are identified in all subjects investigated. While in animal models, the inactivation of one ENaC subunit is associated to specific phenotypes, in humans, the genotype-phenotype correlations are not well established. Hanukoglu et al. evaluated the reported distinct growth and puberty phenotypes in PHA1 patients depending on the degree of functional ENaC impairment (Hanukoglu et al. 2008). In the lungs, ENaC contributes for removal of fluid from the alveolar space by regulating the transepithelial alveolar sodium transport (Hummler and Planes 2010). Accordingly, ENaC plays a role on the pathogenesis of pulmonary diseases such as cystic fibrosis and respiratory distress syndrome in preterm infants. In cystic fibrosis, increase in ENaC activity is increased, contributing to airway mucus dehydration and decreased mucociliary transport. In contrast, in systemic PHA1, the absence of ENaC activity results in increased airway surface liquid (Kerem et al. 1999).

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Animal Models of ENaC Subunits Invalidation Inactivation of the genes encoding the α, β, and γ subunits of the ENaC channel have clearly and unequivocally demonstrated that they are essential for individual survival (Hummler et al. 1996; Barker et al. 1998; McDonald et al. 1999). Models with reduced or completely abolished channel activity have been used to study the minimum activity required for survival or for proper function of sodium reabsorption by a tissue. Mice deficient in the α subunit of the ENaC channel were obtained by homologous recombination. These mice do not have problems during fetal development but they die prematurely within 2 days after birth. This premature death is due to respiratory distress, caused by the complete absence of sodium transport in the airway epithelium (Hummler et al. 1996). In order to study the effects of invalidation of this subunit, particularly at the renal level, heterozygous α ENaC mice were crossed with transgenic mice overexpressing the α subunit only in alveolar cells. These mice survive but rapidly develop signs of very severe PHA1 (mortality close to 50%) with loss of salts in the urine, growth retardation, and metabolic acidosis. As in humans, adults show compensated PHA1 with elevated plasma aldosterone levels (Hummler et al. 1997). Inactivation of the α-subunit of ENaC specifically in the CCD with intact expression in the more upstream parts, the DCT and CNT, allows animal survival as well as maintenance of sodium and potassium balance, even when mice are subjected to different regimes such as sodium restriction, water deprivation, or potassium overload. Thus, ENaC expression in the CCD is not required to regulate water balance but this highlights its importance in more upstream segments of the nephron (late DCT and CNT) (Rubera et al. 2003). Two transgenic mouse lines for the β-subunit were obtained by homologous recombination either by deletion of exon 1 (β ENaC(/) mice) (McDonald et al. 1999)) or by introduction of a neomycin cassette mutation (β ENaC (m/m) mice (Pradervand et al. 1999)). Both of these transgenic techniques lead to the near-total absence of β-subunit expression. The β ENaC(/) mice, despite normal embryonic development, die about 48 h after birth due to severe defects in renal function: large urinary salt loss, low potassium excretion with very high plasma aldosterone. These defects are comparable to acute PHA1, responsible for early death. In the other mouse model β ENaC (m/m), a renal PHA1 phenotype is observed when mice are subjected to a salt-deficient diet with weight loss, hyperkalemia, and salt loss. ENaC channel activity is reduced, and elevated plasma aldosterone levels are measured under normal diet conditions, mimicking a compensated form of PHA1 with dietcompensated salt loss (Pradervand et al. 1999). The study of these models suggests that the β-subunit plays an important role in sodium reabsorption at the renal collecting tubule, particularly during a low-salt diet. The γ ENaC(/) mice, obtained by homologous recombination, show a significant defect in renal function causing premature death within 48 h after birth: Lethargy, growth defect associated with salt loss, potassium retention, and increased plasma aldosterone are also the clinical and biological signs found in PHA1 patients and in these transgenic mice.

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In the lungs, β and γ subunits facilitate pulmonary fluid clearance at birth, whereas in the kidney, sodium reabsorption is under the control of aldosterone, where these subunits appear critical for proper channel function. In the Xenopus laevis oocyte expression system, all three subunits are required for maximal activity. Furthermore, the α subunit has been described as essential for channel function in contrast to the β and γ subunits described as regulators of this channel. The conditional KO mice Scnn1blox/lox and Scnn1glox/lox have been created to further investigate the roles of these subunits in sodium reabsorption (Merillat et al. 2009) but have yet to be exploited.

Secondary PHA1 Secondary PHA1 is characterized by transient mineralocorticoid resistance in infants with less than 7 months of age, associated with malformations in the urinary tract and/or urinary tract infections (Bulchmann et al. 2001; Rodriguez-Soriano et al. 1983; Watanabe 2003). The phenotype is less severe in older infants than in neonates suggesting a pathophysiologic role for the tubular immaturity (Belot et al. 2008). Other pathophysiological hypothesis is the increase in cytokines (mainly the transforming growth factor beta-1-TGF-β1) in response to the urinary tract obstruction, resulting in the downregulation of MR (Klahr 2000; Furness 3rd et al. 1999; Kuhnle et al. 1993; Bogdanovic et al. 2009). Diagnosis of secondary PHA1 includes hyponatremia, hyperkalemia, and metabolic acidosis in the presence of high levels of plasma aldosterone and renin, associated to abnormalities in urine culture and/or ultrasound examination, allowing the differentiation from genetic forms. Transient PHA1 was also observed secondary to congenital jejunal membrane (Nissen et al. 2017), after resection of the ileum and colon in an adult patient (Vantyghem et al. 1999), and in patients treated with immunosuppressors (Deppe et al. 2002). Recent studies challenged the classical classification of PHA1 in two distinct entities. These reports showed a continuum in the PHA1 phenotype between the severe generalized and the mild renal PHA1. While homozygote carriers of a αENaC mutation exhibit a generalized PHA1 phenotype, heterozygote carriers may exhibit a subclinical PHA1 phenotype only with increased sweat sodium and chloride concentrations (Riepe et al. 2009). Moreover, cases of mild forms of PHA1 associated with homozygous ENaC mutations were also described (Dirlewanger et al. 2011).

Differential Diagnosis of PHA1 Differential diagnosis of PHA1 comprises diseases leading to renal salt wasting in the neonatal period, mainly congenital adrenal hyperplasia (CAH) and isolated deficiency in aldosterone synthase (Table 1). Classic CAH due to 21-hydroxylase (21OH) deficiency with salt loss is the main differential diagnosis of PHA1. Classic CAH-21OH is an autosomal recessive disease with an estimated prevalence of 1: 10000 to 1:20000. Defective 21OH-hydroxylation results in decreased glucocorticoid

CYP21A2 mutations Autosomal recessive

Glucocorticoid, mineralocorticoid, and salt supplementation

Genetic cause and inheritance

Treatment

DSD Disorders of sexual differentiation

Increased 17-OH Progesterone

Hormonal alterations

Clinical

Classic form of 21-hydroxylase deficiency CAH Salt wasting 46,XX DSD Hyponatremia Hyperkalemia Hypoglycemia Metabolic acidosis

Glucocorticoid, mineralocorticoid, and salt supplementation

3βHSD2 mutations Autosomal recessive

Increased 17-OH Pregnenolone

3βHSD2 deficiency CAH Salt wasting 46,XY or 46,XX DSD Hyponatremia Hyperkalemia Hypoglycemia Metabolic acidosis

NR3C2 mutations (70% of the cases) Autosomal dominant or sporadic

Salt supplementation

Mineralocorticoid supplementation

Increased plasma aldosterone Increased plasma renin

Renal PHA1 Failure to thrive and salt wasting improvement with age Hyponatremia Hyperkalemia Metabolic acidosis

CYP11B2 mutations Autosomal recessive

Decreased plasma aldosterone Increased plasma renin

Isolated aldosterone deficiency Variant degrees of dehydration Hyponatremia Hyperkalemia Metabolic acidosis

Table 1 Differential diagnosis of PHA1 and other salt wasting syndromes Generalized PHA1 Failure to thrive and lifelong salt wasting Pulmonary and skin phenotype Severe hyponatremia Hyperkalemia Metabolic acidosis Increased plasma aldosterone Increased plasma renin SCNN1A, SCNN1B, and SCNN1G mutations Autosomal recessive Lifelong salt supplementation

Salt supplementation correction of urinary tract disorders

Increased plasma aldosterone Increased plasma renin

Secondary PHA1 Transitory failure to thrive and salt wasting Urinary tract malformation and/or infection Hyponatremia Hyperkalemia Metabolic acidosis

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and mineralocorticoid synthesis and elevated precursors, most notably 17-hydroxyprogesterone (17OHP), and concomitant ACTH-stimulated androgen production. It is characterized by hyponatremia and hyperkalemia in the neonatal period, with different degrees of genitalia virilization in female neonates (DSD 46,XX) (New 2003; Bizzarri et al. 2016; El-Maouche et al. 2017). A complete clinical examination to exclude the presence of ambiguous genitalia and a measure of 17OHP are crucial for differentiating CAH-21OHD from PHA1. CAH due to 3β-hydroxysteroid dehydrogenase deficiency (3βHSD2) is a rare form of CAH responsible by salt wasting in the neonatal period. Abnormal 3βHSD2 activity leads to decreased in aldosterone, cortisol, and androstenedione synthesis and an increase in DHEA production. Neonates affected exhibit hyponatremia and hyperkalemia associated with underdeveloped 46,XY genitalia (DSD 46, XY), and, less frequently, a virilization in 46,XX. The diagnosis is confirmed by high levels of the precursor 17-OH pregnenolone (New 2003; El-Maouche et al. 2017). Isolated aldosterone deficiency is a salt wasting disease due to a defect in the final steps of aldosterone biosynthesis caused by mutations in CYP11B2 (coding for the aldosterone-synthase). Signs of aldosterone deficiency may appear at a few days or weeks of age but some patients are only diagnosed in the early childhood. Diagnosis is confirmed by the presence of increased deoxycorticosterone, undetectable levels of aldosterone in subtype I of the disease, and elevation of the ratio of 18-hydroxycorticosterone to aldosterone in patients with isolated aldosterone deficiency type II (Ulick et al. 1992; White 2004). Finally, patients with antenatal Bartter’s syndrome, an autosomal recessive disease caused by mutations in the potassium channel ROMK, may present with hyperkalemia in the first week of life associated with increased renin and aldosterone (Finer et al. 2003). Usually, the hyperkalemia is transient and patients will develop hypokalemia and metabolic alkalosis.

PHA1 Treatment Treatment of PHA1 is based on the correction of water, electrolytes, and acid-base disturbances. In the acute phase, salt supplementation, hydration, and correction of hyperkalemia and acidosis hyperkalemia may be associated with the administration of fludrocortisone (mineralocorticoid) and hydrocortisone (glucocorticoid) while performing the differential diagnosis with CAH. Extracellular volume expansion and ion exchange resins may be required to normalize sodium and potassium levels. Recognizing a salt wasting syndrome is important for all pediatricians and physicians in neonatal care and pediatric emergency services; adequate volume expansion, salt supplementation, and steroid replacement are crucial in this death risk condition. After the acute phase and confirmation of PHA1 diagnosis, salt supplementation is the basis of the treatment. In renal PHA1, the amount of salt supplementation depends on the severity of the phenotype, but usually 3–20 mEq/kg/d of Na+ are able to remediate the salt loss. Clinical and biochemical improvement are rapidly achieved, with catch-up on growth and development, and electrolytes normalization.

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Renal PHA1 improves with age, and salt supplementation may be discontinued in the majority of patients after 18–24 months of age. Older children are asymptomatic, with normal growth and development, and electrolytes remaining in normal ranges. Some children, however, may exhibit growth on the lower percentiles of the growth curve (Loomba-Albrecht et al. 2010). Symptoms and signs of secondary PHA1 usually improve with the surgical correction of the underlying urinary tract structural abnormalities and treatment of urinary tract infection. In the acute phase and before treatment of the urinary tract disease, patients with secondary PHA1 may need medical care similar to patients with renal PHA1. Treatment of generalized PHA1 is specific for each patient and is usually based on high doses of sodium supplementation (20–50 mEq/kg/d), associated with ion resins and low-K+ diet to reduce potassium levels. The supplementation of high doses of sodium is difficult and requires tube feeding in some cases. So far, no specific drug has been described, but the use of glucocorticoid or indomethacin seems to be beneficial for some patients (Mathew et al. 1993). Recent preliminary in vitro studies showed that tumor necrosis factor (TNF) activates ENaC current through its lectinlike (TIP) domain, and that peptides mimicking the TIP domain are capable to activate wild-type and mutant ENaC (Willam et al. 2017). These data indicate that these peptides could represent a strategy to treat generalized PHA; however, further studies are necessary to evaluate in vivo efficacy and safety of these peptides. The treatment is needed throughout life but the sodium supplementation may decrease with age to 8–20 g NaCl/day. Specific symptomatic treatment is required for pulmonary and dermatological phenotype.

Conclusions PHA1 comprises two clinically and genetically distinct entities. Alterations in genes encoding the ENaC subunits lead to the systemic form of PHA1 with an autosomal recessive inheritance pattern and the kidney-restricted form occurs by inactivating mutations in NR3C2 gene. In both cases, kidney mineralocorticoid resistance leads to impaired sodium reabsorption and potassium secretion in the ASDN cells. The current genetic classification of PHA1 has been challenged by the improvement in case detection and the genetic diagnosis of PHA1 performed in hospital routine, which allowed for the discovery of a continuum of phenotypically forms of aldosterone resistance. Further studies are needed to understand the natural history of the disease in order to provide earlier therapeutic intervention to avoid severe episodes of dehydration in the neonatal period. Routine genetic investigation of PHA1 subjects may be useful in identifying other genes involved in this disease in families without mutations in the MR gene or in the genes encoding ENaC. These studies may provide a better understand not only of the pathogenesis of the disease but also of mechanisms involved in the regulation of transepithelial sodium transport and the regulation of blood pressure. This knowledge may be useful in the development of new tools not only for the management of patients with neonatal salt waist syndromes but also for improved care of patients with arterial hypertension.

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Primary Aldosteronism

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Sheerazed Boulkroun and Maria-Christina Zennaro

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Adrenal Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anatomy of the Adrenal Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vascularization and Innervation of the Adrenal Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Renewal of the Adrenal Cortex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aldosterone Producing Cell Clusters (APCC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Zona Glomerulosa Zonation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Zona Fasciculata Zonation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction Between Wnt/β-Catenin and ACTH/APMc Signaling Pathways . . . . . . . . . . . . . . Aldosterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aldosterone Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Action of Aldosterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Aldosterone Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnostic and Treatment Outcome of Patients with Primary Aldosteronism . . . . . . . . . . . . . . . . . Diagnosis of Primary Aldosteronism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment of Primary Aldosteronism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Outcome of Patients with Unilateral Form of Primary Aldosteronism . . . . . . . . . . . . . . . . . . . . . Etiology and Genetic of Primary Aldosteronism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sporadic Forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Familial Forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Remodeling of Adrenal Glands with APA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zona Glomerulosa Hyperplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Presence of Secondary Nodules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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S. Boulkroun (*) Université Paris Cité, PARCC, Inserm, Paris, France e-mail: [email protected] M.-C. Zennaro Université Paris Cité, PARCC, Inserm, Paris, France Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Service de Génétique, Paris, France e-mail: [email protected] © Springer Nature Switzerland AG 2023 M. Caprio, F. L. Fernandes-Rosa (eds.), Hydro Saline Metabolism, Endocrinology, https://doi.org/10.1007/978-3-031-27119-9_13

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Presence of APCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Presence of Possible APCC to APA Translational Lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prevalence of Somatic Mutations and Associated Correlations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prevalence of Somatic Mutations in APA and Genotype/Phenotype Correlations . . . . . . . . Mutations and Expression Profiles Correlations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutations and Steroid Profiles Correlations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutation and Targeted Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Risk Loci for Primary Aldosteronism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal Models of Primary Aldosteronism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenic Model for Primary Aldosteronism Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion: Toward a Precision Medicine in Primary Aldosteronism . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Primary aldosteronism is the most common form of secondary hypertension. The two main causes are aldosterone producing adenoma (APA) and bilateral adrenal hyperplasia. Over the past 15 years, major advances have been made in our understanding of the disease with the identification of somatic and germline mutations responsible for the development of APA and of familial form of hyperaldosteronism. These mutations affect essentially ionic channels and pumps affecting intracellular ionic equilibrium of the zona glomerulosa cells of the adrenal cortex leading to increased intracellular calcium concentration, and activation of calcium signaling, the main trigger for aldosterone biosynthesis. Recently, the identification of risk loci for the development of PA provides new pathophysiological insight and opens new perspectives for the diagnosis and treatment of PA. The characterization of different mouse models has also contributed to a better understanding of the molecular mechanisms involved in the development of PA. Finally, the use of multiomics signatures combined with machine learning appears to be a promising new tool for improving the diagnosis and management of endocrine hypertension, enabling faster diagnosis and more effective treatment, particularly for patients with PA, who represent up to 10% of the hypertensive population. The next few years should see us move toward a precision medicine in primary aldosteronism. Keywords

Primary aldosteronism · Hypertension · Adrenal gland · Genetic · Calcium signaling

Introduction Arterial hypertension (HT) is a major cardiovascular risk factor that affects up to 45% of the general population. HT is defined as high blood pressure equal or higher than 140/90 mmHg. Up to 53% of patients with hypertension are unaware of their condition and are untreated (Chow et al. 2013). In some cases, patients have no

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symptoms until the development of serious complications in target organs such as the heart, kidneys, and brain. There are various types of treatment; however, optimal blood pressure is not achieved in two-thirds of hypertensive patients, despite the use of different combinations of drugs. The identification of secondary forms of hypertension is essential for a better management of hypertensive patients and prevention of cardiovascular complications. Secondary causes of hypertension include a number of adrenal disorders, in which increased autonomous hormone production increases blood pressure; these include primary aldosteronism, Cushing’s syndrome, and pheochromocytoma. In most cases, these diseases are curable and blood pressure can be normalized with surgery or medication. However, in many cases, these endocrine disorders go undiagnosed. The renin-angiotensin-aldosterone system (RAAS) regulates blood pressure, fluid volume, and the vascular response to injury and inflammation (Ferrario 2006). Its chronic activation leads to the development of persistent hypertension and triggers a cascade of inflammatory, thrombotic, and atherogenic effects, ultimately leading to end-organ damage (Brewster et al. 2003; Cooper 2004). High renin or aldosterone concentrations are predictors of adverse outcome in hypertension (Vasan et al. 2004), heart failure (Tsutamoto et al. 2007; Güder et al. 2007), myocardial infarction (Beygui et al. 2006), renal insufficiency (Tylicki et al. 2005), and influence insulin resistance (Freel et al. 2009). Primary aldosteronism (PA) is the most common form of endocrine hypertension with an estimated prevalence of around 4–6% hypertensives in primary care (Hannemann and Wallaschofski 2012; Monticone et al. 2017) and 10% in referred patients (Hannemann and Wallaschofski 2012). PA is associated with high aldosterone to renin ratio due to suppressed renin and often hypokalemia. Detection of PA is key to targeted management of the underlying disease and prevention of cardiovascular complications. The two major causes of dysregulated aldosterone secretion are unilateral aldosterone producing adenoma (APA) and bilateral adrenal hyperplasia, accounting together for around 95% of the cases. The distinction between unilateral and bilateral forms is important for the targeted management of the disease. Whereas unilateral forms, mainly APA, are surgically correctable, bilateral forms should be treated pharmacologically. Although sporadic forms represent the majority of cases, familial forms (FH) with mendelian distribution account for about 6% of cases (Monticone et al. 2018). During the last decade, major advances have been made in our understanding of the genetic bases of the disease with the identification, in sporadic and inherited forms, of mutations in different ionic channels (KCNJ5 (Choi et al. 2011), CACNA1D (Azizan et al. 2013; Scholl et al. 2013), CACNA1H (Scholl et al. 2015a), and CLCN2 (Fernandes-Rosa et al. 2018; Scholl et al. 2018)) and pumps (ATP1A1 (Azizan et al. 2013; Beuschlein et al. 2013) and ATP2B3 (Beuschlein et al. 2013)) making PA a channelopathy. Less frequently, mutations in CTNNB1 gene, coding for β-catenin, have been reported. Finally, co-occurrence of CTNNB1 mutations and mutations in GNA11/GNAQ genes have been reported in particular forms of PA (Zhou et al. 2016).

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The Adrenal Gland Anatomy of the Adrenal Gland The adrenal glands are endocrine tissues located on top of the kidneys and are involved in body homeostasis and stress response. Adrenal gland is divided into the outer cortex and the inner medulla, surrounded by an external capsule. Although they constitute a single tissue, the cortex and the medulla have different embryonic origins. Thus, the adrenal cortex originates from the intermediate mesoderm, sharing common origin with the gonads, whereas the adrenal medulla originates from the neural crest. The medulla produces catecholamines, epinephrin, and norepinephrine, but also dopamine. Epinephrin is associated with the “fight or flight” response whereas norepinephrine is also involved in the activation of the sympathetic nervous system. The adrenal cortex is composed of three distinct morphological zones with specific functions. Each of the three zones secretes a specific profile of steroid hormones and is under different and independent regulatory control. The zona glomerulosa (ZG), composed of small and clear cells, is located immediately underneath the capsule. The ZG cells are characterized by a specific molecular signature as they express aldosterone synthase, disabled-2 and β-catenin. Their mitochondria are characterized by the lamellar appearance of their crest. This zone produces mineralocorticoids involved in sodium and potassium homeostasis and in the regulation of blood pressure (Yates et al. 2013). The zona fasciculata (ZF), the thickest zone of the cortex, is composed of large and clear cells organized in column, rich in lipid droplets. They are distinguished by the expression of 11β-hydroxylase, encoded by CYP11B1, and aldo-keto reductase family 1 member B7 (AKR1B7). These cells, which are composed of mitochondria with tubular crest and a developed smooth endoplasmic reticulum, are separated by capillary windows. ZF produces glucocorticoid hormones that play important roles in stress response, energy homeostasis, cardiovascular system, and immune system (Yates et al. 2013). The zona reticularis (ZR) is composed of small and eosinophilic cells. These cells have common characteristics to those of the ZF, although they have fewer lipid droplets, more lysosomes, and granules of lipofuscin pigment. This zone produces androgen precursors such as dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione (Yates et al. 2013).

Vascularization and Innervation of the Adrenal Gland The adrenal gland is a richly vascularized organ, which is essential for the supply of hormones to the adrenal gland and also for the secretion of hormones into the bloodstream. The adrenal arteries are divided into three groups. The superior arteries arise from the inferior phrenic arteries, the middle adrenal arteries arise from the abdominal aorta, and the inferior arteries arise from the renal arteries. These different groups of arteries branch off from the capsule before forming a subcapsular arteriolar plexus. The cortical arterioles supply the sinusoidal vessels that cross the cortex and

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form a plexus in the ZR in adults, followed by an anastomosis with the medullary plexus. The medulla is also supplied directly by the perforating arteries, which bypass the plexus of the ZR and also supply the medullary plexus. The venous plexus of the medulla is drained into the central veins and then into the adrenal vein, which empties into the inferior vena cava for the right adrenal vein and into the renal vein for the left adrenal vein. The adrenal gland also contains a subcapsular and medullary lymphatic plexus that allows lymphatic drainage toward the lumbar and para-aortic lymph nodes. The adrenal gland is also a richly innervated organ, allowing the chromaffin cells of the adrenal medulla to be innervated by myelinated preganglionic sympathetic fibers originating from the intermediolateral cellular column of segments T10-11. The adrenal cortex, unlike the adrenal medulla, does not have direct innervation but is innervated by afferent nerves originating from the adrenal medulla (Yates et al. 2013). This innervation is therefore under the control of the sympathetic nervous system and allows innervation of the internal part of the adrenal cortex. The adrenal cortex is also innervated by sympathetic fibers originating from extra-adrenal neurons that form the subcapsular plexus with the blood vessels (Lefebvre et al. 2013).

Renewal of the Adrenal Cortex The adrenal cortex is continuously renewing itself, allowing dying cells to be replaced. This renewal is mainly due to centripetal migration, which corresponds to the continuous production of new cortical cells in the outer part of the gland, which migrate under mitotic pressure toward the inner part of the cortex where they die at the boundary with the medulla. This theory is based on various studies in rodents in which a majority of mitotic cells were localized between the ZG and the ZF, and the presence of more apoptotic cells in the boundary between the ZR and the medulla. The loss of expression of Sonic hedgehog (Shh) in the adrenal gland supports the hypothesis of a centripetal migration of undifferentiated cells located under the capsule of the adrenal gland and that differentiate into steroidogenic cells. Shh is a member of the hedgehog family that plays different roles during embryonic development and also in adults, where it maintains, differentiates, and regulates the stem cell population. Its expression remains limited to subcapsular undifferentiated cells, close to cells expressing the aldosterone synthase in both mice and humans (King et al. 2009; Boulkroun et al. 2011). Conditional loss of Shh expression in the adrenal induces a decrease in capsular cell proliferation and a 50–75% reduction in adrenal cortical thickness (Ching and Vilain 2009). The use of a mouse model, in which the green fluorescent protein (GFP) is expressed only in ZG cells by using a Cre recombinase inserted into the Cyp11b2 locus, has shown that adrenocortical zonation is derived from transdifferentiation and centripetal migration of ZG cells into ZF cells in the adrenal cortex (Freedman et al. 2013). The transcription factor Sf-1 plays an important role in maintaining the ZG; its specific deletion in this zone prevents the conversion of embryonic and mesenchymal stem cells into ZG cells. However, this does not affect ZF formation,

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suggesting the existence of alternative pathways allowing ZF development (Freedman et al. 2013). Although centripetal migration is the most dominant theory, another theory is also being considered. In contrast to mice, where the ZG and the ZF are in direct contact, suggesting differentiation of cells from the ZG to the ZF, the rat adrenal has a layer separating these two zones, the undifferentiated zone. This zone is subdivided into two other zones in contact with the ZG or with the ZF, characterized by the presence or the absence of Shh expression, respectively. The characterization of this zone suggests bidirectional differentiation of Shh-positive cells into cells expressing the aldosterone synthase, while Shh-negative cells develop into cells expressing the 11β-hydroxylase (Yates et al. 2013).

