Molecular Nephrology: Biochemical Aspects of Kidney Function. Proceedings of the 8th International Symposium, Dubrovnik, Yugoslavia, October 5–8, 1986 9783110884746, 9783110111217

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Molecular Nephrology Biochemical Aspects of Kidney Function

Molecular Nephrology Biochemical Aspects of Kidney Function Proceedings of the 8th International Symposium Dubrovnik, Yugoslavia, October 5-8,1986

Editors Z. Kovacevic • W G. Guder

W DE

G

Walter de Gruyter • Berlin • New York 1987

Editors Zoran KovaCevic, Dr. Professor of Biochemistry University of Novi Sad Department of Biochemistry Medical Faculty 21000 Novi Sad Yugoslavia Walter G. Guder, Dr. med. Professor of Clinical Biochemistry and Clinical Chemistry Institut fur Klinische Chemie Städtisches Krankenhaus D-8000 München 81 Federal Republic of Germany Library of Congress Cataloging in Publication Data Molecular nephrology. „International Symposium on Biochemical Aspects of Kidney Function"~Pref. Includes bibliographies and indexes. I. Kidneys—Congresses. 2. Biological chemistry—Congresses. I. Kovacevic, Z. (Zoran), 1935- . II. Guder, Walter G. III. International Symposium on Biochemical Aspects of Kidney Function (8th : 1986 : Dubrovnik, Croatia) [DNLM: 1. Acid-Base Equilibrium—congresses. 2. Biological Transport-congresses. 3. Cell Membrane-physiology-congresses. 4. Kidney—metabolism-congresses. 5. Kidney-physiology-congresses. WJ 301 M718 1986] QP249.M65 1987 599\0149 ISBN 0-89925-400-4 (U.S.)

87-8957

CIP-Kurztitelaufnahme der Deutschen Bibliothek Molecular nephrology : biochem. aspects of kidney function ; proceedings of the 8th internat, symposium, Dubrovnik, Yugoslavia, October 5-8,1986 / ed. Z. Kovafievic ; W G. Guder. - Berlin ; New York : de Gruyter, 1987. ISBN 3-11-011121-7 NE: Kovacevic, Zoran [Hrsg.]

Copyright © 1987 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. Binding: D. Mikolai, Berlin. - Printed in Germany.

FOREWORD The proceedings contain the papers submitted by the participants of the 8th International

Symposium on Biochemical Aspects of Kidney Function, held in Dubrov-

nik in October 1986. The symposium continues a fifteen-year tradition of European renal biochemists, physiologists, clinical chemists and nephrologists who are engaged in research on the biochemical

basis of kidney function. The seven

previous meetings sprang from an idea of Dr. M. Hohenegger of Vienna. The earlier meetings were held in Salzburg (1971), Gdansk (1973), Basle (1975), Ulm (1977), Oxford (1979), Strasbourg (1981) and in Smolenice (1984). They were organized successively by Drs. Hohenegger, Angielski, Dubach and Schmidt, Guder, Ross, Morel and Dzurik. Although they have remained largely European, they were also attended by colleagues from non-European countries, especially from the USA and Canada. Thanks to the fact that the meetings have always had an interdisciplinary character, they have proven very successful for the exchange of ideas and in fostering the study of the kidney in all its complexity. Important progress has been made in understanding the key problems of renal chemistry and in tracing new ways in connecting biochemistry and renal

bio-

physiolo-

gy. This is reflected, first of all, in the elucidation of molecular mechanisms of physiological

phenomena rather than in their mere description.

Heterogeneity

of the kidney, the problem which was in the focus in some earlier symposia, has been greatly overcome thanks to sophisticated techniques of nephron microdissection, isolation of tubule segments and analysis of their enzyme and metabolite profile, physiological

function and especially of the transport processes. Ap-

plication of the techniques of genetic engineering, NMR spectroscopy, and the culturing of tubule cells open new possibilities for solving still existing

pro-

blems in this field. Information about new techniques and new data in such an interdisciplinary meeting leads not only to an increase in knowledge

concerning

the biochemical mechanisms of renal physiology but also to the creation of new concepts regarding the role of the kindney in the physiology of the organism as a whole. Thanks to the broad and at the same time fundamental approach to the questions raised, these proceedings will undoubtedly be of great value to those researchers interested in increasing their knowledge in the expanding field of renal biochemistry, physiology and nephrology.

Novi Sad and Munich

Zoran

December 1986

Walter G. Guder

Kovacevic

VI

OPENING

REMARKS

Dear Colleagues and Friends, On behalf of the Organizing Committee I should like to thank you for coming to Dubrovnik and taking part in this Symposium. All of you who had the opportunity of participating in the earlier meetings know that they have been very

success-

ful in fostering the study of renal biochemistry. I hope that this 8th Symposium will continue this tradition and that it will give you the opportunity of establishing and continuing valuable scientific and satisfying personal

contacts

and of discussing the latest developments in this field. When I accepted the request to organize this symposium I did not realize what I was getting into. This was in the charming atmosphere of Smolenice castle during a nice party and over good wine. There were many problems I could not foresee at the time, but thanks to the co-organizers of the meetinq and my collaborators from Novi Sad, and especially to Dr. Walter Guder, whose help was literally invaluable, it seems to me that we have overcome most of the problems. This meeting would not have been possible without our generous sponsors not only from Yugoslavia but also from abroad. We are very grateful

to all of them, but

I should like to acknowledge in particular the contribution of the German Society for Clinical Chemistry. A special word of thanks is addressed to Professor F. Korber from the editorial

board of the Journal of Clinical

Chemistry

and Clinical Biochemistry who prepared the abstracts so rapidly and successfully. Since the number of participants surpassed our expectations the program of the symposium is quite large. As always we wanted to have as much time for the discussion as for presentation of the papers. Compared to earlier m e e t i n g s , which were held in quiet locations like Reisensburg or Smolenice castle or in an ancient college in Oxford, the present location is livelier. Nonetheless I hope that the lectures we are going to hear will prove more attractive than the beaches of our modern hotels and that we will find enough quiet places to discuss the details of our experimental work. This does not m e a n , of course, that we will not enjoy

Dubrovnik.

Let me say again that I am very pleased to welcome you here and for being given the privilege of opening the 8th International of Kidney

Symposium on Biochemical

Aspects

Function. Zoran

Kovacevic

VII

SPONSORS

We are grateful for the financial support donated by the following

institutions

and firms:

The Self-Managed Community of Interest for Science of Vojvodina The Republic of Croatia Community for Scientific Research German Society for Clinical Chemistry (F.R. of Germany) Interservis, Agrovojvodina

(Yugoslavia)

Dunav-Tisa-Dunav

(Yugoslavia)

Malinoremont-DTD

(Yugoslavia)

Sajamcommerce-Novosadski

sajam

Yugoslav Airlines, JAT Eisai Co. Ltd., Tokyo

(Japan)

(Yugoslavia)

(Yugoslavia)

(Yugoslavia)

CONTENTS

I. FUNCTION OF BRUSH BORDER

MEMBRANE

Hydrolysis of Neuropeptides by Pig Kidney Microvillar Peptidases: The Key Role of Endopeptidase-24.11 S.L. Stephenson, A.J. Kenny

3 +

Identification of Components of Na -D-Glucose Cotransport Proteins in Brush-Border Membranes from Renal Cortex H. Koepsell, M. Neeb, K. Korn, U. Kunz, E. Zoubek

13

Renal Transport and Metabolism of Carnosine in the Rat S. Si 1 bernagl, K. Völker

21 +

Modulation of Renal Apical Membrane Fluidity and Na -Dependent D-Glucose Transport by Fatty Acids in vitro S. Wagner, H. Ebel

27

Renal Metabolism of C-Peptide in Man G. Deferrari, I. Zavaroni, G. Garibotto, C. Robaudo, R. Lugari, A. Tizianello

II. TRANSPORT ATPASES AND METABOLITE TRANSPORTING

33

SYSTEMS

Biochemical Properties of the Proton Translocating ATPase in Rat Kidney Cortex Brush Border Membranes: Effect of Inhibitors, pH Profile and Molecular Weight E. Kinne-Saffran, R. Kinne, E.L. Sattler, G. Doll

43

Aldosterone-Independent Effects of Dietary Potassium on N-EthylmaleimideSensitive ATPase in Distal Nephron Segments of Adrenalectomized Rabbits L.C. Garg, N. Narang

51

+

Electrogenic H -ATPase and Anion Conductance in Pig Renal Cortical Endocytotic Vesicles G. Burckhardt, B. Moewes, I. Sabolic

57

Interaction between Triiodothyronine and Aldosterone in Control of Kidney Na-K-ATPase C. Barlet, A. Doucet

63

Structural Basis for Regulation of Active NaCl Transport in Thick Ascending Limb of Henle's Loop P.L. Jtfrgensen, D.A. Klaerke

71

X Evidence for a Reabsorptive K + Pump in Kidneys of Potassium-Depleted Rats A.I. Katz, M. Hayashi

77

Affinity Labels as Substrates for the Anion Transport Systems at the Contraluminal Cell Side of the Renal Proximal Tubule K.J. Ullrich, G. Rumrich, H. Fasold, L. Zaki

85

A Rapid Method for the Reconstitution of Neutral Amino Acid Transport Systems from Ox Kidney Brush Border Membranes J.D. McGivan, A. Lynch

91

Kinetics of Inhibition of Transmembrane Glucose Transport and of Insulin Binding by a Peptide Isolated from Uremic Plasma S. Milutinovic, I. Samarzija, D. Breyer, V. Molnar, V. Spustova, R. Dz'urik

97

Mediated Transport of Long Chain Fatty Acids by Rat Renal Basolateral Membranes and Evidence for its Regulation M.E. Trimble

103

Maintenance of Differentiated Function of Thick Ascending Loop of Henle (TALH) Cells Following Transfection with "Early Region DNA" of SV 40 Virus D.M. Scott, R. Kinne, C. MacDonald, H. Brzeski

109

NMR Studies on a Renal Epithelial Cell Line: LLC-PK1/CI 4 A.W.H. Jans, E. Kellenbach, J. Luig, P. Raniewski, B. Griewel, R. Kinne ..

115

Respiration of Nephron Segments of the Dog: Effect of Ouabain and DCCD P. Vinay, C. Manillier, A. Tejedor, J. Noël, Y. Boulanger, A. Gougoux

III.

C E L L MEMBRANE P O T E N T I A L

AND TRANSPORT

121

PROCESSES

Electrophysiology of Cell Volume Regulation F. Lang, M. Paulmichl, H. Voelkl, E. Gstrein, F. Friedrich

133

Effects of A 23187, Verapamil, and Quinidine on Electrical Properties of Madin-Darby-Canine-Kidney and Mouse Straight Proximal Tubule Cells M. Defregger, H. Voelkl, M. Paulmichl, F. Lang

141

Effect of Bradykinin and Epinephrine on Electrical Properties of MadinDarby-Canine-Kidney Cells M. Paulmichl, M. Defregger, F. Friedrich, F. Lang

149

Intracellular Na + and K + Activities in Sodium Maleate Treated Frog Kidney Proximal Tubular Cells D.A. Cemerikic, D.M. FilipoviS

157

XI

Peritubular Membrane Potential in Sodium Maleate-Treated Frog Kidney Proximal Tubular Cells •J

D.M. Filipovic, D.A. Cemerikic

163

Prostaglandin Action on Renal Proximal Tubule as Revealed by Electrical Studies of Na + -Glucose Cotransport I. Samarzija

171

IV. ACID BASE REGULATION Failure of the Kidney to Oxidize Fatty Acids during Diabetic Ketoacidosis C. Lemieux, J. Berkofsky, G. Lemieux

179

A Role for Alanine Biosynthesis by the Kidney G. Lemieux, J. Berl'ofsky, C. Lemieux, A. Quenneville, M. Marsolais

185

Effect of Glucocorticoids and Metabolic Acidosis on the Level of Rat Renal Phosphoenolpyruvate Carboxykinase mRNA M. Gallo, R.A. Shapiro, N.P. Curthoys

191

Studies on the Role of y-Glutamyltransferase in Glutamine Metabolism in Rat Proximal Tubules P.D. Dass, E. Bourke

199

Hippurate Synthesis in Rat Kidney Cortex Slices V. Spustovä, M. Gerykovä, R. Dztirik

207

Proliferation of the Endoplasmic Reticulum of the Proximal Nephron Cells during Chronic Metabolic Acidosis and after Treatment with Triamcinolone F. Berthelet, M. Beaudry-Lonergan, M. Bergeron

213

Liver and pH Homeostasis: Regulatory Mechanisms D. Häussinger

221

Role of Acid Base in the Regulation of Urea Synthesis M.L. Halperin, S. Cheema-Dhadli, R.L. Jungas

231

Importance of the Glutamate Dehydrogenase Pathway in Aldosterone Stimulated Ammoniagenesis R. Snart, D.R. Coates

237

Organ Specificity of Glutamine Hydrolysis and Oxidation Z. Kovacevic, 0. Brkljac

245

The Stimulation of the Respiration of Glutamine by Rat Renal Mitochondria in Chronic Metabolic Acidosis D.J. 0' Donovan, R.B. Tobin, M.A. Horrum

251

XII

How Hydrogen Ion Homeostasis Regulates Proximal Tubular Reabsorption in the Dog Kidney J. 0stensen, H. Langberg, F. Kiil

257

Role of Renal Oxygen Consumption Rate in the Adaptive Ammoniagenesis of Acidosis H.G. Preuss, J. Areas, P. Schubert, M. Lenhart, D. Slemmer

263

Renal Adaptation to Lactic Acidosis in the Dog E. Junco, R. Perez, R. Jofre, M. Rengel, S. Alonso, F. Valderräbano

269

V, ACTION OF DRUGS AND METABOLISM Valproate does not Stimulate Renal Ammoniagenesis in the Dog in vivo M. Rengel, J.M. Lopez-Novoa, A. Gougoux, P. Vinay

277

Metabolic Effects of Valproate on Dog Thick Ascending Limbs A. Gougoux, P. Vinay

281

Acceleration of Ammoniagenesis in Isolated Rat Kidney Tubules by the Antiepileptic Drug: Valproic Acid G. Martin, D. Durozard, G. Baverel

287

Substrate-Dependent Effect of Gentamicin on Glucose Formation in Isolated Rabbit Kidney-Cortex Tubules M. Michalik, J. Bryta

293

Urinary Enzymes in Premature Infants: Relation to Gentamicin Therapy V. Marinkovic, M. Stanulovic, S. Jovanovic

299

Characterization of a Soluble 5 1 -Nucleotidase of Rat Kidney M. Le Hir, U.C. Dubach

305

Differences in Adenylate Metabolism in Renal Cortex and Medulla S. Angielski, T. Pawelozyk, D. Bizon, G. Piec, L. Wojnowski, J. Stepifiski

VI, PATHOBIOCHEMICAL

311

MODELS

Studies on the Role of Glycerophosphorylcholine and Sorbitol in Renal Osmoregulation G. Wirthensohn, S. Lefrank, W.G. Guder, F.X. Beck

321

Lipid Peroxidation, an Initial Event in Acute Renal Failure (ARF) W. Joannidis, W. Pfaller

329

XIII

Specific Fructose-1,6-Bisphosphatease Activities in Microdissected Proximal Tubules of Human Kidneys in Function and Dysfunction H. Schmid, A. Mall, H. Bockhorn

339

Intranephron Distribution and Properties of Xanthine Oxidase, Superoxide Dismutase and Guanase Activities in Control and Nephrotic Rats H. Endou, H. Yamada, T. Takahashi, K. Tamura, S. Ito

347

Contrasting Effects of Amino Acid Mixtures on Hypoxic Dysfunction in the Rat Kidney M. Mälyusz, G. Gronow

353

11ß-Hydroxysteroid Dehydrogenase(11-HSD) - its Function in Renal Corticosteroid Metabolism W. Schulz, N. Kobayashi, H. Siebe, K. Hierholzer, I. Lichtenstein

361

Tamm-Horsfal1 Protein and Antibodies to Tamm-Horsfall Protein in the Study of Renal Diseases M. Radonic, D. Cvoriscec, A. Stavljenic, J. Sertic, G. Borso

369

VII. RENAL KALLIKREIN-KININ-SYSTEM, DEVELOPMENTAL ASPECTS OF RENAL METABOLISM Studies on the Renal

Kallikrein-Kinin-System

W.G. Guder, J. Hallbacli, G. Wirthensohn, R. Linke, E. Fink, W. Muller-Esterl

377

Relation between the Renal Angiotensin I Converting Enzyme (CE) and the Inactivation of Kinins by the Kidney J. Marchetti, F. Praddaude, S. Roseau, F. Morel

385

Hormonal Regulation of Postnatal Development of Renal Tubular Transport Processes H. Braunl ich

391

Regulation of Uric Acid Synthesis in Embryonic Liver and Kidney J. Wittmann, A. Mengi, S. KrauBer

397

Role of Adrenal Steroids in the Maturation of Fetal Rat Kidney Na-K-ATPase D. Dobrovic-Jenik, S. Milkovic

403

LIST OF PARTICIPANTS

411

AUTHOR INDEX

415

SUBJECT INDEX

417

I. FUNCTION OF BRUSH BORDER MEMBRANE

HYDROLYSIS OF NEUROPEPTIDES BY PIG KIDNEY MICROVILLAR PEPTIDASES: THE KEY ROLE OF ENDOPEPTIDASE-24.11

Sally L. Stephenson and A. John Kenny MRC Membrane Peptidase Research Group, Department of Biochemistry, University of Leeds, Leeds LS2 9JT, U.K.

Introduction The brush border of the renal proximal tubule is particularly rich in peptidases (table 1). They are integral membrane proteins with their active sites facing the lumen of the tubule (1). Although much is known about their structure and other properties, we have little understanding of the roles of these enzymes, but their location strongly suggests that they are involved in the hydrolysis of peptides present in the glomerular filtrate. Studies with purified enzymes have yielded a good deal of information on the specificity of the microvillar peptidases. This is shown diagrammatically in table 1 along with details of specific inhibitors of these enzymes. In this report we have addressed the question: How are neuropeptides degraded by the combined attack of this group of membrane peptidases? We have incubated a selection of neuropeptides with microvillar membranes in order to assess the rate and mode of attack. The contribution of individual enzymes was determined by including appropriate inhibitors with the membrane fraction. We have shown that endopeptidase-24.11 plays a dominant role in the initial attack of all the susceptible neuropeptides studied. 3 A similar conclusions was reached when [ H]bradykinin was studied at concentrations closer to those existing physiologically (2).

Molecular Nephrology Biochemical A s p e c t s of Kidney Function © 1987 Walter d e G r u y t e r & Co. • Berlin • New Y o r k - P r i n t e d in Germany

4 Table 1.

Peptidases identified in pig kidney microvilli

Enzyme

Specificity (amino acid residue, O)

Effective inhibitors (concentration used in brackets)

Endopeptidase-24.11

-0-0-*-0-

Phosphoramidon (1 nM)

(hydrophobic) Aminopeptidase N

• - 0 - 0 - 0 -

Aminopeptidase A

• - 0 - 0 - 0 -

Aminopeptidase W

0-0-0-0-

(many) Amastatin (1 nM)

(Glu/Asp)

Aminopeptidase P Carboxypeptidase P Dipeptidyl peptidase IV (DPP-IV) Peptidyl dipeptidase A (angiotensin converting) enzyme, PDP)

(Trp) 0-®-0~0(Pro) -0-0-•-0 (Pro,Ala,Gly) 0-*-0-0(Pro,Ala) -0-0-*-* (nonspecific)

Diisopropylfluorophosphate (Dip-F) (0.1 mM) Captopril (1 m-M)

Methods The methods used have been described in detail elsewhere (3). Microvillar membrane vesicles (0.2 - 5 ng of protein) or purified peptidases (50 - 100 ng) were incubated with peptide (0.02 500 nM) at 37°C for various times. Where inhibitors were included they were pre-incubated with membranes or enzyme for 15 min, 20°C before addition of the peptide. (See table 1 for concentration of inhibitors used.) Incubation was terminated and peptide products analysed by h.p.l.c. as described previously.

5 Results Rate of hydrolysis of peptides by kidney microvillar membranes The relative rates, assessed in defined experimental conditions, as t, values are given in table 2. Two of the peptides studied [Arg°]vasopressin and insulin, were essentially resistant to attack in the conditions employed although some degradation was noted after 24 h. Oxytocin was only slowly hydrolysed, with a t^ of about 8 h, while the other peptides were very rapidly hydrolysed with t^ values of 5 - 34 min. Substance P and bradykinin were attacked considerably faster than any other peptide in the group. Table 2. Rates of hydrolysis of peptides incubated with kidney microvillar membranes Rapid (t^ i n ) . cyl-IJ-D-glucopyranoside

The synthesis

of

10-N-(bromoacetyl)amino-1-de-

(BADG) and the labeling procedure has been described

recently (6,7). The labeled proteins were prepared for SDS polyacrylamide gel electrophoresis by incubation for 1 h at 37° C in a buffer containing SDS and

15

mercaptoethanol but no urea (7). Two dimensional gel electrophoresis was performed as described e a r l i e r (9) and

the proteins were e l e c t r i c a l l y transferred

t o n i t r o c e l l u l o s e as reported by Burnette (10).

Results and Discussion To test the hypothesis that p h l o r i z i n binds to the Na+-D-glucose cotransporter at

two

attachment

points,

one f o r

D-glucose and the other f o r

phloretin,

i n h i b i t i o n of p h l o r i z i n binding by D-glucose or phloretin was investigated. For D-glucose (data not shown) and f o r phloretin (Fig.1) inhibition

was

98 + 4.8mM

demonstrated

were

and respective

calculated.

The

data

a competitive type

Kj values

show t h a t

of

of

12.3 + 1.0 mM and

phlorizin

binds

at

the

D-glucose binding s i t e and at the same time also at a closely related hydrophoFig.1. Competitive i n h i b i t i o n of Na+ dependent high a f f i n i t y p h l o r i z i n binding by phloretin. Binding of p h l o r i zin at d i f f e r e n t concentrations was measured in the presence of 100 mM Na+ in the absence of phloretin ( O ) or in the presence of 25 pM ( • ) , ( • ) or 200 pM phloretin

100 pM The

lines in the presented Scatachard p l o t Phlor. bound

have been calculated by linear re2 gression analysis ( r > 0 . 9 4 ) .

