Kinasen in der Regulation des epithelialen Transports [Elektronische Ressource] = Kinases in the regulation of epithelial transport / vorgelegt von Madhuri Bhandaru

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• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2009
• Nombre de lectures: 12
• Langue: English

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Kinasen in der Regulation des epithelialen Transports Kinases in the regulation of epithelial transport DISSERTATION der Fakultät für Chemie und Pharmazie der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften 2009 vorgelegt von Madhuri Bhandaru

Warangal, India

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Tag der mündlichen Prüfung: 15.10.2009 Dekan: Prof. Dr. L. Wesemann 1. Berichterstatter: Prof. Dr. F. Lang 2. Berichterstatter: Prof. Dr. P. Ruth

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To my Father Sri B Siva Rao, Mother Srimati B Sujatha Father-in-law ‘Late’ Sri R Rajeshwer Rao Mother-in-law Srimati R Radha my beloved husband and rest of the family For all their support and encouragement

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS................................................................................ 1

SUMMARY.......................................................................................................... 2

ZUSAMMENFASSUNG .................................................................................... 5

INTRODUCTION ............................................................................................... 8

KIDNEY................................................................................................................................. 8

Water and electrolyte balance ............................................................................................ 8

Excretion of waste products ............................................................................................... 9

Regulation of Acid-Base balance ....................................................................................... 9

Regulation of BP ................................................................................................................ 9

Endocrine regulation and homeostasis ................................................................................. 10

Aldosterone hormone regulation of sodium and potassium............................................ 10

Parathyroid hormone and calcium and phosphate regulation .......................................... 11

Phosphate homeostasis ................................................................................................. 11

SGK1 .................................................................................................................................... 13

Target Proteins ................................................................................................................. 14

+ Role of SGK1 in Aldosterone-Dependent Na Reabsorption .......................................... 14

Mouse phenotype ............................................................................................................. 16

GSK3 .................................................................................................................................... 17

Regulation of GSK3 by the Wnt pathway........................................................................ 17

Regulation of GSK3 by other signalling pathways .......................................................... 18

GSK3 inhibition ............................................................................................................... 19

Mouse phenotype ............................................................................................................. 20

APC ...................................................................................................................................... 21

APC in WNT pathway ..................................................................................................... 21

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Mouse Phenotype ............................................................................................................. 22

SGK3 .................................................................................................................................... 23

Regulation of SGK3 kinase activity................................................................................. 23

Functions .......................................................................................................................... 23

Ion channels.................................................................................................................. 23

Carriers and Pumps ...................................................................................................... 23

Mouse phenotype ............................................................................................................. 24

AIMS OF THE STUDIES ................................................................................ 25

Rationale for the present studies .......................................................................................... 25

a) The role of PKB/SGK resistant GSK3 in renal water and electrolyte excretion as well

as steroid hormone release ............................................................................................... 25

b) SGK1 dependence of renal electrolyte balance and hormone regulation in APC min

mice .................................................................................................................................. 26

c) The role of SGK3 in renal phosphate handling............................................................ 26

MATERIALS..................................................................................................... 28

Equipment ............................................................................................................................ 28

Chemicals ............................................................................................................................. 29

Diets (Altromin, Heidenau, Germany) ................................................................................. 30

Software ............................................................................................................................... 30

Animals ................................................................................................................................ 31

METHODS......................................................................................................... 32

Collection of urine and feces................................................................................................ 32

Preparation of feces .............................................................................................................. 32

Determination of plasma, urinary and fecal concentrations................................................. 32

Blood pressure...................................................................................................................... 33

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Mineralocorticoid Treatment................................................................................................ 33

Determination of plasma volume ......................................................................................... 34

Determination of Bone Density............................................................................................ 34

In vitro experiments in SGK3 .............................................................................................. 35

Statistical analysis ................................................................................................................ 35

RESULTS........................................................................................................... 36

The role of PKB/SGK resistant GSK3 in renal water and electrolyte excretion as well as

steroid hormone release........................................................................................................ 36

SGK1 dependence of renal electrolyte balance and hormone regulation in APC min mice 47

The role of SGK3 in renal phosphate handling.................................................................... 53

DISCUSSION..................................................................................................... 58

The role of PKB/SGK resistant GSK3 in renal water and electrolyte excretion as well as

steroid hormone release........................................................................................................ 58

SGK1 dependence of renal electrolyte balance and hormone regulation in APC min mice 61

The role of SGK3 in renal phosphate handling.................................................................... 63