Aldosterone Producing Cell Clusters (APCC) APCC are structures that develop in the subcapsular part of the adrenal cortex. They are composed in the outer part of ZG-like cells and in the inner part of ZF-like cells. They express uniformly aldosterone synthase but not 11β-hydroxylase, suggesting that this structure produces aldosterone (Nishimoto et al. 2010). These structures are also characterized by the absence of Dab2 expression unlike the ZG (Boulkroun et al. 2010). In human, the adrenal gland presents continuous expression of aldosterone synthase in the ZG in childhood, followed by a loss of continuity of aldosterone synthase expression with an increase in the number of APCC as a function of age. The number of APCC is higher in female adrenals, independently of ethnic origin. The loss of continuity of aldosterone synthase expression in the zona glomerulosa could be due to a negative feedback by APCC and associated aldosterone production (Nanba et al. 2017; Nishimoto et al. 2015, 2016). Interestingly, although the number of APCC increases with age, a decrease in their number and size was observed from the age of 50 and they appeared to detach from the capsule (Hayashi et al. 2019). APCC have a transcriptional signature similar to that of ZG cells. However, these structures differ from ZG cells by the differential expression of 29 genes. The expression of CYP11B2, solute carrier family 35 member F1 (SLC35F1), protein phosphatase 4 regulatory subunit 4 (PPP4R4), and MC2R is significantly higher in APCC compared to the ZG; their expression could be involved in aldosterone biosynthesis. SLC35F1 plays a role in glucose transport, suggesting a role in the metabolism of APCC, while PPP4R4 could be involved in the regulation of aldosterone biosynthesis in APCC by forming a complex with the serine/threonine phosphatase 4 (PP4C) protein, which is involved in different cellular processes such as the regulation of histone acetylation, DNA damage checkpoints, nuclear factor kappa-B activation, and microtubule organization (Nishimoto et al. 2015). The mechanisms leading to APCC development in healthy individuals are still unknown. However, some hypotheses suggest that the appearance of these structures could be linked to a sodium-rich diet, age, or environmental factors (Nishimoto et al. 2016; Bollag 2014). Indeed, the ZG is sensitive to sodium intake; while low sodium

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diet leads to the development of ZG hyperplasia, high sodium diet induces suppression of the renin angiotensin system and therefore a decrease in zona glomerulosa size (Bollag 2014).

Regulation of Zona Glomerulosa Zonation The ACTH/AMPc and Wnt/β-catenin signaling pathways play an important role in the control of the homeostasis and the zonation of the adrenal cortex. The Wnt/β-catenin signaling pathway regulates ZG differentiation but also plays a role in maintaining ZG identity. The inhibition of Wnt4, specifically expressed in the ZG, leads to a reduction in CYP11B2 expression and a decrease in plasma aldosterone concentration, without affecting the ZF (Heikkila et al. 2002). In mice, the constitutive activation of β-catenin induces ectopic differentiation of Cyp11b2 expressing cells in the adrenal cortex with an increase in plasma aldosterone concentration and a decrease in plasma renin concentration, confirming the role of Wnt/β-catenin in ZG zonation (Berthon et al. 2012). Interestingly, Shh expression is correlated with the Axin2 expression, a Wnt/β-catenin target gene, suggesting that differentiation of the ZG could be mediated by the recruitment of stem/progenitor cells via direct regulation of Shh expression by the Wnt/β-catenin pathway (Drelon et al. 2014). This hypothesis is supported by a study showing that the Wnt/β-catenin signaling pathway activates the Shh signaling pathway in the ZG, which in turn induces the expression of the transcription factor Gli1 in capsule cells. Cells expressing Gli1 thus differentiate into cortical cells expressing Sf-1, but also into Shh-positive progenitor cells. Gli-1 activation also allows an increase in Wnt4 and R-spondin 3 expression which enables these cells to go through a rapid expansion and differentiation into steroidogenic cells (Finco et al. 2018). The Wnt/β-catenin pathway is also involved in the acquisition of ZG identity and the repression of ZF since β-catenin controls the expression of the Angiotensin Type I Receptor (AT1R) and CYP11B2, which determine ZG identity, and represses the expression of MC2R and CYP11B1, which are essential in determining ZF identity (Drelon et al. 2014). Indeed, β-catenin binds directly to the regulatory regions of the AT1R gene and to the promoters of the genes coding for the nuclear receptors Nurr1 and Nur77, which stimulate CYP11B2 expression (Berthon et al. 2014). The maintenance of ZG cell identity by β-catenin is also due to an increase in the expression of phosphodiesterase 2A, which inhibits the cAMP/PKA signaling pathway, through its phosphodiesterase activity, inducing the blocking of the transdifferentiation of ZG cells into ZF cells (Pignatti et al. 2020).

Regulation of Zona Fasciculata Zonation ACTH is often considered to be the main regulator of adrenal growth. This signaling pathway regulates cell proliferation, but also the differentiation of ZG cells into ZF cells. Under normal conditions, cell proliferation in the adrenal cortex follows a

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circadian rhythm with a peak observed at 4 am, mainly in the outer part of the ZF and following the circadian rhythm of ACTH, suggesting a role in stimulating proliferation. This observation is supported by other studies showing atrophy of the adrenal cortex in animals having undergone hypophysectomy, or a reduction in the size of the ZF following treatment with dexamethasone, a synthetic glucocorticoid hormone that suppresses the hypothalamic-pituitary-adrenal axis. The excess of ACTH observed in patients with Cushing’s disease or the chronic administration of ACTH in mice induces hyperplasia of the adrenal cortex with expansion of the ZF (Yates et al. 2013). Indeed, long-term exposure to ACTH induces the synthesis of growth factors, such as insulin-like growth factor 2, fibroblast growth factor, and epidermal growth factor, which induce adrenal cell hypertrophy and hyperplasia (Ruggiero and Lalli 2016). Prolonged exposure to ACTH also stimulates the differentiation of ZG cells into ZF cells and has an antiapoptotic effect inducing ZF hypertrophy (Gallo-Payet 2016). Remodeling by ACTH could therefore be due to a combination of different processes such as differentiation of stem/progenitor cells into ZG cells, differentiation of ZG cells into ZF cells, cell proliferation, and a decrease in apoptosis (Yates et al. 2013).

Interaction Between Wnt/β-Catenin and ACTH/APMc Signaling Pathways Recent studies suggest that adrenal zonation is regulated by an interaction between Wnt/β-catenin and ACTH/AMPc signaling pathways. The Wnt/β-catenin signaling pathway, which plays an essential role in the differentiation and maintenance of the ZG, presents a gradient in the adrenal cortex. This gradient is maintained by the presence in the adrenal cortex of the zinc and ring finger 3 (ZNRF3), an E3 transmembrane ubiquitin ligase, which inhibits the Wnt signaling pathway by inducing the endocytosis of the frizzled receptor. Its action is controlled by the R-spondin protein, which induces its clearance from the membrane, thereby neutralizing the negative effect of ZNRF3 on the Wnt signaling pathway. A loss of ZNRF3 expression induces adrenocortical hyperplasia with a loss of the Wnt/β-catenin signaling pathway gradient (Basham et al. 2019). Thus, at the level of the ZG, the Wnt signaling pathway inhibits the expression of MC2R and of targets of the PKA, allowing the ZG maintenance. Interestingly, β-catenin also decreases the expression and interferes with the transcriptional activity of Sf-1, which is activated by PKA in the ZF (Walczak et al. 2014). Moreover, PKA activation inhibits the Wnt signaling pathway in H295R cells through an increase in inactivating phosphorylations and a decrease in activating phosphorylations of β-catenin, but also through an inhibition of Wnt4 expression which is essential in ZG differentiation, inducing a differentiation of ZG into ZF cells (Drelon et al. 2016). Thus, zonation of the adrenal cortex would result from a subtle balance between the two signaling pathways Wnt/β-catenin and ACTH/AMPc.

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Aldosterone Aldosterone regulates blood volume and electrolyte homeostasis, thus playing a major role in the control of blood pressure; it is also involved in the regulation of potassium excretion (Bonvalet 1998). Its biosynthesis is finely controlled, the main regulators being angiotensin II and extracellular potassium concentration. However, many other factors are involved in the regulation of aldosterone biosynthesis such as neuropeptides, catecholamines, prostaglandin, serotonin, and atrial natriuretic peptide.

Aldosterone Biosynthesis Aldosterone is synthesized from cholesterol by a series of specific enzymatic reactions in the ZG. In steroidogenic cells, cholesterol has different origins. It can be synthetized de novo from acetate, or derive from the mobilization of cholesterol esters stored in lipid droplets after uptake of HDL cholesterol via the scavenger receptor, class B type 1 (SR-B1), or from lipoprotein-derived cholesterol esters. Cholesterol is first translocated to the inner mitochondrial membrane by a protein complex comprising the steroidogenic acute regulatory protein (StAR, encoded by STAR) and then converted into pregnenolone by the cholesterol side-chain cleavage enzyme (encoded by CYP11A1). In the outermost ZG, the 3 beta-hydroxysteroid dehydrogenase type 2 (3-betaHSD II, encoded by HSD3B2) and the steroid 21-hydroxylase (21-OHase, encoded by CYP21A2, P450c21) act consecutively to convert, respectively, pregnenolone into progesterone and progesterone into 11-deoxycorticosterone (DOC). Finally, the specific expression of the cytochrome P450 11B2 (aldosterone synthase encoded by CYP11B2) permits the subsequent transformation of DOC into corticosterone, of corticosterone to 18-hydroxyxorticosterone, and finally of 18-hydroxycorticosterone into aldosterone thanks to the 11β-hydroxylase, 18-hydroxylase, and 18-methyl oxidase activities of the aldosterone synthase (Miller and Auchus 2011).

Action of Aldosterone Aldosterone exerts its effect on different tissues such as the heart, brain vessels, and adipose tissue (Zennaro et al. 2009), but its classic targets are epithelial tissues such as the kidney, the colon and the salivary and sweet glands. In the kidney, aldosterone regulates sodium reabsorption and potassium excretion, accompanied by passive water reabsorption in the distal nephron. An increase in aldosterone level leads to an increase in blood volume and therefore an increase in blood pressure. Aldosterone acts by binding to the mineralocorticoid receptor (MR), a ligand binding transcription factor. Aldosterone binding leads to MR translocation into the

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nucleus where it binds to specific response elements, inducing the expression of specific target genes. The MR, encoded by the NR3C2 gene, has the same affinity for cortisol and aldosterone, but the plasma concentration of cortisol is 100- to 1000-fold higher than plasma aldosterone concentration, which would suggest that MR is occupied only by cortisol. To ensure aldosterone binding to MR, aldosterone target tissues express the 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2), which converts cortisol to cortisone, reducing the intracellular concentration of cortisol. This enzyme is expressed in some aldosterone target tissues such as the kidney, colon, salivary, and sweat glands (Farman and Rafestin-Oblin 2001); in tissues that do not express the HSD11B2, MR is a high affinity receptor for cortisol.

Regulation of Aldosterone Biosynthesis The regulation of aldosterone biosynthesis is divided into two key events in steroidogenesis (Hattangady et al. 2012): (1) the “early regulatory step” occurs few minutes after stimulation and involves an increase of the expression and phosphorylation of StAR protein, resulting in the rapid transfer of cholesterol into the mitochondria where it is converted into pregnenolone leading finally to an increase of aldosterone biosynthesis (Cherradi et al. 1998; Arakane et al. 1997); (2) the “late regulatory step” occurs hours to days after stimulation and is mainly due to the increase of expression of enzymes involved in aldosterone biosynthesis (Bassett et al. 2004). Aldosterone biosynthesis is mainly regulated by angiotensin II (AngII), potassium (K+), and ACTH (Connell and Davies 2005). However, there are also local paracrine and autocrine regulatory mechanisms of aldosterone production (Lefebvre et al. 2019).

Regulation by Angiotensin II One of the main regulators of aldosterone biosynthesis is the renin-angiotensin system. Renin is released from the renal juxtaglomerular apparatus in response to different stimuli: (1) a decrease in renal perfusion pressure detected directly by intrarenal baroreceptors, leading to a decrease in intracellular Ca2+ concentration and an increase in renin secretion. Conversely, an increase in renal perfusion inhibits renin secretion; (2) a variation in the sodium load of the macula densa; (3) a stimulation of the sympathetic system: the juxtaglomerular cells are innervated by sympathetic nerve fibers. Activation of the sympathetic renal nerves and stimulation of β-adrenergic receptors increase renin secretion. The initial step is the synthesis of angiotensinogen in the liver. The angiotensinogen is converted to angiotensin I under the action of the renin (Patel et al. 2017). Angiotensin I is then converted to AngII by the angiotensin converting enzyme (ACE), which is released ubiquitously from endothelial cells. AngII has an important effect on vasoconstriction, activation of the sympathetic nervous system, but most importantly, it stimulates aldosterone biosynthesis by the adrenal cortex through binding to AT1R expressed by ZG cells. The AT1R is a seven-transmembrane domain receptor coupled to the Gαq protein. The Gαq protein

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acts by activating phospholipase C, inducing the hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP2) and the generation of inositol triphosphate (IP3) and diacylglycerol (DAG). The binding of IP3 to its receptor (IP3R) located in the endoplasmic reticulum stimulates the release of Ca2+ from the reticulum, thus increasing the intracellular Ca2+ concentration and activating calcium signaling pathway, which involves the activation of calcium/calmodulin-dependent protein Kinase I and II (CAMKI and CAMKII) (Hattangady et al. 2012). AngII also acts by inhibiting the activity of the TWIK-related acid sensitive K+ (TASK) channels and the Na+,K+-ATPase pump, resulting in cell membrane depolarization, opening of voltage-gated calcium channels, Ca2+ entry into the cell, and activation of calcium signaling (Hattangady et al. 2012). The activation of calcium signaling pathway triggers a phosphorylation cascade that leads to a positive regulation of CYP11B2 transcription and aldosterone biosynthesis.

Regulation by Potassium The expression of different types of K+ channels makes K+ the main ionic conductance of ZG cells. As a result, the membrane potential of these cells closely follows the equilibrium potential of K+ over a wide range of extracellular K+ concentrations and ZG cells are considered as sensors of extracellular K+ concentrations and thus very sensitive to small variations in kalemia (Heitzmann et al. 2008). An increase in extracellular K+ concentration induces a decrease in the electrochemical conductance of TASK and TREK (TWIK-related K+ channel) potassium channels, leading to cell membrane depolarization followed by opening of voltage-gated calcium channels and activation of calcium signaling (Bandulik 2017), the main trigger of aldosterone biosynthesis. Regulation by ACTH Under certain conditions, ACTH can also regulate aldosterone biosynthesis. ACTH binds to its receptors, the melanocortin type 2 receptor (MC2R), a 7 transmembrane domain receptor coupled to the Gαs protein. Binding of ACTH to this receptor activates adenylate cyclase (AC), leading to PKA signaling pathway activation. PKA activates StAR either directly by phosphorylating a specific serine (S194 in mice and S195 in human) and thus affecting its function (Baker et al. 2007), or by increasing its expression via the phosphorylation of the transcription factor CREB (cAMP response element binding protein), leading to the increase of cholesterol delivery to the inner mitochondrial membrane. In addition, ACTH increases the expression of other enzymes in the steroidogenic cascade, such as CYP11A1, increasing the amount of precursors for aldosterone biosynthesis (Ruggiero and Lalli 2016; Sewer and Waterman 2003). Role of the β-Catenin in Regulating Mineralocorticoids Biosynthesis β-catenin is a transcription coactivator that induces the recruitment and activation of transcription factors that regulate the expression of the CYP11B2, CYP21A2, and AT1R genes. Although β-catenin is known to interact with the transcription factor TCF/LEF and the CYP11B2 promoter has two TCF/LEF binding sites, β-catenin is

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thought to regulate CYP11B2 expression via nuclear receptors of the NUR family. Indeed, β-catenin induces expression of the NURR1 and NUR77 genes, members of the NGFI-B family, in the H295R human adrenocortical cell line. The NURR1 gene has binding sites for TCF/LEF unlike the NUR77 gene which has four binding sites for AP-1. The β-catenin thus induces their transcription by activating TCF/LEF and c-Jun/c-Fos transcription factors, which are proteins capable of binding to the TCF/LEF and AP-1 response elements, respectively. Unlike the CYP11B2 gene, whose expression is regulated by β-catenin via an indirect mechanism, the expression of the CYP21A2 and AT1R genes is regulated by direct and indirect mechanisms. The CYP21A2 promoter has both TCF/LEF binding sites and response elements for binding transcription factors of the NUR family. Thus β-catenin controls its expression by direct binding to its promoter but also by regulating the expression of NUR family genes. As for the AT1R gene promoter, it has TCF/LEF binding sites allowing direct regulation by β-catenin binding (Berthon et al. 2014).

Regulation by Paracrine/Autocrine Factors The local regulatory mechanisms are likely to modify aldosterone biosynthesis directly, through the binding to specific membrane receptor and activation of second messenger, or indirectly, through the modulation of the adrenal blood flow or by stimulating the secretion of another regulatory factors. The presence of chromaffin cells in adrenal cortex suggests that they may play a role in steroidogenesis. Some factors, co-secreted with catecholamines, have been described to play a role in the paracrine control of the stimulation of aldosterone biosynthesis: these include the arginine vasopressin (AVP), opioid peptides, the vasoactive intestinal peptide (VIP), the pituitary adenylate cyclase activating polypeptide (PACAP), the galanin, the calcitonin gene related peptide (CGRP), and the adrenomedullin. Chromaffin cells produce also ACTH and corticotropin releasing hormone (CRH), suggesting the presence of a local corticotrope axis that could transitorily modulate aldosterone biosynthesis. Endothelin-1, secreted by endothelial cells, is also able to stimulate aldosterone biosynthesis (Nussdorfer et al. 1999). Chromaffin cells are also producing factors that inhibit aldosterone production such as dopamine, somatostatin, neuropeptide Y, or atrial natriuretic peptide (ANP) (Ehrhart-Bornstein et al. 1998). Autocrine growth factors may also play a role in regulating aldosterone biosynthesis: Epidermal growth factor (EGF) is able to stimulate aldosterone synthesis (Kim et al. 1998) whereas transforming growth factor-beta 1 (TGF-β1) has been shown to inhibit it (Gupta et al. 1993). However, the physiological relevance and importance of these different paracrine regulators is still difficult to determine. The role of serotonin (5-HT) in regulating aldosterone biosynthesis has been investigated more extensively (Lefebvre et al. 2019). In mast cell–deficient mice, sodium restriction leads to the activation of adrenal mast cells, possibly contributing to increase aldosterone biosynthesis in this condition whereas no effect is observed in normal salt diet (Boyer et al. 2017). In normal adrenal gland, degranulation of intraadrenal mast cells leads to the release of 5-HT, activation of 5-HT type

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4 receptors, and stimulation of aldosterone biosynthesis (Lefebvre et al. 2001). In clinical trials, the use of 5-HT reuptake inhibitors and of 5-HT agonist increases plasma aldosterone concentrations; however, the lack of specific 5-HT receptor antagonists hampered to evaluate the role of 5-HT in regulating aldosterone biosynthesis (Lefebvre et al. 2019). Leptin, identified as an obesity-associated hormone and produced by adipose tissue, is involved in the regulation of appetite and energy expenditure but also inflammation, insulin and glucose homeostasis, sympathetic activity, and the control of blood pressure (Faulkner and Belin de Chantemele 2019). Several studies have shown an increase of aldosterone levels in obese patients in association with visceral adipose mass. In mice, leptin infusion results in an increase in plasma aldosterone levels in lean as in diet-induced obese mice. Since then, the role of leptin in regulating aldosterone biosynthesis has been explored in different mouse models. In vitro data revealed a dose-dependent effect of leptin on aldosterone release through a calcium-dependent mechanism (Huby et al. 2015).

Diagnostic and Treatment Outcome of Patients with Primary Aldosteronism In 1955, Jerome Conn reported, for the first time, the case of a 34-year-old woman complaining of 7 years of episodic muscle weakness, muscle spasms, and cramps in her hands. Biologically, she presented hypokalemia and alkalosis. In absence of signs of cortisol excess, excessive mineralocorticoid secretion was suspected and confirmed in the urine of the patient. Surgical exploration revealed the presence of a 4 centimeters tumor on one adrenal gland (Conn 1955). PA is characterized by hypertension, high aldosterone concentration, suppressed plasma renin both leading to higher aldosterone to renin ratio, and often hypokalemia. Elevated aldosterone levels in patients with PA are associated with an increased cardiovascular risk compared to patients with essential hypertension (Savard et al. 2013). A higher prevalence of cardiovascular events such as left ventricular hypertrophy, coronary heart disease, myocardial infarction, or atrial fibrillation have been reported in patients with PA compared to patients with essential hypertension paired for age, sex, and blood pressure (Savard et al. 2013). This increase in cardiovascular morbidity is due to cardiac remodeling and myocardial fibrosis, independently of blood pressure (Rossi et al. 1997; Freel et al. 2012). The excessive and autonomous aldosterone production is due mainly to the presence of a unilateral aldosterone producing adenoma or bilateral adrenal hyperplasia.

Diagnosis of Primary Aldosteronism Patients with PA usually have no specific symptoms. Excess of aldosterone leads to hypertension, which may be associated with hypokalemia. The mean age at diagnosis varies between 45 and 55 years (Rossi et al. 2006). Furthermore, the diagnosis of

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hypertension was usually made 5–10 years before the diagnosis of PA, indicating a significant delay between the onset of the disease and its diagnosis. Recent publication of guidelines for the diagnosis, management, and treatment of hypertension recommend to screen for PA patients with sustained blood pressure above 150/100 mmHg for three measurements on different days, hypertension (140/90 mmHg) resistant to three antihypertensive drugs, hypertension associated with the presence of an adrenal incidentaloma, of sleep apnea, or of a familial history of early-onset hypertension or stroke at a young age (before 40 years), and all firstdegree hypertensive relatives of patients with PA (Funder et al. 2016). The diagnosis is made in the presence of high aldosterone, suppressed renin levels, an increased aldosterone to renin ratio, and in some cases hypokalemia, and is confirmed by a suppression test. Four different tests are commonly used to confirm the diagnosis of primary aldosteronism: the oral sodium loading test, the saline infusion test, the fludrocortisone suppression test, and the captopril challenge test. To avoid false positive results, it is recommended, during the confirmatory test, to use the pharmacological agents with minimal or no effects on the renin-angiotensinaldosterone system to control blood pressure. Once PA has been diagnosed, patients undergo adrenal CT with two objectives: (1) rule out a malignant lesion by the size of the tumor and the noninjected density, and (2) point to the etiology of primary aldosteronism by studying the appearance of the adrenals. A single adrenal nodule measuring 10–20 mm in long axis, with the absence of lesion on the contralateral adrenal gland, points more toward a unilateral cause, whereas the presence of two normal adrenals or two hyperplastic adrenals points toward a bilateral cause. However, CT cannot be used to conclude whether the disease is unilateral or bilateral. In fact, if the decision to perform adrenalectomy was based only on CT, 20% of patients with a lateralized form would not have undergone surgery and surgery would have been offered to 25% of patients with a bilateral form (Young et al. 2004). The adrenal vein sampling (AVS) is considered to be the reference test to distinguish unilateral from bilateral aldosterone hypersecretion in PA. Blood is collected from both adrenal veins and the inferior vena cava, below the level of the renal vein. The concentrations of aldosterone and cortisol are then measured. The success of the catheterization of the adrenal veins is assessed by determining the selectivity index (SI), defined as the ratio of the cortisol concentration in the adrenal vein to that of the peripheral vein. This SI must be
2.0 for a nonstimulated AVS. The lateralization index (LI) is defined by the ratio of the aldosterone to cortisol concentration in the dominant adrenal vein to that of the nondominant adrenal vein. Lateralization indices of >2 for unstimulated and >4 for stimulated sampling are recommended by the endocrine society (Funder et al. 2016). However, higher LI correlated with significantly higher clinical and biochemical success rates after adrenalectomy (Williams et al. 2017). However, AVS is an invasive procedure that can be performed only in specialized center, the development of molecular imaging methods could facilitate the identification of lateralized form of PA. Recently, different studies compared the potency of

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two diagnostic imaging procedures with AVS to identify the side of aldosterone lateralization (Wu et al. 2023a; Gao et al. 2023). The first one takes benefit of the expression of the C-X-C chemokine receptor type 4 (CXCR4) in APA to use the positron emission tomography (PET) tracer [68Ga]Ga-pentixafor PET/CT, a specific ligand for CXCR4. Fifty patients diagnosed with PA and ten with nonfunctional adrenal adenoma were enrolled in a prospective study to evaluate its diagnosis efficiency (Gao et al. 2023). The presence of an adrenal adenoma was confirmed by CT on 49 patients, 39 diagnosed as APA and 10 with a nonfunctional adenoma; 11 patients were diagnosed as nodular hyperplasia. The diagnosis was confirmed by performing CYP11B2 immunohistochemistry on 56 adrenals among which 37 presented an APA, 9 an idiopathic adrenal hyperplasia, and 10 a nonfunctional adrenal adenoma. Forty out of forty-three functional nodules (93,0%) showed positive findings and 11 out of 13 lesions without functional nodules (84,6%) showed negative findings on [68Ga]Ga-pentixafor PET/CT, and its detection ability was much better for nodules with a diameter >1 cm. These data suggest that [68Ga]Ga-pentixafor PET/CT could be a promising surgical decision-making tool for patient with PA (Gao et al. 2023). The second study investigated the potential benefit of using the metomidate, a potent inhibitor of CYP11B1 and CYP11B2 enzymes, in the diagnosis of unilateral PA. The metomidate can be 11CH3 labeled and then used as a PET radiotracer combined with high-resolution CT (MTO) to detect adrenocortical tumors expressing these two enzymes (Wu et al. 2023a). Among the 128 patients presented in the study, MTO and AVS classified, respectively, 52% (67/128) and 45% (58/128) of patients as having a high probability of unilateral PA. Concordance of MTO and AVS was observed in 39 patients out of 128 (30%) and 47/128 patients (37%) were diagnosed by MTO or AVS alone. Thus, a total of 86 patients out of 128 (67%) were classified as having unilateral PA and 78 underwent surgery; 69 (88%) achieved complete biological success and 24 (31%) complete clinical success; MTO predicts the biochemical success of adrenalectomy with an accuracy of 72.7% and 65.4%, respectively, and the AVS with an accuracy of 63.6 and 61.5% respectively. Even if MTO was not significantly superior, this study shows that it would be possible to diagnose unilateral APA using MTO when AVS is unsuccessful, not available, or not wished by the patient (Wu et al. 2023a).

Treatment of Primary Aldosteronism In case of lateralized form of PA, the optimal treatment is adrenalectomy, which results in biological recovery and clinical recovery or improvement in terms of blood pressure. In case of bilateral forms, or if the patient refuses surgery, spironolactone, a MR antagonist, is administrated as first-line treatment, with therapeutic adjustment in the case of insufficient efficacy or poor tolerance. To control blood pressure, other antihypertensive drugs can be added: diuretics such as amiloride, which blocks the sodium channel ENaC, angiotensin II receptor antagonists, and calcium channel blockers.