[pmol/mg]

bic protein domain of the Na+-D-glucose cotransporter. To understand the i n t e r action

of

the

covalently

binding

no-1-decyl-B-D-glucopyranoside reversible and covalent "short term"

effects

Na+-dependent p h l o r i z i n

D-glucose

(BADG) with

effects

of

analog

the

10-N-(bromoacetyl)ami-

Na+-D-glucose

BADG were studied.

cotransporter,

Measuring

reversible

i t was found that BADG was a competitive i n h i b i t o r of binding

to brush-border membrane proteins

(data not

shown) and of Na+ gradient-dependent D-glucose uptake into brush-border membrane vesicles ( F i g . 2 ) . The K- values calculated f o r i n h i b i t i o n of binding and transport were 0.45 + 0.05 and 0.30 4 0.01 mM respectively. The data indicate that a f t e r short incubation BADG interacts reversibly with the D-glucose

16 Fig. 2. Competitive inhibition of Na+ gradient-dependent D-glucose uptake by BADG. D-glucose uptake rates into membrane vesicles were calculated from measurements after 1 and 5 s incubation with 0.09 mM D-glucose ( O ) ,

0.18 mM

D-glucose ( • ) or 0.36 mM D-glucose ( A ) The lines have been calculalated by l i 2 near regression analysis (r >0.97).

w>J

BAOG [mM]

binding site of the Na+-D-glucose cotransporter. To find out whether BADG may also bind covalently at the D-glucose binding site i t was tested whether BADG is

able to

irreversibly

inhibit

Na+-dependent phlorizin

incubation at pH 8.5 with 0.1 mM BADG binding sites were irreversibly during

incubation

with

binding.

After

21% of the Na+ dependent

inactivated.

1h

phlorizin

No inactivation was observed

BADG 100 mM D-glucose was present.

Thus

it

if

may be

assumed that the reactive bromoacetyl group at the end of the aliphatic chain of BADG binds

covalently

to the hydrophobic domain close to the D-glucose

binding site. After covalent binding the D-glucose moiety of BADG i s supposed to

remain

reversibly

bound to

the

D-glucose

binding

site

and to

inhibit

+

Na -D-glucose cotransport. To identify D-glucose binding polypeptides of the Na+-D-glucose cotransporter a protein fraction from brush-border membranes of pig kidney cortex (PI, see Methods) in which the concentration of Na+-D-glucose cotransporter was fourfold enriched over that

in the membrane, was incubated

for 1 h at pH 8.5 with 30 uM (14C)BADG. The incubation with BADG was performed in the presence of Na+ or K+ plus either D-glucose, L-glucose or D-mannose. In the

presence

of

80 mM Na+ and 100 mM D-mannose BADG was mainly bound to

polypeptides with molecular weights of about 47 000, 75 000 and 82 000 (Fig.3). BADG binding to these polypeptides was significantly

reduced i f

labeling was

performed in the presence of 100 mM D-glucose. Furthermore, in the absence of D-glucose

labeling +

presence of Na

characterization

of

these

polypeptides

was +

than in the presence of K BADG-labeled

dimensions by f i r s t

membrane

significantly

in

the

(data not shown). For a further

polypeptides

performing isoelectric

higher

were

separated

in

two

focussing and then SDS polyacryl-

amide gel electrophoresis. To identify the polypeptides which had been labeled with

(14C)BADG the proteins were e l e c t r i c a l l y

transferred from the gel to a

nitrocellulose membrane. Then the proteins were stained and autoradiography was performed (Fig.4). From Fig. 4 i t can be seen that in the presence of Na+ and

17

705k

116 k 97 k

BGk

ISh

191.

Fig.3. Covalent labeling of brush-border membrane proteins from pig kidney cortex with BADG. Membrane proteins (PI) were labeled by incubation for 1 h (37°C) at pH 8.5 with 30 pM ( 14 C)BADG and 80 mM Na+ plus 100 mM D-mannose (O) or plus 100 mM D-glucose ( • ) . Then the reaction was stopped and SDS Polyacrylamide gel electrophoresis was performed. In a) the distribution of radioactivity after labeling in the presence of 100 mM D-mannose (O) or 100 mM D-glucose ( • ) i s presented. In b) the difference between BADG labeling in the presence of D-mannose and D-glucose is shown.

pH

7.0

6.0

5.0

I i i i i I i i i i I

7.0

6.0

5.0

I i i i i I i i i i I

^r —116 k — 97 k

ig*#w-'

— 66 k — A5k

Fig.4.

Separation of BADG-labeled membrane polypeptides by two dimensional gel

electrophoresis and their analysis by autoradiography. Membrane proteins were labeled with BADG in the presence of Na+ and D-mannose as in Fig.3. The proteins were separated by two dimensional gel electrophoresis and transferred to a nitrocellulose membrane. In a) the nitrocellulose membrane stained for proteins and in b) an autoradiogram of this membrane is shown.

18

D-mannose five polypeptides were labeled with BADG. Protein spots from labeling experiments performed in the presence of Na+ plus D-mannose or plus D-glucose were dissected activity. Fig.4a

from the

nitrocellulose

In four of the five

(nos. 1,2,3.5)

membranes

and analysed

for

radio-

labeled polypeptides which are indicated

BADG labeling

was

reduced

by more than

in

25% in the

presence of D-glucose. The molecular weights and respective isoelectric points of these polypeptides were: 82 000, pH 5.6; 75 000, pH 5.4; 75 000, pH 6.9 and 47 000 , 5.4. D-glucose protection of BADG labeling was observed in the same polypeptides if labeling experiments were performed with brush-border membranes from

outer

cortex,

inner

cortex

or

outer

medulla

of

pig

kidneys.

When

brush-border membrane polypeptides with molecular weights between 70 000 and 90 000 were extracted from SDS polyacrylamide gels and employed as antigens three monoclonal antibodies were obtained which bind to the BADG-labeled 82 000 ^-polypeptide and crossreact with the BADG-labeled proteins with the molecular weights and isoelectric points of 64 000, pH 5.2 (No. 4 in Fig.4) and 47 000, pH 5.4

(No.5

antibodies

in Fig.4).

In Fig.5

a radioimmune

assay

with

one of

these

is shown which was performed with membrane polypeptides which had

been separated by two dimensional gel electrophoresis.

PH I

Mr —

82k

—

6Z,k

—

¿7k

Fig.5. Crossreaction of a monoclonal antibody with three membrane polypeptides. In a) staining of the membrane polypeptides which have been separated by two dimensional gel electrophoresis and transferred to a nitrocellulose membrane and in b) an autoradiogram of the transferred proteins is shown. The membrane was f i r s t incubated with the monoclonal antibody R3A1 from mouse and then with a second radiolabeled antibody which was directed against mice antibodies.

19 The observed crossreactivity with monoclonal antibodies shows that the 64 000 and 47 000 M r -polypeptides share epitopes with the 82 000 M r -polypeptide. The 64 000 and 47 000 M r -polypeptides may be s p l i t t i n g M

r -polypeptide.

brush-border

products

of the 82 000

However, they are supposed to be already present in the native

membranes

since

BADG-labeling

of

the

47 000

and

64 000

M r -polypeptides was also observed i f membrane vesicles were labeled which had been prepared in the presence of

protease

inhibitors

(data not shown).

In

conclusion three polypeptides with molecular weights and isoelectric points of 82 000, pH 5.6; 75 000, pH 5.4; 75 000, pH 6.9 can be considered as components of the Na + -D-glucose cotransporter in cortex and outer medulla from pig kidney. The 75 000 M r -polypeptide with the isoelectric point of pH 5.4 appears to be identical with a component of the intestinal Na + -D-glucose cotransporter (5).

Acknedgement Supported by the Deutsche Forschungsgemeinschaft (SFB 169).

References 1.

Turner, R.J., A. Moran. 1982. Am.J.Physiol. 242, F406.

2.

Brot-Laroche, E., M.-A. Serrano, B. Delhomme, F. Alvarado. 1986.

3.

Koepsell, H. 1986. Rev. Physiol.Biochem.Pharmacol. J04, 65.

J.Biol.Chem. 26^, 6168. 4.

Peerce, B.E., E.M. Wright. 1985. J.Biol.Chem. 260, 6026.

5.

Wright, E.M., B.E. Peerce. 1986. In: Ion Gradient-Coupled Transport (F. Alvarado and C.H. Van 0s, eds.) Elsevier Amsterdam, p.127.

6.

Neeb, M., H. Fasold, H. Koepsell. 1985. FEBS Lett. JI82, 139.

7.

Koepsell, H., M. Neeb, A. Madrala. 1986. In: Ion Gradient-Coupled Transport (F. Alvarado and C.H. Van 0s, eds.). Elsevier Amsterdam, p. 117.

8.

Ramaswamy, K., B.R. Bhattacharyya, R.K. Crane. 1976. Biochim. Biophys.Acta

9.

Koepsell, H., K. Korn, D. Ferguson, H. Menuhr, D. 011 ig, W. Haase. 1984.

433, 32. J.Biol.Chem. 259, 6548. 10. Burnette, W.N. 1981. Anal. Biochem. 112, 195.

RENAL TRANSPORT AND METABOLISM OF CARNOSINE IN THE RAT

S. Silbernagl with the technical assistance of K. Völker Physiologisches Institut der Universität, D-8700 Würzburg, FRG

Introduction After Gulewitsch and Admiradzibi (1) had succeeded in isolating a new basic substance from the muscle in 1900, it was characterized as B-alanyl-L-histidine later on (2). This dipeptide is found in a wide variety of animals, localized primarily in the skeletal muscle (¿). This has also been shown for the rat (3). However, the small amounts found in other organs may also be of physiological importance. In the mammalian olfactory pathway, for instance, carnosine.seems to be the principal neurotransmitter (4, 5). The kidney seems to be the major site of carnosine breakdown (6, 7, 8). After Abderhalden and Hunter had observed hydrolysis of oligopeptides in the kidney already in 1917 (9), it was only ten years ago when it turned out that tubular brushborder peptidases hydrolyze within the lumen, a process followed by the reabsorption of the free constituent amino acids (reviews: 10, 11). Some oligopeptides, however, resist the attack of the brushborder peptidases (review: \i.) . Nevertheless, L-carnosine is taken up into rabbit renal brushborder membrane vesicles. This transport is inhibited by other peptides but not by free amino acids (13). It has been reported most recently, that another dipeptide, i.e. sarcosyl-glycine, is actively taken up into rat renal brushborder vesicles (14). This transport is driven by a H + gradient directed into the cell (tertiary-active transport) and is mainly localized in the late proximal tubule (14). In the present study, renal reabsorption of L-carnosine was measured and its localization was determined by free-flow micropuncture of rat renal tubules in vivo and in situ. Furthermore, the renal extraction or formation, respectively, of L-carnosine and its con-

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin - New York - Printed in Germany

22 stituent amino acids, B-alanine and L-histidine, were measured at exogenously elevated carnosine plasma levels.

Methods Male Wistar rats of some 300 g body weight (Ivanovas, KiBlegg, FRG) were fed on an Altromin standard diet arid had free access to water. The rats were anaesthetized with 120 mg/kg BW Inactin (Byk-Gulden, Konstanz, FRG). After surgery (15) the rats were infused with Ringers solution (75 ul/min) containing inulin and, after a control period, carnosine, too. Free-flow micropuncture and amino acid analysis, which included L-carnosine, were described previously (15). The arterial blood was taken from the femoral artery. The renal venous blood was obtained by puncturing the vein of the left kidney exposed for micropuncture, with a small injection needle. The puncture site was sealed around the needle by a tissue glue (Histoacryl, Braun-Melsungen, D-3508 Melsungen, FRG). The filtration fraction was calculated from the inulin concentration in arterial and renal venous plasma. The renal plasma flow was calculated from the glomerular filtration rate (inulin clearance) and the filtration fraction.

Results Endogenous carnosine plasma concentrations of up to 3 umol/1 were found , (see Table 1). Endogenous plasma levels of histidine and 8alanine varied between 90 and 110 umol/1, and 4-6 umol/1, respectively. Under these conditions, fractional excretion of carnosine and histidine was always smaller than 1 %, but was 17-56 % for 8alanine. During intravenous infusion of L-carnosine (825 nmol/min/rat of 300 g BW) fractional excretion was not increased, although the plasma concentration was two orders of magnitude higher than under control conditions (see Table 1). This result demonstrates that the renal reabsorption mechanism for carnosine has a relatively high capacity

23 Table 1. Renal Excretion and Extraction of Carnosine and its Constituent Amino Acids Carnosine

Histidine

B-Alanine

traces - 3 max. 0.8

90-110 0.3-1

4 - 6 17-56

Control (n=7) Arterial plasma conc.(umol/1) Fractional excretion (%)

Carnosine infusion (825 nmol/min/300 g BW; n=8) Arterial plasma conc.(umol/1) 243 + 29 236 + 1 4 Fractional excretion (%) Extraction(E) or release(R): absol.(nmol/min/g kidney) relative (%)

0.25 + 0.05 0.31 + 0.03 E 302 + 6 5 E 37 + 6

R 88 + 77 R 13 + 12

61+5 11+2 E 13 + 14 E 5 + 8

+ SEM. GFR=0.96+0.04 ml/min/g kidney; filtration fraction=31+2% if the low endogenous filtered load of such peptides is taken into consideration. At a mean carnosine plasma concentration of 207 + 70 umol/1, freeflow micropuncture samples obtained from the last superficial loop of the proximal convoluted tubule and the earliest superficial loop of the distal convoluted tubule, respectively, revealed a carnosine tubular-fluid/plasma concentration ratio (TF/P) of 1.08 and >

>

4-t O (0 a>

so a. CO e Í . IO CL Í +J ai

IO E X o

sz +J

c


, —. X o E no

CM

Í.

Ì.

>,

1 ai +-> IO

3 a. a.

c o

"O e IO

*

1 di

IO c

,!

ai -C

1 . e tu •t-> +-> IO ï c

44IO

LO

a.

0) +J IO e

LO

co o o

—

O

O

o

O

O

o

O

o

"3"

i ' o

O

O

co1 o

o

n

á ' o

O

o

O

o

o

o

o

o

o

CT> c o

s

OI

o A

CM

o

ir)

4 '

•!->

c o • 1—1

OO C

>

o IO à i U J 1ai m +J n < c t— »—i 4->

A

oo

oo o

LO

CM

G0

o

O

LO

+ 1

LO

LO

i ai c 01 N C a)

IO

i

Ol _l 1

CU

+->

IO

>

IO CT O Li-

X o

1_ io u Q

•—i

ra CO o

+->

3;

m

3

+ 1 LO

r^

•O 4-

IO c O)

00 o Q. ai =3 4— O IO

+1

LO

o

+1

>>

CM

LO CM

A

A

sz

• «

•a o

IO c IO aj +J IO ,—1

>,

V

•o

o o

o o

1 CM 31

»—'

0)

+->

IO c o 4-

Z3

+->

o LO

•o l

t

cz 1

o o • r-l X x: o JD +J So LO IO 1 CM

LO CM

o

^—

• o +J IO c IO

+ 1 CM

*

3 LO

o

O

+ 1

Ü0

LO O

O

co

r—1

CO o o

lo o o

--

—>

m

co

o

o o 3 LO

a> 4-> IO 4—

LO r^

o

CO

>, c

oo

•îl

o u

a; .o IO i

+->

LO

•!->

cu E

«3-

OK

3 1—1 i/i c: o o

o

CM

OK

IO e

e

X


j = s- o o c a.

c IO l. +J

CO

ai

IO

IO

Vf

i

•(-> - e

+->

+J (/I

c IO

o

O

f

co

•o 1 CM

-

CM 1 ai c: CU

ri

IO i—H IO 1 C2 1

IO •C

¿Z

a. IO ci i LO

>>

— T O +J IO c IO

>>

a)

+->

0)

u •P o IO • i-M c sz o •!-> 4 O 00 L/l 1 co •o

ai

+->

IO i—i

>.

X o .a sIO

3

0) c -rH C IO

4->

IO

Z

1

c

a> +J IO c 10 >> o o

c eu o 00 a) 5O 3

-C +J o L/) •rH c:

4O "O

ai o 00 a>

a) o IO o TD O

o 3 ,—i U. 00

e S

1 LO CTI

00

CM

IO

co

CTI

CM

o

LO

CO

O

•«a- co

'

*

CO CVJ

*

'

LO

o

— C o 1 c: i/i c «=f IÖ 00 cu sz O 1 .c 1 +-> UJ Oai OI +-> ra c o 43 t/i a> c 0) N C

,

>.

o

.

CTI O LO oo

LO

CTI co

LO

CSI co *—

'

CJ

i o

ra >> u

i-

+->

u a) .— » eu o 4s-> So ai c o sz

+•>

3

00 CL 3 O sOl O •F -H •1—1 SZ CL

o • 1—I io XI X +J o o >> l/l •F—t en >-. >> c C aj cu sz sz a. CL o O s_ S+-> 4-> CZ C 1 1 «d-

1—1 '—

LO *—

IO X o >">

Ol

>, c= ai

sz a.

o

"O

N 1 X 1—1 1o —1 o >) NI o c N cu c a) S- -Q o 1 3 o ,—i S4— +-> O "O c 1 N CSI IO 1 1 o «a- "O i o N s- IO +-> 1 LO c -—1 1 CSI z '—

co

LO

LO

o o

LO

o

¿1

co

o

o

CTI CD LO o +1 +1 co o A «3- LO

— ,

o LO — , — , co +1 + 1 + 1 f^ CSI CSI CTI

ai IO o sz o o "O E IO N c ai J2 o +-> IO c to ICT^m) of DOCD on oell content of ATP (•), ACP (*), AMP (• ) and total adenylates (o). The control values observed with or without the vehicle of DOCD (ethanol) are also presented. Note the different scale used for cortical tubules and thick ascending lints.

129 Discussion Qxymetric measurements of respiration of dog proximal tubules with ana without auatain demonstrate that the Na+,K+-AXPase activity can account for only 30% of respiration when these secpents are incubated with lactate and glutatiine in KrebsHenseleit saline.

In this in vitro condition, the luten of the tubular segment is

open and the brush border membrane is in contact with the incubaticn mediun.

Thus,

the transcellular sodium transport is not limited by glaterular filtration as in situ.

Therefore the sodiun transport occurs at a faster rate in this preparation

than what is observed in vivo.

Nevertheless, this transport only represents a

surprisingly small 30% of the total energetic expenditure in these nephron segments. This suggests that other major ATP-utilizirtg processes distinct from the Na^K"1"ATPase irust exist in proximal tubular oells.

A proton ATF&se has been described in

these oells, and most specifically in their trush-border membrane (4). This enzyme is in seme aspects similar to the mitochondrial ATPase (phosptarylating F0-Fi structures): both structures insure the transport of protons energetically linked with the hydrolysis (in the membrane) or synthesis (in the mitochondria) of OTP. DOCD is an inhibitor of F 0 unit in both H+ ArPases (5). Our data demonstrate that DCCD has also other less specific effects: at 25 juM, it inhibits the Na+,K+-ATPase in proximal and distal tubules.

Nevertheless, DCCD was able to inhibit a fraction of the ouabain in-

sensitive respiration in proximals, but not in TAL cells.

This suggests that an

ATP-dependent proton purrp exists in the former structures, but not in the latter. In absence of a proton gradient, as realized in suspension of cortical tubules, this ATEase may be responsible for as much as 30% of the respiration of tubules: i.e. an oxygen uptake oarparable to that driven by the Na+,K+-ATEase.

In collecting ducts,

our data suggest that DOCD interferes with glucose entry in the cells, suggesting that a protcn, rather than a sodiun, gradient may be responsible for this process, lactate production, new mainly derived froti endogenous glycogen, is also reduced. We have ascertained that DOCD did not interfere with papilla hexokinase activity. These data are thus oarpatible with a specific effect of DOCD in the distal nephron. However, the very marked effect of DOCD on tissue ATP is such that additional experiments are required to identify possible non-specific effects of DCCD on these segrents.

130 In sutnary, our results suggest that a H+'-ATPase may be active in proximal tubular cells and collecting ducts, but not in thick ascending lints segments of the dog nephron, and that this activity may have significant energetic consequences.

Ackrowlecxpent Ihis work was supported by grant MI7875 awarded by the Medical Research Council of Canada.

C. Manillier and A. Tejedor are scholars of the Groupe de Recherche en

Transport ffembranaire, Université de Montréal.

P. Vinay and Y. Boulanger are

scholars of the Ponds de Recherches en Santé du Québec.

References 1. BalaOan, R.S., L.J. Mandel, S.P. Soltoff, J.M. Storey. Sci. USA. 77: 447-451.

1980. Proc. Natl. Acad.

2. Vinay, P., C. Manillier, L. Lalonde, G. Thibault, Y. Boulanger, A. Gougoux, M. Cantin. Kidney International, (in press) 3. Tejedor, A., J. Noel, C. Manillier, Y. Boulanger, A. Gougoux, P. Vinay. Clinical and Invest. Medicine. 9: A107.

1986.

4. Sabolic, I., W. Haase, G. Burckhardt. 1985. im. J. Physiol. 248; F835-F844. 5. Eanestil, D.D., C.S. Parck, 1981. Am. J. Physiol. 240; C201-C206. 6. Bank, N., H.S. Aynedjian, B.F. Matz. 1985. Am. J. Physiol. 249; F636-F644. 7. Vinay, P., A. Gougoux, G. Demieux. 1981. Am. J. Physiol. 241: F403-F411. 8. Schwartz, A., K. Nagao, M. Nakao, G.E. Lindenmayer, J.C. Allen, H. Matsui. 1986. In "Methods in Pharmacology". Martinez Maldonado M. ed. pp. 361-383. Plenum.

III. CELL MEMBRANE POTENTIAL AND TRANSPORT PROCESSES

ELECTROPHYSIOLOGY

F. L a n g , Inst,

OF CELL

M. P a u l m i c h l ,

of

Physiology,

VOLUME

H.

REGULATION

Voelkl,

E. G s t r e i n ,

University

of A 6 0 1 0

F. F r i e d r i c h . Innsbruck,

Austria

Introduction In

a variety

leads

to

cell

of

cell

volume

tissues, swelling

close

[3,5,9,11,13]). of

KC1

in

and chloride Ehrlich

Canine

Kidney

[2,15]

.

is

to

were

exposed

cell

membrane

and

been

Tumor

test by

cells

volume

cells

the

these

media

with

is

observed. the

loss

Madin of

(EAT,

continuous

of

Darby (PT)

conductive

alterations

during

A for

tubules

activation

conventional

loss

pathways

[5,6],

tissues

of see

decrease,

proximal

respective

from

to h y p o t o n i c potential

(EAT)

decrease

to m e d i a t e

[10] and

media

references

conductive

invoked

whether

hypotonic

(for

chloride

has

cells

to

regulatory

value

regulatory

separate

reflected

potential,

by

of

Ascites

In o r d e r

pathways membrane

this

(MDCK)

of c e l l s

original

potassium

activation

potassium

followed

the

During

intracellular

parallel

to

exposure

of

MDCK

cell and

PT)

recording

of

microelectrodes.

Methods Proximal swiss

straight mice

principally maintained Dulbecco's fetal

tubules

weighing the m e t h o d

of B u r g

prior

the

to

modified

medium

calf

serum,

Streptomycin

[4,8,12]

were

dispersed

3 Na2HP04, plated

were

1.5

on cover

(0.03%

dissected

1 0 - 1 5

experiment (DMEM)

100

U/ml

EDTA

glasses

and

titrated and

from

and

et a l .

at 5 % C 0 2 ,

KH2P04,

g

and

kidneys

perfused

[1,14]. in Ham's

1:1

with

of 10%

air 1 mol/1 again

were

mixture

(in m m o l / 1 )

incubated

cells

F12 medium with

Penicillin 95%

female

following

MDCK

a

of

and and

100

pg/ml

3?°C.