ABBREVIATIONS ........................................................................................... 64

REFERENCES .................................................................................................. 66

AKADEMISCHE LEHRER ............................................................................ 80

LEBENSLAUF .................................................................................................. 81

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Acknowledgements I

ACKNOWLEDGEMENTS As I stand at the threshold of earning my doctorate, I am overwhelmed when I recall all the people who have helped me get this far. First and foremost, I would like to thank my Ph.D. advisor, Professor Dr Florian Lang, for his constant support, guidance and inspiration. Professor Lang is a truly remarkable advisor who gives his students lots of new ideas and interacts closely with them. I have greatly benefited both from his excellent technical advice and from his role model as a successful researcher. I look forward to continuing my association with him in the future. Secondly, I would like to thank Prof Dr Peter Ruth for giving me the opportunity to present my dissertation at Faculty of Pharmacy and Chemistry, Eberhard Karls Universität, Tübingen. I would like to thank Dr Ferruh Artunc for his critical suggestions during my work. My graduate student career has also been enriched by interactions with several talented researchers including Dr. Adriana Magalska, Dr. Diana Sandulache, Dr. Rexhep Rexhepaj, Ciprian Sandu, Dr. Omaima Nasir and Dr. Raja Biswas. I would like to thank my fellow researchers Anand Rotte, Teresa Ackermann, Xuan Nguyen Thi, my friends Venkanna Pasham and Lalitha Biswas whose company I enjoyed a lot during my work. I would like to specially thank Dr. Daniela S Kempe for careful correction of my thesis. I am especially thankful to Elfirade Faber, Gisela Heck, Birgitta Noll and Maria Halter for their technical help and most importantly for their care and support throughtout my Ph.D work. I thank Uwe Schüler and Peter Dürr for their help with the software and instrument installations. I also would like to thank all the secretaries and administrative staff of Prof Lang. I would like to thank all my teachers, from elementary school through graduate school, for providing me with an excellent education. Most important of all, I would like to express my gratitude to my family for being an unstinting source of support and encouragement. My parents have inspired me a lot through their courage in overcoming the challenges of life, taught me the value of education and have worked hard to provide me the very best of it. They have always been there when I have needed them.

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Summary II

SUMMARY Insulin and insulin-like growth factor (IGF-1) regulate renal electrolyte excretion and the signalling includes PI3-kinase, PDK1, PKB and SGK isoforms. Thus SGK isoforms are expected to play a major role in the regulation of renal function which is evident from renal -/- phenotype ofsgk1mice. The end effect of PI3-kinase signalling is phosphorylation of GSK3 by PKB/SGK1 which results in inhibition of its activity. Accordingly, similar/parallel renal phenotype can be -/- KI expected betweensgk1 mice andgsk3mice where GSK3 is resistant to PKB/SGK1 inhibition. The first study was made to elucidate the role of GSK3 in renal electrolyte excretion and hormone release. The plasma aldosterone and corticosterone concentrations were significantly lower while 24-hour urinary aldosterone was significantly higher and urinary KIWT corticosterone tended to be higher ingsk3mice than ingsk3 mice. The possibility of KI WT reduced salt appetite due to low aldosterone was checked ingsk3 mice .Thegsk3 mice KI drank more saline over tap water whilegsk3 mice drank similar amounts of tap and saline KI water.gsk3mice display higher metabolic rate with significantly more food and fluid intake, + + fecal excretion, GFR, urinary flow rate and urinary Na , K and urea excretion with lower + plasma Na and urea concentrations and significantly higher blood pressure. + Enhanced Na excretion may at least partially be due to low aldosterone levels which + should however, not increase but decrease the renal K excretion. Thus, a renal mechanism + involving enhanced ENaC activity presumably causes enhanced renal K secretion as Lithium, an inhibitor of GSK3 is shown to downregulate ENaC expression. Higher fluid intake accounts for higher urinary flow rate but does not reflect decreased urine concentrating KI ability, as urinary osmolarity is increased ingsk3mice. Water deprivation did not abrogate the differences in urine output. GSK3 may participate in the regulation of renal tubular water transport. The unrestrained GSK3 could downregulate NO-synthase which induces thirst. The role of GSK3 in eNOS regulation may further contribute to differences in BP. The results thus KI -/- indicate that the renal phenotype ofgsk3mice is different from thesgk1 mice in several aspects, thereby suggesting a more direct role of GSK3 in renal electrolyte balance. APC fosters degradation of ß-catenin which is known to upregulate a variety of proteins responsible for tumerogenesis. It was suggested and shown that SGK1 is among the proteins that are upregulated. SGK1 expression has also been shown recently to be enhanced Min/+ in gastric glands ofapcA similar upregulation was expected in the kidneys of mice.  2