Analysis of the role of the mineralocorticoid receptor in renal principal cell sodium reabsorption in vivo [Elektronische Ressource] / presented by Caroline Ronzaud

Caro Recio

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 31
• Langue: English
• Poids de l'ouvrage: 11 Mo

Extrait

DISSERTATION


submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany

for the degree of
Doctor of Natural Sciences







Presented by
Graduate Engineer in Biotechnology Caroline Ronzaud
born in Echirolles, France



thOral-examination: 19 of March 2007













Analysis of the role of the mineralocorticoid receptor
in renal principal cell sodium reabsorption in vivo














Referees: Prof. Dr. med. Günther Schütz
Prof. Dr. Blanche Schwappach

Table of contents
TABLE OF CONTENTS
1. SUMMARY ...............................................................................................................................4
1. ZUSAMMENFASSUNG...........................................................................................................5

2. INTRODUCTION .....................................................................................................................7
2.1. The mineralocorticoid aldosterone...................................................................................7
2.2. The mineralocorticoid receptor ........................................................................................8
2.2.1. A member of the nuclear receptor family...........................................................8
2.2.2. Ligand binding specificity...............................................................................10
2.2.3. MR mutations and MR knockout mice ............................................................10
2.3. Renal control of sodium and water balance....................................................................11
2.3.1. The kidney ......................................................................................................11
2.3.2. The aldosterone-sensitive distal nephron (ASDN) ...........................................12
2.4. Regulation of renal transepithelial sodium reabsorption by aldosterone..........................14
2.4.1. The epithelial sodium channel (ENaC) ............................................................14
2.4.2. Regulation of ENaC-mediated renal sodium reabsorption by aldosterone ........15
2.5. Conditional somatic mutagenesis in mice using the Cre-loxP recombination system......17
2.6. Aims of the present study ..............................................................................................18

3. RESULTS ................................................................................................................................20
3.1. Renal principal cell-specific MR inactivation in the mouse............................................20
3.1.1. Generation of mice lacking MR in renal principal cells ...................................21
AQP2Cre3.1.2. Analysis of MR mutant mice.................................................................24
3.2. Generation of an inducible renal principal cell-specific gene inactivation system...........31
T2 3.2.1. Generation of AQP2CreER mice ..................................................................32
T23.2.2. Analysis of AQP2CreER mice......................................................................33
3.3. Identification of aldosterone-regulated genes possibly involved in the control of ENaC-
mediated renal sodium reabsorption .....................................................................................35
3.3.1. Sodium transport in mpkCCD cells is mediated by ENaC and regulated by c14
aldosterone via GR ...................................................................................................35
1 Table of contents
3.3.2. Gene expression profiling of mpkCCD cells ................................................37 c14
3.3.3. Validation of aldosterone-induced genes in microdissected CDs......................42
3.3.4. Effect of MR deficiency on the aldosterone response in vivo...........................42

4. DISCUSSION ..........................................................................................................................44
4.1. Selective inactivation of MR in mouse renal principal cells ...........................................44
AQP2Cre4.1.1. MR mice are viable and show MR deficiency in all principal cells of the
CD and late CNT ......................................................................................................44
4.1.2. The late CNT is an important site of MR-regulated ENaC-mediated sodium
reabsorption..............................................................................................................45
4.1.3. Other possible compensatory actions triggered by increased plasma aldosterone
levels ........................................................................................................................46
4.2. Generation of an inducible renal principal cell-specific gene inactivation system...........47
4.3. Identification of MR-regulated genes potentially involved in the control of principal cell
ENaC-mediated sodium reabsorption ...................................................................................49
4.3.1. Gene expression profiling of mpkCCD cells reveals aldosterone-induced c14
genes potentially involved in ENaC regulation..........................................................49
4.3.2. The regulation of SGK1 and PIM3 by aldosterone is mediated by MR in
microdissected CDs ..................................................................................................51

5. CONCLUSION AND OUTLOOK..........................................................................................53

6. MATERIALS AND METHODS.............................................................................................54
6.1. Materials .......................................................................................................................54
6.1.2. Chemicals and radioactivity ............................................................................54
6.1.3. Enzymes .........................................................................................................54
6.1.4. Bacterial strains...............................................................................................54
6.1.5. Plasmids..........................................................................................................54
6.1.6. DNA microarrays............................................................................................55
6.1.7. Buffers and solutions.......................................................................................55
6.1.8. Genomic PAC library......................................................................................58
6.1.9. mpkCCD cell line........................................................................................58 c14
2 Table of contents
6.1.10. Mouse strains ................................................................................................59
6.1.11. Probes and primers........................................................................................59
6.2. Methods.........................................................................................................................60
6.2.1. Standard technics of molecular biology...........................................................60
6.2.2. Polymerase chain reaction (PCR) ....................................................................64
6.2.3. Modification of a PAC by homologous recombination in E.coli (ET-cloning) .65
6.2.4. RNA analysis ..................................................................................................67
6.2.5. Protein analysis ...............................................................................................68
6.2.6. mpkCCD cell culture...................................................................................70 c14
6.2.7. Gene expression analysis.................................................................................71
6.2.8. Generation of transgenic mice .........................................................................72
6.2.9. Metabolic balance studies................................................................................73
6.2.10. Determination of ENaC and TSC activity......................................................74
6.2.11. Statistics........................................................................................................74

7. REFERENCES........................................................................................................................75

8. PUBLICATIONS.....................................................................................................................83

9. ABBREVIATIONS..................................................................................................................84

10. ACKNOWLEDGEMENTS...................................................................................................88

3 Summary
1.