Cells

13? N a C l , NaOH in

the

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin • New York - Fainted in Germany

3

KC1,

to p H

7.4),

same

medium

134

as

above

f o r at

least

48 hours.

Lettré

subtype,

glycogen

minimum

essential

medium

with

20

mmol/1

3 7 ° C.

into

the

mammalian

were

cultured

salts

buffered

Penicillin

coated

by were

to c o n t r o l

mannitol made

95%

air

performed

at

and

(PD) w a s

(resistance

(20

with

Mfl, t i p

ô

of

30

were

was

part

pipetted

very

NaCl

to was

osmolarity).

Hypotonic identical

otherwise

continuously All

fine

90

similar of

but

< 5

bubbled

experiments

difference

potential

60

supplemented

were

7.4].

potential

determined

100 - 2 0 0

7.35

Streptomycin,

that

mannitol

(pH

Eagles

acid,

solutions

solutions

The

in to p H

dish.

to 5 0 %

5% C02

cells,

100 m g / 1

suspensions

fluid except

The

37°C.

cell

petri

up w i t h o u t

solutions.

G and

of c o n t r o l

extracellular

solutions

membrane

[7]

composition

replaced

Tumor

with Earles

experiments

a poly-L-lysine

Electrolyte

with

6 mg/1

For

free

Ascites

morpholinopropanesulfonic

with 7 X serum, at

Ehrlich

across

tip

the

were cell

microelectrodes

mV).

120

150

'time (s)

Fig.

1:

exposure 2

min,

(closed

Cell

height

to h y p o t o n i c in t h e symbols,

[%

presence n =

of c o n t r o l

(230 m o s m o l / 1 ] (open

13] o f

± SEM)

symbols,

1 mmol/1

in

extracellular n = 4]

barium.

and

PT

during

fluid

for

absence

135

Results As

shown

leads

in Fig.

to

decrease

cell is

During

swelling,

-67 ±

143)

in E A T

cell

membrane

potassium. potassium 0.01

cells and to

and - 5 4 ± 1 mV

+10 ±

over

by m a n n i t o l

(n = 7 )

in

1 mV EAT

2:

Effect

extracellular basolateral increasing mmol/1 and

20 K*

of

fluid cell

membrane

both

hypotonic

of h y p o t o n i c

absence

of

1 mmol/1

to

barium

(Ba)

of

to

slope is

0.66

in

EAT

fluid

of only

extracellular however,

leads

(Fig-

and

4)

hypo

*

The

Replacement

in P T c e l l s

2), and

hypo Ba .MO"3mol/l

20K'

hypotonic

(240

difference cells.

concentration

isotonic

extracellular

=

103)

(Figs. 3

in PT

potassium and

(tk

(n =

of m a n n i t o l ,

cells

(n

cells.

conductance)

cells.

(n = 3 3 )

potential (PD)

1 mV

conductive

numbers

Reduction

20K*

exposure on the

extracellular

in

b: E f f e c t

WW

20 K*

-5? ±

in e x t r a c e l l u l a r

tissue.

barium. potential

in MDCK

0 . 5 3 ± 0.01 in MDCK

"volume"

mmol/1

mainly

slope membrane

in e i t h e r by +9 ±

is

hypotonic

Fig.

1

media

"volume"

membrane

transference

(n = 2 0 )

NaCl

PD

20 K*

of

(n = 3 7 )

to 7 5 - 80 % by o m i s s i o n 1 mV

regulatory

cell

tissues

in P T c e l l s ,

to a d e p o l a r i z a t i o n by

three

respective

0.45 ± 0.02

osmolarity

in t h e p r e s e n c e [n = 3 3 )

hypotonic

The regulatory

in PT c e l l s ,

103)

alters

by

height].

1 mV

in all

40 mmol/1

slightly

followed

to

the

conductance (n =

cells

conditions,

cells The

o f PT

from cell

abolished

control

approaches

30

exposure

(as e v i d e n t

decrease

±

1,

fluid,

mosmol/1) across

a:

of

f r o m 5 to

20

extracellular both

(two original

the

Effect

in the

fluid, presence

recordings).

136

by

+ 26 ±

1 mV

membrane MDCK

cells.

reduced cells

(to

0.07

cell

by + 2 8 (n =

+

+7 ±

(n = 3 2 ] media

cells

1 mV

in

5].

depolarize

EAT cells

(n = 7)

5),

but

in PT

blocker

anthracene-9-C00H

observed

during

hypotonic

cells

exposure

the

cell 3]

not

the

+15 +

MDCK

of

2), 1 mV

1 mmol/1

further 1 mV

cell

(n

by -

membrane

Chloride

the

is MDCK

(Fig.

of

membrane

2).

tk in

potassium

a n d by + 1 4 +

(Fig.

of PT c e l l s a n d

by

presence

abolish

in

depolarizes

cells

3] a n d

the

(Fig.

PT

Cell and

decrease

which

in

In

and

tissues,

hyperpolarize

cells

does

three

(Fig.

4)

to a

barium,

(n - 7 )

6).

in E A T

solutions

(Fig.

pointing

In a l l

+ 3 mV (Fig.

(Fig.

5 and

altered

cells

1 mmol/1

in E A T

hypotonic (n = 4 ]

6).

by

by +31 cells

1 mV

-5 ±

(Fig.

MDCK

in M D C K

12) by

in

EAT

n = 20],

inhibited

membrane 1 mV

13]

barium,

is

(Figs.

to h y p o t o n i c

2),

± 0.01,

cells

significantly

exposure (Fig.

conductance

conductance

in M D C K

is n o t

During

in PT c e l l s

potassium the

(n = 3 2 )

resistance

channel

depolarization

cells

(Fig.

6)

to

media.

0

Bq

1 -101

h

0

I /P°

" B e T

-20-

-30I

-AO-

Q CL

-50-60-

-70J

Fig.

3:

Effect

extracellular EAT barium

1 1| 1 1mfn

cells (Ba)

concentration

of

fluid both

exposure (hypo)

in

to

on cell

hypotonic membrane

the p r e s e n c e

and

recording).

K:

(Original increased

from 5.4

to 20

(240

mosmol/1)

potential

absence

of

extracellular mmol/1.

(PD)

D.7

in

mmol/1

potassium

137

P -¿0

Fig.

4:

resp.) cell

Effect

extracellular membrane

increasing mmol/1

(K)

5:

during

(Ba)

cells

both

(Original

of

fluid in

to h y p o t o n i c (hypo

(PD)

(Original

exposure (HYPO)

the

240 and in

potassium exposure

solution

Effect

extracellular MDCK

fluid

potential

extracellular

extracellular

Fig.

of e x p o s u r e

hypo

to

150

150,

cells.

concentration isotonic

mosmol/1, resp.)

Effect from 5.4

on of

to

20

or

hypotonic

(240

mosmol/1)

recording].

to

on cell

presence

recording).

EAT

( 2 4 0 or

hypotonic membrane

and absence

potential of

(PD)

1 mmol/1

in

barium

138

Conclusion Osmotic

swelling

depolarization

may

conductance. indicate,

depolarizes be

The

in p a r t

that

force)

some

may

blocked

anthracene-9-CODH.

then

by

in p a r t

increases However,

be

exposure

Kept

for diffusive

of

to

to

Effect

of

The

of

could

potassium

support

However, PD

during

may

reflect

properties As

the

activation

of

media

regulation.

be

efflux.

channels.

membrane

The

cannot

efflux

to a p a r a l l e l

hypotonic

cell

(and/or

depolarization

not

the a l t e r a t i o n s

volume

barium

potassium

- selective

that

cell

leads

potassium of

anion

do

This

swelling.

increase

seemingly

swelling

electrical

for an

of

however,

enhanced

force

cells

related

which,

depolarization.

is o b t a i n e d

cells.

conductance

osmotic

the

in m i n d

the

by

The

PT

presence

additional

observations

osmotic

the

channel,

- and of c h l o r i d e

of

alterations directly

The

that

of p o t a s s i u m

for

driving

no e v i d e n c e

assumption must

account

the

conductance.

it

be an a n i o n

and

to a d e c r e a s e

in

is a l t e r e d

conductance

MDCK

due

observations

however,

electromotive

EAT,

in

not

previous

[mV] Fig.

6:

extracellular MDCK

cells

fluid both

anthracene-9-COOH potassium exposure

exposure (HYPO)

in

the

(ANT).

concentration to

hypotonic

of a n t h r a c e n e - 9 - C 0 0 H

to

on c e l l presence Effect

from media

(Original

5.4 both

hypotonic membrane and of to

(240

absence

increasing 20

in t h e

recording).

mosmol/1)

potential

mmol/1 presence

of

(PD) 1

in

mmol/1

extracellular (K) and

during absence

139

studies

from

regulatory K

other

conductance

occur.

However,

inhibitory

effect

volume

due

to be

this

involves

indirect,

decrease

appears

regulation

mechanism

laboratories

volume

does

to

not

barium

a

barium

PT

necessary

activation of

[15],

in

for

1)-

volume

necessarily

on

volume

s u c h as an u n s p e c i f i c

anion

imply

force

the

that

for

to

volume

Rather,

regulation

channel

the

Thus,

regulation

of K c o n d u c t a n c e .

of d r i v i n g

loss

abolishes

(Fig-

the

may

some

activated

be

other during

regulation.

References 1. B u r g , M . , J. Physiol. 2±0,

Grantham, 1293.

M. A b r a m o v ,

2. G r a n t h a m , J.J., C.M. Lowe, A m . J. P h y s i o l . 2 3 2 . F 4 2 3. G r i n s t e i n , S., A m . J. P h y s i o l .

Gstraunthaler, G., Physiol. 246. F536.

W.

5.

Hoffmann,

Molec.

6.

Hoffmann, E.K., I.H. M e m b r . B i o l . 9_1_, 2 2 7 .

7.

Nielsen,

8.

Paulmichl , Arch. 405,

9.

Siebens, 527

10. S i m m o n s , 11. S p r i n g ,

E.K.

K.,

Ussing,

M., 102.

A.W.,

K.,

12. T a u b , M., Nat. Acad. 13.

1985.

A.-C.

1983.

Quart.

Ericson.

1986.

Renal

Geibel,

P.A., M.A. 249, F20.

Physiol.

1977.

Golfand

1984.

9,

R. G r e g e r ,

L.P.

1965.

Am.

J.

1986.

J.

96., 9 5 .

J.

1965.

Gen.

Physiol.

Jr.,

J.

Cole.

Simonsen.

J. M e m b r .

Saier

Am.

167.

F. L a n g .

1965.

1982.

B.R.

E.W.

Hereditas

J. E x p e r .

Linshaw,

8,

L.O.

Kregenow,

L. C h u m a n , M . H . Sci. 76, 3338

H.H.

Physiol.

Lambert,

1966.

P. K o t a n k o .

Gstraunthaler,

F.M.

1964.

14. V o l k 1 , H., J. Arch. 407, 252. 15. W e l l i n g , Physiol.

G.

B. S a k a d i ,

Pfaller,

C. G r a n z o w .

N.L.

M. D e l l a s e g a ,

A. R o t h s t e i n , 246, C204.

4.

J. O r l o f f .

G.

PflLigers

Physiol.

66.,

69., 6 3 .

Biol. Sato.

69., 1979.

167. Proc.

36. F.

Lang.

1966.

Sullivan.

Pflugers

1965.

Am.

J.

EFFECTS

OF A 2318?,

PROPERTIES PROXIMAL

OF

TUBULE

M. D e f r e g g e r , Inst,

for

VERAPAMIL,

AND

QUINIDINE

MADIN-DARBY-CANINE-KIDNEY

ON

AND

ELECTRICAL

MOUSE

STRAIGHT

CELLS.

H.

Voelkl,

Physiology,

M. P a u l m i c h l ,

F.

Lang.

University

of A 6010

Innsbruck,

is k n o w n

to r e g u l a t e

Austria

Introduction Intracellular ductances

calcium

and

transport

n o n e p i t h e 1ia1 calcium A

23187

channels impair has

tissues

can

be

[8]

can

been

verapamil

performed

substances

on

isolated

of

kidney

the

and

references

by

All

transport to

test

potential

by

[9]). blocking have

[1,6,12],

The

the

straight

(PD)

calcium

been

shown

present

these

the

tubules

(MDCK)

to

study

of

across

Kidney

and

ionophore of

influence

proximal

Canine

con-

Intracellular the

substances for

Darby

membrane

of e p i t h e l i a l

[12] and

difference

perfused

of M a d i n

see

quinidine

[4].

cell

in a n u m b e r

be d e c r e a s e d

transepithelial

membrane mouse

(for

raised

and

with

functions

cell

of

the

cells.

Met hod s MDCK-cells mixture medium

were

maintained

of

Dulbecco's

with

10% f e t a l

pg/ml

Streptomycin

Cells

were

KC1, 7.4],

plated

medium

as

tubules weighing method

10

of B u r g

(0.03%

on

cover

at

dissected -

at EDTA

15 g a n d et a l .

and

least

free

48

from

perfused

[2,11],

All

(DMEM)

100 U / m l 5% C02, and

95%

with

incubated hours. kidneys

in

and air

1 mol/1 again

of f e m a l e

following

1:1

Ham's

F12

and

and 137 in

the

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

37°C. to

3 pH

same

straight

swiss

principally were

100

NaCl , NaOH

Proximal

solutions

a

Penicillin

(in m m o l / 1 )

titrated

glasses

for

to t h e e x p e r i m e n t

medium

serum,

1.5 K H 2 P 0 4 ,

above

were

modified calf

[5,7,10]

dispersed

3 Na2HP04,

prior

mice the

continuously

142

bubbled

with

composition

95% of

extracellular The

very

fine

potential

5%

C02

solutions

All

difference

with

2 0 0 Ml], t i p

and

control

fluid.

potential

determined

air

< 5

(pH was

experiments across

7.4).

similar

were

Electrolyte to

mammalian

performed

the c e l l

at

membrane

tip m i c r o e l e c t r o d e s

37°

C.

(PD)

(resistance

was 100

-

mV).

Results In

proximal

amounts

tubules,

extracellular membrane

Fig.

1:

the mouse

Effect

potential of

Kidney

(n=13).

potassium

cell

membrane

PD a c r o s s

to - 6 8 ± 2 mV is

concentration

mainly

of 0.1

(> 7 0 % )

mmol/1

difference

isolated

and

(PD)

perfused

(Original

the b a s o l a t e r a l

As evidenced (Fig.

membrane

the e f f e c t

of

3),

the

basolateral

conductive

to

potassium.

of

1 mmol/1

across

proximal

tracing).

cell

from

the

quinidine

(Q)

basolateral

straight

tubules

of

on

cell the

143

1

mmol/1

(n

=

barium depolarizes

6)

and

quinidine +46

±

depolarizes

2

effect

mV

(Fig-

abolishes

the

2)

potassium

by

+20

is o n l y

10

quinidine

ineffective. of

PD

2

in m o u s e

membrane cell

(n at

10 m m o l / 1 ) . affected

10

pmol/1

1

more

=

20)

the

by

high

pmol/1

A 23187

is w i t h o u t

tubules

in b o t h ,

potas-

extracellular

however, verapamil

verapamil

proximal

the cell

virtually

low e x t r a c e l l u l a r At

1) by

than

and

mV

mmol/1

(Fig.

depolarizes

(20 - 4 0 m m o l / 1 ) , or

+37 ± 2

membrane

significantly

+ 2 mV

slightly

by

conductance.

verapamil

conductance

(5 -

concentrations

pmol/1

is

mmol/1

conductance

on

which

0.1

potassium

concentrations

the c e l l

potassium

the b a s o l a t e r a l

(n = 1 2 ) ,

of b a r i u m .

membrane sium

abolishes

potassium (Fig.

are

significant presence

3).

almost

and

effect absence

verapamil.

Fig.

2:

Effect

difference isolated (Original

of

(PD)

perfused

0.1

across proximal

tracing).

mmol/1 the

verapamil

basolateral

straight

tubules

on

the

cell of

potential

membrane

the m o u s e

of

Kidney

144

In

MDCK

cells

membrane

is

1 mmol/1 (n

= =

preferably

barium

20)

1 mmol/1 (n

and

(Fig-

is a g a i n

+10

conductance

is

transient

Fig.

by

+5

in t h e

(Fig.

from

both

mmol/1

5 to

difference

isolated

6)

and in

of

in t h e

verapamil

and

an

absence of

=

of

absence

(PD)

whereas leads tk

cell the

10

pmol/1

to a

marked

to a p p r o x .

verapamil.

0.9

However,

verapamil

of

potassium

depolarizes

9),

of

mV mV

the

the

of

1

the e f f e c t

A 23187

increase

and

the

is

only

verapamil.

extracellular

10 to 2 0

perfused

(n

than

still

50%). ±

conductance.

depolarizes

verapamil

mV

+15

by + 4 ? ± 2

reduces

2 pmol/1

the

presence

Effect

potential kidney

+1

more

cell

(=

by

membrane

verapamil

1 pmol/1

sustained

3:

altered

0.1

5).

in t h e p r e s e n c e

effect

of

(n = 9 ) a n d

is i n e f f e c t i v e .

hyperpolarization both

significantly

the

potassium

potassium

the cell

mmol/1

and

membrane

abolishes

1 mV

(n=3?)

to

cell

0.01 ±

(Fig.

membrane

quinidine

the

depolarizes

4).

by

1 mV

conductive

virtually

quinidine

membrane

to - 5 3 +

depolarizes

19) w h i c h

barium

cell

PD a m o u n t s

and

across

proximal

presence (Original

potassium

to 40 m m o l / 1 , the b a s o l a t e r a l straight

and absence tracing).

tubules (5 a n d

concentration resp., cell of

on

the

membrane the

mouse

20 m m o l / 1 )

of

145

Fig.

4:

Effect

difference Effect 5.4

of

(PD)

of

1 mmol/1

quinidine

across

the cell

extracellular

potassium

to 2 0 m m o l / 1 ,

in p r e s e n c e

0

membrane

the in

concentration

and absence

WM

I&ok"]

-to-

on

of

potential

MDCK

cells.

altered

from

quinidine.

M

M -20"

PD [mV] , 30 . -40Nö-y

-50

MaSKa

-60-

^

SÖ

-70-

Fig.

5:

Effect

1 min

Effect

difference

\V

(PD)

of

10 p m o l / 1

across

of e x t r a c e l l u l a r

or 4 0 m m o l / 1 ,

resp.

in

the

verapamil cell

potassium presence

on the

membrane altered

in

the

from 5.4

and absence

of

potential

MDCK to

cells. 10,

verapamil.

20

146 Conclusions The

cell

cells, which

membrane

potential

is d e p o l a r i z e d contributes

effect

of

tubules

to

verapamil

their

verapamil

than

by

in

MDCK

in M D C K

cells

- could

at

least

intracellular

calcium

transiently

MDCK

only

cells.

appears since

In

to

be

proximal

verapamil

extracellular

be

due

tubules,

obviously

potassium

not

an

to

a

it

the of

can

effect,

in

effect

is n o t of

effect

of

tubules of

clear,

why

verapamil

in

of

verapamil

conductance,

displaced

concentrations.

The

proximal

reduction

potassium

be

MDCK-

transport.

in p r o x i m a l

the e f f e c t

inhibition

and

depolarizing

However,

reverses

on

concentrations

The

activity.

a direct

action

probably

partially

tubules

and quinidine,

higher

cells. - but

A

proximal

inhibitory

requires

verapamil

23187

in b o t h ,

The

by

high

effect

of

0 -5 Verapam A 23187

-10 -20-

PD [mV] -30-

1

I

f

tg

-40-

, "1 'if

f *

-50-

6:

difference of 2 p m o l / 1

f\

£

fii

'

\ • '

V

,,

i

r

/

\ '

/

-j 'S

?

\

J

\

'

»

I :'»

V

/

-60

Fig.

(

1

Effect (PD)

of

across

A 23187

on

1 the PD

pmol/1 cell in

the

: 1 min

verapamil

on

the

potential

membrane

of

MDCK-cells.

presence

of

verapamil.

Effect

147 quinidine

in PT a n d M D C K

similarly However,

due some

to d i r e c t additional

depolarization, complete

since

inhibition

inefficacy

as

in o t h e r

tissues

inhibition factor

of p o t a s s i u m

must

contribute

the d e p o l a r i z a t i o n of

of A 2 3 1 8 7

potassium

that

sensitive

to i n t r a c e l l u l a r

potassium

conductance calcium

be

in

part

conductance.

to t h e

is m o r e

observed

profound

than

alone.

conductance

in p r o x i m a l

indicate

[3] m a y

convoluted

tubules

in p r o x i m a l

tubules

The could is

not

activity.

Reference s 1. A r r u d a ,

J.A.L.,

S.

Sabatini.

2. B u r g , M . , J. G r a n t h a m , P h y s i o l . 2JL0, 1 2 9 3 3.

Burgess, 317. 67

G.M.,

4. F l e c k e n s t e i n ,

M. A.

5.

Gstraunthaler, G., Physiol. 248. F536

6.

MacLaughlin, M., P h y s i o l . 8, 112

7. P a u l m i c h l , 407. 258 8.

Pressman,

9.

Rasmussen, Pharmacol.

10. T a u b , M., Nat. Acad. 11.

M., B.C.

H., D.M. 95., 111

Geibel, A.

1983.

1986.

Rev.

Jr.,

R. G r e g e r ,

Taylor.

J.

141

Am.

J.

Physiol.

1983.

1985.

G. M a l n i c .

Biochem.

Saier

55.,

1966.

1981.

P. K o t a n k o .

F. L a n g .

Rev.

Waisman.

L. C h u m a n , M . H . Sci. 76, 3338

E.E.,

J. O r l o f f .

Mello Aires,

Annu.

Biol.

52., 3

Pfaller,

M. De

Membr.

Jenkinson.

Res.

F. F r i e d r i c h ,

Vblkl, H., J. Arch. 407, 252.

12. W i n d h a g e r , 519

D.H.

Circ.

W.

1976.

J.

M. A b r a m o v ,

Claret, 1983.

1980.

F.

G.

J.

1985.

Renal

Pflugers

Arch.

45., 501 Physiol. Sato.

Lang.

Ann.

Am.

1979.

1966.

Rev.

Biochem. Proc.

Pflugers

Physiol.

45.,

EFFECT

OF BRADYKININ

AND

EPINEPHRINE

OF M A D I N - D A R B Y - C A N I N E - K I D N E Y

M.

Paulmichl,

Inst,

for

M. D e f r e g g e r ,

Physiology,

ON E L E C T R I C A L

PROPERTIES

CELLS.

F. F r i e d r i c h ,

University

of

Lang.

F.

A 6010

Innsbruck,

Austria

Introduction Bradykinin

and

processes Darby the

epinephrine

in a v a r i e t y

Canine Kidney

established

exhibit

many

study

has

across

known

cells

to

influence

tissues

cell

line form

confluent

of d i s t a l

nephron

performed

and

to

bradykinin

the p l a s m a

membrane

on

of

test

from

monolayers,

which

the

the p o t e n t i a l

subconfluent

Madin-

Cells

[4].

for

transport

including

[1,2,6,7,9,10,11].

properties been

epinephrine

(MDCK)

MDCK

are

of e p i t h e l i a l

The

present

influence

difference

MDCK

of (PD)

cells.