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Outcome of Patients with Unilateral Form of Primary Aldosteronism The surgical treatment of patients with unilateral form of PA should lead to normalization of plasma aldosterone concentration; the persistence of hyperaldosteronism after adrenalectomy suggests that the patients have a bilateral form of the disease, with asymmetrical secretion, rather than a unilateral form. An international multicentric consensus for classifying surgical outcome and follow-up of patients with unilateral primary aldosteronism, the primary aldosteronism surgical outcome (PASO) study has created consensus criteria for clinical and biochemical outcomes and follow-up of adrenalectomy for unilateral PA and has applied these criteria to determine the frequency of remission and to identify preoperative determinants of successful outcome (Williams et al. 2017). Six outcomes were used regarding the clinical (complete, partial, and absent success) and biochemical (complete, partial, and absent success) outcomes. The clinical outcomes were evaluated for 705 patients from different centers, and for 699 of these patients the biological data were available and outcomes could also be evaluated. A number of 257 out of 705 patients (37%) presented a complete clinical success and 334 a partial success (47%) whereas complete biochemical success was observed in 656 out of 699 patients (94%). Younger patients and female patients have a higher chance of complete clinical success and clinical benefit whereas lower levels of complete clinical success was associated with higher levels of preoperative medication (Williams et al. 2017). The PASO consensus allows to establish feasible criteria for the classification of outcomes of adrenalectomy for the treatment of unilateral form of PA. Even if all patients will not benefit to complete clinical success and clinical benefit, screening for primary aldosteronism should be done in all patients following the Endocrine Society guidelines as almost all patients have complete biochemical success. A recent study analyses clinical data of 12 patients with APA without biochemical success after adrenalectomy and investigates the histological and genetic characteristics of their adrenal glands (Hacini et al. 2021). All these patients have been recruited within a single referral center for hypertension. Higher systolic and diastolic blood pressure, higher plasma aldosterone concentration, lower renin levels, lower plasma K+ concentrations, and higher number of antihypertensive drugs were observed in patients with partial or absent biochemical success compared with patients with complete biochemical success. The proportion of males was higher but not significant. In 10 out of 12 cases, 1 functional adenoma of at least 5 mm and expressing CYP11B2 was found in the resected adrenal confirming the diagnosis of APA. In the two last adrenals, one presented a CYP11B2 negative macronodular hyperplasia and three APCC and the other one exhibited a nonexpressing CYP11B2 adenoma and two APCC. Somatic mutations were found in fresh frozen adenoma from four patients, one harboring a KCNJ5 mutations and three CACNA1D mutations. Next-generation sequencing (NGS) performed on CYP11B2-positive adenoma allowed to identified mutations in CACNA1D in three patients, mutations in KCNJ5 in two patients, and a mutation in ATP1A1 in one patient. Interestingly, somatic mutations in APA driver genes were found in all CYP11B2 expressing nodules. These findings confirm the lateralization of aldosterone production in the

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majority of the patients investigated in this study (10 out of 12), suggesting asymmetric bilateral aldosterone overproduction in the context of bilateral adrenal hyperplasia. For the two last patients the absence of an aldosterone producing adenoma in the resected adrenal suggests a misdiagnosis of PA subtyping. The identification of somatic mutations in adrenal gland of those patients suggest common mechanisms underlying APA and bilateral adrenal hyperplasia (Hacini et al. 2021).

Etiology and Genetic of Primary Aldosteronism Sporadic Forms During the last decade, using exome sequencing approaches, recurrent somatic mutations have been identified in APA. These mutations affect essentially genes coding for ion channels (KCNJ5, CACNA1D, CLCN2, and SLC30A1) (Choi et al. 2011; Azizan et al. 2013; Scholl et al. 2013) and ATPases (ATP1A1 and ATP2B3) (Azizan et al. 2013; Beuschlein et al. 2013), regulating intracellular ion homeostasis and membrane potential (Fig. 1). Mutations have also been identified in CTNNB1 gene encoding β-catenin, in GNAQ/11 (Zhou et al. 2021) and in CADM1 (Wu et al. 2023b).

Mutations Affecting Cell Membrane Potential KCNJ5 encodes the GIRK4 (G protein activated inward rectifier potassium channel 4) potassium channel. All the KCNJ5 mutations identified in APA, as well as the various germline mutations identified in FH-III (see below), are located in exon 2; the two most frequent are the p.Gly151Arg and p.Leu168Arg but other mutations located nearby have also been described (p.Gly151Glu, p.Thr158Ala, p.Glu141Gln, Ile157Ser, and delIle157 (Gomez-Sanchez 2014). All these mutations are located in or near the selectivity filter that allows the selective entry of K+ and affect the ion selectivity of the channel with an increase in Na+ conductance (Fig. 1a). This increase in Na+ conductance leads to an influx of Na+ into the cell and consequently to chronic depolarization of the plasma membrane, followed by opening of voltagegated Ca2+ channel, activation of calcium signaling, and increase of aldosterone biosynthesis. Overexpression of different mutations of this channel in an adrenal cortex cell line results in increased expression of CYP11B2 and aldosterone biosynthesis, as well as increasing two of the transcriptional regulators of CYP11B2, NURR1 (encoded by NR4A2) and NOR-1 (encoded by NR4A3) (Monticone et al. 2012; Oki et al. 2012). The α1 subunit of the Na+/K+-ATPase, encoded by the ATP1A1 gene, is a member of the P-type ATPase family and is composed of ten transmembrane domains, M1 to M10, with intracellular N- and C-terminal regions. The mutations in the ATP1A1 gene affect amino acids of the M1, M4, and M9 transmembrane helices. They led to a loss of pump activity and a reduction in its affinity for K+ as well as an inward leak of Na+, inducing cell membrane depolarization (Beuschlein et al. 2013) (Fig. 1a). Electrophysiological studies performed on primary adrenal adenoma cells carrying ATP1A1 mutations revealed inappropriate cell membrane

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depolarization (Beuschlein et al. 2013). Interestingly, expression of mutated α1 subunit of the Na+/K+-ATPase in adrenocortical cell line does not induce an increase in intracellular Ca2+ concentrations, but is responsible for intracellular acidification, due to proton leakage, leading to increased aldosterone production (Stindl et al. 2015). The chloride channel CLC-2, encoded by the CLCN2 gene, is a two-pore homodimeric, voltage-gated chloride channel. Mutations in this gene were first identified to cause germline early-onset PA (see below). Somatic mutations were found in a few number of APA (Rege et al. 2020). The mutations lead to an increased activation of the channel, leading to outflow of Cl from the cell, cell membrane depolarization, opening of voltage-gated Ca2+ channel, activation of calcium signaling, and stimulation of aldosterone biosynthesis (Fig. 1a).

Fig. 1 Pathogenic mechanisms of aldosterone biosynthesis. (a) Mutations in the GIRK4 potassium channel, the α1 subunit of the Na+,K+-ATPase, the CLC-2 chloride channel or the ZnT1 zinc transporter induces modification of the ionic equilibrium, responsible for cell membrane depolarization, leading to the opening of voltage-gated calcium channels, calcium entry into the cells, activation of calcium signaling resulting in increased CYP11B2 expression and aldosterone production. (b) Mutations in the Cav1.3 or Cav3.2 calcium channels or in the PMCA3 calcium pump directly induce calcium entry into the cells, with activation of calcium signaling leading, as described above, to an increase in CYP11B2 expression and aldosterone production. (c) The concomitant presence of mutations in Gαq/11 (yellow star) and β-catenin (green star) proteins induces an increase in the expression of the LHCGR receptor, as well as activation of cAMP/PKA signaling, leading to an increase in CYP11B2 expression and aldosterone biosynthesis. AC Adenylate cyclase, cAMP cyclic adenosine monophosphate, PKA protein kinase A

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Mutations Affecting Directly Intracellular Calcium Concentration Under physiological conditions, the PMCA3 pump, encoded by the ATP2B3 gene, extrudes cytosolic Ca2+ in exchange for two H+. The mutations in PMCA3 affect highly conserved residues located in the M4 transmembrane helix (deletion of an amino acids stretch between leucines 425 and 433), which is thought to be involved in the interaction with Ca2+ (Beuschlein et al. 2013; Fernandes-Rosa et al. 2014). Cells expressing a mutant of the PMCA3 pump exhibited a reduced capacity to extrude Ca2+, suggesting that mutations affecting this pump led to a loss of function (Fig. 1b). In addition, they induce increased Ca2+ influx probably due to the opening of Ca2+ channels activated by depolarization or Ca2+ leakage by the mutated pump. All these mutations induce an increase in intracellular Ca2+ concentrations, followed by activation of calcium signaling and therefore an increase in aldosterone biosynthesis. The CACNA1D gene encodes the α1 subunit of the Cav1.3 L-type voltage-gated calcium channel. The channel is composed of four domains (I to IV), each of which has six transmembrane segments (S1 to S6). The segment S4 is involved in voltage sensitivity, while segments S5 and S6 and the loop between them form the pore. Mutations in this gene induce a gain of function allowing channels to open at more negative potentials, following a shift in the activation threshold potential toward more negative potentials or a reduction in channel inactivation. This leads to an increase in intracellular calcium concentration and therefore activation of calcium signaling (Azizan et al. 2013; Scholl et al. 2013) (Fig. 1b). Less frequent mutations that play an important role in APA development have been identified in the CTNNB1, GNA11/GNAQ, PRKACA, CADM1, and SLC30A1 genes. The CTNNB1 gene codes for the β-catenin. As previously discussed, this protein is a central component of the Wnt/β-catenin signaling pathway, which plays an essential role in adrenal cortex development, ZG differentiation, and steroid hormone biosynthesis (El Wakil and Lalli 2011). These mutations have been found in 2–5% of APA and are more common in females (Scholl et al. 2015b; Teo et al. 2015). The mutations are located specifically in exon 3, which encodes a region important for the regulation of the protein activity. Recently, gain of function mutations in GNA11 or its close homologue GNAQ, coding for small G proteins, have been identified in APA carrying CTNNB1 mutations (Zhou et al. 2021) (Fig. 1c). Interestingly, these mutations were found in patients developing hyperaldosteronism at puberty, pregnancy, or menopause. In ZG, the small GαQ/11 proteins are involved in the response of cells to AngII, inducing activation of calcium signaling and aldosterone biosynthesis. In vitro studies performed on adrenocortical cells have demonstrated the additive effect of GNA11 and CTNNB1 mutations on aldosterone secretion and the modulation of specific genes expression. Among the differentially expressed genes in APA carrying the double mutations, the expression of the LHCGR gene, coding for the luteinising hormone (LH) and chorionic gonadotropin (HCG) receptor, was found to be increased. The overexpression of this receptor in tumors carrying a double CTNNB1 and GNAQ/11 mutation could explain why these patients develop primary aldosteronism at puberty, pregnancy, or menopause (Zhou et al. 2021).

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Mutations in PRKACA, coding for the α catalytic subunit of the protein kinase A, have been described in two APA from a cohort of 122 patients. One of the two mutations, p.Leu206Arg, has previously been described in cortisol producing adenoma whereas the second mutation, p.His88Asp, has never been described before. The p.Leu206Arg mutation induces an increase in the enzymatic activity of the protein and was associated with predominant expression of CYP11B1 in the tumor, while the p.His88Asp mutation causes a significant decrease in the enzymatic activity of PKA. After adrenalectomy, both patients showed reduced number of antihypertensive medications and normalized serum potassium levels (Rhayem et al. 2016). Mutations in CADM1, encoding the Gj alpha-1, formerly connexin 43, was recently found in six patients, three harboring the p.Gly379Arg mutation and three the p.Val380Arg mutation. High expression of CYP11B2 and low expression of CYP11B1 were found in the six APA. These mutations are located within the membrane dimerization domain of the protein. Functional analysis in H295R cells revealed an increase of CYP11B2 expression and aldosterone biosynthesis when mutants of CADM1 were expressed. This was associated with an inhibition of gap junction communication (Wu et al. 2023b). Recurrent in frame deletions in SLC30A1 gene (p.Leu51_Ala57del and p. Leu49_Leu55del), coding for the zinc efflux transporter ZnT1, was identified in five APA. The mutations are located in transmembrane domain II, in close proximity of the zinc binding site. Functional analyses of these mutants in adrenocortical cell line revealed abnormal Na+ conductivity, leading to cell membrane depolarization, opening of voltage-gated Ca2+ channel resulting in an increase of intracellular Ca2+ concentration, which stimulated CYP11B2 mRNA expression and aldosterone biosynthesis (Rege et al. 2022) (Fig. 1a).

Familial Forms Although the majority of the cases are sporadic, PA can be transmitted as a Mendelian trait in familial forms of the disease. Familial forms of PA account for 1–5% of cases and are inherited as an autosomal dominant trait. Four different forms have been described, depending on their underlying genetic defect.

Familial Hyperaldosteronism Type I (FH-I) FH-I, also known as glucocorticoid remediable aldosteronism (GRA), is an autosomal dominant disease with an estimated prevalence of 1% in adult patients (Mulatero et al. 2011; Stowasser and Gordon 2000) and around 3% in pediatric cohorts (Pallauf et al. 2012). It is characterized by a severe hypertension at young age associated to suppressed renin, hypokalemia, and production of hybrid steroids: 18-hydroxycortisol and 18-oxocortisol. Interestingly, aldosterone biosynthesis is blocked by glucocorticoid treatment (Sutherland et al. 1966). From a molecular point of view, FH-I is due to the presence of a hybrid gene in which the CYP11B2 coding sequence is under the control of the CYP11B1 promoter,

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leading to an ectopic expression of the aldosterone synthase in the zona fasciculata. This is due to an unequal crossing-over between the adjacent and highly homologous genes CYP11B2 and CYP11B1 (Lifton et al. 1992). Aldosterone is then regulated by ACTH rather than angiotensin II, resulting in circadian aldosterone production similar to cortisol biosynthesis. In the adult hypertensive population, FH-I is thought to represent 0.5–1.0% of PA with an equal male/female distribution (Mulatero et al. 2011). However, a recent study reports a 3% prevalence of the CYP11B1/CYP11B2 chimeric gene in hypertensive children (Aglony et al. 2011). Different chimeric genes have been described in different pedigrees, suggesting that these abnormalities occur independently (Dluhy and Lifton 1995). FH-I is a rare inherited disorder associated with variable degrees of hypertension between but also within pedigrees, sometimes misdiagnosed as essential hypertension. Nevertheless, FH-I is associated with high morbidity and mortality at an early age. The diagnosis of FH-I should therefore be considered in the presence of certain indicators: a familial history of hypertension and the occurrence of a cerebral hemorrhage before the age of 50, hypertension diagnosed at a young age (before 20 years), as well as hypertension that is difficult to control and hypokalemia (Funder et al. 2008, 2016). Measurement of 18-oxocortisol and 18-hydroxycortisol in urine and dexamethasone suppression test can lead to erroneous diagnoses (Fardella et al. 2001). Genetic diagnosis of FH-I is performed by Southern blot or long-range PCR (Funder et al. 2008). The Endocrine Society recommends to treat patients with FH-I with a synthetic glucocorticoid that have a longer duration of action than hydrocortisone (Funder et al. 2008). Administration of exogenous glucocorticoids suppresses ACTH secretion, thereby reducing aldosterone levels. However, it is important to use the minimum effective dose of glucocorticoids to normalize blood pressure and kalemia in order to avoid suppression of circadian cortisol regulation and the development of iatrogenic Cushing’s syndrome (Funder et al. 2008). The addition of a mineralocorticoid inhibitor may be considered when blood pressure control is unsatisfactory. In children, it is preferable to use eplerenone to avoid the secondary effects of glucocorticoid on growth or the antiandrogenic effects of spironolactone (Funder et al. 2008). Patients with FH-I display an increased cardiovascular risk, increased left ventricular wall thickness, and reduced diastolic function, before the onset of hypertension (Stowasser et al. 2005). They present also high circulating levels of interleukin 6, suggesting a role for inflammation in cardiovascular damage independent of blood pressure (Staermose et al. 2009).

Familial Hyperaldosteronism Type II (FH-II) FH-II was first described in 1991 by Gordon et al. as a familial form of hyperaldosteronism non suppressible by the glucocorticoid with an autosomal dominant transmission (Gordon et al. 1991). The estimated prevalence of FH-II is comprised between 1.2% and 6% in adult patients with PA (Mulatero et al. 2011; Stowasser and Gordon 2000). FH-II is indistinguishable from sporadic forms, can present as

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aldosterone producing adenoma or bilateral adrenal hyperplasia, and is diagnosed on the basis of at least two affected members in the family (Mulatero et al. 2011). In certain families, a link between FH-II and the chromosomal region 7p22 has been identified (Lafferty et al. 2000); however, sequencing of a number of candidate genes present in this region, including RBaK (retinoblastoma-associated Kruppel-associated box gene), PMS2 (postmeiotic segregation increased 2), GNA12 (guaninenucleotide-binding protein α-12), RPA3 (replication protein A3), ZNF12 (zinc finger 12), GLCCI1 (glucocorticoid-induced transcript 1), FSCN1 (Fascin 1) and PRKAR1B (cAMP-dependent protein kinase type I β-regulatory subunit), and of other candidate genes, notably CYP11B2, ATR1, and the tumor suppressor p53, did not identify any mutations responsible for FH-II (Stowasser and Gordon 2000; Jeske et al. 2008; Medeau et al. 2005; Davies et al. 1997). Recently, whole exome sequencing approaches allowed to identify the gene responsible for FH-II. Gain of function mutations in CLCN2 gene, coding for the chloride channel CLC-2, have been identified in patients with FH-II and early-onset PA (Fernandes-Rosa et al. 2018; Scholl et al. 2018). A heterozygous de novo p.Gly24Asp mutation was identified in a patient diagnosed at the age of 9 with severe hypertension, high plasma aldosterone level, and profound hypokalemia. This mutation affects a highly conserved region of the channel, leading to its constitutive opening. Expression of this mutated channel in adrenocortical cells results in increased chloride currents, cell membrane depolarization, opening of voltage-gated calcium channel, and stimulation of aldosterone biosynthesis (Fernandes-Rosa et al. 2018) (Fig. 1a). A germline p.Arg172Gln mutation was identified in affected members of a large Australian family with FH-II previously described by Stowasser et al. (Stowasser et al. 1992). The same mutation was identified in three other cases of early onset of PA, one in a familial context, the three others occurring de novo. Moreover, four other germline variants of CLCN2, p.Met22Lys, p.Tyr26Asn, p.Lys362del, and p. Ser865Arg were identified in four unrelated patients with PA. The mutated channels exhibit a gain of function, with higher opening probabilities at the resting potential of the ZG (Scholl et al. 2018). These results indicate that the CLCN2 gene should be included in the panel of genes that are screened in patients who develop early primary hyperaldosteronism and in families with a diagnosis of FH-II. The mutations may be located in different domains of the CLC-2 channel and therefore affect the function of the channel to different degrees, which explains the phenotypic heterogeneity and the presence of relatively benign cases.

Familial Hyperaldosteronism Type III (FH-III) FH-III was first described in 2008 in a father and his two daughters with severe hypertension at young age (between 4 and 7 years), resistant to treatment and associated with hypokalemia (Geller et al. 2008). They presented characteristic features of hyperaldosteronism, elevated levels of the hybrid steroids 18-oxocortisol and 18-hydroxycortisol, and aldosterone production was not suppressible by glucocorticoids. They exhibited massive hyperplasia of the adrenal

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cortex and bilateral adrenalectomy was performed to control their blood pressure (Geller et al. 2008). Recently, exome sequencing has allowed to identify the genetic origin of FH-III with the identification of the p.Thr158Ala mutation in the KCNJ5 gene (Choi et al. 2011). Similar to the somatic mutations identified in APA, this mutation is located near the GYG motif involved in K+ selectivity of the GIRK4 channel. Functional studies have shown that this mutation results in a loss of selectivity for K+ and a greater influx of Na+ into the cytoplasm resulting in cell membrane depolarization, opening of voltage-gated Ca2+ channels, leading to an increase of intracellular Ca2+ concentration, as well as activation of calcium signaling pathway, resulting in an induction of steroidogenic enzyme expression and an increase in steroid production (Oki et al. 2012) (Fig. 1a). Since this discovery, different germline mutations in KCNJ5 have been described in families with FH-III, however the severity of PA depends on the type of KCNJ5 mutation. Patients with the mutations p.Gly151Arg (identical to one of the recurrent mutations described in APA), p.Thr158Ala, and p.Ile157Ser all present a severe PA phenotype with early and treatment-resistant hypertension whereas patients from three families with FH-III and carrying the KCNJ5 p.Gly151Glu mutation as well as patients carrying the KCNJ5 p.Tyr152Cys mutations show a more moderate phenotype (Choi et al. 2011; Charmandari et al. 2012; Monticone et al. 2013; Mulatero et al. 2012; Scholl et al. 2012). In vitro studies have shown that similar to other mutations, the p.Gly151Glu mutation alters the selectivity of the GIRK4 channel, resulting in Na+ influx and cell membrane depolarization. Electrophysiological studies have shown that the p.Gly151Glu mutation confers a higher Na+ conductance than other mutations, inducing rapid Na+-dependent cell death, which could, in vivo, limit adrenal cortex cell proliferation and the severity of hyperaldosteronism. This could explain the more moderate phenotype of families carrying the p. Gly151Glu mutation and the absence of adrenal hyperplasia (Mulatero et al. 2012; Scholl et al. 2012). The notion of a strict correlation between genotype and phenotype has recently been improved by the description of a patient with sporadic hyperaldosteronism carrying a germline heterozygous p.Gly151Arg mutation, who developed polyuria at the age of 18 months and hypertension with hypokalemia at 4 years. His hyperaldosteronism was successfully treated for seven years with spironolactone without adrenal enlargement (Adachi et al. 2014).

Familial Hyperaldosteronism Type IV (FH-IV) FH-IV is a form of FH not suppressible by glucocorticoids, which have been attributed to germline mutations in the CACNA1H gene. Despite autosomal dominant inheritance of the mutation, clinical differences can be observed between family members, indicating incomplete penetrance of the disease (Scholl et al. 2015a). CACNA1H encodes the α1 subunit of the Cav3.2 voltage-gated T-type calcium channel. The structure of this subunit consists of four homologous domains named I to IV, each containing six transmembrane helices (S1 to S6). A recurrent p.Met1549 mutation in CACNA1H has been identified for the first time in five patients with early-onset hypertension due to PA and in their relatives. The mutation affects the electrophysiological properties of the channels, inducing a

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shift in the sensitivity of the channel toward a more negative current and a change in its inactivation properties, which trigger the channel to open at less depolarized membrane potentials, allowing activation of calcium signaling in the absence of stimulation (Fig. 1b). The expression of the Cav3.2 channel carrying the p. Met1549Val mutation in adrenal cortical cells induced an increase in CYP11B2 expression and aldosterone biosynthesis compared with cells transfected with the wild-type Cav3.2 channel (Scholl et al. 2015a). In a similar study, Daniil et al. identified four germline mutations in the CACNA1H gene in PA patients with different phenotypic presentations (Daniil et al. 2016). Two of the variants, p.Val1951Glu and p.Pro2083Leu, identified in this study are located in the cytoplasmic C-terminal region of the channel. The p. Ser196Leu variant is located in the S4 segment of the domain I of the channel, while the p.Met1549Ile mutation is located in the S6 segment of domain III. The p. Met1549Ile mutation, affecting the same amino acid identified by Scholl et al. in patients with FH-IV, was identified in a patient with early onset of PA and a complex developmental disorder. The p.Ser196Leu and p.Pro2083Leu mutations of the Cav3.2 channel were identified in two patients with FH-II and the p.Val1951Glu mutation in a patient with APA. Electrophysiological analysis of these different mutants revealed significant changes in calcium current properties for all mutants, suggesting a gain-of-function phenotype. Furthermore, expression of different Cav3.2 channel mutants in the H295R_S2 cell line results in increased steroidogenic enzyme expression and aldosterone production following potassium stimulation (Daniil et al. 2016).

Primary Aldosteronism, Seizures, and Neurological Abnormalities (PASNA) Germline mutations in CACNA1D have been identified in subjects with early-onset hypertension, hyperaldosteronism, and cerebral palsy (Scholl et al. 2013). The first subjects presented hypertension from birth, biventricular hypertrophy, high aldosterone levels, a high aldosterone to renin ratio, and hypokalemia at one month. This subject developed also seizures, apparent cerebral palsy, cortical blindness, and complex neuromuscular abnormalities. There was no familial history of early-onset hypertension or seizures. Interestingly, the use of amlodipine, a calcium blocker, normalized blood pressure. A p.Gly403Asp germline mutation in the CACNA1D gene was identified. Somatic mutations in the same residue in the CACNA1D gene (p.Gly403Arg) have been identified in APA (Azizan et al. 2013; Scholl et al. 2013; Fernandes-Rosa et al. 2014). Functional analysis of the channel carrying this mutation showed activation at lower potentials than the wild-type channel (Scholl et al. 2013). The second case described was a subject, diagnosed at birth with cerebral palsy and complex seizures, presented with arterial hypertension, elevated plasma aldosterone, and suppressed plasma renin activity at five years; hypokalemia was reported at eight years. No abnormalities of the adrenal gland were detected by CT scan and no familial history of early-onset hypertension or seizures reported. A de novo germline mutation p. Ile770Met also found in APA was identified. Functional analysis, in H295R cells, of the channel carrying these mutations revealed maximum current amplitudes for less depolarized potentials (Scholl et al. 2013).

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These germline mutations are thought to induce an increase in intracellular Ca2+ concentrations resulting in activation of calcium signaling pathway and an increase in aldosterone production.

Remodeling of Adrenal Glands with APA Zona Glomerulosa Hyperplasia Adrenals with APA displays remodeling of the cortex with increased nodulation and decreased vascularization compared with normal adrenal (Boulkroun et al. 2010). ZG hyperplasia is also described in the majority of adrenals with APA showing CYP11B2 expression, at the mRNA level detected by in situ hybridization, in the ZG, which is not observed in control adrenals (Boulkroun et al. 2010), suggesting that the presence of an APA induces remodeling of the adjacent tissue or that APA develop in a remodeled adrenal. However, study of aldosterone synthase expression by immunohistochemistry in adrenals with APA shows expression in APCC, but no continuous expression in the ZG (Nishimoto et al. 2015). These different results suggest either a problem related to the sensitivity of the antibodies used in immunohistochemistry, or a negative feedback mechanism that would induce post-trasncriptional or post-translational regulation decreasing the synthesis of the enzyme in the adrenal but not in the APCC. It is important to note that APCC themselves carry somatic mutations involved in autonomous aldosterone synthesis (see below).

Presence of Secondary Nodules It has also been shown that adrenals with APA often have secondary nodules, either expressing aldosterone synthase or not. Secondary nodules that express aldosterone synthase present also mutations in the APA driver genes KCNJ5 and CACNA1D (Fernandes-Rosa et al. 2014; Nanba et al. 2016). The presence of secondary nodules that did not express aldosterone synthase in an adrenal with APA could induce errors when identifying mutations by the Sanger method, since sequencing is performed on frozen tissue in the absence of CYP11B2 immunohistochemistry guided sequencing (Nanba et al. 2018). Interestingly, depending on the mutation that is present in the APA, the adjacent tissue is impacted. Indeed, the adjacent tissue of APA carrying KCNJ5 mutations has a different proteomic signature from the adjacent tissue of APA carrying other mutations (Swierczynska et al. 2019).

Presence of APCC As previously mentioned, APCC are structures that develop in the adrenal cortex at the subcapsular level in healthy subjects, but that have also been identified in

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adrenals with APA. These structures express aldosterone synthase and GIRK4 (Yang et al. 2019). Interestingly, these structures also display somatic mutations in APA driver genes. Next-generation sequencing (for more details see below) performed on formalin-fixed, paraffin-embedded APCC identified somatic mutations in 26–35% of APCC. Unlike APA, APCC mainly have mutations in the CACNA1D gene; mutations in ATP1A1 and ATP2B3 genes were also found, whereas mutations in KCNJ5 gene were not found (Nishimoto et al. 2015; Omata et al. 2018). Interestingly, as described in APA, APCC produce 18-oxocortisol (Sugiura et al. 2018) which could be explained by the presence of ZF cells in the inner part of the APCC. Although APCC develop in healthy subjects, APCC accumulation could induce bilateral adrenal hyperplasia and nonlateralized primary aldosteronism. Indeed, a recent study showed that adrenals with bilateral hyperplasia have at least one APCC or aldosterone producing micronodule. Moreover, in these patients, the number of APCC is higher than in control subjects. Omata et al. reported that 58% of APCC had somatic mutations in genes involved in primary aldosteronism, mainly CACNA1D mutations and only one case with a KCNJ5 mutation found in a micronodule (Omata et al. 2018).

