THE EFFECT OF CALCINEURIN INHIBITION ON THE EXPRESSION AND ACTIVATION OF RENAL ELECTROLYTE TRANSPORTERS King Yan Felice Leung A thesis submitted for the degree of Doctor of Philosophy September 2017 Neuroscience, Physiology & Pharmacology, Division of Biosciences University College London 1 Declaration I, King Yan Felice Leung confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. King Yan Felice Leung 2 Abstract Tacrolimus (FK506) is a calcineurin inhibitor (CNI), and the main immunosuppressant used in organ transplantation. It causes complications such as hypertension, hyperkalaemia, hypercalciuria and acidosis. Together with insulin resistance, dyslipidaemia and obesity, they comprise the metabolic syndrome. CNIs induce hypertension through the activation of the WNK-NCC cascade, causing an increase in sodium chloride reabsorption, and phosphorylation is an important post-translational modification that alters the activity of the members in the WNK-NCC cascade. Using quantitative phosphoproteomics and several bioinformatic techniques, a phosphoproteome profile of the renal cortices from FK506-treated mice was generated and phosphoproteins involved in renal tubular transport were identified. In this data, AKT was phosphorylated by FK506 and was suggested to act as the intermediary protein in the calcineurin-WNK cascade. In addition, ERK1/2 was also dysregulated by FK506 and was suggested to regulate NCC through the WNK4-ERK1/2 pathway. The phosphoproteome profile also revealed several FK506-dysregulated phosphoproteins that are involved in sodium, acid-base, glucose and potassium handling. CNIs have been suggested to cause hypercalciuria through a decrease in TRPV5 and calbindin-D28K expression, however, the effect of FK506 on other regulatory and transport proteins involved in calcium handling is unclear. In contrast to previous studies, FK506 did not dysregulate TRPV5 expression, but instead increased the expression of the basolateral calcium transporters, NCX1 and PMCA. Based on these findings, a novel mechanism explaining CNI-induced hypercalciuria, driven by the activation of NCC, is proposed and the relationship between sodium and calcium handling in the DCT is discussed. The effects of calcineurin inhibition on renal electrolyte transporters and their potential regulators induce salt-sensitive hypertension and hypercalciuria. The findings presented in this thesis contribute to the understanding of the underlying mechanism that governs sodium and calcium handling in the DCT and full elucidation of this molecular machinery is of interest and clinical importance. 3 Acknowledgements First of all, I would like to thank my supervisors, Dr. Stephen Benedict Walsh and Dr. Joanne Marks, for their unconditional support and patient guidance during my Ph.D. experience at the Centre for Nephrology in UCL. Their unlimited supply of encouragements allowed me to explore my ideas, which enabled me to achieve goals beyond my beliefs. I cannot stress the appreciation I have for both of them in providing the opportunities for me to work on several different projects and the chance to collaborate with different research groups, broadening my knowledge and sparking my interests in various fields of biology, and more importantly, for believing that I can get the job done. The opportunity to work with Professor Jasminka Godovac-Zimmermann and her research team gave me invaluable insights into the proteomics world. The quote, “You learn something new every day.” applied very literally and without the patient teachings of Dr. Benedetta Lombardi, this project would not have been as successful as it is now and on that note, I cannot emphasise on how appreciative I am of their help. I would also like to thank Dr. Anselm Zdebik for his detailed advice and ideas on the oocyte project. He inspired me to think innovatively and taught me many novel experimental techniques so that I could achieve the goals I originally set out. Special thanks to St. Peter’s Trust and UCL for my IMPACT studentship award, which funded and enabled me to complete these research projects. Furthermore, I would also like to thank my family for their care and willingness to answer every single one of my “emergency” phone calls, despite the time difference between London and Hong Kong. I would also like to thank Dr. Adam Dyer, Anne Kesselheim and Dr. Gregory Jacquillet for their support and ability to provide last-minute accommodations whenever I needed them. Finally, thanks must also go to the colleagues and staff from the Centre for Nephrology, for being the friendliest and most approachable group I have ever worked with; never have they hesitated or failed to answer any of my questions. 