Methods Experiments

were

maintained

prior

Dulbecco's fetal

performed to

modified serum,

Streptomycin

[3,12]

Na2HP04, plated as

[0.03% 1.5

above

for

temperature composed CaCl2,

of

determined 200

Mn,

tip

and

least

again

bubbled

with

very

potential

48

a

and

(in m m o l / 1 )

137

with

hours.

of

F12 medium with

10%

114 N a C l ,

and 37°C. NaCl,

1 mol/1 again

NaOH

in

the the

extracellular 5.4

KC1,

0.8

16 N a H C 0 3 , and 5%

MDCK-cells, mixture

During

Control

0.2 NaH2P04, with

95% air

fine

tip m i c r o e l e c t r o d e s

< 5 mV]

1:1

Penicillin

incubated

37°C.

(in m m o l / 1 ]

0.8 Na2HP04,

continuously

and

in

95% air

titrated

glasses

subconfluent and Ham's

U/ml

at 5 % C 0 2 ,

EDTA

at

was

(DMEM]

100

KH2P04,

on c o v e r

on

experiment

medium

calf

dispersed

the

C02

5.5

100

Mg/ml

Cells 3

KC1 ,

to p H

medium

experiments fluid

was

MgC12,

1.2

glucose

and

PD

(resistance

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

3

7.4),

same

(pH 7 . 4 ] .

[8],

were

was 100

-

150 Hesults During

control

increase to

of

From

slope

depolarizes

this

value,

potassium

conductance) of

cell

application

of

1 pmol/1

increase

membrane indicate

o f tk

Fig.

Effect

of

MOCK

concentration presence increased

the

(Fig.

cell

membrane

Effect

altered from 5.4

and absence

of

(n =

37) and

Thus, with leads

epinephrine the

cell

phentolamine (Fig.

to a

and

potential potassium

on PD both,

depolarizing

2).

depola-

(n = 8 )

cell m e m b r a n e

concentration

%

The

resistance

extracellular The

SO

sustained

1 mV

by + 2.8 ± 0.8 mV

to 2 0 m m o l / 1

potassium

a

±

=

membrane

by a - r e c e p t o r s

on the

(tk

some

increasing

Studies

of e p i n e p h r i n e .

extracellular

to

membrane 1).

5.4 (n =

potassium.

to - 7 2

isoprenaline

of epinephrine cells.

by

1 mV

i.e.

to

Step

from

16 ± cell

leads

of cell

is mediated

of p - r e c e p t o r s by

of

due

membrane

68).

potassium

0.45,

membrane 0.9

+

slope to

is

conductance.

this effect

rization

1:

cell

by

number for

over

decrease

(n =

concentration

epinephrine

the cell

to a p p r o x .

the

potassium that

Stimulation

(PD)

of

1 mV

membrane

amounting

conductance

by a m a r k e d

hyperpolarizes

potassium

the cell

conductance

hyperpolarization an

is - 5 3 ±

a transference

is c a l c u l a t e d

membrane

paralleled

PD

extracellular

20 mmol/1

5).

conditions,

in

the

effect

of

is a m e a s u r e

of

151

increases

the

effect

concentration,

pointing

calcium

blocker

to

channel

- 41

effect

± 3 mV

(n~24),

reduces As

the the

verapamil,

the

transient.

Omission

mmol/1 13)

only

cell

the

bradykinin

(Fig.

hormone

resistance

effect

cell

In t h e of

in t h e c o n t i n u e d effect

on

the

pmol/1

to - 7 0 ±

1

mV

and

increases

tk

the

presence is

of

of only

and addition

epinephrine A second

absence

0.1

by + 2 5 ± 3 mV

absence

The

membrane

verapamil, 3).

bradykinin

calcium

nominal

either

in

of

membrane

is t r a n s i e n t .

significant

of (Fig.

of e p i n e p h r i n e

the

the cell

membrane

of e x t r a c e l l u l a r

effect 6)

cell

membrane

effect

sec.

calcium,

is w i t h o u t

the

chloride

conductance.

chloride

depolarizes

transient

hyperpolarizing

40

extracellular

In the p r e s e n c e

is

EDTA depolarizes

within

same

to e n h a n c e d

hyperpolarizes

4).

altered

verapamil

11).

of e p i n e p h r i n e

bradykinin (Fig.

(n =

of

of 1 (n

=

extracellular (Fig.

application

of e x t r a c e l l u l a r

5)

or

of

the

calcium

PD.

o-20-

PD [mV] o-60-80-

Fig.

2:

potential

Effect (PD)

of a - a n t a g o n i s t

of

of MDCK

epinephrine cells

phentolamine

both,

(EPI) in

(PHENT).

on

the

the

cell

presence

and

membrane absence

152

Fig.

3:

Effect

potential of

of e p i n e p h r i n e

(PD) of M D C K

(EPINEPH)

cells both,

on t h e c e l l

in the p r e s e n c e

membrane

and

absence

verapamil.

M BRADYKININW

Fig.

4:

(PD)

Effect

of

MDCK

concentration presence tk.

cells.

altered

and absence

increasing for

of b r a d y k i n i n

on the c e l l m e m b r a n e

Effect

from 5.4

of

of b r a d y k i n i n .

extracellular

extracellular

to 20 m m o l / 1

potassium

potential potassium

on PD b o t h ,

The d e p o l a r i z i n g concentration

is

in

effect

the of

indicative

153

Conclusions Both,

epinephrine

polarize

the

conductance is

of

Preliminary

both

activates

Fig.

5:

of

clamp

- in

chloride

Effect

of

potential

(PD)

of

MDCK

(1 m m o l / 1

EDTA

added)

effect

[5]

- via

channels,

epinephrine cells

from

in

Possibly

reveal

the

cell of

on

nominal

membrane, p-receptors similarly

cell

absence

by

stores.

concealed

the

the

calcium

bradykinin

effect

(EPI) the

hormones activity.

channels

intracellular

stimulation

an

of b o t h

intracellular at

hyper-

potassium

calcium

indeed

channels

calcium

the

potassium

increases

conductance.

chloride

The

studies

addition

bradykinin,

intracellular

of c a l c i u m

release

and

increasing

sensitive

Epinephrine

by o p e n i n g

by

membrane.

calcium

hormones

Epinephrine enhances

cell

increases

of

epinephrine. and

membrane

patch

activation activity

the

by

mediated

(via a-receptors)

cell

by

the

membrane of

calcium

154

simultaneous

activation

of

hyperpolarization

enhances

efflux

cell,

from

the

chloride-channels secretion

in

epinephrine

and

epinephrine,

bradykinin in

the

effect of

to t h e

the to

of b r a d y k i n i n

has

same of

after

elicits still

vanished

of

chloride Whether

established. verapamil the

and

epinephrine

a second effective

despite

and

intracellular

be

presence

Since chloride

potassium-

hormones.

remains

is

for

stimulation

utilize

bradykinin

of e p i n e p h r i n e

both,

two

hyperpolarization Thus

force

of

these

continuous

application

transient

hyperpolarization. presence

by

mechanisms,

Interestingly,

the

contribute

channels.

driving

activation

epithelia

transducing

induced

may

potassium

the

the

transient even

when

continuous

epinephrine.

IBRAI"

Fig.

6:

potential

Effect (PD)

extracellular

of of

calcium

bradykinin MDCK

cells

(1 m m o l / 1

(BRA) in EDTA

on

the

the

cell

nominal

added).

membrane

absence

of

155

References 1. B r o w n , C.D.A., 649, 427 2. C u t h b e r t , 1984. Br.

N.L.

Gstraunthaler, G., P h y s i o l . 248» F 5 3 6

4.

Handler, Physiol.

H.A.,

8. P a u l m i c h l , Arch. 405. Paulmichl, 406. 367

M., D.H.,

11. S i m m o n s , 43. 2 2 2 5

N.L.,

G. M.

P. K o t a n k o . J.P.

Lang.

R.J.

Defregger,

Y. M a r u y a m a .

L. C h u m a n , 76, 3338

Johnson.

1987

M.H.

(in

19B4.

1986.

Rugg.

Jr.,

G.

Spayne.

1985.

Am.

J.

1980.

Am.

J.

Pflügers Field.

F. L a n g .

E.L.

Saier

M.

Nature

J.A.

Acta

preparation)

1986.

Miller,

F. L a n g .

Brown,

Biophys.

Margolius,

Gstraunthaler,

C.D.A.

Biochim.

M. P a u l m i c h l .

J.F. Kachur, 21, 1073

10. P e t e r s e n ,

M., Sei.

Perkins,

M. D e f r e g g e r ,

M., 102

H.S.

Pfaller,

M. PauIrnich 1, F.

7. M ü s c h , M.W., Clin. Invest.

12. T a u b , Acad.

W.

J.S., F.M. 2 3 8 . Fl

6. L a n g , F., 4 0 7 . 158

9.

1981.

A.W., P.V. Halushka, J. P h a r m a c . 8 2 , 5 8 7

3.

5. K o l b ,

Simmons.

1985.

1983.

Arch.

693

1984. Sato.

J.

Pflügers

Pflügers

307.

Arch.

Fed.

Proc .

1979.

Proc.

INTRACELLULAR N a + AND K + ACTIVITIES IN SODIUM MALEATE TREATED FROG KIDNEY PROXIMAL TUBULAR CELLS

D. Cemerikic and D. Filipovic Dept. of Pathologic Physiology.Faculty of Medicine,Beograd,Yugoslavia

Introduction

Administration of maleate produces features that closely resemble the Fanconi syndrome. It has been shown that maleate induced proximal tubular defects are due to metabolic disturbances in tubular cells (1,2) or due to an impairment of the peritubular Na + -K + -ATPase (3),while on the other side it was suggested a modification of luminal cell membrane permeability along the whole nephron (4) or the leak of substrates by diffusion across injured proximal tubular cells (5). The purpose of the present work is to study the effects of maleate on cellular cation composition in proximal tubular cells. Present experiments showed ouabain like maleate effects on cellular cation activities in proximal tubule.

Materials and Methods

Experiments were performed at room temperature in isolated doubly-perfused kidneys of Rana esculenta prepared according to Wang et al (6). The dorsal surface of the kidney was exposed for micropuncture and continously superfused with oxygenated amphibian Ringer. Both perfusions,aortic kept at about 1.5 ml/min and portal vein kept at about 1 ml/min,arranged by gravity flow systems,were checked by infusion of 0.05 ml 5% FDC green. Amphibian Ringer of the following composition (in mmol/1) was used: NaCl 90,KC1 2.5,NaHC0 3 lO.Nah^PO,, 0.5,CaCl 2

1.8,MgCl 2

1,glucose 2.2,dextran (m.w. 80 000) 15g/l and heparin 2000 U/l. The solution was equilibrated with 99% 0 ? - l % C0_ and had pH 7.6. If applicable,sodium maleate -3 -2 was added at a concentration of 10

or 10

mol/1 to the Ringer which was then

buffered to pH of 7.6 by NaOH IN. Peritubular cell membrane potential (PD) was measured with conventional 3 mol KC1 microelectrodes (tip diameter less than 1 ^im,input resistance as tested in Ringer 40-60 Mft,tip potential less than 5 mV) made from thick wall borosilicate

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin - New York - Printed in Germany

158 capillary tubing with internal glass fibre (Federick Haer & Single barrelled N a

+

Co,Brunswick,Ma,USA).

selective microelectrodes with a tip diameter less than 1

pra were made from the same glass,prepared using highly selective liquid ion exchanger

(7). Siliconization of the microelectrodes was the same as described by

Fujimoto and Kubota (8) and Kubota et al (9). The characteristics of N a +

selec-

tive microelectrodes were the following: slope -5*4 + 0.86 (n=l8) .selectivity to N a + over K + 15 + 1.7 (n=l8),input resistance as tested in Ringer 2-5 x lO^^Jl , response time of 1 second order,as calibrated in pure single salt calibration solutions of NaCl and KC1. Intracellular N a + activity,a* +,was calculated direcNa tly from the calibration curves of each microelectrode in pure NaCl solutions after correction for the peritubular membrane PD and for interference due to cellular potassium. Double Barrelled K + selective microelectrodes,with a tip d i ameter less than 1 jjm,were made from same glass,and their manufacture and siliconisation were similar to those described by Fujimoto and Kubot^ (8) and Kubota et al (9). Input resistance of the K + selective barrel as tested in Ringer 9 was 6 x 10 H , r e s p o n s e time within 1 second,the slope was -58 + 0.5 lectivity to K

+

over N a

+

(n=12),se-

was 48 + 4.6 (n=12),as calibrated in pure single salt

calibration solutions of KC1 and NaCl. Intracellular potassium

activity,a*+,was

calculated from the calibration curves of each microelectrode in pure KC1 solutions . By Ag-AgCl wires the microelectrodes were connected to a probe of a very high input impedance dual electrometer (Analog Devices AD 515

L,Norwood,Mass,USA)and

the voltage signals were recorded by a 2 channel recorder (model

GmbH,Linseis,

Selb,FRG). The ground electrode was Ag-AgCl wire connected with the kidney

sur-

face by a 3 mol KC1 3% agar bridge. All results are expressed as mean + SEM. ComDarison between groups was made u sing unpaired "Student's t" analysis.

Results

As shown in Fig 1 is the control measurement of a?", + of 12 mmol/1 at peritubular Na PD of - 7 0 mV,including calibration procedure of Na Fig 2 represents the control measurement of

selective

microelectrode.

of 67.5 mmol/1 at peritubular

membrane PD of - 6 8 mV,as recorded with double barrelled K

selective

microelec-

trode. Table 1 summarizes the measurements pf peritubular membrane PD.a.1, + and Na i -3 -2 in control conditions and during 1-3 hours of perfusions of 10 and 10 mol/1 maleate Ringer. Under control conditions,at peritubular membrane PD of -71 m V , a J a + was 10.9 mmol/1 and a * + was 65 mmol/1. B o t h , 3 ^ + and ajj,+ ,were above

159

Fig.1. Control measurement of intracellular sodium

activity.

Fig.2. Control measurement of intracellular potassium

activity.

160

the electrochemical equilibrium. For sodium ion it was 49.3 mV and for potassium ion it was - 9 0 mV. Perfusion of 10

3

mol/1 maleate Ringer for 1-3 hours depola-

Table 1. Peritubular Membrane Potential,Cellular N a + and K + Activities in Sodium Maleate Treated Frog Kidney Proximal Tubular

PD mV

Cells

a* + mmol/1 Na

K

mmol/1

Control

& 71,.0 + 2..5 (8)

10..9 + 0..5 (5)

65 + 3.6 (3)

10~ 3 mol Na-maleate

48..1 + 3..9 (10)

14..2 + 1..1 (5)

55 + 1.8 (5)

10

-2

P < mol Na-maleate

0,.001

37,.8 + 4..9 (9) P
2 mm Hg H C O 3 - mM

Plasma lactate ymoles/ml Plasma pyruvate ymoles/ml Plasma alanine ymoles/ml GFR ml/min Renal blood flow ml/min Lactate excretion ymoles/min

Control 7..36 + 0..02 40..5 23..0

1 . .0 ± 0..7 +

Lactic acidosis 7..07 + 0..03 * 42.4 ± 1 . ,5 12.,5 + 0..7 *

1 . .12 +

0..13

9.,40

+

0.,62 *

0..062 +

0..006

0..205 +

0.,033*

0..401 +

0..039

0..476 +

0..036*

37..1 208..5 0..31

1..6 ± 12.1 +

35,.1

± 1 , .4

188..3

+

6,.7

+

0..01

25..5

+

7,.9 *

Lactate extraction ymoles/min

42..9

+

7 .3

185 .0

+

34 .2 *

Alanine production ymoles/min

6 .0

+

0 .5

16 .3

+

1 .7 *

* = significantly different from control (P < 0.05).

187 Data obtained at the end of lactic acid infusion are presented in Table 1.

It can be seen that the animals were severely acidotic

with plasma bicarbonate at 12.5 mM. plasma lactate at 9.4 ymoles/ml. 3 fold.

There was marked elevation of

Alanine production rose almost

Renal hemodynamics were not modified by the infusion of

lactic acid.

If the production of alanine was the same in both

kidneys as was the renal extraction of lactate, the production of alanine was responsible for the transformation of 9% of extracted lactate.

In these experiments, the sum of alanine produced and

lactate excretion in the urine would be responsible for riddance of 23% of extracted lactate per minute. DOG : Na LACTATE INFUSION

Figure 1.

Renal production of alanine by the left kidney during infusion of sodium lactate.

188 Figure 1 shows renal alanine production during the infusion of sodium lactate.

It can be seen that at plasma concentration of

lactate approaching 20 ymoles/ml, the renal production of alanine is close to 25 vimoles/min.

This establishes that the renal pro-

duction of alanine is conditioned by the plasma lactate concentration according to the reaction pyruvate + glutamate 1 [ LDH lactate

alanine + a-ketoglutarate GPT

under the influence of lactate dehydrogenase aminotransferase

(LDH) and alanine

(GPT).

Discussion The generation of alanine by the kidney is a well known phenomenon which was first studied in detail by Pitts and Stone in 1967 (2) . This operation was thought to play no special role in acid-base status or aminoacid metabolism.

The generation of alanine by the

kidney cannot play an important role in tissue metabolism since alanine is readily available from muscle which is the major source of this amino acid (8) In most clinical situations where lactic acidosis develops, lactate will originate from muscle through anoxia or some other metabolic dysfunction

(9).

The liver can metabolize lactate to CC>2 (10).

In the present study, we demonstrate that during lactic acidosis the kidney is able to transform lactate into alanine and that this operation can be significant in allowing the organism to get rid of overproduced lactate.

The latter is a good source of pyruvate.

Glutamate which may originate from glutamine will permit the generation of alanine under the activity of alanine aminotransferase (GPT) (9).

The reaction

pyruvate + glutamate «

• alanine + a-ketoglutarate

is freely reversible and the equilibrium ratio is close to unity. However, the concentration of the reactants will influence the reaction to go forward or backward.

In the present case, lactate

will generate sufficient pyruvate to forward the reaction towards alanine production.

It is significant that in our study, plasma

189

pyruvate concentration rose three fold during lactic acid infusion. The production of alanine by the kidney could prevent undesirable accumulation of lactate in various tissues during lactic acidosis. However, this mechanism cannot correct the state of acidosis and accumulated protons will have to be neutralized by body buffers. It is of great interest that a useful role can be attributed to the synthesis of alanine by the kidney, a phenomenon previously thought to be irrelevant.

Summary In the present work, we demonstrate that the renal generation of alanine during lactic acidosis can play an important role in preventing accumulation of lactate in various tissues.

Acknowledgements The present work was supported by the Medical Research Council of Canada grant MT 7874. The authors wish to thank Sylvie Desjardins, Suzanne Carioto and Roger Langlais for their precious assistance.

References 1.

Fass, S.J., M.R. Hammerman and B. Sacktor. 1977. Transport of amino acids in renal brush border membrane vesicles. Uptake of the neutral amino acid L-alanine. J. Biol. Chem. 252, 583-590.

2.

Pitts, R.F. and W.J. Stone. 1967. J. Clin. Invest. 46_, 530-538.

3.

Lemieux, G., A.-L. Kiss, C. Lemieux, R. Joffre Ibanez and M. Rengel Aranda. 1985. Renal tubular biochemistry during acute and chronic metabolic alkalosis in the dog. Kidney Int. 27, 908-918.

Renal metabolism of alanine.

190

4.

Vinay, P., E. Allignet, C. Pichette, M. Watford, G. Lemieux and A. Gougoux. 1980. Changes in renal metabolite profile and ammoniagenesis during acute and chronic metabolic acidosis in dog and rat. Kidney Int. \1_, 312-325.

5.

Pitts, R.F. 1966. The renal metabolism of ammonia. Physiologist 97-109.

6.

Lotspeich, W.D. and R.F. Pitts. 1947. The role of amino acids in the renal tubular secretion of ammonia. J. Biol. Chem.

The

168 , 611-622.

7.

Pitts, R.F. 1971. Metabolism of amino acids by the perfused rat kidney. Am. J. Physiol. 220, 862-867.

8.

Chang, T.W. and A.L. Goldberg. 1978. The origin of alanine produced in skeletal muscle. J. Biol. Chem. 253, 3677-3684.

9.

Lemieux, G., E. Junco, R. Perez, E. Allignet, C. Lemieux, M. Rengel Aranda and F. Valderrabano Quintana. 1986. Renal metabolism during four types of lactic acidosis in the dog including anoxia. Can. J. Physiol. Pharmacol. £4, 169-175.

10. Lemieux, G., P. Vinay, G. Baverel, R. Briere and A. Gougoux. 1979. Relationship between lactate and glutamine metabolism in vitro by the kidney. Differences between dog and rat and importance of alanine synthesis in the dog. Kidney Int. 16, 451-458.

EFFECT OF GLUCOCORTICOIDS AND METABOLIC ACIDOSIS ON THE LEVEL OF RAT RENAL PHOSPHOENOLPYRUVATE CARBOXYKINASE mRNA

M. Gallo, R.A. Shapiro and N.P. Curthoys Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261

In

response to the

onset of

exhibits a rapid and pronounced metabolize

plasma

ammoniagenesis excretion

of acids

an

and

this

(1).

The

expendable

conserves

resulting a-ketoglutarate

the

increase in its ability to

glutamine

provides

metabolic acidosis, associated

cation

sodium

that

and

rat kidney extract and

increase

in

facilitates

the

potassium

ions•

is largely converted to glucose.

adaptive process, the rat renal

The

As part of

phosphoenolpyruvate carboxykinase

(PEPCK) activity is increased within 4 to 6 h and reaches a plateau that is

3-fold greater than

activity is

due to

normal within

a rapid increase

2 d

(2).

in the

The increased

level of

the

PEPCK

PEPCK mRNA

(3,4). Renal synthetic

PEPCK

activity

glucocorticoids

is

also

(5).

Increased

Renal

nuclei

pretreated with

dexamethasone

increase in the

rate of [ 32 P]UMP incorporation into

mRNA

(6).

Increases

The in

translation. gene

increased

the cytosolic

or

by

with

rate

of

level

isolated

cAMP exhibit

transcription of

administration

PEPCK

mRNA

a

accounts

for

and

rate

its

the of

Thus, the two effectors induce the expression of the PEPCK

through transcriptional regulation.

than with either treatment alone.

When the

two effectors were

the inductive

effect

of

increased more

Thus, glucocorticoid induction of the

renal PEPCK may be mediated through cAMP. characterized.

rats

7-fold

hybridizable PEPCK

given together, the rate of PEPCK mRNA synthesis was not

which

from

4- to

of

acidosis

In contrast, the mechanism by is

mediated

has

not

been

In this study, a hybridization assay was used to compare

the effects of glucocorticoids and of metabolic acidosis on the relative level of rat renal PEPCK mRNA.