Presence of Possible APCC to APA Translational Lesions Possible APCC to APA translational lesions, also called pAATL, have also been described. pAATL are micronodules close to the capsule and composed of a subcapsular part similar to APCC and a more inner part similar to an APA but without encapsulation. These structures produce aldosterone but also 18-oxocortisol in the APCC-like region. Analysis of three pAATL by NGS identified in the inner part the presence of KCNJ5 mutations in two pAATL but not in the APCC region, suggesting that KCNJ5 mutations would occur in a second stage after micronodule development. The third pAATL presented an ATP1A1 mutation in the two parts of the pAATL, suggesting a clonal origin (Nishimoto et al. 2016). The pAATL also showed CACNA1D and ATP2B3 mutations (Nishimoto et al. 2017). Taken together, these observations have led the authors to suggest that pAATL derived from APCC.

Prevalence of Somatic Mutations and Associated Correlations Prevalence of Somatic Mutations in APA and Genotype/Phenotype Correlations The prevalence of the somatic mutations identified in APA has been established from various studies on cohorts of more than 100 patients with APA (Beuschlein et al. 2013; Fernandes-Rosa et al. 2014; Boulkroun et al. 2012; Akerstrom et al. 2012; Azizan et al. 2012; Rossi et al. 2014; Kitamoto et al. 2015). Two consecutive multicenter studies performed within the European Network for the Study of Adrenal Tumors (ENS@T) evaluated the genetic spectrum and clinical

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correlates of adrenals with APA (Fernandes-Rosa et al. 2014; Boulkroun et al. 2012). A first analyses on 380 subjects with APA assessed that the prevalence of KCNJ5 mutations was approximately 34% and that these mutations were more frequent in female and in younger patients, and were associated with higher plasma aldosterone levels (Boulkroun et al. 2012). The second study performed on 474 subjects with APA found mutations in 54.2% of APA. Among the tumors investigated, 38% carried a mutation in the KCNJ5 gene, 9.3% in the CACNA1D gene, 5.3% in the ATP1A1 gene, and 1.7% in the ATP2B3 gene (Fernandes-Rosa et al. 2014). Patients with mutations in the KCNJ5 gene were predominantly female and younger patients, while APA with mutations in the CACNA1D gene were smaller than APA with other types of mutations (Fernandes-Rosa et al. 2014). In this study, the authors identified ten new mutations in the CACNA1D gene, underlining the need for extensive genotyping of this gene in APA. Similar results have been reported from the analysis of different cohorts (Azizan et al. 2013; Beuschlein et al. 2013; Williams et al. 2014); although the prevalence of KCNJ5 mutations varies between studies, the frequency of KCNJ5 mutations reaches 70% in Japanese studies (Kitamoto et al. 2015; Taguchi et al. 2012). Before surgery, there was no association of mutation status with plasma aldosterone or renin activity, the aldosterone to renin ratio, or the number of medications taken before surgery. In follow-up, there was also no correlation with blood pressure outcome such as blood pressure, treatment score, cure, or improvement of hypertension (Fernandes-Rosa et al. 2014). Another multicenter study showed that ATP1A1 and ATP2B3 mutations were more frequent in male and associated with higher aldosterone level and lower kalemia (Beuschlein et al. 2013). A meta-analysis of 1636 patients from 13 studies showed a prevalence of KCNJ5 mutations of 43%, with much higher frequencies (up to 77%) in East Asian population compared with European populations. APA carrying KCNJ5 mutations were associated with larger tumors, higher plasma aldosterone concentration compared to APA without mutations, and were mainly composed of ZF-like cells, whereas mutations in ATP1A1, ATP2B3, and CACNA1D were mainly composed of ZG-like cells. Mutations in ATP1A1 were found to be more frequent in male and mutations in CACNA1D gene associated with smaller adenoma size (Lenzini et al. 2015). In a more recent study, CYP11B2 immunohistochemistry guided next-generation sequencing (NGS) was performed on 75 adrenals with APA from White American population. A panel of ten genes including genes involved in APA development (KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CACNA1H), in other adrenal diseases (PRKACA, PRKAR1A, and ARMC5), and hotspots in the oncogenes GNAS and CTNNB1 was sequenced by this approach. This study identified somatic mutations in 88% of APA, a much higher prevalence than in studies analyzing frozen tissue by Sanger sequencing. A 43% of APA have a mutation in KCNJ5, 21% in CACNA1D, 17% in ATP1A1, 4% in ATP2B3, and 3% in CTNNB1 (Nanba et al. 2018). Interestingly and in contrast to the results obtain in European and Asian population, CYP11B2 immunohistochemistry guided NGS performed on 79 African American patients with APA showed that mutations in CACNA1D were the most frequent events with a prevalence of 42%, mutations in KCNJ5 were found in 34% of cases, mutations in ATP1A1 in 8%, and mutations in ATP2B3 in 4% (Nanba et al. 2019).

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Finally, another study applying the same sequencing strategy using a panel including KCNJ5, ATP1A1, ATP2B3, CTNNB1, CACNA1D, APC, CACNA1H, PRKACA, AND ARMC5 found somatic mutations in APA driver genes in around 94% of APA. Mutations in KCNJ5 gene were the most frequent with a prevalence of 44%, followed by mutations in CACNA1D in 27% of APA, in ATP1A1 in 13%, and ATP2B3 in 10% (De Sousa et al. 2020).

Mutations and Expression Profiles Correlations Although APA with KCNJ5 mutations are larger, they proliferate less compared to other mutational groups (Yang et al. 2019); however, their proliferative index is positively correlated with tumor size. They have also a higher percentage of clear, ZF–like cells compared to the ATP1A1 and CACNA1D mutations groups. Altogether, these data suggest that the size of KCNJ5-mutated APA is not due to proliferation but rather to their cellular composition. Furthermore, the overexpression of VSNL1 (visinin-like 1), which has a calcium-induced antiapoptotic role, in APA with a KCNJ5 mutation could be responsible for a decrease in apoptosis in these APA and an increase in their size (Williams et al. 2012). Although transcriptome analyses do not all allow to distinguish APA harboring KCNJ5 mutations from other APA (Boulkroun et al. 2012), KCNJ5 mutated APA exhibit a number of genes that are differentially expressed compared with other mutations groups. The expression of CYP11B1 and CYP17A1 is higher compared with APA carrying ATP1A1 and CACNA1D mutations (Azizan et al. 2012; Monticone et al. 2015). In contrast, CYP11B2 and GIRK4 protein expression is lower in APA with KCNJ5 mutation compared to other mutational groups (Yang et al. 2019; Akerstrom et al. 2015), although expression of CYP11B2 and KCNJ5 mRNA does not correlate with mutational status, suggesting post-transcriptional or post-translational regulation (Monticone et al. 2012; Boulkroun et al. 2013). However, other studies show a high expression of aldosterone synthase in APA with KCNJ5 mutations (Monticone et al. 2012). Interestingly, proteome analysis of six APA also shows that APA with KCNJ5 mutation differ from other by decreased expression of enzymes responsible for glucocorticoids and androgen synthesis (11β-hydroxylase, 17α-hydroxylase, and AKR1C3) and increased expression of the aldosterone synthase, while the expression of the cholesterol side chain cleavage, 3β-HSD, and 21-hydroxylase remained unchanged (Swierczynska et al. 2019).

Mutations and Steroid Profiles Correlations The steroid profile obtained from the analysis of adrenal vein blood samples or peripheral blood samples constitute a good predictor of mutation status. APA with KCNJ5 mutation secrete more aldosterone, 18-oxocortisol, 18-hydroxycortisol, 11-deoxycorticosterone, and corticosterone than APA with other mutations. APA with CACNA1D mutation had lower concentrations of aldosterone, corticosterone,

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and 11-deoxycorticosterone than APA harboring mutations in ATPases (Williams et al. 2016). In APA with KCNJ5 mutations, 18-oxocortisol concentrations were found to correlate with adenoma size (Tezuka et al. 2019). Interestingly, APA carrying KCNJ5 p.Leu165Arg mutations induce higher production of 18-oxocortisol compared to APA carrying the p.Gly151Arg mutation (Tezuka et al. 2019). The presence of the hybrid steroids 18-oxocortisol and 18-hydroxycortisol can be explained by the presence of cells expressing the aldosterone synthase but also the 11β-hydroxylase and the 17α-hydroxylase (Nakamura et al. 2016). Studies have shown plasma steroid profiles that are highly specific to APA harboring KCNJ5 mutations, with artificial intelligence approaches allowing them to be correctly identified in most cases (Williams et al. 2016). These biomarkers, combined with other tumor characteristics, could make it possible to develop decision-making algorithms to identify certain genetic types of APA, thereby avoiding the need for adrenal vein catheterization.

Mutation and Targeted Treatment Knowledge of the pharmacology of mutated channels and pumps is useful for the interpretation of experimental and clinical data and may also be relevant for the development of new therapies in APA. Somatic KCNJ5 mutations have been shown to confer pathological Na+ permeability to mutated GIRK4 channels (Choi et al. 2011), leading to cell membrane depolarization and increased aldosterone production via Ca2+-dependent mechanisms (Monticone et al. 2012; Oki et al. 2012). The increase in intracellular Ca2+ concentration is thought to be due to the opening of voltage-gated Ca2+ channels. Interestingly, in H295R cells, high intracellular Na+ concentrations would prevent Ca2+ export via the Na+/Ca2+ exchanger (NCX) resulting in an influx of Ca2+ through NCX which would then function in reverse transport mode (Tauber et al. 2014). While the wild-type GIRK4 channel is inhibited by tertiapin-Q, mutated channels are only weekly inhibited. In vitro studies have even shown that GIRK4 channel harboring the p.Leu168Arg mutation is inhibited by amiloride, and even more potently by the L-type Ca2+ channel antagonist verapamil. Channels carrying the pGly151Arg or the p.Thr158Ala mutants are also inhibited by verapamil, but less potently (Tauber et al. 2014). Verapamil would not only act on aldosterone production by blocking the mutated channel but also by inhibiting voltage-gated Ca2+ channels activated by depolarization. These results point to the possible influence of verapamil administration on the diagnosis of primary aldosteronism. Calcium antagonists are used in the treatment of patients with primary aldosteronism; they reduce not only blood pressure but also plasma aldosterone levels (Aritomi et al. 2012). In one of two patients with a germline mutations of CACNA1D, use of the calcium channel blocker amlodipine normalized blood pressure and resolved biventricular hypertrophy (Scholl et al. 2013). These data raise the possibility of a specific treatment for patients with KCNJ5 and CACNA1D mutations. It would be particularly relevant to identify biomarkers associated with

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mutation status, allowing patients to be stratified and targeted treatment with verapamil or a calcium antagonist (alone or in combination with amiloride) to be administrated with surgery. In the future, it should be possible to detect the presence of somatic mutations in circulating free DNA derived from APA cell turnover. In particular for KCNJ5 mutations, which represent the majority of cases, this would constitute a sensitive and noninvasive screening method for APA.

Risk Loci for Primary Aldosteronism Although germline and somatic mutations have been identified in familial forms of PA and in APA, the causes underlying a large proportion of cases of PA are still unknown and the existence of common mechanisms involved in the development of APA and bilateral adrenal hyperplasia has been evoked. A genome-wide association study (GWAS) performed on a discovery cohort of 562 patients with PA and 950 controls from the Paris Prospective Study III allowed to identify three loci on chromosomes 1, 13 and X at a genome-wide significance, and a fourth locus on chromosome 11 (Le Floch et al. 2022). The associations found in chromosomes 1, 11, and 13 were replicated in a second cohort and confirmed by a global metaanalysis involving 1162 cases and 3296 controls. Subanalyses performed for men, women, APA, and BAH revealed that the association on chromosome 13 was specific to men and appear stronger in bilateral adrenal hyperplasia than in APA. Within the four main loci associated with PA at genome-wide significance level in the full discovery dataset and/or in one stratified analysis, a total of 51 protein coding genes were located within a 1 Mb interval around the identified variants. The lead single nucleotide polymorphism (SNP) on chromosome 1, as well as the other SNPs of the locus, cluster within the CASZ1 gene while the locus on chromosome 13 lies upstart of RXFP2. Interestingly, SNPs at CASZ1 and SNPs in linkage disequilibrium with SNPs at the RXFP2 locus associate with different blood pressure–related traits (Ehret et al. 2016; Irvin et al. 2019; Surendran et al. 2020). CASZ1 is a zinc finger transcription factor; interestingly, CACNA1D and KCNK3 genes, two channels involved in human and mouse primary aldosteronism, are regulated by CASZ1. The expression of CASZ1 has been recently identified as differentially expressed between APCC and ZG cells in the adrenal cortex (Nishimoto et al. 2015). RXFP2 codes for the G protein–coupled, seven-transmembrane receptor for the relaxin family peptide, insulin-like peptide 3 (INSL3), that signals through Gαs to increase cAMP (Bathgate et al. 2013), which acts as second messenger for several aldosterone secretagogues. Expression of both CASZ1 and RXFP2 was found in adrenals. Functional analyses performed in H295R_S2 cells showed that overexpression of CASZ1 and RXFP2 significantly affect steroid production, without modifying cell proliferation. Their expression significantly reduced aldosterone biosynthesis under basal and stimulated conditions, without affecting cortisol biosynthesis. Interestingly, only CASZ1 overexpression reduced aldosterone synthase expression. CASZ1 and RXFP2 could influence the function of the adrenal gland and risk alleles on chromosomes 1 and 13 could increase susceptibility to develop PA by modifying

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basal and stimulated production of mineralocorticoids in the adrenal gland. Moreover, data on RXFP2 suggest a mechanism whereby a genetically determined reduction in basal or AngII-stimulated aldosterone production by the zona glomerulosa leads to PA through lifelong increased stimulation of the adrenal cortex to ensure appropriate aldosterone levels. More recently, a second GWAS of PA was conducted in the Japanese population and a cross-ancestry meta-analysis combined with UK Biobank and FinnGen cohorts to identify genetic variants that contribute to PA susceptibility (Naito et al. 2023). Ten loci with suggestive evidence were identified in the Japanese GWAS and five genome-wide significant loci in the meta-analysis in chromosomes 1, 7 11, 12, and 13, including three of the suggested loci identified in the Japanese GWAS. The strongest association identified was found in an intronic region of WNT2B, reinforcing the implication of the Wnt/β-catenin pathway in the primary aldosteronism pathogenesis. Remarkably, this study identified two loci discovered in the previous study, the locus mapped to RXFP2 on chromosome 13 and the locus mapped to LSP1 in chromosome 11 (Le Floch et al. 2022; Naito et al. 2023). The identification of susceptibility genes involved in the development of primary aldosteronism will provide new pathophysiological insight and open new perspectives for the diagnosis and treatment of arterial hypertension.

Animal Models of Primary Aldosteronism To decipher the molecular mechanism responsible for the development of PA, different mouse models have been generated; they have provided new insights in adrenal pathophysiology. The invalidation of kcnk3 and kcnk9 genes coding for the two-pore domain potassium channels Task1 and Task3, respectively, lead to the development of hyperaldosteronism or low renin hypertension (Heitzmann et al. 2008; Davies et al. 2008; Guagliardo et al. 2012; Penton et al. 2012). The deletion of Task1 leads to the development of severe hyperaldosteronism independent of salt intake, hypokalemia, low renin arterial hypertension, and aberrant expression of aldosterone synthase in the ZF. Interestingly, hyperaldosteronism was fully remediable by glucocorticoids. While before puberty this phenotype was observed in both male and female mice, after puberty it can only be observed in females, suggesting the existence of compensatory mechanisms in males after puberty. The expression of Task3, in male mice after puberty, compensates for the loss of Task1 expression (Heitzmann et al. 2008). Task3 knockout mice display low-renin salt-sensitive hypertension, suppressed plasma renin, and inappropriate aldosterone secretion when mice are fed with a high salt diet (Penton et al. 2012). This was associated with an abnormal calcium signaling activation in the ZG (Penton et al. 2012). Remarkably, deletion of both task1 and task3 leads to a mild hyperaldosteronism and absence of aldosterone level suppression by a high sodium diet whereas aldosterone biosynthesis is stimulated in response to a low sodium diet (Davies et al. 2008). The specific deletion of these two channels specifically in ZG cells,

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using a cre-recombinase, was driven by cyp11b2. These mice develop mild hyperaldosteronism leading to chronic blood pressure elevation (Guagliardo et al. 2019). The generation of a knock-in mouse model expressing a constitutively open CLC-2 chloride channel (Clcn2op) provided some insight into the role of this chloride channel in the pathogenesis of primary aldosteronism (Göppner et al. 2019). The constitutive expression of the Clcn2op leads to an increase of chloride conductance of ZG cells leading to a huge cell membrane depolarization and an increase in intracellular Ca2+ concentration. Male and female mice presented high plasma aldosterone level, associated with low renin activity, hypertension, and hypokalemia; however, despite an increase in the number of Cyp11b2-positive cells in Clcn2op/op mice, no major morphological changes were observed in adrenal gland and no adenomas were detected. Interestingly, mice in which a missense mutation (Clcn2R180Q/+) was introduced to mimic the most common mutation found in FH-II developed a mild form of hyperaldosteronism with increased aldosterone to renin ratio and elevated blood pressure levels, these effects being more pronounced in male than in female (Schewe et al. 2019). These mice recapitulate all the features of human primary aldosteronism, demonstrate the role of CLC-2 chloride channel in aldosterone biosynthesis, and constitute valuable models to study the pathological mechanism underlying the disease (Göppner et al. 2019; Schewe et al. 2019). Using the CRISPR/Cas9 approach, Seidel et al. have generated a Cacna1hM1560V/+ mouse model to recapitulate the features of FH-IV (Seidel et al. 2021). Although Cacna1hM1560V/+ mice have elevated baseline and peak ZG calcium levels, no major modifications were observed in their adrenals and they displayed normal plasma and urinary electrolytes. An increase in Cyp11b2 mRNA expression was observed only in male mice, however, the number of Cyb11b2-positive cells was unchanged, suggesting that zona glomerulosa cells express higher level of the enzyme. Only a mild increase in plasma aldosterone concentration was observed in male mice, associated with a modest increase in blood pressure. This mouse model of FH-IV presented a mild primary aldosteronism due to higher baseline and peak intracellular calcium concentrations in the ZG, due to gain of function of Cav3.2 channel (Seidel et al. 2021). Recently, chemogenetic tools were used to generate a mouse model expressing, specifically in the adrenal cortex, a Gαq coupled designer receptor exclusively activated by designer drugs (DREADD) (Taylor et al. 2020). It is important to remember that aldosterone biosynthesis is mainly regulated by AngII activation of the Gαq signaling pathway. A mouse model expressing a gain of function mutant of the AngII receptor type 1A presented a moderate and stable increase in blood pressure, with low renin and inappropriate normal aldosterone, mimicking low-renin human hypertension (Billet et al. 2007). The activation of this receptor led to adrenal cortex functional disorganization with an extension of Cyp11b2 expression in the ZF. These mice displayed also an increase in plasma aldosterone concentration and a decrease in plasma renin concentration in female mice. Surprisingly, male mice exhibited a milder phenotype compared to female mice. Interestingly, mutations in GNA11 and GNAQ genes associated with mutations in CTNNB1 gene have recently been identified in APA (Zhou et al. 2021). These mutations were found almost exclusively in female with primary aldosteronism developing at

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puberty, pregnancy, and menopause. This model in which we could induce “on demand” Gαq signaling pathway, leading to the activation of Ca2+ signaling, will be a useful tool for the study of primary aldosteronism and provide a preclinical model for investigating potential therapeutic targets (Taylor et al. 2020). Interestingly, whereas all these mouse models develop hyperaldosteronism, the presence of adrenal tumors was never observed, suggesting that modification of intracellular ion balance alone is not sufficient to induce both increase of aldosterone production and adenoma development. The β-catenin plays an important role in adrenal cortex development and in the maintenance of ZG differentiation. Its activation is observed in two-third of APA (Boulkroun et al. 2011) and mutations in CTNNB1 gene are found in 5% of APA. The generation of a mouse model expressing an constitutively active form of the β-catenin in the adrenal cortex provide new evidence for a role of Wnt/β-catenin pathway in the development of PA (Berthon et al. 2012). These mice presented adrenal hyperplasia and dysplasia as well as increased cell proliferation and anarchic vascular architecture. Moreover, they showed also increased Cyp11b2 expression with ectopic expression in the ZF leading to an increase in aldosterone production, a decreased plasma renin concentration, and development of hyperaldosteronism. The development of tumors was observed only in rare cases with features of carcinoma rather than adenoma (Berthon et al. 2012). Transcriptomic analysis performed on APA and control adrenal identified the retinoic acid receptor alpha (RARα) as involved in nodulation (El Zein et al. 2019). Interestingly, investigation of the adrenal phenotype of Rarα total knockout mice revealed zona fasciculata disorganization associated to modifications in the extracellular matrix and architecture in 12 and 52 old male and female mice. No major modification of aldosterone biosynthesis was observed. Although the morphological phenotype was observed in both genders, molecular exploration revealed alterations in Wnt and VegfA signaling pathways only in 12 weeks old male mice as well as a reduced transcriptomic expression of some steroidogenic enzymes in these mice and an increased proliferative index; however, no molecular abnormalities could be observed in female mice or male Rarα knockout mice at 52 weeks. These results suggest that Rarα contributes in a sex- and time-dependent manner to the normal morphology and functional zonation of the adrenal cortex by modulating both Wnt and VegfA signaling. The authors propose a model in which a homeostatic equilibrium between retinoic acid, Wnt, and Vegf signaling pathways is required for a normal development of the adrenal cortex. Dysregulation of this equilibrium in adult adrenal cortex may contribute to abnormal cell proliferation and APA development, creating a propitious environment for the emergence of specific driver mutations in APA (El Zein et al. 2019).

Pathogenic Model for Primary Aldosteronism Development Although the role of the mutations identified in PA in promoting autonomous aldosterone production has been clearly established, their role in promoting cell proliferation and adenoma development is still a matter of debate.

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Some studies suggest that the genes involved in aldosterone biosynthesis are not involved in cell proliferation and favor a model in which the development of APA is associated with two events. The first event corresponds to the activation of a signaling pathway involved in cell proliferation in ZG cells, such as the Wnt/β-catenin signaling pathway, inducing abnormal cell proliferation. This abnormal cell proliferation would lead to the occurrence of somatic mutations in APA driver genes, such as the KCNJ5 gene, inducing activation of the calcium signaling pathway and an increase in CYP11B2 expression and aldosterone production. Indeed, this “two-hit model” hypothesis is based on the observation of different mutations in different nodules of the same adrenal with APA (Fig. 2). It is corroborated by the study of a young patient with primary aldosteronism associated with bilateral macronodular adrenal hyperplasia. The identification of lateralized aldosterone secretion on AVS led to adrenalectomy with clinical and biochemical cure. Pathological analysis of the adrenal gland revealed the presence of three nodules, one of which expressed aldosterone synthase and carried a KCNJ5 mutation. Interestingly, the patient also had familial adenomatous polyposis and carried a germline mutation in the adenomatous polyposis coli (APC) gene, a tumor suppressor gene. In the adrenal gland, all three nodules showed biallelic inactivation of the APC gene, suggesting that these mutations were involved in adenoma formation, while the KCNJ5 mutation appeared later and was responsible for aldosterone production (Vouillarmet et al. 2016). Furthermore, the observation of cellular, molecular, and genetic heterogeneity in the same APA also suggests the acquisition of somatic mutations involved in autonomous aldosterone production in a preexisting nodule (Nanba et al. 2016). It could also be suggested that APA could originate not from zona glomerulosa cells but from cells from the capsule or Shh-positive undifferentiated cells that have the capacity to differentiate into steroidogenic cells (Yates et al. 2013) and whose loss of the control of cell proliferation could be involved in the development of the adenoma followed by the appearance of somatic mutations in genes involved in autonomous aldosterone production. On the other hand, other studies suggest that APA derived from APCC, the APCC model hypothesis (Fig. 2). This hypothesis is based on the fact that APCC carry somatic mutations in genes involved in primary aldosteronism, but also on the observation that pAATL carrying somatic mutations have a subcapsular structure similar to APCC and an internal structure similar to APA. However, as discussed above, APCC mainly carry CACNA1D mutations, unlike APA, which tend to carry mutations in the KCNJ5 gene, suggesting that only APA carrying a KCNJ5 mutations develop more rapidly into APA.

Conclusion: Toward a Precision Medicine in Primary Aldosteronism Overs the last 15 years, the development of new approaches such as exome sequencing and CYP11B2 guided NGS has allowed the identification of somatic mutations in more than 90% of APA and of new familial form of hyperaldosteronism. The

Fig. 2 Pathogenic model for primary aldosteronism development. Two different models may explain APA development in the adrenal cortex. Upper panel. “The Two-hit model” hypothesis: Abnormal cell proliferation in the zona glomerulosa, due to genetic, risk allele, or environmental factors, may favor the occurrence of somatic mutations in APA driver genes (KCNJ5, CACNA1D, ATP1A1, ATP2B3, CLCN2n ZnT1, and CADM1). Lower panel. “APCC model” hypothesis: occurrence of somatic mutations in different APA driver genes (KCNJ5, CACNA1D, ATP1A1, and ATP2B3) lead to the development of aldosterone producing cell clusters (APCC). APCC could evolve into possible APCC-to-APA translational lesions (pAATL) and then into APA. In the two models, activation of calcium signaling, due to the presence of somatic mutations, leads to increased CYP11B2 expression and autonomous aldosterone production

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identification of germline mutations in familial forms of PA represents the opportunity for transferring knowledge to clinical practice for early diagnosis and rapid management of affected subject. The clinical implications of identifying somatic mutations in APA will certainly take longer time to be implemented. The majority of mutations identified in PA affect channels and pumps that affect ionic equilibrium and highlighted the central role of ZG cell membrane potential, modification of intracellular calcium concentration, and calcium signaling in the pathogenesis of PA. Most importantly, the identification of mutations in CTNNB1 and GNAQ/GNA11 responsible to peculiar form of PA developing at puberty, pregnancy, and menopause suggests that PA should be sought in patients presenting with arterial hypertension during puberty or pregnancy, enabling rapid diagnosis and the proposal of curative treatment by ablation of the affected adrenal gland. The recent identification of risk loci for the development of PA constitutes a major advance and provides new pathophysiological insight and opens new perspectives for the diagnosis and treatment of PA. Omics biomarkers have been tested in large cohorts of hypertensive patients to identify multiomics signature of PA and other forms of endocrine hypertension as part of the Horizon 2020 ENSAT-HT project (www.ensat-ht.eu). The development of a machine learning pipeline provided promising classification outcomes and the reduced features have the potential to contribute as clinical biomarkers for diagnosing hypertension subtypes. Remarkably, the use of a multi-mics approach outperformed the use of individual omics, suggesting that the use of different biomarkers can compensate for individual variability and analytical problems. Omics biomarkers appear to be a promising new tool for improving the diagnosis and management of endocrine hypertension, enabling faster diagnosis and more effective treatment, particularly of patients with PA, which account for up to 10% of the hypertensive population (Reel et al. 2022). The next few years should see us move toward a precision medicine in primary aldosteronism.