4 Abbreviations ACE Angiotensin-converting enzyme ADCY Adenylyl cyclase AKT Protein kinase B AMPK Adenosine monophosphate-activated protein kinase ANG Angiotensin AP3D1 AP-3 complex subunit delta-1 AQP2 Aquaporin-2 ASPA Animals (Scientific Procedures) Act ATP Adenosine triphosphate ATR Angiotensin II receptor AVPR2 Arginine vasopressin receptor 2 AWERB Animal Welfare and the ethical review body Broad-Complex, Tramtrack and Bric a brac-Cullin3- BCR RING-box protein 1 BK “big” K+ channel BSA Bovine serum albumin B-WNK3 Brain-WNK3 CACNA1E Calcium voltage-gated channel subunit α1E CaMK-II Calcium/calmodulin-dependent protein kinase II CaN Calcineurin CANX Calnexin CASK Calcium/calmodulin-dependent serine protein kinase CaSR Calcium-sensing receptor CD Collecting duct CD8 Cluster of differentiation 8 CDK Cyclin-dependent kinases CDPK Calcium-dependent protein kinase CK-II casein kinase II CLCN5 Hydrogen/chloride exchange transporter 5 CLCNKB Chloride voltage-gated channel Kb CNI Calcineurin inhibitor CNT Connecting tubule CUL3 Cullin-3 5 CYPIIB2 Aldosterone synthase CyA Cyclosporine A DCT Distal convoluted tubule DGKQ Diacylglycerol kinase theta DNA Deoxyribonucleic acid DTT Dithiothreitol Epilepsy, ataxia, sensorineural deafness and salt- EAST syndrome wasting renal tubulopathy EGF Epidermal growth factor EMA Epithelial membrane antigen ENaC Epithelial sodium channel eNOS Endothelial nitric oxide synthase ER Endoplasmic reticulum ERK1/2 Extracellular signal-regulated kinase 1 and 2 FGF23 Fibroblast growth factor 23 FHHt Familial hyperkalaemic hypertension FK506 Tacrolimus FKBP FK506 binding protein GSK3 Glycogen synthase kinase 3 HA Human influenza hemagglutinin HCD Higher energy collision dissociation HDL-C High-density lipoprotein cholesterol HEK 293H Human embryonic kidney 293 cells HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HSD High sodium chloride diet HSP90AB1 Heat shock protein HSP 90-β IDH Isolated dominant hypomagnesaemia IGF1 insulin-like growth factor 1 IL Interleukin IMAC Immobilised metal affinity chromatography IP Intraperitoneal IP3R Inositol 1,4,5-trisphosphate receptors IRAK2 Interleukin-1 receptor-associated kinase 2 KCC Potassium chloride cotransporter 6 KCNJ Inward-rectifying potassium channel KLHL3 Kelch-like protein 3 Klotho β-glucuronidase klotho KS-WNK1 Kidney specific-WNK1 Kv1.1 Shaker-related voltage-gated potassium channel LAT4 L-type amino acid transporter 4 LB Lysogeny Broth LCM Laser capture microdissection LC-MS/MS Liquid chromatography-mass spectrometry LKB1 Liver kinase B1 MAPK Mitogen-activated protein kinase MAPK8IP4 Mitogen-activated protein kinase 8-interacting protein 4 MDCK Madin-darby canine kidney MO25 Mouse embryo scaffolding protein mDCT Mouse distal convoluted tubule MR Mineralocorticoid receptor MS Mass spectrometry mTOR Mechanistic/mammalian Target Of Rapamycin Na+/K+-ATPase Sodium-potassium adenosine triphosphatase NaPi Sodium phosphate cotransporter NBCe1 sodium bicarbonate cotransporter 1 NCC Sodium chloride cotransporter NCX1 Sodium/calcium exchanger 1 Nedd4 E3 ubiquitin-protein ligase NFATc Cytoplasmic nuclear factor of activated T cells NHE Sodium/hydrogen exchanger NHERF Sodium/hydrogen exchanger regulatory factor NKCC Sodium chloride potassium cotransporter NKCC2 Sodium chloride potassium cotransporter 2 NLK Nemo-Like Kinase NO Nitric oxide OSR1 Oxidative stress responsive kinase 1 PBS Phosphate-buffered saline PCR Polymerase chain reaction 7 PCT Proximal convoluted tubule PI3K Phosphatidylinositol 3-kinase PKA/C Protein kinase A/C PMCA Plasma membrane Ca2+ ATPase PNST Post-nuclear supernatant PPIase Peptidylprolyl cis-trans isomerase PsHP Pseudohypoparathyroidism PTH Parathyroid hormone PTH1R Parathyroid hormone 1 receptor PTK Protein tyrosine kinase PVDF Polyvinylidene difluoride RAAS Renin-angiotensin-aldosterone system RFx[V/I] Arg-Phe-Xaa-Val/Ile RLK Receptor-like kinase RLU/s Relative light units per seconds ROMK Renal outer medullary K+ channel SLC Solute carrier family SLC41A1 Solute carrier family 41 member 1 RTK Tyrosine kinase receptor Quantitative reverse transcription polymerase chain RT-qPCR reaction R-WNK3 Renal-WNK3 RyR Ryanodine receptor SDS Sodium dodecyl sulphate Sodium dodecyl sulphate-polyacrylamide SDS-PAGE electrophoresis SERCA Sarco endoplasmic reticulum calcium ATPase 2b SGK1 Serum and glucocorticoid-regulated kinase 1 SGLT2 sodium glucose cotransporter 2 SGPP1 Sphingosine-1-phosphatase 1 SNP Single nucleotide polymorphism SPAK STE20-like proline-alanine rich kinase SPNA2 Spectrin α-chain STRAD STE20 related adapter 8 TAE Tris-acetate-EDTA TALH Thick ascending limb of the loop of Henle TBS Tris-buffered saline TEAB Triethylammonium bicarbonate TM Transmembrane TMT Tandem mass tags TNF-α