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

192

Materials and Methods

from New

Gene screen and [a-32p]dCTP (>600 Ci/mmol) were purchased England Nuclear. of cDNA from

The pPCK2 plasmid (4), which contains 600

that correspond to the 3'

Dr.

Richard

Hanson.

solubilization in

end of rat PEPCK mRNA,

Rat

renal

diluted with 25 mM sodium phosphate, using

a

Bio-Rad dot-blot

irradiation (8). specific

RNA

quanidinium thiocyanate

was

(7).

base pairs

was obtained

isolated The RNA

pH 6.5 and spotted on

apparatus.

The

samples

following

samples were Gene Screen

were fixed

by UV

The pPCK2 plasmid was labeled by nick-translation to a

activity

of

10® dpm/lig

(9).

The

hybridization

procedures were carried out as described by Amasino (10). then exposed to Kodak X-omat film for 1 to 6 h at -70°C. autoradiogram was scanned with a Helena R & D

and

wash

The blot was The resulting

densiotometer.

Results Total RNA was isolated from a control rat and analyzed analysis (Fig. 1A).

The amount

linearly with respect to

of [

by dot-blot

P]-labeled pPCK2 bound increased

the amount of RNA spotted.

The slope

of the

plot is a measure of the relative level of the PEPCK mRNA. Acute

acidosis was induced

intyCl/kg body wt.

Within 1

by stomach

loading rats with

h, the arterial blood pH

7.26.

By 2 h, the pH was decreased to less than 7.1 where

for at

least 4

amount of

h.

In contrast,

NH4HCO3 or NaCl

had no

stomach loading effect on

blood

20 mmol

was decreased to it remained

with an

equivalent

acid-base balance.

Total RNA samples were isolated from rats at various times after stomach loading.

The increase

onset of acidosis

in relative levels of PEPCK

is shown in Fig. IB.

The initial

mRNA following the increase in PEPCK

mRNA occurred 2 h after stomach loading;

i.e., at a time when

had

Over the next 4 h the relative

already reached its minimal level.

level of PEPCK mRNA increased 4-fold.

blood pH

The increased level is sustained

193

in rats that were made chronically acidotic by feeding NH4CI for 3 d.

The magnitude of the changes observed in

to account

for the increase

(3,5) and the slower

in the

normal rats Thus, the

relative rate

of

PEPCK synthesis

increase in specific activity (2).

difference in the amount of and rats that

There was

pPCK2 that hybridized to RNA were stomach

loaded with

the load of Nlfy"" or

no

isolated from

NH4HCO3 1

onset of acidosis, and not

to 6

this study are sufficient

or NaCl.

Cl~ ions, is

responsible for the increase in PEPCK mRNA.

HQ RNA

HOURS

Fig. 1. Dot-blot analysis of the relative level of PEPCK mRNA. Panel A: Samples containing 0.5 to 2.0 yg of total RNA isolated from control rats were spotted on Gene Screen, hybridized with [ ^ P ] - l a b e l e d p pcK2 and exposed to film. The relative area of the resulting autoradiograph was determined by densitometric analysis. Panel B: Increase in renal PEPCK mRNA following onset of acidosis. Rats were stomach loaded with 20 mmol NHCl/kg body wt. and sacrificed at the indicated times. Chronic acidosis (72 h) was produced by providing rats with 0.28 M NH4CI as their sole source of drinking water. The amount of PEPCK mRNA is expressed relative to the level contained in RNA isolated from control rats. Each point represents the mean ± S.E. of triplicate determinations of two separate preparations of RNA.

The 3-fold

plasma

concentration

following

adrenalectomized

the rats

were

of corticosterone

onset used

of to

acidosis insure

is

increased

(11). that

the

2- to

Therefore, effect

of

194

acidosis on the level of hormones (Table I).

PEPCK mRNA was not mediated

adrenalectoraized

that observed in normal animals.

rats is very similar to

the increases in PEPCK mRNA that NH4CI were very similar. only

by glucocorticoid

The basal level of PEPCK mRNA in

Furthermore,

occur in response to various

doses of

In both sets of animals, the administration of

2 mmol NH^Cl/kg body wt.

was sufficient

to produce a

slight but

significant increase in the relative level of the PEPCK mRNA. caused

the blood

pH and

values of 7.43 ± 0.03 23.9 ±

HCO3" concentration to decrease

and 29.4 ± 0.6 mM

4.7 mM, respectively.

Thus, the

to values

This dose

from control

of 7.36 ±

increase caused

0.01 and

by metabolic

acidosis is independent of glucocorticoid secretion. Table I Comparison of the Effect of NH^Cl-induced Acidosis on the level of PEPCK mRNA in Normal and Adrenalectomized Rats NH4CI Load

Normal

mmol/kg body wt

Adrenalectomized

Relative Level PEPCK mRNA

2

1.55 ± .06

1.71 ± .30

5

2.56 ± .09

1.83 ± .17

15

2.86 ± .17

3.06 ± .47

Rats were stomach loaded with NH^Cl 4 h before they were sacrificed. The amount of PEPCK mRNA is expressed relative to the level contained in RNA isolated from control rats. Each point represents the mean ± S.E. of triplicate determinations of two separate preparations of mRNA. Rats were treated

with dexamethasone and made acidotic

with NH4CI in order to determine if their effects on the of PEPCK mRNA were additive.

The treatment

by loading

relative level

of rats with maximal doses

of both dexamethasone and NH^Cl produced an induction that was

the same

as that caused when

However,

either treatment was given individually.

when the two stimulators were given at doses that

individually produced

195

less

than

half-maximal

Therefore,

acidosis

responses,

and

their

glucocorticoids

effects may

were

act

synergistic.

independently

to

initiate the same intracellular response which promotes transcription of the PEPCK gene.

Table II Effect of Dexamethasone and of Metabolic Acidosis on the Level of Rat Renal PEPCK mRNA Treatment

A.

B.

Relative Level PEPCK mRNA

- control

1.0 ± .16

- 15 mmol NH 4 C1

3.25 ± .25

- 2 mg Dexamethasone

3.06 ± .12

- combined

3.28 ± .21

- 5 mmol NH4CI

1.55 ± .05

- 0.01 mg Dexamethasone

1.68 ± .11

- combined

3.42 ± .49

Rats were injected intraperitonially or stomach loaded with the indicated amounts of dexamethasone or NH^Cl per kg body wt., respectively, and sacrificed approximately 5 h later. Each point represents the mean ± S.E. of triplicate determinations of two separate preparations of RNA.

Discussion

By comparing the time

course for

the onset of

systemic metabolic

acidosis and the induction of PEPCK mRNA it may be possible to determine if alterations In pH or HCO3 - concentration participate directly mechanism of induction. in

the

blood

parameters

in the

If acidosis is a direct mediator, then changes should occur

coordinately

with

However, rats that were loaded with 20 mmol NltyCl/kg body wt.

induction. exhibit a

196

slight decrease in blood pH after 1 h

and a maximal effect within

2 h.

In contrast, the induction of PEPCK mRNA is initiated between 1 and

2 h

after

two

stomach

loading.

Thus,

the

apparent

lag

between

the

processes suggests that acidosis may stimulate the release of a specific hormone or the

synthesis of

an intracellular

mediator

(3)

that

that regulates

induction of PEPCK. Iynedjian

and

Hanson

translatable PEPCK mRNA in

have

shown

response to

the

increase

acute acidosis is

in

unaltered by

adrenalectomy, hypophysectomy or thyroid-parathyroidectomy.

The results

obtained

release

in this

study

confirm the

conclusion that

the

glucocorticoids is not the direct mediator of PEPCK induction acidosis.

However,

the

experiments

carried

out

to

of

caused by

determine

the

additive effect of the two stimulators suggest that they may act through a

common mechanism.

contains

The 5' regulatory region of the hepatic PEPCK gene

a unique sequence

transcription release of

(12).

an

that is

Thus,

alternative

synthesis of cAMP

essential for cAMP

acidosis compound

may cause that also

which in turn mediates the

stimulation of

the synthesis stimulates

and/or

the

renal

tissue-specific induction

of PEPCK. The amount is also

of the mitochondrial glutaminase present

increased

in response

to acidosis

(13).

within kidney

Induction

of

the

glutaminase results from an Increase in its rate of synthesis

(14) that

correlates with an increased level of translatable glutaminase

mRNA (J.

Tong,

R.A.

Shapiro, and

N.P. Curthoys,

submitted

for

publication).

Similar to PEPCK (15), the increase in the glutaminase also within the

proximal

convoluted

segment

of the

renal

However, the 20-fold increase in glutaminase synthesis over a period of 5 d.

occurs only

nephron

(16).

occurs gradually

The difference in apparent rates of induction is

probably due to a difference In the relative stability of the two mRNAs. The glutaminase mRNA has an

apparent half-life of 3.3 d.

from the time course for induction degradation

of the

(Fig. 1), one can estimate

PEPCK mRNA occurs

Under these conditions, the same

In contrast,

with a

Increase in

half-life of the rate of

that the only

2 h.

synthesis of

the two mRNAs would result in a much more rapid approach of the level of the PEPCK mRNA rat

contains a

to a new steady state. factor that causes

Serum derived

an increase

in the

from an acidotic amount

of the

glutaminase contained in primary cultures of rat renal epithelial

cells

(P.D.

Pinnadwage,

submitted for publication). factor

Isolated from acidotic

T.

Frielle

proximal

and

N.P.

tubular

Curthoys,

It will be interesting to determine if the serum also

causes an

induction

of the

renal PEPCK.

Acknowledgement

This research was supported in part by NIH grant A M 16651.

References

1.

Tannen, R.L., S. Sastrasinh. 1984. Kidney Int. 2^, 1.

2. 3. 4.

Alleyne, G.A.O., G.H. Scullard. 1969. J. Clin. Invest. 48, 364. Iynedjian, P.B., R.W. Hanson, 1977. J. Biol. Chem. 252, 8398. Cimbala, M . A . , W . H . Lamars, K . Nelson, J.E. Monahan, H. Yoo-Warren, R.W. Hanson. 1982. J. Biol. Chem. 257, 7629. Iynedjian, P.B., F.J. Ballard, R.W. Hanson. 1975. J. Biol. Chem. 250, 5596.

5. 6.

Meisner, H., 421.

D.S. Loose,

R.W. Hanson.

1985.

7.

Chirgwin, J.M., A.E. Przybyla, R.J. MacDonald, W . J . Rutter. 1979. Biochemistry 18, 5294.

8.

Church, G.M., W . Gilbert. 1984. 1991.

9.

Rigby, P.W.J., M . Duckmann, C. Rhodes, Biol. 113, 237.

Proc. Natl.

Biochemistry 24,

Acad. Sei. U S A 81^,

P . Berg. 1977. J. Mol.

10.

Amasino, R.M. 1986. Anal. Biochem. 152, 304.

11.

Welbourne, T.C. 1976. Endocrin.

12.

Short, J.M., A . Wynshaw-Boris, H.P. Short, R.W. Biol. Chem. 261, 9721.

13.

Curthoys, N.P., T. Kuhlenschmidt, S.S. Godfrey, R.F. Weiss. 1976. A r c h . Biochem. Biophys. 172, 162.

14.

Tong, J., 139.

15.

Burch, H.B., C. Narins, C. Chu, S. Fagioli, S. Choi, W. McCarthy, O.H. Lowry. 1978. Am. J. Physiol. 235, F246.

16.

Curthoys, N.P., O.H. Lowry. 1973. J. Biol. Chem. 248, 162.

G. Harrison,

1071.

N.P. Curthoys.

1986.

Hanson. 1986. J.

Biochem. J.

233,

STUDIES ON THE ROLE OP Y-GLUTAMYLTRANSFERASE IN GLUTAMINE METABOLISM IN RAT PROXIMAL TUBULES P.D. Dass, E. Bourke Nephrology Division, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30329

Introduction The possible role of the membrane-bound enzyme Y-glutamyltransferase (Y-GT, E.C.2.3.2.2.) in renal glutamine metabolism and related ammoniagenesis has not as yet been resolved (1 ). In particular dispute is the contribution of Y-GT to the augmented glutamine ammoniagenesis seen in acidosis (1-3). In an effort to resolve these issues we studied the role of Y-GT in L-glutamine metabolism by separated renal proximal tubules from non acidotic and chronic NH^Cl - acidotic rats. Materials and Methods Rat renal proximal tubules prepared as described by Vinay et. al. (4) were incubated for 30 min in KRB, pH 7.4, as previously described ( .5). Substrates when added were always at 2mM final concentration. When used the Y-GT inhibitor AT-125 (L-[ S,5S] - a- amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid) (6), or the enzyme modulators hippurate and maleate (7) were added to the incubation 5 min. prior to the addition of substrate. When glutathione (GSH) was used as substrate, dithiothreitol (2mM) was added (5). y-glutamyl glutamine was monitored isotopically (8). Other assays were as previously described (5). Results and Discussion The effects of AT-125 (0.5mM) on glutamine metabolism were studied in tubules from non acidotic and acidotic rats. AT-125 inhibits

Y-GT but not PDG

(3). In t h e

presence

of AT-125, GSH utilization was 99.5% inhibited in both non acidosis and acidosis (5). Since Y-GT is the only enzyme known to initiate GSH degradation, effective inhibitory

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

200 concentrations of AT-125 were clearly achieved. Under these conditions there was a significant inhibition of glutamine utilization, glutamate formation and ammonia p r o duction in both non acidosis and acidosis (fig.l). The p o s s i b i l i t y of impaired membrane transport has not been seen at the concentrations used (9). The absence of change in gluconeogenesis suggests that glutamine availability to the intramitochondrial pathway was not impaired. The results indicate that r-GT plays a significant role in rat proximal tubular glutamine metabolism. In absolute terms however the decrease in glutamine utilization by AT-125 was similar in acidosis vs non acidosis. (-114.8 + 34.5 vs -128.6 + 8.0 nmoles/mg protein/ 30 min, N.S.) as was ammonia formation 103.3 + 27.8 vs 119.5 + 11.2 nmoles/mg protein/30 min, N.S.). It would thus appear that Y-GT does not make a major contribution to the augmentation in glutamine ammoniagenesis seen in the proximal tubules of chronically acidotic rats in vitro. NONACIDOSIS

Fig. 1

GLUTAMINE N MOLES

/ MG

PROTEIN

MIN.

m rï-i ' •ii

à •Jfi GLUTAMINE

/ 30

GLUTAMATE

• P < 0.05

AMMONIA

P < 0.005

WITH

REF.TO

ACIDOSIS GLUTAMINE

GLUCOSE

BAR

ON

LEFT

Hippurate is a physiological modulator of Y-GT (2,7) . Urinary hippurate excretion increases in acidosis (2). There was an increased glutamine utilization and product formation under both acid base states when hippurate (4mM) was added to tubules in vitro (fig. 2) . In absolute terms however the increase in glutamine utilization was similar in acidosis vs non acidosis (-131.9 + 53.0 vs -188.1 + 10.8 N.S.) and ammoniagenesis was less (249.9 + 30.4 vs 407.9 ± 2 . 2 p 0.6mM Na 2 HP0 4 > 3.ImM NaH 2 P0 4 , O.OlmMNa acetate, pH 7.4) maintained at 37°. The intestinal sections were incubated,with shaking,for 15min after which they were removed from the medium. A 200^1 sample of medium was taken from each vessel and pipetted into separate spectrophotometer cuvettes. Nessler's reagent (2ml) was added and the colour allowed to develop for 10 min. The absorbance at 420nm was used to determine the concentration of ammonia, using a previously determined standard curve. This simplified routine was adopted after showing that it gave the same results as those obtained when ammonia was first displaced from the medium with saturated potassium carbonate. The kidney was excised,decapsulated and sliced (0.5mm) using a Stadie-Riggs microtome. Two slices from each side of the kidney were weighed, then incubated with 10ml intestinal buffer and the ammonia released into the medium in a 15min period was determined as prevlouly described.

Table 1

Intestinal and renal glutaminase activity (GLU) )imole/mg protein/h. tyrosine amino transferase activity (TAT) jimole/g protein/min and glutamate dehydrogenase activity (GDH) /imole/g protein/min measured in kidney and intestinal homogenates of adult rats. The results represent the mean ± S.E. (n) for five sections of the small intestine numbered 1 to 5 from stomach to caecum,the colon and kidney.

GLU. Section 1

TAT.

GDH.

B.4 • 0.5 (6)

.49 • .03 (8)

175 «

32 (6)

2

7.4 + 0.5

.38 • .03

165 •

38

3

6.4 • 0.7

.46 • .05

216

33

4

6.3 + 0 . 4

.75 + .09

258 •

34

5

4.7 + 0 . 6

.60 + .10

217 •

29

Colon

1.2+0.1

.77 • .11

233 +

79

Kidney

6.0 • 0.6

1.59 • .24

336 +

76

Bilateral adrenalectomy was performed on adult rats under ether anaesthetic. Following surgery animals were maintained on standard Labsure PMD diet with 0.9% saline as drinking water. Aldosterone was injected intraperitoneally 4h before killing.

239

Intestinal tissue removed after incubation was placed on an enamel tray and the mucosal cells removed using the edge of a microscope slide. The mucosal tissue was suspended in 5ml distilled water and homogenised for 15s using a polytron homogeniser. Kidney slices were placed into 10ml distilled water,cut into small pieces and then homogenised for 30s. In each case homogenates from the four animals were pooled and kept on ice prior to enzyme assay. Crude homogenate (200JJ1) was pipetted into the outer chamber of a modified Warburg flask together with 500pl 0.1SM phosphate buffer containing lOmM glutamine (pH 7.5). In the central well of the flask was pipetted 500>J1 HC1 (0.01N) to trap the liberated ammonia. The flask w a s placed in a waterbath, at

Fig. 1 Shows the more uniform distribution of glutaaate dehydrogenase activity along the length of the small intestine and eolon in adrenalectomlsed animals. Following aldosterone treatment (25yg/100g).injected intraperltoneally.the enzyme is predominantly stimulated In the more distal sections of the small intestine and in the colon.

37°, and shaken for 30min. On removal»saturated potassium carbonate (1ml) was added to the outer chamber and the flask rapidly sealed and left for lh at room temperature. The flask was then opened and a IOOJJI sample taken from the central well and added to 2ml Nessler's reagent In a spectrophotometer cell. After lOmln the absorbance at 420nm was determined against a blank containing IOOJJI HC1 (0.01N) in 2ml Nessler's reagent. The protein concentration of the homogenate w a s determined and glutaminase activity calculated and expressed in terms of jjmol ammonia released/gm proteln/h. Tyrosine aminotransferase activity was measured using the method of Granner and Tomkins (5) and glutamate dehydrogenase activity using the method conforming to the recommendations of the Deutsche Gesellschaft fur klinische Chemie(6).

240 Results

Intestinal glutamlnase, glutamate dehydrogenase and tyrosine aminotransferase avtlvltles measured In Intestinal mucosa homogenates, prepared using adult male rats, are comparable with those measured in kidney homogenates (table 1). However, the distribution of intestinal enzyme activity varies along the length of the intestine. Glutamlnase activity is highest in proximal sections of the small intestine, progressively decreasing in more distal sections and is low in the colon. In contrast glutamate dehydrogenase and tyrosine amino transferase activities are highest in more distal sections.

Fig. 2

Shows the m o r e u n i f o r m distribution of glutaminas« and tyrosine aminotransferase activities a l o n g the length of the small intestine and c o l o n In adrenalectomlsed animals. Following aldosterone treatment (25yg/100g),Injected lntraperltoneally,the glutamlnase activity is Increased throughout the length of the small Intestine but the tyrosine aminotransferase la predominantly stimulated in the more distal sections of the small Intestine and i n the colon.

Adrenalectomy causes a decreased activity of all three enzymes (figs 1 and 2) such that their activity becomes uniform throughout the length of the intestine. Subsequent aldosterone treatment (25pg/100g) Increases glutamlnase

241

activity throughout the intestine but glutamate dehydrogenase activity and tyrosine aminotransferase activity only in more distal sections. Kidney values are also lower in adrenalectomised animals and increase following aldosterone treatment. Ammonia release from isolated sections of rat Intestine (7) is also found to be uniform throughout the length of the Intestine in adrenalectomised rats (fig 3). Subsequent aldosterone treatment increases the ammonia release in the more distal sections.

This stimulated increase is entirely sodium dependent as has

previously been shown (8) to be the case for the kidney (fig 4).

Fie 3 &

Ammonia release lumole/gm tissue/15 mini from isolated sections ol the small intestine and colon taken from newly weaned animals

II

III

IV

V

Colon

S E C T I O N

Discussion

The characteristic aldosterone stimulated intestinal ammonia release is sodium dependent and occurs principally in distal sections of the Intestine. The pattern of response is closely paralleled by stimulated tyrosine amino transferase and glutamate dehydrogenase activities. It is therefore believed that these enzyme responses are specifically linked to mineralocorticoid activity. The role of glutamate dehydrogenase in renal ammonia production has been well characterised. It maintains an equilibrium by which ammonia production is determined directly by sustrate concentrations. Its importance lies inppooiding

242 an easy pathway for the disposal of glutamine or glutamate into energy for ion pumps or the synthesis of glucose. In contrast stimulated glutaminase activity occuring in proximal sections of the intestine does not lead to an Increased ammonia release, a fact that is taken to confirm our view (8) that neutral ammonia release may be limited by membrane solubility and that an Increased ammonia release in such situations requires a stimulataion of the Na/NH^+ exchange system. These proximal sections have the highest concentration of glucocorticoid receptor (9) so the normal distribution of glutaminase activity is believed to be a specific response to glucocorticoid. At the concentration of aldosterone used in the present work these glucocortcoid receptors would be saturated and this may be the reason why aldosterone may be more potent than the glucocorticoid (3). It is well known that several days may be required for an animal to fully respond to an acidotic condition. This delay may be understood in terms of the refractory state of aldosterone responsive tissues in animals in which aldosterone secretions are low. If animals are maintained on a high salt diet (10) at least 27h is required before the intestine responds to aldosterone, a time lag that is believed to be required for replacement of unresponsive villus cells by activated crypt cells. Some comparable adaptive response may be supposed to exist in the kidney.

Fig. 4

X JU

02 051015 2025 10 20 Aldosterone mjecied (/«fl/100 9 body wt ) Ttw r»to of »m moni» relet*« (me*n ± t.e. of me*n) from thirty-two or forty-tight kidney «lie** Mken from group* of four or aix normal or ad renn loot ornimi (Ari) rut* injected with 10010%fctOHin 0 0% saline in tlio abocne« or prewneo of »Wort crono (0 2-20 ¿rg/IOOg body wt.) .Th® kidney •lice« wen incubtted in Kreb» Minger (tml.) for 10 min »t 37 *C. The mult» have bwn Mprand in torma of tmmoni» relegarti per 300 incubation medium.

243

It is concluded that while, in the short term, the major hormone controlling renal and intestinal ammoniagenesis may be the glucocorticoid. In more severe acidosis it is likely that aldosterone plays an important role.

We wish to thank the A.R.C. for support.

References

1.

Whelbourne, T.C.