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Vasan RS, Evans JC, Larson MG, Wilson PWF, Meigs JB, Rifai N, et al. Serum aldosterone and the incidence of hypertension in nonhypertensive persons. N Engl J Med. 2004;351(1):33–41. Vouillarmet J, Fernandes-Rosa F, Graeppi-Dulac J, Lantelme P, Decaussin-Petrucci M, Thivolet C, et al. Aldosterone-producing adenoma with a somatic KCNJ5 mutation revealing APC-dependent familial adenomatous polyposis. J Clin Endocrinol Metab. 2016;101(11):3874–8. Walczak EM, Kuick R, Finco I, Bohin N, Hrycaj SM, Wellik DM, et al. Wnt signaling inhibits adrenal steroidogenesis by cell-autonomous and non-cell-autonomous mechanisms. Mol Endocrinol. 2014;28(9):1471–86. Williams TA, Monticone S, Crudo V, Warth R, Veglio F, Mulatero P. Visinin-like 1 is upregulated in aldosterone-producing adenomas with KCNJ5 mutations and protects from calcium-induced apoptosis. Hypertension [Internet]. 2012; Disponible sur: http://www.ncbi.nlm.nih.gov/entrez/ query.fcgi?cmd¼Retrieve&db¼PubMed&dopt¼Citation&list_uids¼22331379 Williams TA, Monticone S, Schack VR, Stindl J, Burrello J, Buffolo F, et al. Somatic ATP1A1, ATP2B3, and KCNJ5 mutations in aldosterone-producing adenomas. Hypertension. 2014;63 (1):188–95. Williams TA, Peitzsch M, Dietz AS, Dekkers T, Bidlingmaier M, Riester A, et al. Genotype-specific steroid profiles associated with aldosterone-producing adenomas. Hypertension. 2016;67(1): 139–45. Williams TA, Lenders JWM, Mulatero P, Burrello J, Rottenkolber M, Adolf C, et al. Outcomes after adrenalectomy for unilateral primary aldosteronism: an international consensus on outcome measures and analysis of remission rates in an international cohort. Lancet Diabetes Endocrinol. 2017;5(9):689–99. Wu X, Senanayake R, Goodchild E, Bashari WA, Salsbury J, Cabrera CP, et al. [11C]metomidate PET-CT versus adrenal vein sampling for diagnosing surgically curable primary aldosteronism: a prospective, within-patient trial. Nat Med. 2023a;29(1):190–202. Wu X, Azizan EAB, Goodchild E, Garg S, Hagiyama M, Cabrera CP, et al. Somatic mutations of CADM1 in aldosterone-producing adenomas and gap junction-dependent regulation of aldosterone production. Nat Genet. 2023b;55(6):1009–21. Yang Y, Gomez-Sanchez CE, Jaquin D, Aristizabal Prada ET, Meyer LS, Knosel T, et al. Primary aldosteronism: KCNJ5 mutations and adrenocortical cell growth. Hypertension. 2019;74(4): 809–16. Yates R, Katugampola H, Cavlan D, Cogger K, Meimaridou E, Hughes C, et al. Adrenocortical development, maintenance, and disease. Curr Top Dev Biol. 2013;106:239–312. Young WF, Stanson AW, Thompson GB, Grant CS, Farley DR, van Heerden JA. Role for adrenal venous sampling in primary aldosteronism. Surgery. 2004;136(6):1227–35. Zennaro MC, Caprio M, Feve B. Mineralocorticoid receptors in the metabolic syndrome. Trends Endocrinol Metab. 2009;20(9):444–51. Zhou J, Lam B, Neogi SG, Yeo GS, Azizan EA, Brown MJ. Transcriptome pathway analysis of pathological and physiological aldosterone-producing human tissues. Hypertension. 2016;68 (6):1424–31. Zhou J, Azizan EAB, Cabrera CP, Fernandes-Rosa FL, Boulkroun S, Argentesi G, et al. Somatic mutations of GNA11 and GNAQ in CTNNB1-mutant aldosterone-producing adenomas presenting in puberty, pregnancy or menopause. Nat Genet. 2021;53(9):1360–72.

Mineralocorticoid Receptor and Aldosterone: From Hydro-saline Metabolism to Metabolic Diseases

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aldosterone and Mineralocorticoid Receptor in the Regulation of Electrolytes and Water Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Renal Handling of Electrolytes and Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Renal Ion Channels by the Aldosterone-Minealocorticoid Receptor Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dysregulation of Renal Mineralocorticoid Receptor Signaling Pathways . . . . . . . . . . . . . . . . . . . . . Regulation of Aldosterone Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dysregulated Mineralocorticoid Receptor Signaling in the Kidney and Clinical Consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extra-Renal Mineralocorticoid Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mineralocorticoid Receptor in the Vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overactivation of Vascular Mineralocorticoid Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mineralocorticoid Receptor in Cardiac Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mineralocorticoid Receptor Overactivation in the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Mineralocorticoid Receptor in Adipose Tissue Physiology . . . . . . . . . . . . . . . . . . . . . . . . Mineralocorticoid Receptor in Adipose Tissue Dysfunctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mineralocorticoid Receptor Antagonists in Cardiovascular and Renal Dysfunctions . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The steroid hormone aldosterone regulates electrolyte homeostasis and blood pressure by activating mineralocorticoid receptor (MR) in in the distal nephron. In the last 10 years, a number of preclinical studies has shown that MR is A. Armani · M. Caprio (*) Laboratory of Cardiovascular Endocrinology, IRCCS San Raffaele, Rome, Italy Department of Human Sciences and Promotion of the Quality of Life, San Raffaele Roma Open University, Rome, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2023 M. Caprio, F. L. Fernandes-Rosa (eds.), Hydro Saline Metabolism, Endocrinology, https://doi.org/10.1007/978-3-031-27119-9_14

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expressed also in extra-renal tissues such as heart, adipose tissue and vasculature, providing strong evidence that dysregulated extra-renal MR activation contributes to the development of hypertension, obesity, insulin resistance and heart disease. Notably, mice with overexpression of adipose tissue-specific MR display obesity and typical features of metabolic syndrome. On the other hand, preclinical studies in mice prone to develop obesity, atherosclerosis, heart failure and insulin resistance have observed that pharmacological blockade of MR is able to prevent development of such derangements. Increased circulating aldosterone levels have been observed in patients with obesity and metabolic syndrome, suggesting that overactivation of MR may have a causal role in the occurrence and/or progression of metabolic and cardiovascular dysfunctions also in humans. MR antagonist (MRA) treatment has shown remarkable efficacy in pathological conditions such as chronic HF, primary aldosteronism and chronic kidney disease, although clinical use of these drugs has been limited by the occurrence of hyperkalemia and other side effects. The novel non-steroidal MRAs provide reduced risk of hyperkalemia and represent attractive tools to treat diabetic patients with the cardio-renal syndrome. Keywords

Electrolytes · Aldosteronism · Obesity · Cardiovascular disease · Insulin resistance

Introduction The kidney has a critical role in regulating water and electrolyte homeostasis (Hoenig and Zeidel 2014). Electrolytes are involved in crucial biological processes such as enzymatic reactions, cardiovascular (CV) and nerve function, muscle contraction, regulation of water and acid-base balance, and maintain whole-body homeostasis overall. Derangements in electrolyte balance can lead to life-threatening complications, and comprehension of physiologic mechanisms that regulate renal electrolyte handling is crucial to adopt proper treatments aimed at correcting electrolyte imbalance (Kraft et al. 2005). Sodium represents the most abundant ion in the extracellular fluids and remarkebly contributes to blood osmolarity. The renal mineralocorticoid receptor (MR)/aldosterone system activity promotes sodium reabsorption in the distal convoluted tubule and in the renal collecting ducts, resulting in water retention, and stimulates potassium secretion by the kidney (Terker and Ellison 2015). The steroid hormone aldosterone, by activating the renal MR, favors sodium reabsorption and conservation, with subsequent vasconstricition and increase in blood pressure. Activation of the renin angiotensin system (RAS) and circulating potassium are the main contributors to the regulation of aldosterone secretion by the adrenal cortex (Lefebvre et al. 2019). Dysregulated aldosterone production and/or MR functional derangements in the kidney result in life-threatening diseases such as primary

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aldosteronism (PA), Liddle syndrome and pseudohypoaldosteronism type I (PHA I) (Enslow et al. 2019; De Sousa et al. 2019; Furgeson and Linas 2010). Intensive preclinical research have shown that MR, in addition to the kidney, is expressed in several tissues and organs such as vasculature, heart, adipose tissue (AT), brain and immune cells. Murine models lacking or overexpressing MR in specific tissues have provided information on the role of MR particularly in the development of CV and metabolic dysfunctions (Feraco et al. 2020). In preclinical studies, AT-specific overexpression of MR has shown to promote obesity, glucose metabolism alterations and other metabolic features typical of metabolic syndrome (MetS) (Urbanet et al. 2015). In mice, increased activity of cardiomyocyte-specific MR resulted in the development of arrhythmias and altered cardiac function (Ouvrard-Pascaud et al. 2005). Notably, these effects were independent on changes in blood pressure, suggesting a direct effect of MR activity on specific molecular pathways and cellular functions of extra-renal tissues. On the other hand, pharmacological MR blockade in numerous murine models of obesity, atherosclerosis and cardiac and renal diseases has shown that MR blockade has protective effects, at least in preclinical models, on AT and glucose metabolism, cardiovascular and renal functions (Feraco et al. 2020). Interestingly, MR blockade in obese mice has been shown to counteract white adipose tissue (WAT) expansion and impaired glucose tolerance by stimulating metabolic activation of brown adipose tissue (BAT) (Feraco et al. 2020). A number of studies have observed that BAT activation is associated to protection against AT and glucose dysfunctions, and pharmacological MR antagonism might be a novel approach to fight obesity and type 2 diabetes (Cinti 2012). Human studies have revealed an increase in circulating aldosterone levels in subjects with obesity and MetS, suggesting that overactivation of MR is an important contributor to the development of metabolic and cardiovascular (CV) complications, and indicating that treatment with MR antagonists (MRAs) may tackle AT and glucose dysfunction as well as cardiovascular (CV) derangements (Feraco et al. 2020). In subjects with PA, MRA treatment has shown beneficial effects on blood pressure (BP) control, insulin resistance (IR) and visceral obesity, together representing key features of the MetS (Feraco et al. 2020). Steroidal MRAs have found clinical application in in the treatment of several diseases such as chronic heart failure (HF), PA and and resistant hypertension, although side effects such as gynaecomastia and erectile dysfunction in men, amenorrhoea in pre-menopausal women, and hyperkalaemia have limited the clinical use of these drugs (Bauersachs et al. 2015; Bramlage et al. 2016). Importantly, the landmark trial RALES showed that spironolactone treatment led to an impressive reduction of mortality in subjects showing HF with reduced left ventricular ejection fraction (LVEF), and other trials (EPHESUS and EMPHASISHF) revealed protective CV effects also for eplerenone (Bauersachs et al. 2015). However, patients needed careful monitoring of serum potassium levels and renal function, due to the hyperkalemic effect associatd with steroidal MRA tretament. In recent preclinical and clinical studies, the novel non-steroidal MRA finerenone has showed a reduced risk of hyperkalemia, suggesting the application of this drug also in vulnerable subjects, i.e. diabetic patients with chronic kidney disease (CKD) (Bramlage et al. 2016; Al Dhaybi and Bakris 2020).

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Aldosterone and Mineralocorticoid Receptor in the Regulation of Electrolytes and Water Homeostasis Renal Handling of Electrolytes and Water The kidneys maintains an equilibrium between consumption and excretion of electrolytes and water. These two organs regulate intake of water and electrolytes to regulate the osmolality and body fluids volume (Hoenig and Zeidel 2014). Reduction in water and electrolytes is balanced by increases in intake and retention; on the other hand, excesses of both are excreted via urine (Hoenig and Zeidel 2014). It is generally assumed that regulation of electrolyte intake affects fluids volume and osmolality. Particularly, it is observed that sweating or diarrhea lead to increased osmolality which, in turn, results in water retaining mediated by release of antidiuretic hormone or stimulation of thirst which both restore a normal range of osmolality (Hoenig and Zeidel 2014). The kidneys regulate the levels of electrolytes by filtering salts from blood, and returning them to the blood, or excreting into the urine, in the case that electrolytes are present in excess. The most abundant are sodium, potassium, chloride, magnesium and calcium (Hoenig and Zeidel 2014; Blaine et al. 2015). Electrolytes have a key role in many biological processes, such as conduction of electrical impulses in neurones and muscles, regulation of enzyme activity and cellular signaling pathways. Moreover, electrolytes that are present in plasma influence osmotic presssure and modulate the passage of water between the cell and the extracellular environment (Hoenig and Zeidel 2014). Perturbations in electrolyte balance can alter body fluid volumes, BP, and acid-base balance. Among the most important electrolytes, sodium with its high concentrations in the extracellular environment, compared with the intracellular environment, contributes to regulate extracellular fluid volume. In the kidney, filtration of sodium take places at the glomerulus. Sodium reabsorption occurs in the proximal and in the distal convoluted tubule through sodium-chloride cotransporters whose activity is regulated by aldosterone (Hoenig and Zeidel 2014). Condition of reduced serum levels of sodium (less than 135 mmol/L) is termed as hyponatremia which has neurological manifestations, such as headache, confusion, nausea, deliriums. On the contrary, sodium levels greater than 145 mmol/L in the serum lead to hypernatremia whose symptoms include thirsty, weakness, nausea, and vomiting (Bockenhauer and Zieg 2014). Potassium, another key electrolyte for the body, is present at higher concentrations inside the cell. Activity of the sodium-potassium adenosine triphosphatase pumps sodium out and potassium into the cells through a process requiring energy that is provided by the hydrolysis of adenosine triphosphate (ATP). Potassium filtered at the glomerulus is reabsorbed at the proximal convoluted tubule and at the loop of Henle through both transcellular and paracellular pathways. Potassium secretion, which occurs at the distal convoluted tubule, is stimulated by aldosterone (Palmer and Clegg 2016). Disturbance in potassium levels result in cardiac arrhythmias. Hypokalemia, defined as serum potassium less than 3.6 mmol/L, is associated with higher mortality rates due to incresed risk of ventricular and atrial fibrillation (Palmer and Clegg 2016). Other manifestation include muscular cramps, weakness,

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paresis, constipation, respiratory insufficiency. Also hyperkalemia, which takes place when serum potassium levels rise above 5.5 mmol/L, can favor cardiac arrhythmias, and it is associated with significantly increased mortality. Hyperkalemia is a frequent alteration in subjects with CKD, cardiovascular disease (CVD), diabetes mellitus (DM), or pharmacologicl treatments used in subjects with CKD represent additional risk factors for hyperkalemia (Palmer and Clegg 2016). Calcium plays a prominent role in a variety of biological processes including skeletal mineralization, muscle contraction, transmission of electrical signals, blood coagulation and secretion of hormones. Bones and teeth represent a reservoir of calcium. Calcium absorption from the intestine is modulated by vitamin D whose deficiency can result in reduced uptake of calcium and, in turn, in enhanced depletion of skeletal calcium reserves, leading to osteoporosis. In the kidney, parathyroid hormone (PTH) and vitamin D promote calcium reabsorption (Hoenig and Zeidel 2014). Calcitonin opposites the effects of PTH by counteracting the efflux of calcium from bone stimulated by PTH (Naot and Cornish 2008). Deficiency of PTH causes an imbalance of calcium and phosphorus, leading to hypocalcemia and hyperphosphatemia, and leads to twitching, muscle spasm, kidney disfunction and neurocognitive disorders (Rubin 2020). Hypercalcemia can commonly derive from primary hyperparathyroidism (PHPT) in subjects with autonomous secretion of PTH from dysfunctional parathyroid glands deriving from hyperplasia, adenoma, or from carcinoma of parathyroid glands. Subjects with PHPT may show gastroenterological and neurological disturbances (Han et al. 2020). In addition to the regulation of cell osmotic gradient, heart rate and bone metabolism, as above described, other key physiological processes are modulated by electrolytes such as magnesium and the anionic electrolytes bicarbonate, chloride and phosphorus. Magnesium stabilizes the DNA conformation, partecipates to DNA repair mechanisms and serves as a cofactor for many enzymes. Deficiency of magnesium is associated with increased incidence of cardiac arrhythmias and hypertension (Blaine et al. 2015). Bicarbonate regulates the acid-base balance, chloride balances cations in the extracellular fluid and this results in electrical neutrality of the fluid outside the cells. Phosphate is present in the structure of the phospholipids, ATP and nucleotides, which are fundamental molecules for cell structure and function (Blaine et al. 2015).

Regulation of Renal Ion Channels by the Aldosterone-Minealocorticoid Receptor Signaling Activity of MR/aldosterone system regulates electrolyte homeostasis in the kidney. The intracellular MR mediates the effects of the hormone aldosterone, which stimulates sodium retention and, in parallel, potassium and proton secretion in the distal nephron (Terker and Ellison 2015). In an evolutionary perspective, aldosterone-MR axis was a key step to allow ion balance during the life transition from the water to the land. MR probably appeared a long time before aldosterone, in bony and cartilaginous fishes. The lungfishs have gills and lungs and represents the first creatures which have expressed aldosterone, providing evidence that the ability to produce aldosterone develops when vertebrates move to terrestrial life (Funder 2017).

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MR shows comparable affinity for aldosterone and glucocorticoids, and the higher concentrations of circulating glucocorticoids, compared with those of aldosterone, would lead to prevalent occupancy of MR by these hormones. In the epithelial tissues, as observed in the distal nephron, selective activation of MR by aldosterone is allowed by the expression of 11b-hydroxysteroid dehydrogenase type 2 (11-HSD2) which converts glucocorticoids into inactive metabolites (Funder 2017). Aldosterone-MR signaling stimulates expression of the Na+-K+-ATPase, localized at the basolateral membrane, and the epithelial sodium channel (ENaC) which is localized at the apical membrane (Terker and Ellison 2015). Activity of Na+-K+-ATPase, which moves 3 sodium ions (Na+) out of the cell and, in parallel, transports 2 potassium ions (K+) into the cell. ENaC activity allows Na+ reabsorption in the distal nephron, and overactivation of this channel may result in hypertension (Terker and Ellison 2015) (see below). MR/aldosterone system increases transcription of Na+-K+-ATPase and ENaC through a mechanism that implies translocation of the aldosterone-MR complex into the nucleus and complex-regulated transcription of subunits that assemble to form these systems of ion transport (Terker and Ellison 2015). In addition to the transcriptional regulation, aldosterone is able to enhance ENaC and Na+-K+-ATPase through stimulation of trafficking of preexisting subunits to the membranes (Ruhs et al. 2017). Notably, several preclinical studies have observe a rapid increase in ion transport activity of Na+-K+-ATPase and ENaC within few minutes after aldosterone stimulation, suggesting that nongenomic effects contribute to modulate activity of these ion transporters (Ruhs et al. 2017). Serum- and glucocorticoid-induced kinase 1 (SGK1) has been identified as a target gene of MR which has the ability to stimulate ENaC activity by increasing apical membrane abundance of the channel (McCormick et al. 2005). A number of studies over the last 20 years have revealed that there are several mechanisms by which SGK1 modulates ENaC function (McCormick et al. 2005). SGK1 has been shown to phosphorylate the E3 ubiquitin ligase neural precursor cell-expressed developmentally down-regulated protein (Nedd 4–2). This enzyme interacts with ENaC promoting ubiquitination and subsequent internalization of the channel, thus affecting ENaC turnover. In fact, many ubiquinated proteins undergo degradation by the proteasome or the by lysosome. Phosphorylation of Nedd4–2 by SGK1 reduces interaction with ENaC and internalization of the channel with subsequent increase in ENaC abundance at the plasma membrane (Valinsky et al. 2018). SGK1 has been also shown to phosphorylate with-no-lysine kinase 4 (WNK4), removing the inhibitory activity of this kinase on ENaC. In addition, SGK1 is able to phosphorylate ENaC and increase its function, as well as may promote transcription of α-subunit of ENaC (Fig. 1). In the distal nephrone, SGK1 also regulates function of the renal outer medullary potassium channel (ROMK), a potassium secretory channel which is located at the apical membrane (Valinsky et al. 2018). Cell culture experiments have shown that SGK1 promotes trafficking of this channel, leading to increased amount of ROMK in the plasma membrane. This effects may occur because phosphorylation of WNK4 by SGK1 prevents endocytic degradation of ROMK, increasing expression of ROMK at the apical membrane (Fig. 1). In addition to the modulation of ENaC and ROMK, studies on the role of SGK1 as mediator of

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Fig. 1 Regulation of ENaC and ROMK function by MR signaling in the distal nephron. SGK1 represents a target gene of MR and stimulates ENaC activity by increasing apical membrane abundance of this ion channel. The enzyme Nedd4-2 interacts with ENaC and promotes its turnover. Phosphorylation of Nedd4-2 by SGK1 reduces interaction with ENaC and promotes interaction between Nedd4-2 and 14-3-3, resulting in decreased internalization of ENaC and increased abundance of this ion channel at the apical membrane. SGK1 has also been shown to increase ENaC function by direct phosphorylation of the channel. In addition, SGK1 can phosphorylate WNK4 and reduce its inhibitory activity on ENaC. SGK1 regulates ROMK function, in a similar manner, through phosphorylation of WNK4 by preventing endocytic degradation of this ion channel and increasing abundance of ROMK at the apical membrane

aldosterone signaling have revealed that this kinase is able to regulate activity of other families of ion channels, although there are still few information on the physiological effects of such modulation (Valinsky et al. 2018). Cell-based experiments show that SGK1 is able to increase the membrane expression of the apical calcium channel transient receptor potential vanilloid 5 (TRPV5) (Valinsky et al. 2018).

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Aldosterone treatment increases the expression of TRPV4 in mouse renal cells, and phosphorylation of TRPV4 by SGK1 results in increased activity and stability of this channel. Other studies have shown that aldosterone led to increased expression of transient receptor potential melastatin 7 (TRPM7), a magnesium permeable channel, at the plasma membrane, through MR and SGK1-dependent mechanisms (Valinsky et al. 2018). Regulation of chloride transport by SGK1 has been also investigated in studies indicating that SGK1 is capable of modulating chloride transport through molecular mechanisms which increase abundance of ClC-Ka/ barttin channel at the plasma membrane. This regulation may require reduced interaction of barttin with Nedd4–2, as previously observed by studies on the regulation of ENaC (Valinsky et al. 2018). Although extensive research has investigated the role of MR/aldosterone in regulating activity of sodium and potassium renal channels, further work is required to explore the MR/aldosterone-mediated signaling pathways which modify transport of calcium, magnesium, chloride in the kidney and affect balance of these electrolytes.

Dysregulation of Renal Mineralocorticoid Receptor Signaling Pathways Regulation of Aldosterone Production It is well known that secretion of aldosterone from adrenal cortex is stimulated by RAS, potassium and, to a lesser extent, by the adrenocorticotropic hormone (ACTH). Increased synthesis of aldosterone is promoted by elevation of intracellular calcium levels, which stimulates cholesterol uptake and phosphorylation of enzymes involved in aldosterone synthesis. Increase in calcium concentration also results in upregulated expression of the adrenal steroidogenic enzymes (Lefebvre et al. 2019). As a response to reduced blood volume or to dehydration, the juxta-glomerulus apparatus of the kidney releases renin which cleaves the angiotensinogen producing angiotensin I subsequently converted into angiotensin II (Ang II) by the angiotensin-converting enzyme (ACE). In the zona glomerulosa, Ang II binds AT1 receptor and activates a molecular pathway which stimulates calcium release from the endoplasmic reticulum (Lefebvre et al. 2019). In addition, Ang II-mediated modulation of the potassium channels TASK (TWIK-related acid-sensitive potassium channel), GIRK4 (G-proteinactivated inward rectifier potassium channel) and the Na+-K+ ATPase leads to depolarization of the cell membrane and stimulation of voltage-dependent membrane calcium channels, futher increasing rise in calcium intracellular concentrations (Lefebvre et al. 2019). Enhancement of calcium signaling promotes activity of the enzyme cholesterol ester hydrolase and increases the expression of steroid acute regulatory protein (StAR) which allows transport of cholesterol into the mitochondria. Calcium signaling also activates transcription factors regualting expression of the CYP11B2 gene which encodes the aldosterone synthase. Production of glucocorticoids and aldosterone is also regulated by the hypothalamic-pituitary axis through secretion of the ACTH produced by the pituitary (Lefebvre et al. 2019). ACTH binds the melanocortin receptor 2 in the adrenal cortex, increasing intracellular levels of

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cholesterol as well as its entry in the mitochondria, and increasing expression of steroidogenic enzymes. Particularly, aldosterone is stimulated by calcium influx in the zona glomerulosa through ACTH-mediated activation of L-type Ca2+ channels (Lefebvre et al. 2019). Interestingly, a number of studies have shown that aldosterone production is also regulated by intraadrenal signals released from chromaffin cells, adipocytes, and immune system cells localized in proximity to adrenocortical cells. Notably, dysregulated production of these intraadrenal mediators is suggested to contribute to the hypersecretion of aldosterone and development of aldosterone-producing adenomas (APA). Mast cells infiltrate many tissues and release numerous factors such as cytokines, growth factors, proteolytic enzymes (Lefebvre et al. 2019). In the adrenal gland, mast cells are found close to cells which produce aldosterone, suggesting a role for these cells in the regulation of aldosterone synthesis. In cell culture experiments, medium condition from the mast cells was able to increase expression of CYP11B2 and aldosterone production. Other experiments have shown that release of serotonin from mast cells promotes aldosterone secretion (Lefebvre et al. 2019). Endothelial cells (ECs) forming the vascular network in the adrenal cortex may also modulate aldosterone production, as suggested by experiments with endothelial cell-conditioned medium which increase secretion of aldosterone in the adrenocortical H295R cell line (Lefebvre et al. 2019). Chromaffin cell have been identified in the vicinity of cells producing aldosterone in the zona glomerulosa and this suggests a potential regulatory role for these cells which are able to produce epinephrine, norepinephrine and many neuropeptides. To confirm this hypothesis, in vitro experiments have shown that norepinephrine was able to stimulate aldosterone secretion from cultures of adrenocortical cells (Lefebvre et al. 2019). Recent studies on the function of leptin, released from the adipocytes and with a key role in the homeostatic control of AT, have revealed that this adipokine regulates aldosterone synthesis by acting directly on adrenocortical cells, which co-express CYP11B2 and leptin receptors, by increasing CYP11B2 expression and aldosterone production (Huby et al. 2015). To demonstrate this regualtory mechanims mediated by leptin, the study reported that mice with deficiency of leptin (ob/ob mice) or leptin receptor (db/db mice) did not display increased expression of CYP11B2 or high circulating aldosterone levels. Leptinmediated regulation of aldosterone production provides a mechanism which explains the link between obesity and hyperaldosteronism which may contribute to endothelial dysfunction, hypertension, and increased expression of profibrotic markers in the heart (Huby et al. 2015) (see below section “Mineralocorticoid Receptor in Adipose Tissue Dysfunctions”). Such detrimental effects on the vasculature and the heart expand the impact of aldosterone-MR signaling further the electrolyte balance and, as discussed below, can be attributed to the activation of extra-renal (vascular and cardiac) MRs (see section “Extra-Renal Mineralocorticoid Receptor”).

