Role of the transient receptor potential canonical 6 ion channel in genetic and acquired forms of proteinuric kidney disease [Elektronische Ressource] / presented by Clemens Claudius Möller

Clemens Brentano

Sujets

• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 39
• Langue: English
• Poids de l'ouvrage: 20 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
Dipl.-Biotechnol. Clemens Claudius Möller
Born in Karlsruhe
Oral Examination: 17.10.2007



Dissertation

Role of the Transient Receptor Potential Canonical 6 ion channel
in genetic and acquired forms of proteinuric kidney disease
of the Ruperto-Carola University of Heidelberg, Germany
for the Degree of
Doctor of Natural Sciences






















Referees: Prof. Dr. Michael Wink
Prof. Dr. Jochen Reiser
Born in Karlsruhe
Oral Examination: 17.10.200 TABLE OF CONTENTS I

Table of Contents

Acknowledgments............................................................................. IV

Publications......................................................................................... V
Summary............................................................................................ VI
.
Zusammenfassung........................................................................... VII
Abbreviations................................................................................. VIII

I. INTRODUCTION............................................................................... 1

I.1. Overview of kidney structure and function...................................... 1
I.1.1. The glomerular filtration barrier............................................................ 4

I.2. Glomerular kidney disease................................................................. 6
I.2.1. Genetic forms of glomerular kidney disease......................................... 9
I.2.2. Acquired forms of glomerular kidney disease..................................... 13
I.2.3. Focal segmental glomerulosclerosis (FSGS)....................................... 13
I.2.4. Model systems for the study of glomerular kidney disease................. 15

I.3. Transient Receptor Potential (TRP) channels................................ 18
I.3.1. The TRPC subfamily of ion channels.................................................. 21
I.3.2. Transient Receptor Potential Canonical 6 (TRPC6)............................ 22

I.4. Specific aims....................................................................................... 25

Appendix Chapter I................................................................................................... 26

Moller, C. C., Pollak, M. R., & Reiser, J. (2006). The genetic basis of human
glomerular disease. Adv Chronic Kidney Dis, 13(2), 166-173.

II. TRPC6 MUTATIONS IN GENETIC FORMS
OF PROTEINURIC KIDNEY DISEASE....................................... 27

II.1. Rationale............................................................................................. 27

II.2. Materials and Methods..................................................................... 27
II.2.1. Patient families.................................................................................... 27
II.2.2. Sequence alignment............................................................................. 30
II.2.3.ite-directedmutagenesis.................................................................... 30
II.2.4. Patch-clamp electrophysiology............................................................ 31

II.3. Results................................................................................................. 31
II.3.1. Families with TRPC6-related FSGS.................................................... 31
II.3.2. Localization of mutations in the TRPC6 sequence..............................33
II.3.3. Electrophysiological analysis of mutant TRPC6 channels.................. 34

II.4. Discussion........................................................................................... 36
II.4.1. Clinical presentation of TRPC6-related FSGS.................................... 36
II.4.2. Nature of TRPC6 mutations................................................................ 37
-/-II.4.3. TRPC mice – a suitable model for TRPC-related FSGS?.................39
II.4.4. Consequences for the diagnosis of FSGS............................................ 40
TABLE OF CONTENTS II

III. TRPC6 ASSOCIATON WITH THE
GLOMERULAR FILTRATION BARRIER.................................. 41
1
III.1. Rationale............................................................................................. 41
1
III.2. Materials and Methods..................................................................... 41
III.2.1. Cell Culture.......................................................................................... 41
III.2.2. Immunohistochemistry and Immunocytochemistry............................ 41
III.2.3. RT-PCR............................................................................................... 42
III.2.4. Immunogold and Transmission Electron Microscopy......................... 42
III.2.5. Coimmunoprecipitation....................................................................... 42

III.3. Results................................................................................................. 43
III.3.1. TRPC6 expression pattern in the kidney glomerulus.......................... 43
III.3.2. TRPC6 association with the glomerular slit diaphragm...................... 44
.
III.4. Discussion........................................................................................... 48
III.4.1. Implications of the localization of TRPC6 in the glomerulus............. 48
III.4.2. Implications of the interaction of TRPC6 with podocin, nephrin....... 49

Appendix Chapters II and III...................................................................................52

Reiser, J., Polu, K. R., Moller, C. C., Kenlan, P., Altintas, M. M., Wei, C., et al.
(2005). TRPC6 is a glomerular slit diaphragm-associated channel required for
normal renal function. Nat Genet, 37(7), 739-744.

IV. TRPC6 INDUCTION IN ACQUIRED FORMS
OF PROTEINURIC KIDNEY DISEASE....................................... 53

IV.1. Rationale............................................................................................. 53

IV.2. Materials and Methods..................................................................... 53
IV.2.1. Human kidney biopsies....................................................................... 53
IV.2.2. Quantitative real-time PCR................................................................. 54
IV.2.3. Immunohistochemistry and Immunocytochemistry............................ 55
IV.2.4. Podocyte cell culture............................................................................55
IV.2.5. C5b-9 treatment................................................................................... 55
IV.2.6. Induction of passive Heymann nephritis (PHN).................................. 56
IV.2.7. I puromycin aminonucleoside (PAN) nephropathy...........56
IV.2.8. Fluo-4 calcium imaging....................................................................... 56
IV.2.9. Cytochalasin D treatment.................................................................... 57
IV.2.10. GFP-TRPC6 overexpression in vitro................................................... 57
IV.2.11. In vivo gene delivery............................................................................ 57
IV.2.12. Assessment of proteinuria and albuminuria........................................ 57

IV.3. Results................................................................................................. 58
IV.3.1. TRPC6 induction in human acquired glomerular diseases.................. 58
IV.3.2. TRPC6 induction in the C5b-9 cell culture model of
membranous disease............................................................................ 59
IV.3.3. TRPC two animal models of acquired
glomerulardisease............................................................................... 61
2+IV.3.4. OAG-induced Ca influx upon PAN-mediated podocyte injury........63
TABLE OF CONTENTS III

IV.3.5. Rapid-onset proteinuria after TRPC6 overexpression in vivo............. 63
IV.3.6. Effects of TRPC6 overexpression on the podocyte actin
cytoskeleton......................................................................................... 65

IV.4. Discussion........................................................................................... 67
IV.4.1. Dual role for TRPC6 in genetic and acquired forms of
proteinuri