1976.

J. Endocrin. 99, 1071.

2.

Yoshimura, H.,M. Fujimoto, J. Sugimoto.

1962.

Japan J. Physiol.

_12, 143. 3.

Whelbourne, T.C..D. Francoeur. Endocrin.Meta.Gastro.

4.

Windmueller, H.G.

Amer. J. Physiol.:

In Glutamine Metabolism in Mammalian Tissues,

1984.

Ed Haussinger, E.,H. Sies. 5.

1977.

2(1) , E56. Springer-Verlag (Berlin), 61.

Granner, D.K.,G.M. Tomkins.

1970

In Methods in Enzymology,

Ed Taber H.,C.W. Taber. Academic Press (New York), 6.

Deutschen Gesellschaft fur Klinische Chemie.

7.

Coates, D.R.,R.S. Snart.

8.

Snart, R.S..E. Taylor.

9.

Pressley, L..J.W. Funder.

10.

Crocker, A.,K.A. Munday.

Chem. Klin. Biochem.

1972.

H), 182. 1984. 1978.

J. Physiol. 354, 1. J. Physiol. 274, 447.

1975. 1969.

Endocrin. 97, 588. J. Physiol. 202, 329.

633. Z. Klin.

ORGAN SPECIFICITY OF GLUTAMINE HYDROLYSIS AND OXIDATION

Z.Kovacevic, O.Brkljac Institute of Biochemistry, Medical Faculty, 21000 Novi Sad, Yugoslavia

Introduction There is no doubt that glutamine deamidation is of great importance for the regulation of acid-base balance.Kidney and liver belong to the organs with high capacity for glutamine hydrolysis which is catalysed by two different glutaminase isoenzymes located in the mitochondria. There are the following known similarities between these two isoenzymes: Pi-dependence, high pH optimum and intramitochondrial localization. It is reported that both isoenzymes are associated with the inner side of the inner mitochondrial membrane (1,2,3). There are also quite large differences. Contrary to liver glutaminase, kidney enzyme has much lower Km for glutamine, it is strongly inhibited by glutamate, it is not activated by NH^ and it is not hormone sensitive.Physiological function of kidney glutaminase is production of ammonia in acidosis,whereas the function of liver glutaminase is not well understood.lt could have a role in the regulation of plasma glutamine concentration and in systemic pH homeostasis.lt should be emphasised that the liver mitochondrial membrane is much more permeable to glutamate than the kidney one (4,5,6). In addition to these findings our recent investigations revealed that there are two more significant differences which can not be explained simply on the ground of different kinetics of liver and kidney glutaminase.

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

246

Material and Methods Kidney and liver mitochondria were isolated as described elsewhere^). The incubation medium used for the study of respiration, enzyme activity,and metabolic pathways consisted of 100 mM KC1, 10 mM Tris-Cl,20 mM MOPS,10 mM Pi(K+salt),4 mM Mg:i 2 , and 2mM EDTA. The final pH was adjusted with HC1 or Tris.Oxygen consumption was measured by a Clark oxygen electrode.The final volume of the incubation medium was 3,3 ml,the amount of the mitochondria was 3-4 mg protein, and the temperature was 28°C. The activity of glutaminase was estimated by measuring glutamate or ammonia production, 14 or the radioactivity of U C glutamate liberated by the hydrolysis of U 14C glutamine after separation on the column of Dowex 1 x 8 .

Results and Discussion Effect of pH on glutamine hydrolysis and oxidation.- Hydrolysis of glutamine (Fig.l) and oxidation of the produced glutamate(7) in rat kidney mitochondria appears even at a low extramitochondrial pH which is very far from the pH optimum of the enzyme.This depends on the existence of pH gradient since the addition of an uncoupler (carbonylcianide-p-trifluorometoxyphenylhydrazone,FCCP) strongly inhibits hydrolysis and oxidation at low pH.However,liver glutaminase activity and oxidation of glutamine,which is stimulated by NH 3 ,is much more sensitive to the decrease of pH(8) although there is evidence of the existence of pH gradient as well (Fig.2). Intensity of glutamine oxidation in rat liver and kidney mitochondria.- Glumatine is much better respiratory substrate for kidney than for liver mitochondria.This suggests that the fate of glutamate, which is produced by glutamine hydrolysis,is different in the kidney compared to liver.In relation to this the following differences were found.Oxidation of glutamine in kidney mitochondria is much faster than in liver mitochondria even if liver glutaminase is activated by ammonia.Liver mitochondria always respire slower in the presence of glutamine than glutamate whereas the opposite is true for kidney mitochondria(Fig.3).However, the

247 succinate • »rotenone El rotenone o20 i/I in CTl o E

E 10

o^ £c C«j So

•o c 2-E

"O

S 3 E S310 nj o

se *E

•

FCCP * rotenone

3 E 3 S

o40 4, Ol Q5 E Q-

«

Control Hyperprotelndiet

100

S.J

Liver q. 40

•O ä

3o

C C J ;, o 20 Olü >>

j

oi tS

fi fo & E 20 >.o

50

3S2 5 °

oc

I Kidney

Liver

Liver

Fig.3. Oxidation of glutamine and glutamate in rat kidney and liver mitochondria.

• •

&

Control »BCP

SAO

Kidney

J

Glutamine Glu

Fig.4. Hydrolysis of glutamine in rat liver and kidney mitochondria and oxidation of glutamine and glutamate in liver mitochondria. The rats were kept on high protein diets(egg white) for seven days.

This should be especially true in the presence of aminooxyace-

5 §i

tate which inhibits the trans-

ai

amination pathway of glutamate

CÜ

oxidation but it is not(Table 1).

H

On the grounds of all these observations one could suggest

Ol C

i

Glutamine Glu

that rat liver glutaminase is sensitive to both intra and extramitochondrial pH changes, whereas kidney glutaminase is

Fig.5. Effect of bromocresol purple on respiration of rat liver mitochondria with glutamine and glutamate.

sensitive to the changes in intramitochondrial pH only.This enables inhibition of the liver enzyme by the decrease of extramitochondrial and extracellular pH,whereas the kidney enzyme,

249

Table 1. Effect of Aminooxyacetate on the Respiration of Rat Liver Mitochondria in the Presence of Ammonia

Substrates

Glutamine + NH.C1 4 Glutamate Proline Proline + NH 4 C1

Oxygen uptake (ngatom O/min.mg.protein) +AOA -AOA 47.4 69.7 63. 5 62.0

45.2 30.3 63. 5 54.0

Inhibition (%)

5 56 0 14

under these conditions,remains fully active. This finding and the striking difference between ability of rat liver and kidney mitochondria to oxidize glutamine,can not be explained solely on the basis of kinetic properties and activities of their glutaminases,but by different submitochondrial location of the isoenzymes or by different rates of efflux of glutamate from the mitochondria.This is supported by the fact that the liver enzyme has not been successfully isolated and purified by now suggesting that this is an integral membrane protein. We propose that the liver enzyme faces both intra- and extramitochondrial space, whereas the kidney enzyme faces the intramitochondrial space only. Glutamate which is produced by liver glutaminase leaves the mitochondria very rapidly. This enables utilization of glutamate for metabolic processes other than oxidation. Also,this could explain why the liver enzyme is not inhibited by glutamate. There is also the possibility that in the case of liver there is more closer interaction between glutaminase and the glutamate carrier or between glutaminase and glutamate dehydrogenase or aspartate aminotransferase. Depending on which kind of interaction predominates glutamate will be preferentially exported to the cytosol or preferentially oxidized through the deamination or the transamination pathway. Glutamate produced by kidney glutaminase accumulates inside the organelles reaching a high enough level to become the substrate for oxidation. Therefore, glutamate formed from glutamine in the kidney is mostly

250

o x i d i z e d , i n a c i d o s i s through deamination pathway. In o t h e r words o x i d a t i o n i s almost the only f a t e of g l u t a m a t e g e n e r a t e d by the h y d r o l y s i s of g l u t a m i n e i n the kidney m i t o c h o n d r i a . This i s e s p e c i a l l y true in metabolic a c i d o s i s (11).

References 1. C u r t h o y s , N . P . . S . S . G o d f r e y . 1 9 7 6 . I n : R e n a l Metabolism i n R e l a t i o n to Renal Function (U.Schmidt and U . C . D u b a c h , e d s . ) Huber, Bern,pp.346-356. 2. M c G i v a n , J . D . , N . M . B r a d f o r d , A . J . V e r h o e v e n , A . J . M e i j e r . 1 9 8 4 . I n : Glutamine Metabolism i n Mammalian T i s s u e s (D.Häussinger and H.Sies,eds.) Springer-Verlag,Berlin,pp.122-137. 3. K o v a c e v i c , Z . , J . D . M c G i v a n . 1 9 8 3 . P h y s i o l . R e v . 6 3 , 5 4 7 - 6 0 5 . 4. B r a d f o r d , N . M . , J . D . M c G i v a n . 1 9 7 3 . B i o c h e m . J . 1 3 4 , 1 0 2 3 - 1 0 2 9 . 5. K o v a c e v i c , Z . 1 9 7 5 . B i o c h i m . B i o p h y s . A c t a 2 9 6 , 3 2 5 - 3 3 4 . 6. S c h o o l w e r t h , A . C . , K . F . L a N o u e , W . J . H o o v e r . 1 9 8 3 . J • B i o l . C h e m . 2 5 8 , 1735-1739. 7. K o v a c e v i c , Z . , K . B a j i n , 0 . B r k l j a c . 1 9 8 5 . I n : K i d n e y Metabolism and F u n c t i o n ( R . D z u r i k , B . L i c h a r d u s and W.Guder,eds.) M a r t i n u s Nijhoff Publishing,Dordrecht-Boston,pp.61-69. 8. V e r h o e v e n , A . J . , J . F . v a n I w a a r d e n , S . K . J o s e p h , A . J . M e i j e r . 1 9 8 3 . Eur.J.Biochem. 133,241-244. 9. K o v a c e v i c , Z . 1 9 7 8 . I n : B i o c h e m i c a l Nephrology(W.Guder and U.Schmidt,eds.),Huber,Bern,pp.254-264. 10. H o e k . J . B . . R . M . N i o g u . 1 9 8 0 . J . B i o l . C h e m . 2 5 5 , 8 7 1 1 - 8 7 1 8 . 11. L o m b a r d o , J . V . , A . R i s q u e z , M . M c C a r t y , H . G . P r e u s s . 1 9 8 1 . K i d n e y Int.19,540-552.

THE STIMULATION OF THE RESPIRATION OF GLUTAMINE BY RAT RENAL MITOCHONDRIA IN CHRONIC METABOLIC ACIDOSIS

D.J. 0 1 DONOVAN Department of Physiology, University College, Galway, Ireland R.B. Tobin, M.A. Horrum Veterans Administration Medical Center, Omaha, Nebraska, U.S.A.

Introduction Glutamine is an important metabolic fuel for many tissues (1).

In addition it

can traverse cell membranes easily, thus making it readily available for metabolism within the cell.

It is readily deamidated by many tissues and the result-

ant glutamate can prime the tricarboxylic acid cycle after deamination (2). A great deal of attention has been devoted to the disposal of ammonia which results from glutamine deamidation. role in cell metabolism.

The other product, glutamate, plays a vital

Little is known about the effects of glutamine on oxi-

dative metabolism within the cell.

Respiratory fuels, which are products of

glutamine metabolism, alter the rate of ammonia production from glutamine.

Thus

the lowering of the intracellular concentration of¿-ketoglutarate (3) will accelerate ammoniagenesis from glutamine.

Furthermore metabolic acidosis (4, 5) or

in vitro acidification of the medium (6, 7) will decrease et -ketoglutarate concentration by accelerating its oxidation. The deamidation of glutamine is an anaerobic pathway and olism is aerobic.

further metab-

Thus, tissues incubated with glutamine alone should have a

lower rate of respiration than substrates being metabolised by aerobic pathways. We compared the respiratory pattern of glutamine with a number of substrates formed during the normal metabolism of glutamine (Fig. 1).

We reasoned that the

oxygen consumption with glutamine could not be greater than that of glutamate, which is formed by anaerobic deamidation of glutamine.

It should also be less

than c-ketoglutarate and succinate, which can be formed from glutamate. was true for renal mitochondria from control rats. ic acidosis the pattern was changed.

This

However, in chronic metabol-

Acidosis decreased oxygen consumption by

renal mitochondria respiring in the presence of glutamate,ol-ketoglutarate, and succinate - and increased it in the presence of glutamine.

Thus in acidosis the

Molecular Nephrology Biochemical A s p e c t s of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

252

GTP ^ ^ ^ SUCCINATE

FADHa

FUMARATE

SUCC1NYL CCJNYL CoA Co C0 2+ NADH

f

o2 to about 70 mm Hg. Bicarbonate reabsorption rose to the values obtained before bicarbonate loading. This experiment permits the conclusion that filtered load and plasma pH rather than PC02 are the two main determinants of bicarbonate reabsorption during acute changes in acid/base balance. It should be noted that no Tm for bicarbonate is reached in experiments performed at constant plasma pH. Fig 2 (upper and middle right panels) show that metabolic alkalosis greatly depressed sodium (and chloride) reabsorption. Reestablishment of control plasma pH only partly restored sodium reabsorption. At equal plasma pH and filtered load (or GFR) sodium reabsorption was almost 50% greater before than after bicarbonate loading. Fig 2 (bottom panels) shows the relationships between water and bicarbonate, respectively water and sodium reabsorption. As expected for isotonic reabsorption the ratio, which is an estimate of the reabsórbate concentration, equals plasma concentration of sodium, provided the Donnan factor is 0.98, which is a realistic value in volume-expanded dogs. Similar calculations for the relationship between water and bicarbonate reabsorption indicate a reabsórbate concentration of 42 mM at P

HC0

3 0

m M

a n d

5 8

"

6 0

m M

a t

P

5 2

hco

loading at plasma pH 7.5 and 7.8.

3

m M

d u r i n ,

3

bicarbonate

DISCUSSION At high P H C 0 , bicarbonate concentration along the proximal tubules may fee close to plasma values. The concentration

261

> pH 7.5 [ P C 0 2 4 2 m m H g

PHC03

i pH 7.8 [ PCO2 4 2 m m H g

P H C O 3 50 m M l

30mM]

> pH 7.5 [ PCO2 7 0 m m H g

P H C O 3 50 m M ]

«• •

/

%

500 Filtered

Fig. 2.

Effects

1000

1500

bicarbonate,

of

altering

2000

2500

2000

pmol - m i n ' 1

GFR.

Upper

panel

Filtered

single

experiment.

Lower

4000

•

*

6000

8000

s o d i u m , timol • m i n " 1

panels

data

from

6

dogs.

262

difference

for b i c a r b o n a t e

may t h e r e f o r e be about loading.

b e t w e e n r e a b s ó r b a t e and t u b u l a r

12 m M b e f o r e and

In these c a l c u l a t i o n s

6-8 mM during

it is a s s u m e d

bicarbonate

that all c h a n g e s

p r o x i m a l t u b u l a r w a t e r r e a b s o r p t i o n p r o c e e d s a c r o s s the junction. cells, would

If w a t e r

reabsorption

the c o n c e n t r a t i o n be

fluid

tight

partly takes place t h r o u g h

d i f f e r e n c e a c r o s s the tight

the

junction

higher.

I n h i b i t i o n of sodium r e a b s o r p t i o n d u r i n g m e t a b o l i c a l k a l o s i s due b o t h to reduced bicarbonate

bicarbonate

concentration

normalization reabsorption increased

r e a b s o r p t i o n and

in the tubular

fluid.

to

is due to r e d u c e d o s m o t i c d r i v i n g concentration

force b e c a u s e fluid.

reabsorbed

of

e l e v a t i o n of

m o r e than two m o l e s of N a C l HL.U for each m o l e of R a H C 0 3 , as a l s o found during

in pH of p r o x i m a l

(Fig.1)

metabolic

tubular

cells. T r a n s c e l l u l a r

difference

alkalosis

than tubular

intracellular

is reduced

During

lowering

h o m e o s t a s i s . M o r e d e t a i l s are presented

difference

falls.

for

the This

renal h y d r o g e n

elsewhere

rather

bicarbonate

t u b u l e s as fluid

of GTB and

more

disturbances of GFR,

in the tubular

regulations

During

increases

b e c a u s e the c o n c e n t r a t i o n

concentration

hypothesis unifies

for

the

by the h y d r o g e n

ions d e c l i n e s a l o n g the p r o x i m a l

bicarbonate

whereas

presumably

e x c h a n g e may be regulated

t h a n the sodium g r a d i e n t . reabsorption

pH

change

difference

remains unaltered.

pH. At least during a c i d / b a s e

sodium/hydrogen

hydrogen

cell m e m b r a n e ,

for sodium

in a

bicarbonate

seems to d e p e n d on the c o n c e n t r a t i o n

ions a c r o s s the luminal

concentration

for

acid/base

We c o n c l u d e that a c h a n g e in plasma pH is r e f l e c t e d reabsorption

of

During

loading about one m o l e of N a C l w a s r e a b s o r b e d

r e d u c t i o n of GFR.

hydrogen

high

NaCl

in the tubular

each mole of NaHCC>3 . Under the other d e v i a t i o n s balance without

is

After

of p l a s m a pH the reduced w a t e r and

bicarbonate

bicarbonate

were

in

ion

(1,2).

References

1. Kiil,F. 1986. In: Kidney Hormones (J.Fisher ed.) Academic Press, London, vol.3, p.101 2. 0stensen,J., H. Langberg, F. Kiil: Acta Physiol.Scand. (in press)

ROLE OF RENAL OXYGEN CONSUMPTION RATE IN THE ADAPTIVE AMMONIAGENESIS OF ACIDOSIS

H.G. Preuss, J. Areas, P. Schubert, M. Lenhart, D. Slemmer Department of Medicine

(Nephrology), Georgetown University

Medical Center, Washington, DC 20007

Introduction This laboratory hypothesized previously that renal oxygen consumption

(QO2) plays an important role in regulating renal

ammoniagenesis through its effect on the pyridine nucleotide system

(1,2).

An earlier study utilizing isolated canine

renal tubules showed a significant positive correlation between QO2 and ammoniagenesis. dinitrophenol

In the same study, addition of 2,4-

(DNP) increased QO2 and ammoniagenesis, while

amytal and cyanide decreased both parameters

(3).

In contrast

to in vitro experiments, the role of QO2 in ammoniagenesis regulation in vivo was less certain. The purpose of the present investigation was two fold: first, to determine if renal QOj was increased in intact acidotic dogs with elevated ammoniagenesis compared to control or alkalotic dogs; and second, to follow the effects of augmenting QO2 through infusions of DNP on renal ammoniagenesis in intact alkalotic dogs.

During the course of our studies, we noted an

enhanced QO2 by the remaining kidney within hours after unilateral nephrectomy.

Therefore, we also investigated ammonia-

genesis in the remaining kidney following removal of a single kidney.

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

264

Methods Our experiments were performed on mongrel dogs of either sex weighing at least 15 kg. Dogs were made chronically acidotic with oral NH 4 C1 (30 dogs) and acutely alkalotic with HCOj" infusions (16 dogs). In general, dogs were handled as previously described (4). In 6 separate experiments, chronically acidotic dogs were rendered normal or alkalotic with HCO^ infusions (4) in order to compare parameters in the same dog. In 17 dogs, the left renal artery was exposed and traced with minimal manipulation from aorta to kidney. A number 25 curved butterfly needle was introduced into the renal artery close to the aorta. The needle was connected through a length of flexible, fine bore tubing to a syringe infusion pump which delivered saline containing heparin at a slow rate to keep the needle patent. Following a 1-hour equilibration period, the study was begun. After collecting blood and urine during two consecutive 20 min control periods (periods 1 & 2), 100 mg of 2,4 dinitrophenol (DNP) dissolved in saline plus heparin (neutralized to pH 7.4) was given into the left renal artery over 10 minutes. Following another 10 minute equilibration period, bloods and urines from 2 consecutive test periods (3 & 4) were collected. In 3 studies on unilaterally nephrectomized normal dogs, the operation was performed after 2 control periods were obtained. Following the removal of the right kidney, 2 consecutive 20 minute test periods were run between 1 - 2 h later. Measurements were carried out as previously described (4). Blood 0 2 content was estimated using a Lex-C^-Con-K, Lexington, Massachusetts.

Results In 30 acidotic dogs with an average pH of 7»22 ± .02 (SEM) and bicarbonate of 13.0 } 0.6 (SEM), ammonia production was 39.8 ymol/min + 1 . 9 (SEM) and Q0 2 averaged

265

169 umol/min + 11 (SEM). In 16 normal dogs receiving i.v. NaHCOj with an average pH of 7.48 ± .02 (SEM) and bicarbonate of 23.6 mEq/L ± 1.1 (SEM), the ammonia production was significantly lower, 19.7 ymol/fnin ± 1.3 (SEM) than in acidosis (p2 (1-3,5). In support of the first proposal, a significant positive correlation between QO2 and ammoniagenesis has been found in incubating canine renal tubules (3). In support of the second proposal, addition of oxidizable substrates decrease and metabolic inhibitors increase ammoniagenesis (2). The role of oxidative metabolism in the regulation of ammoniagenesis in intact dogs is less certain. We know that changes in in vitro renal QC^ are not evident during acidosis when renal ammoniagenesis increases (6). Indeed, renal Q0 2 changes little under most circumstances. Infusion of a major renal fuel such as lactate, which creates augmented renal extraction, does not increase QC^. This suggests that renal oxygen uptake is fairly stable in the intact dog (7). Nevertheless, the possibility remained that small, difficult to detect alterations in QC>2 during acidosis increase ammoniagenesis greatly and account for some adaptation. In 30 chronically acidotic dogs, the average QC>2 was significantly less than 16 acutely alkalotic dogs that were producing considerably less renal ammonia. In addition, acute infusions of HCO^ into acidotic dogs considerably lowered ammoniagenesis despite no change or even perhaps an increase in Q0 2 . When DNP was infused into alkalotic dogs, QC^rose.

Although

DNP is best known for enhancing renal QO2 (8-11) , it has other metabolic and physical effects. It is well accepted that DNP uncouples oxidative phosphorylation, reducing ATP and perhaps other energy source availability (12). In addition, the rate of transfer through the electron transport system is hastened allowing faster reoxidation of NADH to NAD + .