Dysregulated Mineralocorticoid Receptor Signaling in the Kidney and Clinical Consequences The physiological importance of the MR/aldosterone system is demonstrated by clinical syndromes which derive from the dysregulated MR signaling such as

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Liddle’s syndrome, PHA I, Addison’s disease and PA. Liddle’s syndrome is a genetic disease characterized by hypertension, hypokalemic metabolic alkalosis, suppressed secretion of aldosterone (Enslow et al. 2019). This disorder derives from mutations of the subunits of ENaC which alter their rate of ubiquitination and degradation and lead to increased amount of this channels at the apical membrane, resulting in sodium retention, incresae in plasma volume and high BP, paralleled by urinary potassium and hydrogen ion wasting which leads to hypokalaemic alkalosis (Enslow et al. 2019). ENaC consists of three subunits (α, β and γ) and missense or frameshift mutations within the motif (PPPxY) in the intracytoplasmic portion of β or γ subunits impair binding to the ubiquitin-protein ligase Nedd4-2 with subsequent reduced degradation of the channel and increase in expression of the ENaC at the apical membrane. In addition, mutations which increase ENaC activity by favoring the open conformation of the channel have been also identified in subjects with Liddle’s syndrome (Enslow et al. 2019). Enhancement in sodium absorption results in increased driving force for secretion of potassium and hydrogen ions which is observed in this disease. Increased BP is detected by the juxtaglomerular apparatus, and results in decreased renin levels that along with hypokalemia dampen aldosterone production. Suppression of RAS can explain the lack of response to mineralocorticoid synthesis inhibitors as well as to MR antagonists, whereas it has been observed a response to ENaC blockers triamterene and amiloride (Enslow et al. 2019). Hypertension is present in the vast majority of subjects with Liddle’s mutations although the severity of hypertension is not uniform in all patients. Others symptoms can include left ventricular hypertrophy, retinopathy and nephrosclerosis (Enslow et al. 2019). Hypertension-mediated complications in Liddle’s syndrome patients include renal, cardiovascular disease, and cerebrovascular disorders. Patients with this syndrome often die due to HF, myocardial infarction and cerebrovascular consequences. Patients with Liddle’s syndrome are treated with the blockers of ENaC, potassium-sparing diuretics, amiloride or triamterene that counteract hypertension and hypokalemic metabolic alkalosis. A low-sodium diet is also recommended to increase the efficacy of the therapy (Enslow et al. 2019). Impact of alterations of the aldosterone/MR axis on fluid and electrolyte homeostasis, as well as on the regulation of blood pressure is demonstrated also by other renal disorders. PHA types I and II represent genetic disorders characterized by hyperkalemia but derive from different causes (Tajima et al. 2017). PHA I is a rare disease which results in loss of weight, signs of failure to thrive, vomiting and dehydration, hyperkalemia, paralleled by increased plasma levels of renin and aldosterone. Autosomal recessive PHA I results from loss-of-function mutations whitin one of the three genes (SCNN1A, SCNN1B and SCNN1G) encoding the subunits of ENaC (Zennaro et al. 2020). The autosomal dominant PHA I represents the most frequent form of the disease which results from loss-of-function mutations in the NR3C2 gene enconding MR. Concerning the autosomal dominant PHA I, mutations associated with disease have been identified in all exons of the NR3C2 gene. The effects of many mutations of MR fuction have been studied in vitro and showed that either ligand or DNA binding can be altered (Zennaro et al. 2020). The

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autosomal recessive PHA I represents a more severe salt-wasting syndrome which may result in collapse, cardiac arrhythmias and cardiac arrest. Diagnosis in the early stages of the disease is a critical factor for survival of the patients who need supplementation of high doses of sodium. Therefore, comparison of the dominant and recessive form of PHA shows that patients with autosomal recessive PHA I display more severe symptoms and do not spontaneously improve (Zennaro et al. 2020). PHA II results from mutations in the serine-threonine kinases WNK which modulate activity of ion transporters such as Na+ -Cl cotransporter (NCC) and the ROMK channel in the distal nephron (Furgeson and Linas 2010). PHA II is a disorder characterized by hyperkalemia and hypertension, metabolic acidosis, reduction excretion of potassium, hypercalciuria, and low plasma levels of renin (Furgeson and Linas 2010). Aldosterone circulating levels may be either low or normal. Subjects with PHA II show hypertension deriving from increased Na+ reabsorption through NCC. WNK4 negatively regulates NCC, and mutated WNK4 stimulates NCC activity. These subject also display increased chloride reabsorption in the distal nephron whic results in reduced electronegativity of the lumen and decreaed electrochemical gradient for potassium secretion (Pathare et al. 2013). Mutations in WNK4 may reduce TRPV5 activity and reduce calcium reabsorption in the distal convoluted tubule, leading to urinary calcium loss and osteoporosis. In summary, it can be suggested that increased activity of NCC, which leads to increased reabsorption of sodium and chloride, plays a critical role in the development of hypertension in PHA II, and mutations in WNK1 and WNK4 may promote NCC function (Pathare et al. 2013). Impaired MR function, with subsequent alterations in electrolyte balance, can derive from failure in production of aldosterone in the adrenal glands (Barthel et al. 2019). In subjects with primary adrenal insufficiency (PAI), reduced production of aldosterone and glucocorticoids results in manifestations such as weakness, weight loss, anorexia, salt craving, nausea, vomiting, orthostatic hypotension, musculoskeletal pain, orthostatic hypotension which results from dehydration, anaemia, vitiligo (Barthel et al. 2019). Additional signs cal also include hyponatremia, hyperkaliemia, and hypoglycemia after physical exercise due to impaired epinephrine production. PAI, also termed as “Addison’s disease” is frequently caused by autoimmunity and genetic forms. In addition, neoplastic, or dysmetabolic diseases, as well as use of some drugs which inhibit adrenal steroidogenesis or surgical procedures result in adrenal damage (Barthel et al. 2019). More than half of the patients with autoimmune Addison’s disease show also other autoimmune diseases such as autoimmune thyroid disease, autoimmune gastritis, type 1 diabetes mellitus, celiac disease. In subjects with mineralocorticoid deficiency, treatment with fludrocortisone allows to counteract imbalance of fluids and electrolytes (Barthel et al. 2019). On the other hand, PA rises from autonomous production of aldosterone, which is not modulated by blood volume and salt status in the adrenal cortex. Such dysregulated aldosterone release results in enhancement of aldosterone-MR signaling and, in turn, leads to sodium retention and increased excretion of potassium and hydrogen, with subsequent hypertension, suppressed renin levels, hypokalaemia and metabolic alkalosis. Compared to subjects with primary hypertension, patients with

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PA matched for age and blood pressure display increased risk of CVD. Particularly, PA patients are at an elevated risk of myocardial infarction, atrial fibrillation, HF, stroke and renal disease (Zennaro et al. 2020). PA derives from unilateral aldosterone-producing adenoma (APA) or bilateral adrenal hyperplasia (BAH), which are two conditions termed as idiopathic hyperaldosteronism. PA results from mutations in the genes involved in the regulation of intracellular calcium signalling, inside the zona glomerulosa of the adrenal gland. These mutations increse intracellular calcium levels which, in turn, stimulate the expression of the aldosterone synthase CYP11B2 and aldosterone production. As recently disussed by De Sousa et al., APA is associated with mutations in the genes KCNJ5 and CACNA1D, encoding ion channels, and in the genes ATP1A1 and ATP2B3 encoding ATPases (De Sousa et al. 2019). Other mutations have been identified in the gene CTNNB1 encoding β-catenin, as well as in the gene PRKACA, which encodes the subunit α of the protein kinase A. Somatic mutations are identified in APA and in BAH. Majority of PA cases are due to sporadic mutations, nevertheless familial forms of PA have been identified. Mutations of the KCNJ5 gene encoding the potassium channel GIRK4 reduce the selectivity of the this channel and lead to increased influx of sodium. Mutations in the gene ATP1A1 encoding the α1 subunit of the Na+/K+ ATPase also result in increased concentrations of intracellula sodium, whereas mutations in the chloride channel CLC-2, result in increased activation of the channel (De Sousa et al. 2019). In the glomerulosa cells, each of these mutations results in membrane depolarization which favors opening of voltage-gated calcium channels. Increase in calcium intracellular concentrations stimulates the calciummediated signaling pathway which promotes aldosterone synthesis. Notably, mutations in the genes CACNA1H and CACNA1D encoding voltage-gated calcium, can directly increase intracellular levels of calcium and stimulate aldosterone production (De Sousa et al. 2019). Notably, certain somatic mutations have been identified also in aldosterone-producing cell clusters (APCCs) found in normal adrenal glands. Presence of mutations is expected to trigger an autonomous production of aldosterone, and it has been suggested that expansion of APCCs with accumulation of further mutations may lead to APA or BAH (De Sousa et al. 2019). Comparison between PA subjects and patients with essential hypertension has revealed an increased risk of cardiovascular disease in the patients with PA, suggesting that increased levels aldosterone may favor altered CV function regardless of hypertension (Zennaro et al. 2020). Hyperaldosteronism also results in glucose metabolism disorders, and can favor DM development (Okazaki-Hada et al. 2020). In PA, hypokalemia can alter insulin secretion, and high levels of aldosterone can by itself impair β-cell function and promote IR (Okazaki-Hada et al. 2020). Over the last 10 years, numerous studies have shown that MR is expressed in extra-renal organs and tissues, including AT, heart, and the vasculature. Activation of extra-renal MR may contribute to the cardiovascular and metabolic disorders observed in PA (Byrd et al. 2018). In addition, increased plasma levels of aldosterone have been detected in patients with obesity and MetS, further suggesting that extra-renal MR dysfunction has a causal role in the development of metabolic and CV dysfunctions (Feraco et al. 2020). Studies with murine models overexpressing or lacking MR in specific tissues

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have investigated the role of MR both in the physiological extra-renal tissues function and in the pathophysiology of hypertension, obesity, IR and atherosclerosis (see below).

Extra-Renal Mineralocorticoid Receptor Mineralocorticoid Receptor in the Vasculature Expression of MR has been detected in the vessels, both in the vascular smooth muscle cells (VSMCs) and ECs, and many studies suggest that MR is able to modulate vascular contractile properties and affects important processes which are deeply involved in the pathophysiology of CVD (Feraco et al. 2020). Studies by the group of Iris Jaffe have revealed that VSMC MR modulates vascular tone without causing vascular morphology changes or alterations in renal sodium reabsorption, suggesting a directly contribution of VSMC MR to the regulation of systemic BP (McCurley et al. 2012). In these studies, aged VSMC MR knockout (KO) mice displayed reduced BP, but did not show impaired sodium handling in the kidney, or altered vascular structure. Interestingly, reduction in in BP was observed in VSMC MR KO mice, compared with control mice, after 7 months of age. The observed decrease in vascular myogenic tone and BP was mediated by reduced expression of the L-type Ca2+ channel Cav1.2. Molecular data showed that MR activity reduces levels of the micro-RNA miR-155 in VSMCs. Cav1.2 represents a target of the miR-155, and miR-155 has been shown to reduce Cav1.2 transcript (DuPont et al. 2016). Accordingly, in VSMC MR KO mice, expression of Cav 1.2 was downregulated by miR-155 due to the absence of MR activity, resulting in blunted LTCC-mediated vasoconstriction. Other studies have investigated the role of EC MR in the regulation of vascular tone and BP. Analysis of transgenic mice overexpressing EC MR revealed increased BP, with no indication of endothelial dysfunction, of alterations of vasculature morphology, or impaired sodium transport in the kidney (Feraco et al. 2020). Nevertheless, no changes in BP were observed in mice lacking EC MR, and the physiological impact of EC MR on BP remains still unclear (Feraco et al. 2020). Although aldosterone is known to classically activate MR through transcriptional control of MR target genes, rapid non-genomic actions of aldosterone have also been reported to affect vascular reactivity (Ruhs et al. 2017). In VSMCs, non-genomic aldosterone signaling leads to vasoconstriction through complex signaling pathways involving cyclic adenosine monophosphate, Na+/H+ exchanger (NHE), insulin-like growth factor-1, c-Src, phosphoinositide 3-kinase, and platelet-derived growth factor receptors. Particularly, aldosterone stimulates reactive oxygen species (ROS) production through a molecular pathway involving c-Src that leads to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation (Feraco et al. 2020). ROS are generated by NADPH oxidase and reduce the availability of intracellular nitric oxide (NO), synthesized by the endothelial nitric oxide synthase (eNOS) (Feraco et al. 2020). NO has a central role in activating soluble guanylyl cyclase (sGC) to generate cyclic guanosine monophosphate (cGMP), and reduced levels of NO are expected to decreased cGMP production and protein

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kinase G activation, resulting in reduced VSMC relaxation. Decreased NO production in aldosterone-treated VSMCs has been observed to stimulate vascular contraction (Feraco et al. 2020). In ECs, the role of MR in regulating NO levels is still controversial since some studies show that aldosterone stimulates NO production, while others reveal reduced eNOS activity which is mediated by the increase in ROS production (Feraco et al. 2020). In ex vivo experiments with rat arterioles, Heylen et al. showed that aldosterone treatment results in vessel relaxation, whereas aldosterone induces a vasoconstrictor response in endothelium-denuded vessels, suggesting that MR activity in VSMCs and ECs, also in the presence of physiological concentrations of aldosterone (0,1–1 nM), may have opposite effects on vascular tone (Heylen et al. 2009). Regulation of vascular function by MR has been investigated in human studies. Effects of aldosterone have been analyzed on resistance arteries in healthy subjects by Romagni and collaborators who measured forearm blood flow (FBF) by using venous occlusion plethysmography. Aldosterone treatment reduced FBF after few minutes, revealing that aldosterone was able to induce rapid vasconstriction (Romagni et al. 2003). In another study with healthy subjects, aldosterone infusion alone did not modify FBF, although coinfusion of Aldosterone acutely enhanced vasodilatatory response to exogenous NO (Nietlispach et al. 2007). These data are in contrast with the study perfomed by Farquharson and Struthers who observed that aldosterone treatment led to reduced endothelium-dependent vasodilatation (Farquharson and Struthers 2002). In summary, these studies show discrepant results, although we can admit that aldosterone infusion at high doses may not be the proper approach to study the effects of MR activation on the vessels. In alternative to aldosterone infusion, treatment with MRAs may represent a more suited strategy to identify the effecte of the physiological activity of the vascular MR. In healthy older adults, response to treatment with the MRA eplerenone has shown a reduction in endotheliumdependent relaxation, paralleled by a reduction in eNOS activity (Feraco et al. 2020). Eplerenone did not affect endothelium-independent dilation, which excluded MR antagonism-mediated changes in the VSMC function. The study suggested that MR function regulates vascular tone in healthy subjects, and may modulate eNOS function and endothelial function. In summary, the issue of the physiological role of vascular MR still remains open. Another aspect which can complicate data interpretation in these study is that glucocorticoids (GCs) levels were not investigated in the enrolled subjects. In preclinical studies, glucocorticoid receptor (GR) activation has been show to affect vascular properties of resistance arterioles and GCs are able to activate MR, although expression of 11-HSD2 in the vessel is expected to allow selective activation by aldosterone (Feraco et al. 2020).

Overactivation of Vascular Mineralocorticoid Receptor There is a large body of evidence supporting a crucial role for altered vascular MR signaling in hypertension, endothelial dysfunction and atherosclerosis (Feraco et al. 2020). As above mentioned, mice with conditional overexpression of the MR in ECs

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develop higher BP without showing vascular morphological alterations, indicating that specific MR overexpression in the endothelium affects BP independently of salt status alteration and vascular remodeling. These effects were reverted by pharmacological MR blockade (Feraco et al. 2020). In EC cultures, MR overactivity induced by high concentrations of aldosterone has been shown to enhance ROS levels and reduce activation of eNOS reducing NO bioavailability which, in vivo, may have a causal role for vasoconstriction (Feraco et al. 2020). In a mouse model of endothelial dysfunction induced by obesity, ex vivo experiments with aortae from these mice treated with eplerenone showed that MR blockade reduced endothelial dysfunction (Feraco et al. 2020). Similar results were observed also in transgenic mice lacking EC MR: depletion of MR in ECs protected mice against endothelial dysfunction induced by obesity (Feraco et al. 2020). The very interesting concept is that MR activity was able to promote endothelial dysfunction in the presence of obesity. Another study confirmed the key role of EC MR in altering vascular function in obese mice. In fact, mice fed an obesogenic diet showed aortic stiffness which was associated with increased expression of ENaC, reduced activity of eNOS and oxidative stress (Feraco et al. 2020). Mice fed a high fat diet (HFD) with depletion of EC MR were protected against aortic stiffness, decreased production of NO and oxidative stress, indicating that EC MR has a crucial role in the regulation of vessel contractility (Jia et al. 2016). Also in humans, excess of adiposity has been associated to the development of arterial stiffness, which predicts cardiovascular mortality (Feraco et al. 2020). Jia and colleagues suggested that EC MR mediates the link between obesity and endothelial dysfunction, and identified EC MR as a target to counteract alterations in vascular contractile function (Jia et al. 2016). High levels of circulating aldosterone detected in obesity and MetS suggest that overactivation of aldosterone-MR signaling may favor the development of vascular alterations associated with the above-mentioned pathological conditions. Plasma aldosterone represents a predictor of atherosclerotic plaque progression and preclinical studies have indicated that activation of vascular MR promotes atherosclerosis development (de Rita et al. 2012). Cell-based experiments have shown that treatment with aldosterone raises EC expression of the intercellular adhesion molecule-1 (ICAM-1) and favors monocyte binding to ECs which, in vivo, may represent the initial stage in the plaque formation (Caprio et al. 2008). Involvement of MR in these effects of aldosterone was confirmed by cotreatment with MR antagonist which prevented up-regulation of ICAM-1 and monocyte adhesion. Experiments with the transgenic apolipoprotein-E knockout (ApoE KO) mice, a well-known murine model to study atherosclerosis, treated with aldosterone confirmed data obtained with EC cultures (Marzolla et al. 2017). In these mice, aldosterone promoted plaque formation and favored a plaque phenotype associated with ruptured and characterized by high amount of lipids and macrophage infiltration. In vivo contribution of ICAM-1 was confirmed in double ApoE/ ICAM-1 KO mice which displayed resistance to develop aldosterone-mediated atherosclerosis (Marzolla et al. 2017). ApoE KO mice treated with MRAs show decreased atherosclerosis, accordingly (Feraco et al. 2020). Overactivation of VSMC MR may result in increased oxidative stress which damages function of sGC and, in the end, reduce cGMP availability with subsequent

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impaired vascular relaxation (Feraco et al. 2020). In VSMC cultures, MR overactivation induced by high levels of aldosterone promotes expression of genes involved in vascular fibrosis (collagen type I and type III), inflammatory response (IL-6, cytotoxic T-lymphocyte–associated protein 4) and vascular calcification (bone morphogenetic protein 2, alkaline phosphatase, parathyroid hormone receptor 2) (Jaffe and Mendelsohn 2005). Interestingly, ex vivo experiments with mouse aortas showed that MR-regulated expression of a subset of genes, among those analyzed in the study, was further stimulated by endothelium damage and oxidative stress (Newfell et al. 2011), reinforcing the concept that metabolic stress factors may promote MR activity which, in turn, leads to severe tissue function derangements. These studies identify VSMC MR as a crucial player in the vascular remodeling, a process which takes place for instance after endothelial damage triggered by insults from CV risk factors such as smoke, dyslipidemia, hypertension. MR activation effects on vascular remodeling were studied in a mouse model of wire-induced carotid injury model, a well-known model to study vascular responses to the endothelial damage (Jaffe et al. 2010). In this model, aldosterone treatment, at a dose that does not alters BP, promoted VSMC proliferation, and extracellulr matrix (ECM) deposition. Importantly, in the absence of vascular injury, aldosterone alone was not able to stimulate cell proliferation and ECM production, supporting the assumption that MR activity, to induce severe alterations, requires various insults i.e. endothelial damage (Jaffe et al. 2010). The study by Jaffe et al. showed that placental growth factor (PGF), a secreted protein member of vascular endothelial growth factor (VEGF) family, is a gene regulated by the transcriptional activity of the vascular MR. Aldosterone increases vascular levels of the PGF transcript. Importantly, aldosterone-mediated vascular remodeling was prevented in mice with genetic depletion of PGF (Pgf KO mice), showing that PGF is a crucial player in mediating aldosterone-enhanced vascular injury. PGF binds and promotes inflammatory cell chemotaxis, a fundamental step in atherogenesis (Jaffe et al. 2010). Promotion of chemotaxis by PGF is allowed by the expression of the transmembrane VEGF type 1 receptor in monocytes and can contribute to leukocyte recruitment in the early stages of plaque formation (McGraw et al. 2013). These data suggest that PGF may represent an attractive target to fight early steps of plaque formation. Endothelial dysfunction is associated with CVD and is commonly found in subjects with HF (Feraco et al. 2020). Spironolactone administration to patients with congestive HF placed on conventional pharmacological therapy revelaed that MRAs improved endothelial function (Abiose et al. 2004). Particularly, this study showed that spironolactone improved the endothelium-dependent brachial artery vasodilation, probably through an enhancement of activity of eNOS and incraesed production of NO. Obesity is another condition which is associated with altered endothelial function (Feraco et al. 2020). Notably, obesity is characterized by high circulating levels of aldosterone which might affect vascular function through ovearctivation of vascular MR. Eplerenone treatment of subjects showing wide variability in obesity revealed that improvements in endothelial function were associated with more marked obesity and higher fasting glucose levels, indicating

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that benefical effects of MR antagonism on the vascular function were positively associated with AT abundance (Hwang et al. 2013). These mentioned human studies show that terapies with MRAs may represent an effective treatment to dampen endothelial dysfunction probably mediated by overactivation of vascular MR, suggesting that dysregulated MR activity in the vessel, as indicated by several preclinical studies, leads to detrimental effects on vascular function.

Mineralocorticoid Receptor in Cardiac Physiology At the end of the 80s, a study by Pearce and Funder revealed the presence of MR in human cardiomyocytes (Pearce and Funder 1988). More recently, microarray analysis of the cardiac transcriptome of mice overexpressing MR in the cardiomyocyte (MR-cardio mice) treated with aldosterone has shown that MR is able to modulate many different classes of genes encoding growth factors (connective tissue growth factor, hepatocyte growth factor), ion (potassium, calcium) channels and cell adhesion molecules (cadherin 4, integrin β6) (Messaoudi et al. 2013). Notably, aldosterone treatment modulated many genes in these mice, indicating responsiveness of cardiomyocytes to aldosterone, although these cells express low levels of 11-HSD2. In another study, treatment of neonatal rat ventricular cardiomyocyte cultures with aldosterone was able to affect the expression of cardiac voltage-operated calcium channels and accelerate contraction rate (Lalevee et al. 2005). Calcium channels play a crucial role in normal cardiac function. They partecipate to the generation and conduction of the action potential and to the contraction. Activity of the L-type Ca2+ channels stimulates calcium flux into the cell which generates contraction, whereas the T-type Ca2+ channels mainly regulate the conduction of the action potential (Shorofsky and Balke 2001). At a molecular level, aldosterone significantly increased transcript levels of α1C and β2 subunits of the L-type Ca2+ channel, as well as expression of α1H isoform of the T-type Ca2+ channel, with subsequent rising of the L- and T-type Ca2+ currents (Shorofsky and Balke 2001). Other studies provided evidence for non-genomic effects of aldosterone on ion fluxes affecting Na+-K+-2Cl cotransporter and NHE activities, leading to rapid sodium influx and increased cell volume of rat ventricular cardiomyocytes (Matsui et al. 2007). It has been suggested that these effects of aldosterone may protect cardiomyocytes against dehydration in the presence of high extracellular concentration of Na+(Matsui et al. 2007). However, the supraphysiological concentrations of aldosterone used in these experiments (100 nM) do not allow to infer the role of MR in the physiology of the cardiomyocyte in terms of MR-regulated gene expression and spontaneous contraction. In vivo studies performed by different research groups in cardiomyocyte MR KO mice show normal heart development and systolic function and no changes in diastolic function in the absence of any surgical or pharmacological stress intervention (Fraccarollo et al. 2011) suggesting that lack of MR function does not alter cardiomyocyte differentiation and contractile function, at least under basal conditions.