267 Despite the rise in Q0 2 , ammoniagenesis did not increase. However, DNP caused a decrease in RBF and GFR in our studies. Accordingly, we factored our results by GFR to compare the control and test situations at similar delivery rates of substrate and sodium. Such a practice is common among investigators, since it is accepted that changes in RBF (Gin loading) (4) and GFR (13) influence ammoniagenesis. We found an increased ammoniagenesis. We hasten to add that the magnitude of change did not approach that noted in chronic acidosis. Three dogs undergoing unilateral nephrectomy also showed enhanced Q0 2 in the remaining kidneys without a significant elevation in renal ammoniagenesis. The inability of raised renal QC>2 to enhance ammoniagenesis to the magnitude seen in acidosis and the lack of an obvious increase in QC>2 during acidosis (6) indicate that regulation of ammonia metabolism occurs via another mechanism. We suggest substrate competition for oxidation (1-4). Because increased renal substrate removal brought about via exogenous infusions does not enhance renal QC>2 (7) and DNP augments Q0 2 (11), this suggests that kidneys of intact dogs have a relatively fixed QC>2 due to a rate limiting supply of ADP (3,13). The ability of sodium reabsorption to influence Q0 2 could be based on the production of ADP from ATP'ase as the ion is transported. Recently, Halperin et al (14) accepted a somewhat similar hypothesis, i.e., that the extent of ATP production, which is estimated by QO 2 , limits renal ammonia*genesis from glutamine. In summary, these new findings support our previous hypothesis that the means by which glutamine replaces lactate and perhaps other substrates as the preferential renal fuel when the renal oxidative capacity is limited provides the basis for the augmented ammoniagenesis during acidosis (1-3,5).

This grant was supported by NIH grant No. AM27433

268

References 1. 2. 3.

Preuss, H.G.: 1968. J. Lab Clin. Med. 72,370. Preuss, H.G.: 1980. Life Sei. 27,2293. Preuss, H.G., K. Baird, H. Goldin: 1974. J. Lab. Clin Med. 83,937.

4. 5.

Lombardo, J.V., A. Risquez, M. McCarthy, H.G. Preuss: 1981. Kidney Int. 19, 540. Preuss, H.G., F.R. Weiss: 1971. Am. J. Physiol. 221,458.

6.

Cohen, J.J.: 1960.

7. 8.

Levy, M.N.P 1962. Am. J. Physiol. 202,320. Fujimoto, M., F.D. Nash, R.H. Kessler: 1964. Am. J. Physiol. 206 ,1327. Martinez-Maldonado, M, G. Eknoyan, W. Suki: 1970. J. Physiol. 219,1242.

9.

Am. J. Physiol. 199,560.

10.

Mudge, G.H., J.V. Taggart:

11.

Strickler, J.D., R.H. Kessler: 1963. Am. J. Physiol. 205,117. Krebs, H.A.: 1959. In: Ciba Found. Symp. Regulation Cell Metabolism, Little Brown, Boston, MA p 1. Lemieux, G, P. Vinay, P. Cartier: 1974. J. Clin. Invest. 53,448. Halperin, M.L., R.L. Jungas, C. Pichette, M.G. Goldstein: 1982. Can J. Physiol and Pharmacol. 60,1431.

12. 13. 14.

1950.

Am. J. Physiol. 161,173.

RENAL ACftFIAXICN TO LACTIC ACIDOSIS IN THE DOG

E. Junoo, R. Pérez, R. Jofre, M. Rengel, S. Alonso, F. Valderratano Nephrology Service, Hospital Provincial, Madria, Spain P. Vinay, A. Gougoux Departnent of Medicine, University of Montreal, Montreal, Canada H3C 318

Introduction The renal amtcniagenic response to acute metabolic (ISH4CI or HC1 induced) acidosis (1,2) ca: to respiratory acidosis (3) by the kidney of the dog has teen extensively described.

During these circunstances, the renal glutanine metabolitir. is

increased through a direct pH-dependent stimulation of the flux through the glutamate oehyorogenase pathway (4).

In the present paper, we explore in the dog the renal

response to the more natural lactic acidosis and we oarpare the effect of this acio-base disorder in presence or aDsenoe of a significant systemic hypoxemia.

Our

data demonstrate that renal anncniagenesis increases acutely during lactic acidosis by a mechanist! different from that used in non-lactic metabolic acidosis: the renal response now involves a transamination pathway activated by lactic acid accuoilation, and this response is similar with or without systemic hypoxia.

Material and methods Experiments were performed on 24 cfcgs in normal acid-base status.

The dogs

were anesthetized, intubated and ventilated to maintain the initial pCD2 around 35 ntrHg.

The dogs received an isotonic solution containing mannitol (2.5%) and saline

(0.45%) adjusted at pH 7.4. Fallowing freeze-clanping of a piece of the right kidney and right nephrectcny, the renal extraction or production of metabolites by the left kidney was followed by obtaining four 10 m m . urine collections and three arterial ana renal venous blood samples.

In 9 dogs, lactic acidosis was then induced by

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

270 infusing lactic acid (5 mEq/kg) while in another group of 15 dogs, a ocmparable lactic acidosis was induced by changing the expired air for an hypoxic gas mixture (9% 02 91% N2).

After a 30 min. equilibration period, five additional arterial and

renal venous blood sarrpies and four urine collections vrere obtained before a piece of the renal kidney was freeze-ciampea. Blood, urine ana tissue measurements vrere performed as previously described (1-3). In order to ccnpare the renal utilization or production of each metabolite at similar levels of tubular work and AIP turnover, the data were expressed by 100 ml GFR (5). The statistical analysis was performed using a Student "t" test for paired values using an alpha of 0.05.

Results Table 1 presents critical parameters measured in the arterial blood before and after lactic acidosis induction in both groups of dogs. Table 1: Arterial blood values and GFR before and after induction of lactic acidosis

Arterial blood values

Lactic acid infusion Control Lactic acidosis

Blood pH Plasma HCD3 mM Blood pC>2 iimHg

7.34 ± 0.01 7.14 ± 0.02* 21 ± 0.7 15 ± 0.5 *

Lactate GFR

1.4 ± 0.22 33.0 ± 2.00

nM ml/min

6.0 ± 0.6 * 30.0 ± 2.0 *

Hypoxic lactic acidosis Control lactic acidosis 7.32 ± 0.01 7.17 ± 0.02* 15 ± 0.08* 19 ± 0.6 137 ±11.0 29 ± 2.2 * 1.0 ± 0.2 30.0 ± 2.2

4.3 ± 0.6 * 17.0 ± 3.0 *

Data are means ± SEM; * = p>0.05. All dogs were in oarparable acid-base status before induction of lacticidania.

Both

lactic acid infusion and hypoxemia elicited a similar acidosis with elevation of plasma lactate to 6 ana 4.3 mM respectively.

TVs only difference between the two

271 situations is the PO2 which fell below 30 itmHg in hypoxemic dogs.

The GFR was not

significantly influenced by lactic acid infusion but was significantly reduced by hypoxic lactic acidosis. In table 2, the production or extraction of critical metabolites by the left kidney before ana after induction of lactic acidosis is presented.

It is clear that renal

attmoniagenesis and glutamine uptake are stimulated by lactic acid infusicn. tfcwsver, this is mostly die to an increment in synthesis ana release of alanine in the renal vein. A stoechicnetry of 1 extra glutamine extracted for the production at 1 extra alanine plus 1 extra amnonium is new seen. A considerable lactate extraction by the kidney was sinultaneousiy observed. TaDle 2: Renal production ana extraction of metabolites

Production (+) or Extraction (-) of Metabolites by the Left kindey

Lactic acid infusion Cfcntrol

Hypoxic lactic acioosis

Lactic acidosis

Control

Lactic acidosis

+ 56.4 ± 6.1

+ 70.7 ± 4.9

*

+ 77.0 ± 11

Glutamine

- 37.5 ± 6.2

- 56.6 ± 6.7

*

- 60.0 ±

5.3 - 98.0 ± 7.2

*

Alanine

+ 22.7 ± 2.5

+ 41.2 ± 3.7

*

+ 44.0 ±

6.3 + 87.0 ± 23.0

*

Lactate

-136.3 ±2.7

-396

*

-119

± 73

± 14

+103.0 ± 21

*

Antonia

-329

± 73

*

Data are means ± SEM; * = p

•

CONTROL

H

+GENTAMICIN

« 60 40

20

LACT

PYR

PROP

DHA

FRU

2-OG

MAL

SUC

CIT

ICIT

O 60

£ 20 O u z> O 0L

Fig. 1. Effect of Gentamicin on Glucose Formation in Rabbit Kidney-Cortex Tubules Incubated with Various Substrates Isolated kidney tubules were incubated for 60 min with corresponding substrate and either without or with 5 mM gentamicin. Values represent means _+ S.E. for 5 - 7 separate experiments. Substrates listed are: IACT, 1-lactate; PYR, pyruvate; PROP, propionate;FRU, fructose; DHA, dihydroxyacetone; 2-OG, 2- oxoglutarate; MAL, 1-malate; SUC, succinate; CIT, citrate; ICIT, isocitrate.

296 Table 1. Effect of Gentamicin on lactate plus Pyruvate Production and Intracellular ATP Content in Kidney Tubules Incubated with Dihydroxyacetone or Fructose Lactate and pyruvate as well as intracellular ATP were determined following 6 0 min incubation of renal tubules in Krebs-Henseleit bicarbonate buffer with the corresponding substrate. Results are given as means _+ S.E. for three separate preparations. Substrate

Gentamicin

Dihydroxyacetone Fructose

+ +

a

lactate plus Pyruvate Intracellular ATP (/umol/g dry weight) 4 8.3 3.0 50.2 ± 6.7 6 8.5+1.6 53.0 + 2.0a

3.14+0.34 3.23 + 0.40

P 25 KD) and heat s e n s i t i v e . Less clear were the r e s u l t s obtained with

2

4

6 MG CYTOSOI

8

10

2

4

6

8

10

MG CYT0S0L

Fig. 3: Effect of cytosoi on nuclear and microsomal 11-HSD. B = 1 uM. c o s u b s t r a t e (NADPVNADPH) = 1 mM. protein = 0.5 mg/ml (nuclei) or 0.2 mg/ml (microsomes)

365 B - Metabolism in %

Incubation Time Pig. 4: E f f e c t of c y t o s o l i c p r o t e i n on n u c l e a r 11-HSD a f t e r p r e i n c u b a t i o n of n u c l e i ( 1 mg) w i t h B (1 pM). A f t e r 60 min ( a r r o w ) 6 mg of c y t o s o l or NADP* (1 mM) w a s added and t h e i n c u b a t i o n c o n t i n u e d f o r 15 min.

microsomes s i n c e t h e a b o v e m e n t i o n e d m a n e u v e r s w e r e

inefficient.

T h e c o n c e p t of r e t e p t o r hormone complex f o r m a t i o n and s u b s e q u e n t

transformation

i n t o t h e n u c l e u s t e m p t e d us t o p e r f o r m a n o t h e r s e r i e s of e x p e r i m e n t s . r e c e p t o r hormone t r a n s l o c a t i o n nuclear

Could

in our c e l l f r e e s y s t e m f a c i l i t a t e a c c e s s t o

11-HSD? A s shown in Fig. 4 n u c l e a r f r a c t i o n s w e r e i n c u b a t e d w i t h B o v e r

t>0 min w i t h o u t e x o g e n o u s NADP*. T h e basal r e a c t i o n r e a c h e d a p l a t e a u a t

roughly

30 min w i t h o u t f u r t h e r i n c r e a s e of B - m e t a b o l i s m . We assumed t h a t e n d o g e n o u s was used up by t h i s time. A d d i t i o n of e x o g e n o u s i n c r e a s e of B - m e t a b o l i s m i n d i c a t i n g t h a t the cytosol

NADP +

(lO*3

NADP*

M) l e d t o a prompt

11-HSD was i n t a c t . A l s o , a d d i t i o n

of

i n s t e a d of NADP* r e e s t a b l i s h e d n u c l e a r c o n v e r s i o n of B.

Discussion

11-HSD which has been r e p o r t e d t o be p r e s e n t in v a r i o u s s p e c i e s and t a r g e t of g l u c o c o r t i c o i d a c t i o n

(comp. ( 1 1 ) ) has been i n v e s t i g a t e d in t h e p r e s e n t

w i t h k i d n e y t i s s u e in o r d e r t o l o c a l i z e the e n z y m e w i t h i n the

intracellular

s t r u c t u r e s , t o a n a l y z e i t s k i n e t i c p r o p e r t i e s and t o e l u c i d a t e a p o s s i b l e

inter-

a c t i o n w i t h c y t o s o l i c g l u c o c o r t i c o i d r e c e p t o r s . T h e e n z y m e could be l o c a l i z e d n u c l e a r and microsomal f r a c t i o n s of r a t r e n a l c o r t e x . T h i s l e a d s t o the w h e t h e r or n o t t h e r e e x i s t t w o i n t r a c e l l u l a r s i t e s of

organs

studies

in

question

11-HSD which p e r h a p s

differ

366

in their biological significance. Other authors suggest that the enzyme Is l o c a lized in the microsomal tabs near the outer nuclear membrane (11). Further e x p e r i ments with monoclonal antibodies are planed to shed more light on this still u n resolved problem. Mitochondria which are known to be involved in the synthesis of steroid hormones were inactive in metabolizing B under our nonfortlfied in vitro

conditiones. A t

least 11-HSD-activity seems to be absent in mitochondria as we could show in experiments with mitochondrial fractions of different purity (see Fig. 2: for a detailed discussion see (6)). Enzyme kinetic studies with membrane bound 11-HSD from both nuclei and microsomes revealed Km-values of 10"7 M. whereas in the solubllized form the Km increased to 10"6 M. The lower a f f i n i t y of the extracted 11-HSD may be due to conformational changes caused by the solubilization process. A possible interaction between 11-HSD and cytosolic steroid hormone receptors was studied in recombination experiments with cytosol. In the case of nuclei a dose dependent and saturable stimulation of B-metabolism was observed. Results obtained by ultrafiltration and trypsinization suggest that the stimulation is not simply caused by the addition of cosubstrate present in cytosol but is exerted by protein(s). However, the concept that glucocorticoid receptors alone facilitate the access of B to nuclear metabolizing sites is hampered by the experiment described in Fig. 4. Even in the state of cosubstrate depletion reached at about 30 min of incubation B-metabolism could be stimulated by the addition of cytosol. This led us to consider the possibility that the macromolecular f a c t o r ( s ) responsible for turning on the enzymatic system might regenerate endogenous cosubstrate . i.e. NADPH. In fact we could show that cytosol is able to oxidize NADPH. Therefore we suggest that at least one component of the stimulatory e f f e c t on nuclei Is due to a NADP* regenerating system, e.g. thioredoxin or other reducing enzymes, present in cytosol. In contrast to nuclei NADP* stimulated 11-HSD in microsomes was partially inhibited bv cytosol. Again we tested the concept whether or not binding of B to cytosolic glucocorticoid receptors might inhibit access to 11-HSD localized in the endoplasmic reticulum. As in the case of nuclei the inhibition e f f e c t on microsomes was dose dependent, but not yet saturated at 10 mg of cytosolic protein. U l t r a f i l t r a tion experiments gave less clear results, since the inhibitory f a c t o r ( s ) were found in both, retentate and filtrate, respectively. If the inhibition of microsomal 11-HSD would be related to glucocorticoid receptors, this e f f e c t should be reduced or eliminated when cytosol was preincubated. since glucocorticoid receptors have a h a l f l i f e time of some 20 min. However, the inhibition e f f e c t was still retained even after 60 min of preincubation of cytosol at 37 °C. Therefore the possibility that proteases might interfere will have to pursued. The results so far obtained

367 do not provide evidence for an interaction of nuclear or microsomal 11-HSD with cytosolic glucocorticoid receptors. However, this question too, will have to be studied in experiments with glucocorticoid receptor modifying agents such as molybdate and ATP as well as with purified glucocorticoid receptor preparations.

References

1. Hierholzer. K.. I. Lichtenstein. H. Siebe. D. Tsiakiras, I. Witt. 1982. Klin. Wochenschr. 60. 1127. 2. Hierholzer, K., M. Schöneshöfer. H. Siebe, D. Tsiakiras, P. Weskamp with tech. ass. of I. Lichtenstein. 1984. Pflügers Arch. 400. 363. 3. Siebe. H.. D. Tsiakiras, K. Hierholzer with techn. ass. of I. Lichtenstein. 1984. Pflügers Arch. 400, 372. 4. Hoyer. G. A.. D. Tsiakiras, H. Siebe, K. Hierholzer. 1984. Pflügers Arch. 4QQ. 377. 5. Chao. H. P. 1985. Thesis. 6. Kobayashi, N.. W. Schulz. K. Hierholzer with techn. ass. of I. Lichtenstein and H. Siebe: Pflügers Arch, (in press). 7. Lowry. O. H.. N. J. Rosebrough. A. L. Farr. R. J. Randall. 1951. J. Biol. Chem. 193. 265. 8. Bradford. M. M. 1976. Anal. Biochem. 72, 248. 9. Loewenstein. J.. H. R. Schölte, E. M. W i t - P e e t e r s . 1970. Biochim. Biophys. Acta 223. 432. 10. Schulz. W.. N. Kobayashi. K. Hierholzer. 1986. Biol. Chem. Hoppe-Seyler 367 (Suppl.). 271. 11. Monder. C.. C. H. L. Shackleton. 1984. Steroids 44, 383.

TAMM-HORSFALL PROTEIN AND ANTIBODIES TO TAMM-HORSFALL PROTEIN IN THE STUDY OF RENAL DISEASES

M. Radonic Department of Medicine. Clinical Hospital Centre. Zagreb. Medical Faculty University of Zagreb. YU-41000 Zagreb. Kispaticeva 12 D. Cvoriscec. A. Stavljenic. J. Sertic Institute for Clinical Laboratory Diagnostics. Medical Faculty University of Zagreb. Clinical Hospital Centre. Zagreb. YU-41000 Zagreb. Kispaticeva 12 G. Borso Department of Medicine. Clinical Hospital Centre. Zagreb. Medical Faculty University of Zagreb. YU-41000 Zagreb. Kispaticeva 12

Introduction Tamm-Horsfall protein (THP) is a renal epithelial mucoprotein of high molecular mass (7 x 10^ daltons) derived from the tubular cells of the ascending thick limb of Henle's loop. Under physiological conditions it is present in the human urine and represents a major component of urinary casts. Although the precise physiological function of THP has not been definitely elucidated, its specific localization and its chemical and physical properties suggest that it might play a role in the regulation of membrane transport of chloride ions and associated cations and that it may be involved in the pathologic processes of different renal diseases (1). Urinary excretion of THP appears to be fairly constant and no significant diurnal variations in the excretion of THP have been observed (2). In most of the patients with chronic renal failure the excretion of THP is decreased in proportion to the decrease in glomerular filtration rate (GFR) (2.3). In patients with cadmium nephropathy and Fanconi syndrome. an increased excretion of THP relative to GFR has been observed (2). Increased excretion of THP has been also observed in Balkan endemic nephropathy (4). Although several reports dealing with the clinical significance of the determination of urinary excretion of THP as a diagnostic procedure have been reported so far. many questions on its pathophysiological and clinical significance remain still open. The aim of this work was to examine urinary excretion of THP as a potential clinical tool in recognizing and differentiating various

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

370 renal diseases. Apart from this, antibodies to THP in sera of healthy persons and patients with various renal diseases were investigated.

Materials and Methods Excretion of THP was investigated in the following groups: healthy adult subjects (n-32). non renal adult patients (n-57). patients affected by different renal diseases (n-105) and patients treated with bone marrow transplantation (BMT) who received cyclospoMn (n-21). THP in urine samples was assessed by electrophoresis in 1% agarose gel containing antiserum to THP (4.5). The presence of antibodies to THP in sera of healthy subjects (n-50) and patients with various renal diseases (n-50) was tested by several methods: countercurrent Immunoelectrophoresis. reverse rocket Immunoelectrophoresis. rocket Immunoelectrophoresis and reverse radial immunodiffusion (6).

Results The excretion of THP in healthy adult persons and non renal adult patients is presented on Table 1. Table 1. Excretion of THP in Healthy Adult Persons and in non Renal Adult Patients Number

THP range

mg/24 h x

Healthy adult persons

32

15.5-54.9

36.6

Non renal adult patients

57

3.0-45.9

18.1

The results obtained for healthy adult persons do not differ from the reports in literature. The excretion of THP in patients suffering from various renal diseases is summarized on Table 2.

371

Table 2. Excretion of THP in Various Renal Diseases Diagnosis

Number

THP range

Glomerular diseases

15

Tubulo-interstitial diseases

mg/24 h X

3..6 - 39.6

21.3

2..5 - 88.1

20.5

Acute pyelonephritis

17

3.• 3 - 88.1

24.0

Chronic pyelonephritis

18

2..5 - 42.5

20.5

Vesicouretheral reflux

5

4,.0 - 41.7

16.5

Analgesic nephropathy

1

Sponge kidneys

3

4..0 - 47-3

19.3

10

4 .0 - 26.5

14.1

3

4 .0 - 72.7

27-5

Lower urinary tract infection

12

4 .0 - 25.2

13-8

Chronic renal failure

21

4 .0 -

7.1

Other renal diseases Nephrolithiasis Renal cystic disease

9-2

4.9

In the group of patients suffering from glomerular diseases without azotemia the mean excretion value of THP was lower compared to the mean excretion value of THP in healthy adult persons. In the group of patients with tubulo-interstitial diseases the mean excretion value of THP was also lower than the mean value obtained in the control group. However, considering the peak value in this group one can find the highest values in the group of patients with acute pyelonephritis and with polycystic kidneys. In patients with lower urinary tract infection the peak value over 25.2 mg/24h THP was not found pointing thus to the differencei between lower and upper urinary tract infections. In patients with chronic renal failure excretion of THP was rather low as expected due to the nephron loss in the advanced renal disease. The excretion of THP in patients treated with BMT who received cyclosporin was monitored five days before and at least 60 days after BMT. An extremely high excretion of THP was observed in four patients. The excretion of THP in one of them is presented on Fig. 1. as an example being not much different from the remaining three cases. Fig. 1. Excretion of THP in patient treated with BMT who received cyclosporine

372 THP mg/gCr

BMT

DAYS AFTER BMT

Investigation of the presence of antibodies to THP in sera has shown that antibodies to THP were present not only in patients with renal diseases but also in healthy persons. The results of this investigation are in press.

Discussion We investigated the excretion of THP in normal and different pathologic conditions with the aim to get some more ligjit on the clinical meaning of the changes in excretion of THP in so-called usual renal diseases and in the rarer renal affection such as administration of cyclosporin to patients treated with BMT. On the other hand we expected to find some answers about pathophysiological significance of THP in these renal diseases. Excretion values of THP in healthy adult subjects obtained by electrophoresis of THP in 1% agarose gel containing antiserum to THP were within the range from 15.5 to 54.9 mg/24 h. the mean value being 36.6 mg/24 h. These results are in agreement with the data in literature In non renal adult patients excretion of THP were within the range from 3-0 to

373 45.9 mg/24 h. mean value being 18.1rag/24h. Considering excretion of THP in various renal diseases it has been observed that there is no difference between glomerular and tubulo-interstitial diseases regarding the mean value of the THP excretion. But in acute pyelonephritis and polycystic renal diseases the peak values are different. No significant difference has been found in patients with vesicouretheral reflux although it has been expected. In patients with lower urinary tract infection the mean value of THP excretion was smaller than in patients with kidney infections. However, investigation of a larger number of patients with lower urinary tract infection is necessary to draw conclusion whether excretion of THP could be useful as a diagnostic tool in differentiating between lower and upper urinary tract infections. Nephrotoxicity of cyclosporin is well-known. The major information in nephrology is derived from the kidney transplantation e.g. from patients with previously renal lesions. The excretion of THP during administration of cyclosporin in patients with previously intact kidney has not been reported in literature so far. Among 21 patients treated with BMT who received cyclosporin, high excretion of THP was observed in four of them. From these preliminary results it may be presumed that cyclosporin may affect a part of the nephron where synthesis of THP takes place. Further investigation is needed to clarify the exact meaning of this phenomenon. Investigating antibodies to THP in sera of healthy persons and patients with various renal diseases many questions arise such as whether antibodies to THP found in sera are antibodies to THP synthetized in the ascending thick limb of Henle's loop or to the THP-like proteins, and whether the presence of THP in the pericellular interstitium stimulates the production of antibodies to THP as postulated. The meaning of the presence of antibodies to THP in sera not only in patients with renal diseases but also in sera of healthy persons remains to be explained.

Conclusion Changes in the excretion of THP reflect pathological processes occurring in the ascending thick limb of Henle's loop. The exact pathobiochemical meaning of this finding has not been elucidated as yet. Results reported here indicate that the determination of excretion of THP could be usefull in detecting damaged nephrons in various renal diseases. In renal diseases with obscure pathogenesis and in the treatment with toxic drugs the measurement of THP may be helpfull in early detecting of pathological processes in the nephron. The finding of antibodies to THP in sera of healthy persons represents challenge for further investigations.

374 References 1. Hoyer.J.R.. M.W.Seiler. 1979. Pathophysiology of Tamm-Horsfall Protein. Kidney Int.16.279-289. 2. Grant.A.M.S.. L.R.I. Baker. A.Neuberger. 1973- Urinary Tamm-Horsfall glycoprotein in certain kidney diseases and its content in renal and bladder calculi. Clin.Sei.44.377-380. 3. McKenzie.J.K..R.Patel. E.G.McQueen. 1964. The Excretion Rate of Tamm-Horsfall Urinary Mucoprotein in Normals and in Patients with Renal Disease.Aust.Ann. Med.13.32-394. Cvoriscec.D.. A.Stavljenic. M.Radonic. 1985. Tamm-Horsfall Protein in Balkan Endemic Nephropathy. J.Clin.Chem.Clin.Biochem.23.177-181. 5. Cvoriscec.D.. A.Stavljenic. B.Pende. M.Cvetkovic. 1984. Tamm-Horsfall Proteindetermination and Reference Value. Acta Pharm.Jugosl. 34.43-49. 6. Sertic.J. Doprinos otkrivanju i dokazivanju antitijela protiv Tamm-Horsfallova proteina. Magistarski rad. Zagreb. 1985.

VII. RENAL KALLIKREIN-KININ-SYSTEM, DEVELOPMENTAL ASPECTS OF RENAL METABOLISM

STUDIES ON THE RENAL KALLIKREIN-KININ SYSTEM

W.G. Guder, J. Hallbach, G. Wirthensohn Inst. Clin. Chem., Bogenhausen City Hospital, Munich, FRG R. Linke Inst. Immunol. University, Munich, FRG E. Fink, W. Müller-Esterl Dept. Clin. Biochem., University, Munich, FRG

Introduction Several observations indicate that kininogenase activity found in urine is derived from renal tissue (1). Tomita et al. were the first to show that this serine protease is located in the distal nephron of rabbit kidney (2). In accordance with this finding renal kinin was found to be released into urine during passage through the distal nephron (3), indicating that urinary kinin likewise originates from renal tissue. In spite of these findings, however, the physiological role of the renal kallikrein-kinin system is far from being well understood (for review see (1)). Here we would like to summarize the present state of our studies on the localization and function of this system as studied by collaborative projects of the Sonderforschungsbereich 207 "Extracellular Limited Proteolysis" in Munich.

Methods Kininogenase activity of kallikrein was measured in single and pooled microdissected mouse nephron segments as described elsewhere (4), applying a bradykinin radioimmunoassay (5,6). Maximal activity was obtained by a combination of lyophilization,

deoxycholate and trypsin treatment of tubule segments (4). Renal

kininogen was measured by incubating homogenates of human medullary or papillary tissue or human papillary tubule suspensions with pig pancreatic kallikrein or human renal cortical homogenate in the presence of inhibitors of renal kininases. Immunohistochemical demonstration of kininogen was performed in baboon

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

378 kidney using antibodies against human plasma kininogen obtained in rabbits (7,8). Papillary tubules were prepared from rat and human kidney by collagenase treat32 ment and incubated with hormones in the presence of P i to study the effect of bradykinin on phospholipid turnover (9).

Results and Discussion Localization of k a l l i k r e i n along the mouse nephron: Kininogenase activity of microdissected mouse nephron segments was found only in the distal convoluted (DCT) and cortical collecting tubule (CCT) (Fig. 1). A time and tubular length dependence, however, revealed a large scatter of results with single segments. This disappeared when pooled samples were used (4). This indicated that v a r i a b i l i t y of results was due to large internephron heterogeneity. Therefore additional microdissection studies were performed. Distal tubule was further separated into four different segments starting from the macula densa to the straight segment of the cortical collecting tubule.

I

£

Gl

Fig. 1

PCT PR MAL CAL DCT CCT

Distribution of k a l l i k r e i n activity along the mouse nephron. Kininogenase was determined after freezing and thawing of freshly dissected tubule segments.

379 Fig. 2

Distribution of kallikrein activity in the distal convoluted tubule and cortical collecting tubule of the mouse nephron. Kininogenase was measured after lyophilization and deoxycholate treatment in single dissected tubule segments (means + SEM)

40

O

E

20

m.

m

As can be seen from Fig. 2, kininogenase activity was almost absent from the first portion of the distal convoluted tubule indicating that distal convoluted tubule cells are devoid of kallikrein. The consecutive segment, however, contained the highest kininogenase activities, amounting to 33 fmoles/mm length. Segments including branching points (CNT) had lower activities and straight segments of the consecutive collecting tubule (CCT) contained only little amounts of kallikrein. This observation indicates that kallikrein in the mouse nephron is localized exclusively in the final segment of the distal convoluted tubule next to the joining of a CCT, but absent from the straight segment following the branching points. This contrasts to findings in the rabbit nephron (2,6). Notably, the distribution patterns of connecting tubule cells (4) and adenylate cyclase activity (19) are also dissimilar in the two species. Thus our results suggest that renal kallikrein in the mouse nephron is located in the so-called granular portion of the distal convoluted tubule containing highest concentration of connecting tubule cells. Regulation of renal kininogenase activity in mouse DCT Several manipulations were shown to modify renal and urine kallikrein activity leading to the conclusion that this enzyme might be involved in renal sodium balance, renin secretion, blood flow and osmoregulation (1). In order to test the effects of various sodium and potassium loading states, which have been shown to change the connecting tubule morphology (11) and enzyme activities

380

(12), kininogenase activity was measured after 14 days of various diets in microdissected DCT segments (Fig. 3). Interestingly high sodium loads did not alter tubular kallikrein activities, unless potassium was changed simultaneously. Typically a diet poor in potassium and rich in sodium led to a significant reduction of kininogenase activity. Potassium rich diets increased tubular kallikrein activity fivefold as compared to potassium poor diet (Fig. 3). Similar observations have been made in rat and human urine in vivo, leading to the conclusion that potassium regulates renal kallikrein activities and secretion indepen-

sI

dent of the action of aldosteron and sodium load (13,14). This does not exclude a mineralocorticoid effect on kallikrein activity shown by Marchetti et al. in adrenalectomized rabbits (6) and by Overlack et al. in rats (14). The mechanism of this potassium effect seems to involve cellular hypertrophy including increased synthesis of basolateral Na + , K + -ATPase (12).

1

30

25

20

15

10

.. .

sodium

rich

potassium

poor

n=

Fig. 3

47

rich control 25

control

poor

control

rich

52

48

Effect of different dietary sodium and potassium loads on kallikrein activity in mouse DCT segments. Kininogenase was measured before (open bars) and after (hatched bars) trypsin treatment.

Localization of renal kininogen When anti-human kininogen antibodies were applied to cortical and medullary sections of baboon kidney, immunoreactivity was restricted to cortical and medullary collecting tubule cells. This finding was confirmed by studies on human medullary ti ssue and tubu1es: homoQenates of human papi1 la released kinin upon in-

381

cubation with kallikrein (Fig. 4). Kinin release was observed for only 10 min., then the kinin concentration decreased, although kininases were inhibited by the presence of o-phenantroline/EDTA and/or captopril. As shown by Marchetti et al. (15) kinin decrease was caused by a peptidase located in collecting tubule cells separate from kininase I and kininase II. Surprisingly 0,25 mmol/l HgClg completely inhibited the kinin degrading activity. Under these conditions endogenous papillary kinin release amounted to 8 - 26 pmol/mg protein. In order to exclude that the kinin precursor was derived from extratubular cells or even plasma, tubule suspensions were prepared from a human kidney papilla by collagenase treatment. By this way kinin releasing capacity was increased three- to fourfold-, confirming the tubular origin of the kinin containing substrate. In subsequent experiments it was demonstrated that the same amount of kinin was released by adding cortical homogenate from the same kidney. This kinin-liberating capacity of renal cortex was completely suppressed by the addition of aprotinin (J. Hallbach, unpublished observation). From these results we conclude that kininogen-like material is located in the cortical and medullary collecting tubule which might serve as a substrate for renal kallikrein to release kinin(s).

O

Homogenate

alone

•

Homogenate plus pig kallikrein

O

H o m o g e n a t e plus 2000 fmol kinin/ml

pancreatic brady-

SO

180 time ( m i n )

Fig. 4

Demonstration of a papillary kininogen and kininase in human kidney

Localization and mechanism of kinin action Kinin receptors have been localized in rabbit medullary collecting tubules (16) and interstitial cells (17). In order to study the mechanism of action of bradykinin, inner medullary tubules obtained from rat kidney were incubated with

382 several hormones, and phospholipid turnover was studied by measurement of Pj incorporation into different tubular phospholipid fractions (9). As can be seen from Fig. 5, bradykinin stimulated the turnover of phosphatidylinositol while vasopressin, phenylephrine and angiotensin II had no effect. This effect was independent of glucose which stimulated incorporation rates into all phospholipid fractions. Hence bradykinin most probably acts on the collecting tubule cells by the phospholipase C-mediated release of inositol polyphosphates thereby increasing cytosolic calcium concentration.

PI

PC 1500

JJà as. ,

c

'¡>

0 Q.

DPI

01

ia

TPI n. s.

1a !

ï

s î

(11) (8) (6) (5) (6)

Fig. 5

Ji

-*

Z*

FT

V?

(11) (8) (7) (5) (6)

(10) (7) (6)

(7) (6) (5)

32 Effect of hormones and glucose on P incorporation into phosphatidylinositol of papillary tubule suspension (9). PC = phosphatidylcholine, PI = phosphatidylinositol, DPI = phosphatidyl inositol-4-phosphate, TPI = phosphatidylinositol-4,5-bisphosphate.

Conclusions The present results demonstrate that the active kallikrein-kininogen-kinin-kininase system is located along the distal segments of mammalian nephron. Kallikrein is found at a crucial point where the distal convoluted tubule enters a collecting tubule. Here it seems to be regulated by dietary potassium content. Kininogen, on the other hand, is located in the subsequent nephron segment of the primate kidney. Since kallikrein, kinin (3) and kininogen (19) are present in urine, formation of kinin is supposed to occur during the passage of urine through the collec-

383 ting system and urinary tract. This would imply that kinin acts from the luminal site on its target cells. Kallikrein, but not kinin, has recently been shown to reduce sodium transport by its luminal action in bladder epithelia (18). Although the specificity of this effect has to be demonstrated, a direct tubular action of kallikrein on luminal sodium channels would be in line with the observed site of kallikrein secretion. Bradykinin, however, needs to be present at the peritubular site to act on the collecting tubule cell and renal interstitial cells. This apparent discrepancy may be explained by assuming kallikrein to be released on both sites of the connecting tubule. This assumption is supported by the finding that renal kallikrein and kinin appear in renal lymph

(1). The complex functional interrelationship between distal tubular kallikrein, plasma and tubular kininogen and renal transport and blood flow regulation, however, has to be clarified by future experiments.

Acknowledgement The authors gratefully acknowledge the help of K. Shimamoto, Saporo, Japan and F. Morel, Paris, France, for providing samples of their bradykinin antisera. The studies reported herein were supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 207, project B1, B2, B5 and C6. The technical assistance of S. von der Burg and G. Adams and the secretarial help of S. Lefrank and E. Vollrath during preparation of this manuscript are also gratefully acknowledged.

References 1. Scigli, A.G., 0.A. Carretero. 1986. Kidney Int. 29, 120-130. 2. Tomita, K., H. Endou, F. Sakai. 1981. Pflügers Arch. 389, 91-95. 3. Scigli, A.G., R. Gandolfi, O.A. Carretero. 1978. Am. J. Physiol. 234.F36-F40. 4. Guder, W.G., J. Hallbach, E. Fink, B. Kaissling, G. Wirthensohn. 1987. Biol. Chem. Hoppe Seyler, submitted. 5. Shimamoto, K., T. Ando, T. Nahao, S. Tanaka, M. Sakuma, M. Miyahara. 1978. J. Lab. Clin. Med. 91^, 721-728. 6. Marchetti, J., M. Imbert-Teboul, F. Alhenc-Gelas, J. Allegrini, J. Menand, F. Morel. 1984. Pflügers Arch. 401, 27-33.

384 7.

Müller-Esterl, W., G. Rauth, F. Lottspeich, I. Kellermann, A. Henschen. 1985. Europ. J. ßiochem. V49, 15-22.

8.

Sternberger, L.A. 1979. Immunocytochemistry. 2°^ ed. John Wiley & Sons,Inc. N.Y.

9.

Wirthensohn, G., W.G. Guder. 1986. Fresenius Z. Anal. Chem. 324, 343- 344.

10. Chabard&s, D., M. Imbert-Teboul, M. Gagnan-ßrunette, F. Morel. 1979. In: Biochemical Nephrology (W.G. Guder and U. Schmidt, eds.). H. Huber, Bern, pp 447-453. 11. Kaissling, B. 1985. Klin. Wchschr. 63, 868-876. 12. LeHir, M., B. Kaissling, U.C. Dubach. 1982. Cell Tissue Res. 224, 493-504. 13. Miller, D.H., J.G. Lindley, H.S. Margolius. 1985. Proc. Soc. Exper. Biol. Med. J80, 121-125. 14. Overlack, A., A.G. Scigli, O.A. Carretero. 1984. abstract in: "Kinin 84", Savannah. 15. Marchetti, J., F. Praddande, S. Roseau, F. Morel. This volume. 16. Tomita, K., J.J. Pisano. 1984. Am. J. Physiol. 246, F 732-737. 17. Zusman, R.M., H.R. Keiser. 1977. J. Biol. Chem. 252, 2069-2071. 18. Lewis, S.A., W.P. Alles. 1986. Proc. Natl. Acad. Sei. 83,5345-5348. 19. Weinberg, M.S., P. Azar, W.M. Trebbin, R.J. Solomon. 1985. Kidney Int. 28, — 975-981.

RELATION BETWEEN THE RENAL ANGIOTENSIN I CONVERTING ENZYME (CE) AND THE IN ACTIVATION OF KININS BY THE KIDNEY.

3. Marchetti, F. Praddaude, S. Roseau and F. Morel Laboratoire de Physiologie Cellulaire, Collège de Berthelot, 75231 Paris Cedex 05, France.

France,

11, Place

Marcelin

Introduction The exact physiological role of the renal kallikrein kinin system is not entirely clear, despite numerous studies which suggest that kinins a f f e c t electrolyte and water transport. A first approach towards a better understanding of the role of this system was to identify the nephron segment in which the kinins are formed, or more precisely the segment in which kallikrein is synthetized, and then to study the differences in its activity during various physiological states. It is now well established that kallikrein is located in the connecting tubules (I-**) and it is possible to show that the kallikrein content of these tubules changes under different physiopathological conditions (4,5). However, the intrarenal activity of kinins may depend not only on the kinin-generating mechanism (i.e., on kallikrein activity), but also on the inactivating mechanism (i.e., the kininase activity). Kininase II is one of the kininases which might play a part in kinin inactivation. This enzyme has been intensively studied, because in addition to hydrolyzing kinins, it converts angiotensin I into angiotensin II (6). Kininase II is generally referred as angiotensin I converting enzyme (CE). Although the presence of CE in the kidney is now clearly demonstrated (7), its role and its localization along the nephron are not yet fully known, in particular its role in the renal kallikrein-kinin system and its localization in the distal parts of the nephron (8). The purpose of this paper is to report the state of our research related to the contribution of CE to the kinin inactivation by the kidney and to the measurement of its activity along the nephron.

Molecular Nephrology Biochemical Aspects of Kidney Function © 1987 Walter de Gruyter & Co. • Berlin • New York - Printed in Germany

386

Materials and methods The kininase and CE activities were determined on isolated fragments of microdissected rabbit nephron. The micromethods used for these measurements have been previously reported (9). Kininase activity was quantified by determining the rate of lysyl-bradykinin hydrolysis. The technique used for measuring the CE activity was a miniaturized version of RYAN's method (10). This procedure measured the rate of hydrolysis of a tritiated tripeptide , pH|-hippuryl-glycylglycin (^H-HGG). ^H-HGG (about 20 Ci/mmole) was synthetized and tritiated as previously described (11) and kindly supplied by Dr J.P. MORGAT (CEA, Saclay, France). Captopril was a gift from SQUIBB (Paris, France). Purified CE from human kidney was generously supplied by Dr F. ALHENC-GELAS (U 36, INSERM, Paris).

Results and discussion The analysis of the distribution of kininase activity along the rabbit nephron shows that the glomerulus and the both early (proximal tubule) and terminal (medullary collecting tubule) nephron segments have a kininase activity. The kininase activity of the medullary collecting tubule (MCT) is much lower than that of the proximal tubule (9). The kininases located in the proximal tubule are probably involved in the hydrolysis of extra-renal kinins filtered by the glomerulus, whereas kininases present in the MCT might catabolize kinins formed by the kidney kallikrein at and/or beyonds its production site, the connecting tubule. To establish whether or not CE contributes to kinin inactivation by the kidney, the e f f e c t of compounds capable of inhibiting CE was investigated on kininase activity of the glomerulus, proximal convoluted tubule (PCT), pars recta (PR) and MCT. The compounds were chelating agents (since CE is a metalloenzyme) and captopril which is a powerful inhibitor

of

CE (10).The inhibitory effects obtained

with

captopril were compared to those produced on a purified CE preparation from human kidney. Chelating agents, such as EDTA and orthophenanthroline completely inhibited kininase activity of the glomerulus, PCT and PR. But, although used together and at the high concentration of 10'^M, EDTA and orthophenanthroline did not

a f f e c t kininase activity of the MCT (Table 1). Consequently, the

activity present in the MCT could not be due to CE.

kininase

387

Table 1. Effect of EDTA and orthophenanthroline (lO-^M) on the kininase activity of the glomerulus, PCT, PR and MCT. Control Glomerulus PCT PR MCT

177 + 8 428 + 2« 675 + 4 6 90+3

EDTA + o-phenanthroline 10 i 7 8 +7 14 t 10 99 i 7

The hydrolysis of lysyl-bradykinin was performed in the presence of a mixture of EDTA and orthophenanthroline at Kininase activity (mean + SEM) is expressed as femtomoles of lysyl-bradykinin hydrolyzed per minute and per millimeter of tubule. For each segment and for each experimental condition, the measurements were performed on 7 to 8 samples. The captopril e f f e c t on the kininase activity of

the glomerulus, PCT and PR is

shown in the Table 2. These three activities, totally inhibited by the chelating agents, were differently affected by captopril. Captopril failed to inhibit the activity of the glomerulus and PCT, even at the high concentration of 5.10-5m. Only the activity

of

the PR was reduced by captopril. However, this reduction was

smaller than that measured on a purified CE preparation from human kidney. For low concentrations of

captopril (5.10"°M and 5.10-7M), the inhibition of the PR

kininase activity was about one third of that obtained on a purified CE preparation. For higher concentrations (5.10-^ and 5.10-^M), this ratio increased up to 57% and 70% respectively, indicating that at these concentrations, captopril simultaneously affected CE and other kininases present in the PR.

Table 2. Effect of captopril (% inhibition) on glomerulus, PCT, PR and extract of purified CE. Captopril (M)

5.10-8

Glomerulus PCT 12,3 t 7,8 PR Purified CE 38,3 + 6,3

5.10-7

27,6 + 9,2 84,0 + 3,5

the

5.10-6

4,8 0 47,5 98,7

+ 2,3 + 0 + 10,4 + 1,2

kininase

activity

of

the

5.10- 5 5,5 0 69,7 98,9

+ 2,6 J 0 i 15,9 + 1,1

The results are expressed as percentage of inhibition. In both control and inhibited activities were measured on 6 to 8 samples for each condition.

388

Consequently, the following conclusions can be drawn from the results obtained with captopril. - Kinin hydrolysis involves different enzymes in the glomerulus, PCT and PR. - The fact that captopril had no inhibitory e f f e c t in the glomerulus or PCT, indicates that CE does not contribute to inactivate kinins in these segments. - Only CE which is present in the PR, may contribute to the kinin hydroysis in the rabbit nephron. But, this contribution is not major. It would be only of 30% about, assuming that captopril, used at low concentrations, specifically inhibits the kininase activity due to CE. However, the previous observations showing that the kininase activity in the glomerulus, PCT and MCT is not due to CE, do not preclude its presence in these parts of nephron. Indeed, the measurements of the activity performed along the rabbit nephron show that, in addition of the PR, the glomerulus and PCT have a CE activity. But, no statistically significant activity was found in the terminal nephron segments (Fig. 1). This figure also shows that the highest activity was measured in the PR. It was about b times higher than that measured in the first millimeters of PCT.

1

Gl

PCT

Pfl

TDL

CAL OCT CCT MCT

Fig.l : Distribution of CE along the rabbit nephron. In these experiments, the CE activity in the PCT was always measured in the two first millimeters that followed the glomerulus, and the activity of the PR in the first millimeters preceeding the thin ascending limb.

389 The CE activity was measured by determining the rate of hydrolysis of a synthetic tritiated substrate, hippuryl-glycylglycine (^H-HGG). So, to insure that HGG hydrolysis indeed reflects CE activity in the PCT and PR, we checked that chelating agents and low concentrations of captopril similarly inhibited the -^H-HGG hydrolysis produced by the nephron segments and that catalyzed by purified CE. Chelating agents (EDTA and orthophenanthroline at 10-^M) completely suppress the hydrolysis of HGG produced by the PCT and PR fragments and by the extract of purified CE. These activities were also similarly affected by captopril concentrations as low as lO-^M. At

the inhibition was nearly total whatever the activity

measured (Table 3). Consequently, the rate of HGG hydrolysis by the PCT and PR is an accurate reflexion of CE activity of these segments.

Table 3. Effect of captopril (% inhibition) on the CE activity of the PCT, PR and purified CE. Captopril