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Mineralocorticoid Receptor Overactivation in the Heart It is well known that renin-angiotensin-aldosterone system (RAAS) overactivation promotes the development of cardiac remodeling and CVD (Ferrario and Strawn 2006). Aldosterone, as final effector of RAAS, is known to promote cardiac fibrosis, hypertrophy and atrial arrhythmias, and the RALES and EPHESUS trials provided strong evidence of the cardioprotective effects of MRAs (see below section “Mineralocorticoid Receptor Antagonists in Cardiovascular and Renal Dysfunctions”) (Bauersachs et al. 2015). Dysregulated MR activity has been associated with cardiac arrhythmias and HF (Fig. 2). As observed in immunohistochemical and in situ hybridization studies, increased expression of MR transcript and protein has been observed in the cardiomyocytes of patients with congestive HF (Yoshida et al. 2005). Higher levels of MR have also been detected in atrial cardiomyocytes of patients with atrial fibrillation, compared to subjects with normal sinus rhythm (Tsai et al. 2010). Cell-based experiments showed that depolarization of HL-1 myocytes, by using rapid electrical-field stimulation, raised MR expression and activity. Aldosterone in these cells enhanced inward T-type Ca2+ (ICaT) current, promoting the intracellular calcium load (Tsai et al. 2010). These modulation of calcium currents reflected changes in gene expression of α1G and α1h subunits of the T-type Ca2+ channel, confirming the ability of MR to modulate ion channel expression in the cardiomyocyte and lead to electrical remodeling (Tsai et al. 2010). These data suggest that treatment with MRA potentially represents a strategy to counteract calcium overload and atrial fibrillation. Another study that performed whole cell patch-clamp measures on ventricular cardiomyocytes isolated from mice with renal salt-losing phenotype and upregulated circulating levels of aldosterone revealed large values of L-type Ca2+ currents associated with high plasma aldosterone levels (Perrier et al. 2005). On the contrary, the same study showed that L-type Ca2+ currents were reduced in cardiomyocytes isolated from mice with salt-retaining phenotype and low plasma levels of aldosterone. These data suggested that modulation of cardiomyocyte MR activity by aldosterone is able to affect also L-type Ca2+ channel function, a regulation which may have important consequences for excitation-contraction coupling. Alterd activity of the cardiac ryanodine receptor (RyR) can contribute to arrhythmia development. This channel localizes in the sarcoplasmic reticulum (SR) and allows the release of the calcium from the SR lumen into the cytosol which, in turn, determines activation of the myofilaments and contraction (Shorofsky and Balke 2001). By using a patch clamp based approach, a study by Gomez et al. observed that aldosterone treatment induced delayed afterdepolarizations during action potential recordings in isolated rat ventricular myocytes as well as in ventricular myocytes derived from transgenic mice expressing human MR (hMR mice) in the heart (Gomez et al. 2009). Although cardiac RyR expression was not altered, activty of this channel was increased and these changes were probably mediated by downregulated abundance of FK506-binding proteins, which are know to associate to RyR and regulate its activity in a macromolecular complex. Interestingly, reduced

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Fig. 2 Detrimental effects of MR overactivation in the heart, kidney, and adipose tissue. MR overactivation results in increased generation of oxidative stress and contributes to the development of fibrosis and inflammation in the heart and in kidney. Enhancement of adipose tissue-specific MR activation leads to dysregulated adipogenesis, oxidative stress, inflammation, and alterations in adipokine expression

physical association between FKBP12, FKBP12.6 and RyR, which potentially alters RyR activity, was also detected in myocytes after aldosterone treatment and in hMR mice (Gomez et al. 2009). Accordingly, another study performed with transgenic mice with overexpression of MR in cardiomyocytes showed heart arrhythmias leading to defects in cardiac function and a high frequency of death. Surviving

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mice showed abnormal electrocardiogram and ventricular arrhythmias, associated with prolonged action potential duration and increased whole-cell calcium current. Lethality due to overexpression of MR was prevented by pharmacological treatment with spironolactone (Ouvrard-Pascaud et al. 2005). On the other hand, cardiomyocyte MR KO mice reveal normal cardiac function. As previously mentioned, these mice showed neither alterations in systolic and diastolic pressures, heart rate, and LV contraction, nor cardiac fibrosis without surgical manipulation (Lother et al. 2011). Nevertheless, surgical manipulation aimed to induce HF in these mice showed that MR has an important contribution in cardiac remodeling. Left coronary artery ligation is a very well-known technique used to study cardiac acute injury and chronic remodeling (Reichert et al. 2017) and cardiomyocyte MR KO mice that underwent this procedure displayed reduced progressive ventricular dilation, improved cardiac function associated with reduced cardiac hypertrophy and ECM accumulation, and enhanced neovascularization in the healing myocardium. Expression of genes associated with pathological hypertrophy, stiffness, and fibrosis was significantly decreased, as well ROS production (Fraccarollo et al. 2011). Particularly, cardiomyocyte MR ablation led to reduced expression of β-myosin heavy chain, ACE (genes involved in the hypertrophy development), and CTGF, collagen, fibronectin, vimentin (genes involved in fibrosis). Compared with control mice, infarcted cardiomyocyte MR KO mice showed reduced myocardial expression of the NADPH oxidase subunits Nox2 and Nox4, as well as decreased production of superoxide anion. Cardioprotection deriving from absence of cardiomyocyte MR was also mediated by increased degradation of IκBα protein and subsequent enhancement of nuclear factor-κB (NF-κB) activity promoting early inflammatory activation and healing, through induction of the chemoattractant RANTES and subsequent accelerated recruitment of monocytes and macrophages into the damaged myocardium (Fraccarollo et al. 2011). In another study, cardiomyocyte MR-null mice that underwent transverse aortic constriction, another experimental technique commonly used to study cardiac hypertrophy and HF development, showed protection from left ventricular dilatation and dysfunction. LVEF as well as cardiac levels of phosphorylated ERK1/2 were higher in cardiomyocyte MR-null mice after pressure overload, and ERK1/2 activation was proposed to counteract cardiac dilatation and failure after pressure overload (Lother et al. 2011). Cardiomyocyte MR function in the development of cardiac fibrosis (Fig. 2) has been widely explored by Young and collaborators in the deoxycorticosterone (DOC)/salt model. Cardiomyocyte-MR null mice did not show alterations in cardiac function, compared with control mice with intact expression of cardiomyocyte-MR, although depletion of cardiomyocyte MR resulted in reduced cardiac expression of fibrotic markers (Rickard et al. 2012). Cardiomyocyte-MR null mice challenged with DOC/salt for 8 weeks displayed protection against cardiac fibrosis, as confirmed by a reduction of transforming growth factor-β (TGF-β1) and integrin-β1, and showed a parallel increase in the expression of the antifibrotic factor TGF-β inhibitor decorin in cardiac tissue (Rickard et al. 2012). A higher activity of matrix metalloproteinase2 /matrix metalloproteinase-9 in these mice could also contribute to the absence of

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fibrotic response. In addition, macrophage infiltration was reduced in the myocardium of cardiomyocyte-MR null mice (Rickard et al. 2012). This study clearly demonstrated a crucial role for cardiomyocyte MR in cardiac tissue inflammation and remodeling in a context of high salt. Overall, studies in transgenic mice with altered function of cardiomyocyte MR show that overexpression of MR in the heart contributes to arrhythmia, and mechanical or fibrotic insults promote activation of MR resulting in cardiac fibrosis and remodeling.

Role of Mineralocorticoid Receptor in Adipose Tissue Physiology In 2007, Caprio et al. showed that the white adipocyte expresses MR and activity of this receptor regulates adipogenic differentiation (Caprio et al. 2007). Cultures of white murine preadipocytes (3T3-L1 or 3T3-F442A preadipocytes) were induced to differentiate in the presence of aldosterone and revealed a significant increase in adipogenic markers, indicating that MR activation promotes white adipocyte differentiation in vitro. Particularly, aldosterone increased transcript levels of aP2, adiponectin, leptin, and resistin in 3T3-L1 cells. In murine adipose cell cultures, abundance of MR was increased in differentiated adipocytes, compared with undifferentiated preadipocytes. In addition, a dose-dependent effect of aldosterone was observed on intracellular triglyceride amount and glycerol-3-phosphate dehydrogenase (G3PDH) activity, which are considered two indexes of adipose conversion (Caprio et al. 2007). Aldosterone enhanced G3PDH activity and cell triglyceride abundance, confirming that MR activation favors white adipogenesis in mouse preadipocyte cultures. In this study, preadipocyte cultures were treated with aldosterone to selectively activate MR and exclude GR (Caprio et al. 2007). On the contrary, experiments of MR knock-down or pharmacological antagonism of MR showed inhibition of in vitro adipogenic differentiation (Caprio et al. 2011). This antiadipogenic effects of MR blockade were observed also in human adipocyte cultures (Caprio et al. 2011). Accordingly, experiments with cultures of adipocytes derived from newborn homozygous MR KO mice showed that absence of MR led to impaired lipid accumulation and reduced protein expression of the adipogenic markers CCAAT/enhancer-binding protein (C/EBPα) and aP2 (Hoppmann et al. 2010). The AT lacks the enzyme 11-HSD2 and this suggests that AT-specific MR function is predominantly affected by circulating GCs in vivo. Thus, in vivo adipogenesis is expected to be modulated by GCs whose AT-specific levels are regulated by the enzyme 11-HSD1, which converts cortisone (in humans) or 11-dehydrocorticosterone (in rodents) to active GCs capable of stimulating GR (Feraco et al. 2020). In experiments with preadipocyte culture, GSs have been shown to affect human adipogenesis. It should be noted that standard adipogenic cocktails used in 3T3-L1 cells include the GC agonist dexamethason (Armani et al. 2010). As observed by Park et al., dexamethason was able to promote adipogenesis through activation of GR which, in turn, up-regulates expression of adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ), C/EBPα, C/EBPβ, C/EBPδ, which are involved in the early stage of adipogenic

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differentiation (Park and Ge 2017). Data by Lee et al. have revealed that knockdown of GR counteracted differentiation in cultures of primary human preadipocyte. Noticeably, in the same study, the knockdown of MR did not alter adipocyte differentiation (Lee and Fried 2014). These study confimed that the involvement of the interplay between MR and GR in regulating adipogenesis remains to be further investigated. In cultures of differentiated adipocytes, activation of MR by aldosterone led to increased expression of leptin, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), lipocalin-like prostaglandin D2 synthase (PTGDS), indicating that MR is capable of affecting the inflammatory status of AT (Urbanet et al. 2015; Hirata et al. 2009). Additional work showed that MR regulates molecular pathways involved in ROS production and autophagy. Particularly, autophagy in eukaryotic cells is a process which regulates organelle and protein turnover and contributes to maintain tissue homeostasis (Mizushima and Levine 2010). Autophagy consists in a sequence of subsequent steps leading to fusion of the autophagosome, containing organelles and proteins, with the lysosome, and subsequent degradation of the contents sequestered by the autophagosome. The degradation products of autophagy can be used in the cytoplasm to build proteins and macromolecules (Glick et al. 2010). Increased amount of autophagosomes has been detected in 3T3-L1 preadipocyte cultures as well as in primary mouse embryonic fibroblasts induced to differentiate. Importantly, knockdown experiments aimed to remove function of genes involved in the autophagic process (autophagy-related genes (atg) 5, atg 7) resulted in altered adipogenesis, suggesting that authopagy plays a key role in adipocyte differentiation (Armani et al. 2015). A study by Armani et al. has shown that MR activity regulates autophagic flux in 3T3-L1 and in primary murine adipocyte cultures. Activation of MR by aldosterone led to increased autophagy. On the contrast, pharmacological blockade of MR resulted in reduced autophagic flux, both in adipocyte cultures and in mice treated with MRAs which displayed reduced WAT expansion (Armani et al. 2014). These data indicated that MR is able to regulate adipogenesis by modulating the autophagic flux. Two studies performed with transgenic mice lacking MR expression specifically in the adipocyte (adipo-MR KO mice) have provided additional information on the physiological role of MR in the AT (Feraco et al. 2020). Adipo-MR KO mice carrying genetic ablation of the MR gene in mature adipocytes did not showed changes in body weight, glucose tolerance, adipocyte size, or altered formation of AT. In WAT of adipo-MR KO mice, expression of adipokines and inflammatory markers was not changed (Feraco et al. 2018). As observed by Feraco et al., administration of an obesogenic diet led to increased fat mass and enlarged adipocyte size both in adipo-MR KO and control mice, suggesting that MR, expressed by the mature adipocyte, does not regulate cell size and fat mass (Feraco et al. 2018). These two studies suggest that MR has an irrelevant role in the regultion of physiological functions of mature adipocytes. On the other hand, it must be considered that the transgenic mice of both studies expressed Adipoq-Cre which is under the transcriptional control of adiponectin regulatory elements, leading to removal of the MR gene only in differentiated adipocytes (Feraco et al. 2020). It cannot be

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excluded that in vivo MR function may be less prominent in differentiated adipocytes, compared with the early stages of adipogenesis. These studies were not able to prove that MR regulates the early phases of adipogenic differentiation in vivo. Notably, transgenic mice expressing Cre driven by preadipocyte-specific promoters are not available and this make it impracticle to investigate the real impact of MR in adipogenesis (Feraco et al. 2020). As above discussed, preclinical studies show that GR plays a relevant role in regulating adipogenesis in vitro (Feraco et al. 2020). In vivo experiments with adipocyte-specific GR KO mice have shown that ablation of GR in mature adipocytes did not alter body weight or AT formation, suggesting that GR plays a non-essential role in white adipogenesis in vivo (Desarzens and Faresse 2016). As observed for adipocyte MR KO mice, removal of the GR gene is regulated by adipoq-Cre and takes place in the mature adipocyte, suggesting that GR is still allowed to regulate molecular pathways involved in adipocyte differentiation, without alterations during adipogenesis in adipocyte GR KO mice (Desarzens and Faresse 2016). Mammals show two different types of AT with different morphology and functions (Cinti 2012): WAT and BAT. WAT is primarily deputed to store lipids in the form of triglycerides. Function of BAT is to burn fat and convert chemical energy into heat (Cinti 2012), through uncoupling protein 1 (UCP1)-mediated respiration, termed as uncoupled respiration. BAT has a crucial role in regulating thermogenesis and its activity confers anti-obesity effects in small mammals. In addition to BAT, a number of studies have shown the presence of inducible brown-like adipocytes termed “beige” or “brite” (Kajimura et al. 2010). These cells are detectable in WAT of mice after cold exposure or treatment with β3-adrenergic agonists. The beige adipocytes have morphology similar with that of classical brown adipocytes and display thermogenic activity. β-adrenergic stimulation results in the acquisition of brown fat features by WAT in term of increased UCP1 expression and incresed uncoupled respiration (Cinti 2012). Such process is known as “browning” and numerous studies have show that BAT activation and/or browning protects against weigh gain, fat mass expansion and associated dysregulated metabolism of glucose in mice. Brown adipocytes have been discovered also in adult humans and activity of brown fat is inversely associated with BMI, indicating that activation of brwn adipocytes might counteract obesity and diabetes (Cinti 2012). Lombes and collaborators showed that MR is expressed in BAT, observing that MR activity inhibits expression of UCP1 which confers thermogenic function to the brown adipocyte (Viengchareun et al. 2001) (Fig. 2). In hibernoma-derived T37i brown adipocytes expressing functional MR, aldosterone treatment was able to reduce isoproterenol-stimulated UCP1 transcript levels. Noticeably, the treatment of T37i cells with cycloheximide did not change the inhibitory effects of aldosterone on UCP1 mRNA levels, indicating that de novo protein synthesis was not required to damp UCP1 transcript levels (Viengchareun et al. 2001). In accordance with these data, we have shown that pharmacological antagonism of MR results in increased expression of UCP1 and other brown adipocyte-specific markers in BAT of mice fed a HFD (Armani et al. 2014). More recently, Thuzar et al. revealed that MR blockade promotes BAT activation also in humans (Thuzar et al. 2018). The study showed that

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2 week-treatment of healthy humans with the MRA spironolactone enhanced activity of BAT, as observed by using FDG-PET-CT scanning of the neck and upper chest, and such increase in BAT activity was associated with an increased thermogenic activity of the supraclavicular and upper chest regions. The authors of the study also showed that MR antagonism led to reduced lipid biosynthesis, as a likely consequence of thermogenic activation, suggesting that MR antagonism-mediated enhancement of BAT activity promotes a shift in energy utilization which favors energy dissipation, through thermogenesis, rather than nutrients storage (Thuzar et al. 2018). These data by Thuzar et al. suggest that BAT-specific MR activity is able to affect physiological adaptation to cold and modulate thermogenic activity of BAT, Indeed, MR overactivation in BAT might blunt heat dissipation and promote lipid storage.

Mineralocorticoid Receptor in Adipose Tissue Dysfunctions Higher expression levels of MR detected in AT of human subjects and mice that exhibit obesity suggests that overactivation of MR may contribute to AT dysfunctions and metabolic comorbidities. A study by Urbanet et al. has verified this hypothesis by generating transgenic mice overexpressing adipocyte-specific MR (Adipo-MROE mice) (Urbanet et al. 2015). These mice showed increased body weight and adipocyte size, increase in visceral AT and IR. Plasma insulin levels and HOMA index were increased in Adipo-MROE mice which also displayed hypertriglyceridemia and hypercholesterolemia, compared with control mice. Insulin sensitivity was further worsened by HFD administration in Adipo-MROE mice. Notably, this study indicates that adipocyte-specific MR overexpression may per se cause obesity and MetS features, showing that MR activity, in the adipocyte, affects AT as well as glucose and lipid metabolism. Adipo-MROE mice revealed higher expression of PTGDS, an enzyme involved in adipogenesis, although the effects of its activity are controversial (Urbanet et al. 2015). Reduced expression levels of PTGDS have been shown to stimulate adipogenesis, whereas another study by Fujimori et al. has observed that this enzyme promotes adipocyte differentiation through enhancement of PPARγ-mediated transcription activity (Chowdhury et al. 2011; Fujimori et al. 2012). Urbanet et al. have shown that MR activation, in 3T3-L1 adipocytes, led to increased expresion of PTGDS which, in turn, favored adipogenesis. Co-treatment of adipose cells with aldosterone and the PTGDS inhibitor AT56 reduced the adipogenic activity of aldosterone. Reduced transcript levels of PPARγ could mediate the antiadipogenic effects of PTGDS inhibition in adipocyte cultures co-treated with AT56 (Urbanet et al. 2015). In summary, this study proposed the involvement of the pathway MR-PTGDS-PPARγ in the regulation of white adipocyte function, suggesting that dysregulated activity of this pathway can alter WAT metabolism. Interestingly, another study has shown that PTGDS in BAT is required to utilize lipids as fuel for thermogenesis, and PTGDS expression is positively associated with BAT activity. Mice lacking PTGDS displayed increased lipid content in BAT and, in parallel, increased expression of genes involved in

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glucose uptake and glycolysis. Noticeably, these mice revealed increased glucose utilization by BAT with subsequent improvement in glucose tolerance (Virtue et al. 2012). Together, these data suggest that PTGDS may affect AT metabolism and glucose uptake, and increased function of PTGDS in BAT of Adipo-MROE mice might contribute to glucose metabolism dysfunctions. Activity of MR in AT regulates leptin expression (Guo et al. 2008). Leptin is an adipokine mainly involved in regulation of appetite and energy homeostasis. In addition, leptin shows a crucial role in several processes such as inflammation, angiogenesis, immune function, and reproduction. Leptin affects insulin sensitivity and lipid metabolism, and high levels of leptin are positively associated, in obese subjects, with the occurrance of metabolic alterations such as IR and CVD (Sweeney 2010). Increase in circulating levels of leptin in obesity are ascribed to leptin resistance in these subjects and many studies have shown that elevated leptin levels correlate with hypertension, atherosclerosis, stroke, MI (Sweeney 2010). Interestingly, preclinical data demonstrated that leptin is able to directly regulate adrenal CYP11B2 expression and aldosterone secretion by the adrenal cortex. As shown by Huby et al., leptin treatment led to increased aldosterone levels and higher adrenal expression of CYP11B2 in mice (Huby et al. 2015), with no changes in plasma concentrations of Ang II or potassium levels, which represent well recognized modulators of aldosterone secretion. Leptin regulation of aldosterone production is independent from RAS activation, since leptin-induced aldosterone increase was not impaired in mice treated with the inhibitor of the angiotensin type I receptor losartan (Huby et al. 2015). Accordingly, leptin-deficient (ob/ob) mice did not display increased levels of aldosterone and up-regulation of CYP11B2 expression, indicating a key role for leptin in stimulating aldosterone production. Infusion of leptin in these mice resulted in increase of adrenal CYP11B2 expression and aldosterone levels, to confirm the involvement of leptin in aldosterone production (Huby et al. 2015). These preclinical data support the existence of a link between AT and adrenal glands in obese subjects. The model suggests that leptin stimulates production of aldosterone which, in turn, stimulates adipocyte MR transcriptional activity and further promotes MR-mediated leptin transcription. As discussed above, adipocyte MR overactivation is expected to promote adipocyte dysfunctions and favor IR and MetS features, at least in mice (Urbanet et al. 2015). Huby et al. confirmed in vivo observation by analyzing cultures of human adrenal zona glomerulosa cells (HAC15 cells) stimulated with leptin. An increase in expressiono of CYP11B2 and secretion of aldosterone was observed in a leptin dose-dependently manner in these cells. Increased expression of CYP11B2 was suppressed by co-treatment with a leptin receptor antagonist but was not alterd by administration of losartan (blocker of Ang II receptor). At a molecular level, cell culture experiments showed that leptin treatment led to an incraese in Ca2+ signaling, as also oberved upon activation of AngII signaling in adrenocortical cells. Administration of a calcium chelator to HAC15 cells repressed leptin-induced stimulation of CYP11B2 expression (Huby et al. 2015). Altogether, these observations suggest that the effects of leptin on aldosterone production might provide a mechanism which explains the high levels of this steroid hormone in obese subjects.

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In obesity, dysfunctional adipocytes are characterized by increased expression of pro-inflammatory adipokines such as IL-6, plasminogen activator inhibitor-1, TNF-α and MCP-1, paralleled by a decreased expression of anti-inflammatory adipokines such as adiponectin and interleukin-10 (Nakamura et al. 2014). In addition to leptin, expression of IL-6, TNF-α and MCP-1 is also under the control of MR in the adipocyte (Fig. 2), and increased levels of aldosterone detected in obesity and MetS may stimulate MR-mediated expression of these inflammatory adipokines (Guo et al. 2008). A number of studies has demonstrated that obesity is associated with systemic and local increase of oxidative stress in AT resulting in alterd secretion of adipokines (Feraco et al. 2020). Particularly, WAT of obese mice shows higher expression of NADPH oxidase subunits and the transcription factor PU.1, which promotes transcription of the NADPH oxidase, with a parallel reduction in antioxidant enzymes. Moreover, in obese AT, infiltrating macrophages contribute to the production of inflammatory cytokines and ROS (Feraco et al. 2020). A potential link between ROS and monocyte chemoattraction may be the observed ROS-induced increase in expression of MCP-1 which recruits inflammatory cells. In addition, ROS production results in lipid peroxidation which releases metabolites that act as chemoattractants (Curzio et al. 1987). Adipocyte MR activation in obese mice may contribute to promote MCP-1 expression and, in turn, monocyte recruitment through increased ROS production (Fig. 2) (Guo et al. 2008). Genetically obese mice show higher expression of PU.1 and the NADPH oxidase subunits p22 and p47, which are important sources of ROS, reduced expression of catalase and Cu, Zn-SOD which act as ROS-eliminating enzymes, as well as an increase in mitochondrial hydrogen peroxide levels, as a result of mitochondrial dysfunction (Hirata et al. 2009; Lefranc et al. 2019). Treatment of obese mice with MRA resulted in suppressed expression of ROS-generating enzymes paralleled by enhanced expression of ROS-eliminating enzymes. Administration of MRA blunted the high levels of H2O2 levels and counteracted mitochondrial alterations, also reducing macrophage infiltration (Hirata et al. 2009). Data by Lefranc et al. also observed that MR-induced oxidative stress in perivascular AT of obese mice could contribute to the impairement in obesity-induced anticontractile effects on the vasculature, indicating that MR activity in the adipocyte may affect AT funtion and, in addition, contractile funtion of vessels (Lefranc et al. 2019). Healthy perivascular AT exerts a protective anticontractile effect on the vasculature. Obesity is associated with perivascular AT dysfunction and with attenuation of its anticontractile effect on adjacent vessels. In the model proposed by Lefranc et al., MR-mediated AT dysfunctions, including oxidative stress, lead to altered vascular activation of the RhoA/Rho kinase (ROCK) pathway which, in turn, results in increased vascular tone (Lefranc et al. 2019). Involvement of adipoyte-MR in inducing vascular dysfunctions was demonstrated by observing that MRA treatment of mouse mesenteric arteries with perivascular AT, in ex vivo experiments of wire miography, was able to recover the anticontractile properties of the fat (from obese mice) and prevented MYPT1 (myosin phosphatase target subunit 1) and MLC (myosin light chain) phosphorylations in the vessel, dampening vascular activation of ROCK and reducing the contractile response (Lefranc et al. 2019). Accordingly,

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obese Adipo-MROE mice display altered vascular contractility, associated with increased generation of hydrogen peroxide in the adipocyte, and anticontractile effects of the AT were observed in wild-type mice but not in Adipo-MROE mice (Nguyen Dinh et al. 2016). Together, these studies showed that perivascular AT with adipocyte MR overactivity leads to the loss of the anticontractile effects and alters functional properties of the adjacent vasculature. Indeed, these findings indicate that adipocyte MR plays a role in the cross-talk between adipocytes and the vasculature and suggest a key role for adipocyte MR in the development of obesity-associated CV alterations. Expansion of visceral AT has been associated with alterd lipid profile and increased risk of CVD, and adipose MR activity in obesity, by modulating adipokine expression (Fig. 2), may affect vascular function and systemic metabolism (Nakamura et al. 2014). In obesity, increased production and release of pro-inflammatory adipokines such as TNF-a, IL-6, IL-8, and concomitant reduction in adiponectin secretion lead to IR and altered vascular relaxation (Nakamura et al. 2014). Adiponectin expression is regulated by adipocyte MR, whose activty may be damped by the high circulating levels of aldosterone detected in obese subjects (Feraco et al. 2020; Hirata et al. 2009; Guo et al. 2008). MR overactivation may blunt production of adiponectin, potentially affecting the development of IR and atherosclerosis. Reduction in adiponectin is associated with increased uncoupling of eNOS, vascular production of superoxide and subsequent reduction in bioavailability of NO, favoring endothelial dysfunction and atherogenesis (Li et al. 2014). In obesity, overactivation of adipocyte-MR is expected to increase leptin expression which may favor vascular alterations. This is suggested by in vitro experiments with leptin-treated ECs displaying ROS production and enhanced expression of the macrophage-attracting chemokine MCP-1. In addition, leptin has been shown to increse the production of pro-inflammatory cytokines in monocyte cultures, and in vivo observations in mice with hyperleptinemia revealed altered response to vascular injury with increase in neointimal thickness and luminal stenosis (Schafer et al. 2004). Together these data propose a model which identifies a causal link between MR-induced leptin production and CV complications in obesity. Accordingly, Huby et al. observed that leptin infusion in mice, which showed enhancement in leptin-induced aldosterone production, led to reduction in endothelium-dependent relaxation (Huby et al. 2015). In these mice, MR blockade with spironolactone was able to prevent vascular alterations, confirming that detrimental effects on the vasculature were mediated by the high levels of circulating aldosterone. In the same study, leptin infusion also promoted cardiac expression of collagen 1α1, collagen 3α and periostin, and MRA treatment blunted these effects, indicating that leptin induced MR-mediated fibrotic effects, reinfrocing the concept that leptin-induced increse in circulating levels of aldosterone represents a risk factor for CVD (Huby et al. 2015). In obese mice, MR antagonism prevents the expression of pro-inflammatory mediators and ROS production in AT, thus suggesting that blockade of adipocyte MR has protective effects on adipocyte function. This is demonstrated in experiments

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with adipocyte cultures. In fact, aldosterone treatment in differentiated 3T3-L1 adipocytes led to increased mRNA levels of NADPH oxidase subunits p22 and p47, inducing in parallel decreased expression of catalase and Cu, Zn-SOD, two enzymes deputed to remove the ROS. Such effects were countercated by co-treatment with the MRA eplerenone, confirming that involvement of MR activity in the regulationof enzymes partecipating in the modulation of oxidative stress (Hirata et al. 2009) which favors AT dysfunctions (Fig. 2). Interestingly, alterations of autophagy are involved in the pathogenesis of various human diseases, including CVD and obesity, and changes in the autophagic flux have been reported in AT of obese patients and animal models of obesity (Armani et al. 2015). As above mentioned, pharmacological modulation of MR affects the autophagic flux in adipocyte cultures (Armani et al. 2014). Impaired autophagy in the adipocyte affects AT and glucose metabolism. Adipocyte-specific atg7 KO mice with defective autophagy in the adipocyte are resistant to diet-induced obesity and show reduction in WAT size and increased activity of interscapular BAT, as well as incresaed insulin sensitivity (Armani et al. 2015). These transgenic mice display WAT with morphological and molecular characteristics of BAT, such as multilocular lipid droplets, high number of mitochondria, increased expresssion of UCP1, and PPARγ coactivator 1alpha (PGC1a) which drives activation of mitochondrial biogenesis (Armani et al. 2015). Thus adipocyte-specific atg7 KO mice show both increased activity of BAT and the occurrence of WAT browning which confer protection against dysregulated AT and glucose metabolism. Interestingly, autophagy not only affects WAT metabolism, but also classical BAT function in mice. A recent study has shown that induction of ATG7 in the BAT of mice leads this tissue to acquire features of WAT, activating a process termed as “BAT whitening” (Deng et al. 2020). The authors also observed that treatment of mice with the synthetic GC dexamethasone stimulated ATG7 expression in BAT and enhanced whitening, whereas administration of the autophagy inhibitor chloroquine counteracted the effects of dexamethasone. Together, these data suggest that inhibition of the autophagic flux preserves BAT function and can also stimulate WAT to acquire brown fat properties, and indicate that modulation of autophagy may be a potential target to fight obesity. Increased adipose autophagic flux has been detected in obesity and dysfunctional autophagy in AT might promote low-grade inflammmation and dysregulated expansion of fat, promoting development of AT dysfunction and obesity-related comorbidities. Numerous studies have shown protective effects of browning on AT and glucose metabolism, at least in murine models, whereas such beneficial effects have not yet been confirmed in humans (Feraco et al. 2020). Our findings have indicated that treatment of HFD-induced obese mice with MRAs was able to preserve these animals from weigh gain and WAT expansion (Armani et al. 2014). In addition, MRA administration improved glucose tolerance and stimulated BAT activity as well as browning of inguinal WAT (iWAT), suggesting protective effect of MRA-induced enhancement of the brown and brite adipocytes on AT and glucose homeostasis (Fig. 3). Increased metabolic activity of BAT and iWAT was detected by using gene, protein expression analyses and positron emission tomography-computed tomography (PET/CT) techniques to evaluate 18F-Fluorodeoxyglucose (18F-FDG)

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Fig. 3 Activation of brown adipose tissue by mineralocorticoid receptor antagonists (MRA). MR blockade has been shown to increase UCP1 function and thermogenic activity of brown adipose tissue (BAT). In mice, MRA-induced thermogenic activation of BAT results in protection against obesity and impaired glucose tolerance. MRA treatment leads to BAT activation also in humans, but the protective effects of this process induced by MRA still await demonstration

upatke in fat depots. Importantly, in MRA-treated obese mice, a reduced autophagic flux in BAT and iWAT was observed (Armani et al. 2014). In vitro experiments showed that metabolic changes of AT in mice treated were mediated by specific blockade of the adipocyte MR, and MRA administration reduced autophagy with subsequent up-regulation of UCP1 protein (Fig. 3), indicating that adipocyte-specific modulation of autophagy is able to regulate thermogenic activation of brown and brite adipocytes, and confirming the results of other studies which proposed enhancement of brown fat mediated by autophagy dowregulation (Armani et al. 2014). Several studies with obese mice have demonstrated the beneficial effects of MRA treatment, showing that MRA administration led to improved IR, reduced plasma TG levels and macrophage infiltration, and prevented dysregulation of various genes

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involved in ROS production and inflammatory cytokines. Pharamacological MR blockade has also been shown to increase circulating adiponectin, resulting in potential anti-diabetic and anti-inflammatory properties (Hirata et al. 2009; Armani et al. 2014). In mice fed a HFD supplemented with fructose and showing features of MetS, i.e. body weight gain, IR, high BP, altered lipid profile, and fatty liver, treatment with MRA was able to reduce epididymal fat weight and BP, and circulating levels of triglycerides, free fatty acids, leptin, and total cholesterol. MRA also improved glucose tolerance and IR induced by the obesogenic diet (Wada et al. 2010). A recent study performed by our group has investigated the metabolic effects of the novel non-steroidal MRA finerenone in mice fed an obesogenic diet, expanding informations about MR-regulated molecular pathways in AT (Marzolla et al. 2020). Our findings confimed that MR blockade was able to promote BAT activity and such effects are mediated by the blockade of brown adipocyte-specific MR which stimulates activity of the AMP-activated protein kinase (AMPK) and, in turn, activates adipose triglyceride lipase (ATGL) function, leading to enhanced expression of UCP1. Several studies show that AMPK modulates lipogenesis and UCP1 expression, and AMPK activation has been shown to activate ATGL which, in turn, promotes UCP1 activity in murine adipocytes (Ahmadian et al. 2011). Thermogenesis of BAT is stimulated by lipolysis which is a process regulated by hormone-sensitive lipase (HSL) and ATGL (Cannon and Nedergaard 2004). Both these lipases release fatty acids (FAs) which activate UCP1 resulting in mitochondrial uncoupling respiration (Cannon and Nedergaard 2004). Importantly, finerenone treatment led to specific activation of ATGL, but did not affect HSL activity. We proposed that FAs derived from ATGL could activate PPARγ and PPARα which have been shown to bind and activate UCP1 promoter (Marzolla et al. 2020). Our model suggested that finerenone-induced ATGL activation might stimulate UCP1 expression in the brown adipocyte. In summary, MR antagonism by finerenone treatment enhanced BAT activity, through increase in ATGL-mediated lipolysis, which was associated with improved glucose tolerance, confirming that MR blockade preserves glucose homeostasis in obese mice (Marzolla et al. 2020). Interestingly, another recent study by Ferguson et al. has observed that adipocyte MR KO (AMRKO) mice fed a HFD, compared to control mice with an intact MR gene, showed reduced WAT mass, decreased insulin levels, reduction in HOMA-IR and hepatic lipid accumulation (Ferguson et al. 2020). These data are in contrast with studies performed by our laboratory and others that observed no protection against obesity and glucose intolerance induced by HFD in adipocyte MR KO mice (Feraco et al. 2020). In addition, data provided by Ferguson et al. did not show BAT activation or browning of WAT in AMRKO mice, showing a different response to the lack of MR function compared with mice treated with MRA displaying induced brown fat activation (Ferguson et al. 2020). AMRKO (adiponectin-Cre) mice are expected to retain MR expression in the preadipocyte, whereas mice treated with MRA may lose MR expression in preadipocytes; and impaired MR function at early stages of adipocyte differentiation in MRA-treated mice might explain the “thermogenic” effects of MRA administration. In summary, treatment of obese mice with MRA has been shown beneficial effects on the metabolism of these animals by counteracting body weight gain, WAT

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expansion, altered adipokine expression, and alterations in glucose homoestasis. In contrast, MRA treatment in humans did not display comparable efficacy results to those observed in mice (Feraco et al. 2020) (Fig. 3). In obese subjects, administration of spironolactone did not show positive effects on BMI, insulin sensitivity or endothelial function (Garg et al. 2014). Alterations in insulin sensitivity, ectopic fat accumulation, and chronic inflammation as well as increased RAAS activation, which potentially has a causal role in impaired insulin sensitivity and inflammatory marker upregulation, have been deteced in HIV-infected individuals (Srinivasa et al. 2018). In these patients, prone to metabolic dysfunctions, MRA treatment was able to improve intramyocellular lipid content inside the skeletal muscle, MCP-1 and highdensity lipoprotein levels. Nevertheless, MRA did not improve insulin sensitivity, neither did it reduce body weigh and fat mass (Srinivasa et al. 2018). Other studies report evidence that MRA treatment, in PA patients, has been shown to improve insulin sensitivity (Catena et al. 2006) and reduce visceral AT fat depots (Karashima et al. 2016). Interestingly, a recent study by Johansen et al. has shown that treatment with the MRA eplerenone, in subjects with type 2 diabetes, was able to reduce pericellular fibrosis of the subcutaneous AT, suggesting that MR antagonism may counteract the ECM remodeling of AT and positively affect AT function in diabetic patients (Johansen et al. 2020). Altogether, these findings suggest that the metabolic effects of MRA have yet to be clarified in humans. However, it can not be ruled out that dosages, duration of treatment of MRA or eligibility criteria for subjects to be enrolled may have affected the results of the mentioned studies. Diverse complexity of the molecular pathways or the existence of species-specific molecular networks regulating AT metabolism might also explain the different metabolic response of AT to MRA in mice and humans. It has been suggested that pharmacological strategies involving combinations of different drugs, targeting simultaneously multiple pathways, may represent an efficient approach to tackle AT and glucose metabolism dysfunctions in humans (Feraco et al. 2020). As above mentioned, a recent study by Thuzar et al. have observed that 2 weeks pharmacological MR antagonism is able to affect BAT metabolism in healthy humans, leading to enhancement of metabolic and thermogenic functions of the supraclavicular BAT in response to cold exposure, and indicating that MR blockade, in humans subjects, might be more effective in modulating activity of BAT rather than WAT, which effectively was not affected in this study (Thuzar et al. 2018). The inverse association between BAT and obesity detected in numerous studies has suggested that the activation of this thermogenic tissue may provide protective effects against obesity, dislipidemia and type 2 diabetes, and BAT activation has been shown to protect mice against AT and associated dysfunctions. In humans, the ability of BAT in preserving AT function and glucose homesostasis has still to be demonstrated. In the study by Thuzar et al., treatment with MR was applied to non-obese subjects and did not reduce adiposity, neither did it improve insulin sensitivity, thus additional trials with obese subjects and/or type 2 diabetes are needed to understand the metabolic effects of MRAs on fat tissue and glucose metabolism.

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Mineralocorticoid Receptor Antagonists in Cardiovascular and Renal Dysfunctions Preclinical studies has demonstrated the involvement of extra-renal MR in molecular pathways contributing to obesity, IR, hypertension, atherosclerosis and HF (Feraco et al. 2020). As above mentioned, administration of MRAs has shown favorable effects in murine models of obesity, MetS, hypertension, renal and CVD. In humans, the steroidal MRAs spironolactone, canrenone and eplerenone have found application in diseases such as PA, hypertension and HF (Bramlage et al. 2016). Current guidelines suggest to add spironolactone or eplerenone to ACE inhibitors- and β-blockers background therapy for the management of HF patients. Nevertheless, only a low percentage of HF patients is treated with MRAs in clinical practise, and this is mainly due to the side effects, associated with MRA-based therapies, which can reduce patients adherence to these terapies (Bramlage et al. 2016). In fact, spironolactone has been shown to antagonize both MR and the androgen receptor (AR), with subsequent gynaecomastia and erectile dysfunction in men. As above discussed, in the kidney MRA treatment promotes sodium excretion and a parallel retention of potassium, potentially resulting in hyperkalaemia, which represents a life-threating condition especially in patients with impaired kidney function. Noticeably, compared with spironolactone, eplerenone is able to block MR activity with a higher specificity than that displayed for AR and GR, but on the other hand it shows a binding affinity to MR less potent than spironolactone (Bramlage et al. 2016). Treatment with spironolactone or eplerenone have been shown protective effects in murine models of cardiac diseases, counteracting cardiac hypertrophy and the development of HF (Bauersachs et al. 2015). In experimental models of renal disease, MRAs was shown to prevent renal damage by reducing glomerulosclerosis (Barrera-Chimal et al. 2019). MR activation results in increased expression of NADPH oxidase subunits in cardiac and renal tissues, promoting generation of oxidative stress which contributes to the development of fibrosis and inflammation (Bauersachs et al. 2015). Increased activity of NADPH oxidase and enhanced ROS formation have been observed in the cardiac tissue of TG(mRen2)27 rats, which display increased plasma aldosterone levels and BP, as well as cardiac hypertrophy and fibrosis (Habibi et al. 2011). Treatment with a low dose of spironolactone did not reduce BP but was able to decrease oxidative stress and fibrosis markers, and improve diastolic dysfunction, suggesting that the observed positive effects of MRA were independent of changes in BP (Habibi et al. 2011). In a murine model of HF, eplerenone administration resulted in reduction in cardiac oxidative stress, evaluated by 3-nitrotyrosine staining, which was associated with decrease in myocardial fibrosis and myocyte apoptosis, and reduction in ICAM-1 expression and macrophage infiltration (Kuster et al. 2005). Contribution of MR activity to renal inflammation was observed in rats treated with N(omega)-nitro- L-arginine methyl ester (L-NAME) that developed progressive renal injury associated with increased inflammation and activation of RAAS in the renal cortex (Ikeda et al. 2009). Alterations in systolic BP, urinary protein excretion, and serum creatinine, as well as glomerulosclerosis, interstitial fibrosis,

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and macrophage infiltration were prevented by spironolactone treatment in these animals. In another study, an increased expression of podocyte damage and oxidative stress markers were detected in the glomeruli of diabetic rats that developed proteinuria (Toyonaga et al. 2011). Adminstration of MRA was able to reduce podocyte injury as well as renal oxidative stress marker expression, further suggesting a causal role for MR-mediated ROS production in the development of renal damage. In the vessel, activation of MR, through ROS generation, has been proposed to play a role in eNOS uncoupling, with subsequent reduced producion of NO and impaired endothelial function. In hypertensive rats, in addition to ant-inflammatory and anti-fibrotic effects, the observed renoprotective effects of eplerenone treatment, in term of improved glomerulosclerosis and reduced urinary protein, have also been attributed to increased expression of eNOS and improvement in endothelial function (Kobayashi et al. 2005). Indeed, these preclinical studies have shown that MRAs exert protective effects which are independent on changes in BP, suggesting direct effects of MR blockade on cardiac and renal tissues (Bauersachs et al. 2015). Protective properties of MRAs have been evaluated in clincial trials on patients with cardiac and/or renal disease. In Randomized Aldactone Evaluation Study (RALES), which was peformed in subjects with HF and reduced LVEF ( 35%) receiving standard therapy, spironolactone treatment was able to reduced all causemortality, frequency of hospitalization and symptoms of the HF (Bauersachs et al. 2015). The Eplerenone Post-Myocardial Heart Failure Efficacy and Survival Study (EPHESUS) showed that MRA treatment resulted in decrease of mortality from any cause and CV cause, and reduction in hospitalization for CV events in patients with LVEF 10 pg/dl), further evaluations are needed to discern whether the disease is pituitary or ectopic: • High-Dose Dexamethasone Suppression Test (8 mg/day): In the pituitary form of CS, the administration of high-dose DEX results in a significant reduction in cortisol levels (suppression cut-off: 20% within 30–60 min) and ACTH levels (peak >50% within 15–30 min) with high sensitivity and specificity; on the other hand, ectopic ACTH-secreting cells do not retain either CRH or desmopressin receptor: leaving cortisol and ACTH levels generally unaffected by stimulation with both agents. In case CD is suspected, or if second-line tests are discordant, pituitary MRI should be performed, while taking into account that the pituitary location is often not visible on MRI; furthermore, the incidental finding of a pituitary adenoma has been observed in 10–20% of the healthy population: therefore, a certainty cut-off of 6 mm, in association with biochemical confirmation, has been considered as highly suggestive for pituitary CS. If the adenoma’s diameter is less than 6 mm, bilateral inferior petrosal sinus sampling (BIPSS) is the investigation of choice. The procedure is based on the principle that, in the case of pituitary disease, the ACTH concentration in the petrous sinuses, which collect blood from the pituitary gland, must be greater than that present in the peripheral vein (periphery). Specifically, a baseline central to periphery ACTH ratio >2 (>3, in case of previous CRH stimulation) indicates pituitarydependent ACTH syndrome. In contrast, a low gradient suggests an ectopic source of ACTH. Although invasive in nature, this test is characterized by a very high diagnostic accuracy and has also been demonstrated to facilitate the localization of pituitary ACTH adenomas in case of unclear MRI findings. When suspecting an ectopic ACTH secretion, axial imaging with CT or MRI should be performed in tandem with functional imaging, such as Ga-68DOTATATE PET-CT, to precisely localize the source of ectopic ACTH (Isidori et al. 2015b).

Treatment Cushing’s Disease: Pituitary adenomectomy currently represents the first-line treatment in ACTH-secreting adenomas. The primary goal of surgical treatment is to normalize cortisol secretion and achieve surgical radicality with minimal hypopituitarism to ensure long-lasting biochemical and clinical remission (Fleseriu and Biller 2022). The probability of surgical radicality in the case of microadenoma is estimated between 65 and almost 90%. Still, in the case of macroadenoma (>10 mm), it is significantly reduced (up to 12.5% in most centers). Regardless of the size of the adenoma, possibly predictive elements of failure include tumor invasiveness (dura, cavernous sinuses), tumor localization in the pituitary stalk or an ectopic site, inability to visualize the tumor during surgery, the lack of histological demonstration of the presence of tumor tissue in the removed material. Young age at diagnosis is also a predictor of the risk of relapse. However, the best predictor of long-term disease remission is the early biochemical evidence of hypocortisolism (cortisol levels 140/90 mmHg and has been identified as a major determinant of increased mortality risk in patients suffering from endogenous hypercortisolism (Pivonello et al. 2016). In CS, the increase in blood pressure follows a typical pattern, with a comparable rise in systolic and diastolic blood pressure levels. Moreover, a constant and early finding is represented by the loss of nocturnal physiological decrease in blood pressure levels (“non-dipper” pattern), likely due to the disruption in cortisol circadian rhythm (Barbot et al. 2019). Nevertheless, the various subtypes of CS exhibit some differences in the hypertensive phenotype: specifically, patients with adrenal-derived CS exhibit a greater degree of blunting of nocturnal dipping, as well as a tendency toward higher blood pressure levels compared to patients with ACTH-dependent CS; this finding could

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be ascribed as much to a difference in adrenal secretion of androgens and sex steroids (generally suppressed in cortisol-secreting adrenal tumors) as to a partial preservation of cortisol secretory circadianity in mild, pituitary-derived forms. Hypertension caused by long-term treatment with exogenous glucocorticoids warrants specific mention since it occurs much more rarely (about 20% of cases) and largely depends on the dose, administration route, duration, and steroid formulation. Though surprising, this finding is likely referable to the lack of affinity of the main formulations used in clinical practice (i.e., dexamethasone, prednisolone, triamcinolone) for the mineralocorticoid receptor, resulting in the absence of the apparent mineralocorticoid excess typically observed in endogenous CS (Isidori et al. 2015c).

Treatment The most effective therapeutic strategy in managing CS-related hypertension is the normalization of cortisol secretion, typically achieved via surgical removal of the tumor responsible for the disease or, in cases where surgery is ineffective or contraindicated, by appropriate medical treatment. The significant increase in mortality found in hypertensive CS patients appears to be correlated with the duration of the exposure to glucocorticoid excess, with increased mortality seen in patients with symptoms lasting more than three years prior to surgery (Lambert et al. 2013). Disease remission does not always result in normalized blood pressure levels, as hypertension has been shown to persist in 25–54% of patients in remission from CS, likely due to cortisol-mediated irreversible structural cardiovascular changes and vascular remodeling (De Leo et al. 2010). • Surgery: The surgical removal of the ACTH-secreting pituitary tumor via transsphenoidal adenomectomy currently represents the first line of treatment for CD and results in long-term remission in up to 70% of cases. However, blood pressure levels do not always normalize after disease remission, as hypertension persists in about 40% of patients achieving long-term remission. In this regard, a key role is played by the duration of untreated hypercortisolism. In support of this hypothesis, data from studies of pediatric CD patients has shown complete blood pressure normalization within one year of achieving remission in almost all cases, although residual impairment of arterial distensibility appears to persist in the long term. In addition, studies on adult CD patients have shown that hypertension can persist even 30 years after disease remission regardless of surgical treatment, highlighting that a duration of hypercortisolism longer than three years before diagnosis is independently associated with the persistence of hypertension, as well as increased cardiovascular mortality (Isidori et al. 2015c). As for ectopic CS, surgical resection of the neuroendocrine malignancy would theoretically represent the first line of treatment; however, this is not always feasible due to the frequently aggressive behavior of the ACTH-secreting tumor leading to a generally poor prognosis. For this reason, consistent data regarding the response of hypertension to surgical treatment in ECS are scarce, though most

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case series have reported an overall improvement in blood pressure levels (Alexandraki and Grossman 2010). In ACTH-independent forms of CS sustained by a cortisol-secreting tumor, unilateral laparoscopic adrenalectomy represents the gold standard of treatment, resulting in definitive resolution of hypercortisolism in the absence of recurrence. Nevertheless, hypertension has been described to persist following adrenalectomy in a variable percentage of cases, ranging from 32% to 42%, in published series: factors associated with the persistence of hypertension include concomitant obesity, advanced age, long duration, and greater severity of hypercortisolism (Clemente-Gutierrez et al. 2021). Lastly, bilateral adrenalectomy results in a prompt resolution of cortisol excess. Still, it is generally reserved for bilateral nodular adrenal hyperplasia associated with ACTH-independent CS, emergency cases needing immediate resolution of hypercortisolism, non-resectable and/or occult sources of endogenous hypercortisolism due to the invariable need for lifelong replacement therapy and to the increased risk of developing Nelson’s syndrome. Retrospective data from 68 patients treated with bilateral adrenalectomy for ACTH-dependent hypercortisolism from an occult source (26 ECS, 42 CD) showed a post-surgical improvement in blood pressure levels in 64% of patients (Alexandraki and Grossman 2016). • Medical therapy: Increasing evidence has shed light on the potential use of cortisol-lowering drugs in treating hypertension in CS patients. Adrenal-directed drugs: – Ketoconazole: Data regarding the effectiveness of steroidogenesis inhibitor ketoconazole on hypertension in CS patients mainly stems from retrospective studies; in most reports, ketoconazole has proven especially effective, improving blood pressure levels in 40–60% of patients (up to over 80% of cases in one instance), while maintaining an acceptable safety profile (Nieman 2019); – 11β-hydroxylase inhibitors: Regarding CS-associated hypertension, the employment of 11β-hydroxylase inhibitors, such as metyrapone and osilodrostat, is more controversial. They have been shown to potentially worsen hypertension and induce hypokalemia by increasing cortisol and aldosterone precursors with mineralocorticoid activity, thus requiring concurrent treatment with potassium and spironolactone (Feelders et al. 2019). However, these effects are generally counterbalanced by the reduction in UFC, resulting in overall neutralizing or beneficial effects on BP (Valassi et al. 2012). Accordingly, during a recent prospective, open-label, singlearm, multicenter phase III/IV clinical trial aimed at investigating metyrapone’s efficacy and tolerability in a wide CS population, metyrapone treatment led to a decrease in antihypertensive medications in 31.2% of hypertensive patients. In contrast, a dose increase was required in 15.6% (Pivonello et al. 2022). Osilodrostat, on the other hand, has recently emerged as a promising therapeutic alternative in patients with CS of different etiologies; as the literature surrounding its use is steadily growing, most of the currently available data derives from the LINC studies, aimed at assessing the long-term efficacy and

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tolerability of osilodrostat in patients with CD. Regarding CS-related hypertension, osilodrostat treatment was accompanied by a generally neutral effect on blood pressure levels: after 48 weeks of treatment, 40% of hypertensive patients experienced a marked decrease in blood pressure levels – leading to a reduction and/or interruption of antihypertensive drugs – whereas an equal percentage increased dose or BP-lowering drugs (Fleseriu et al. 2022). – GR antagonists: Mifepristone and relacorilant act by blocking the binding between cortisol and the glucocorticoid receptor, leaving endogenous cortisol secretion unaffected. As a result, their use leads to excessively increased circulating levels of cortisol, which can, in turn, inappropriately activate the mineralocorticoid receptor, resulting in worsening blood pressure levels and the potential onset of hypokalaemia and peripheral edema. At the same time, the excessive GR blockade can induce a direct decrease in blood pressure levels, making the overall net effect challenging to predict. Moreover, clinical experience with mifepristone is still limited: published series have reported a clinical improvement in about half of hypertensive CS patients treated with mifepristone, with a more pronounced effect in patients also suffering from hyperglycemia. It should be noted, however, that mifepristone alone was rarely able to normalize blood pressure levels, often requiring concomitant antihypertensive drugs. Data on the effects of relacorilant on CS-related hypertension are still scarce and mostly derive from the phase II study investigating its safety and efficacy in CS patients. In this regard, relacorilant treatment has been associated with beneficial effects regarding hypertension, with 41.7% and 63.6% of hypertensive patients experiencing a decrease of 5 mmHg in either mean 24-h SBP or DBP from baseline following low-dose and high-dose treatment, respectively (Pivonello et al. 2020; Isidori et al. 2015c). – Mitotane: Mitotane is sporadically employed in severe cases of CS, mainly as an alternative to bilateral adrenalectomy. Its effects on blood pressure levels have been explored primarily in the context of EAS, in which it has been demonstrated to improve blood pressure levels in 63% of hypertensive patients (Donadille et al. 2010). Centrally directed drugs: Pituitary-targeting drugs, including the dopamine agonist cabergoline and, more recently, somatostatin analog pasireotide, represent an effective secondline treatment in CD patients not achieving remission after transsphenoidal surgery; in regard to managing hypertension, both drugs have proven effective in reducing blood pressure levels, either as single agents or in combination (Feelders et al. 2019). – Pasireotide: Early data from the phase III study investigating safety and tolerability of pasireotide highlighted a significant decrease in blood pressure levels in hypertensive CD patients, irrespective of whether patients were taking antihypertensive drugs at baseline. Multiple multicenter, prospective studies have since confirmed these findings, reporting a significant improvement in pressor levels following pasireotide treatment, which was further enhanced by the therapeutic association with cabergoline or ketoconazole (Isidori et al. 2015c).

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– Cabergoline: Treatment with dopamine-agonist cabergoline, both as monotherapy and as an adjuvant drug, has been demonstrated to significantly improve the pressor profile in CD patients. The decrease in blood pressure levels occurs early after treatment initiation and has been observed to lead to normalized values in most patients after long-term treatment. Interestingly, the improvement of hypertension is maintained even in patients experiencing treatment escape; this finding suggests a direct, beneficial effect of cabergoline on blood pressure irrespective of cortisol secretion, which is likely attributable to a vasodilatory effect mediated by the dopamine receptors expressed in the vascular system (Pivonello et al. 2009). Whenever the resolution of hypercortisolism, either via surgical or medical means, does not result in BP normalization, additional antihypertensive treatment is required (Barbot et al. 2019). As mentioned above, while mineralocorticoid receptor blockade is generally unable to fully revert hypertension in CS patients (Williamson et al. 1996), the administration of MR antagonists – spironolactone or eplerenone – at standard doses is regarded as an appropriate adjuvant treatment in patients with hypertensive CS. It is mainly used as an add-on treatment to counteract potential side effects of cortisollowering drugs, mainly hypokalemia and peripheral edema. In light of the involvement of the angiotensin pathway in the development of hypertension in CS, as well as their natriuretic effect (via enhancement of renal 11β-HSD2 activity), ACE-I or angiotensin receptor blockers (ARB) should be considered as a first-line therapeutic option, both in monotherapy and in combination with calcium-antagonists and/or a mineralocorticoid receptor antagonist, depending on disease severity and association with hypokalemia (Isidori et al. 2015c).

Potassium Balance in Cushing’s Syndrome Epidemiology Potassium (K) is the most abundant intracellular cation and one of the main determinants of intracellular osmolality. The ratio between intracellular and extracellular concentrations of K strongly influences cell membrane polarization, affecting critical biological processes, such as nerve impulse conduction and the contraction of skeletal and myocardial muscle cells. Therefore, even relatively minor alterations in serum K concentration can be associated with significant clinical manifestations. Hypokalemia is defined by serum potassium levels lower than 3.6 mEq/L and represents a common electrolyte disorder occurring in up to 21% of hospitalized patients and 2% to 3% of outpatients (Viera and Wouk 2015). Decreased potassium levels can be observed in any patient suffering from severe CS, with no significant difference in prevalence between male and female patients. However, hypokalemia is more frequently observed in the context of ectopic ACTH

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secretion (Pivonello et al. 2016). In the general population, and even more in patients with CS, hypokalemia has been shown to be a major determinant in cardiovascular mortality, representing a risk factor for acute heart failure, ventricular arrhythmia, and sudden death (Takagi et al. 2009).

Pathophysiology The main mechanisms involved in the pathogenesis of hypokalaemia can be summarized as follows: • Low dietary intake of K (