Effects of vitamin D receptor activators on vascular calcification in patients with CKD Joanne Louise Laycock A thesis submitted for the degree of Doctor of Philosophy UCL Declaration I, Joanne Louise Laycock 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. 2 Abstract Chronic kidney disease (CKD) patients are at high risk of vascular calcification due to abnormal mineral metabolism, and potentially their treatment with vitamin D receptor activators (VDRAs; calcitriol, alfacalcidol and paricalcitol). The effect of VDRAs on calcification is not fully understood. This thesis compares physiological doses of VDRAs on vascular smooth muscle cell (VSMC) calcification in vessels from children and adults with CKD. The inferior epigastric artery was harvested during renal transplantation and vessel rings cultured in different calcium (Ca) and phosphorous (P) media with physiological doses of VDRAs. The Ca load and alkaline phosphatase (ALP) activity were quantified and histological analysis performed. In children’s vessel rings, calcitriol increased Ca load by 3.6 fold and ALP activity by 2.2 fold compared to vehicle in high Ca and P medium, but two distinct groups of responders and non-responders to calcitriol were noted. Alfacalcidol increased Ca load by 3.4 fold but had no effect on ALP activity and paricalcitol had no effect on Ca load or ALP activity in any vessel rings. Patient variation was observed: this was independent of dialysis status and renal diagnosis. None of the VDRAs tested affected Ca load or ALP activity in adult vessel rings. VSMCs were explanted and their dose dependent responses to calcitriol (1, 10 and 100nM) documented. 100nM calcitriol elicited the greatest upregulation in vitamin D dependent gene expression, including the vitamin D receptor (VDR) whose expression was greater in VSMCs explanted from dialysis patients than non-renal controls. Expression of two VDR isoforms (VDR-A and VDR-B) were shown by Western blot analysis. VDR-A expression as a percentage of total VDR was 30+6.5% in control VSMCs compared to 65+12% in dialysis VSMCs. VSMCs in which 100nM calcitriol activated the vitamin D response element (VDRE) luciferase, expressed 70+17% VDR-A compared to 6.8+2.5% in VSMCs with no VDRE luciferase activation. Patient variation in VDR isoform expression may contribute to the individual patient’s responses to VDRAs in both vessel rings and VSMCs. 3 Acknowledgements This thesis would not have been possible without the help, support and encouragement from my supervisors’ Dr Rukshana Shroff and Professor Cathy Shanahan. Thank you for your patient explanations, your infectious passion for science and your honest feedback (however brutal) which was much valued. I am also grateful to my master student Annelies DeMare, for being a conscientious learner who was dedicated and hard working in the lab. I’d like to thank Kids Kidney Research for funding this PhD. I am thankful to the surgeons and staff at both Great Ormond Street Hospital and the Royal Free Hospital who enabled me to obtain valuable human tissue for this research project. Thanks to all the members of the Shanahan lab for their support, and for the many friendships formed that have made this PhD an enjoyable experience. To mention just a few, Rosie was the first to welcome me to the lab and often brightened my day with her singing, or a trip to the pub. Cheers! Thanks to my qPCR guru Gosia for sharing both her wisdom and her Harry Potter boxset. In any IT emergencies, Jayanta was VERY happy to come to the rescue with his technology know how. To all the girls in the lunch club, thanks for sharing your delicious food, although I haven’t forgotten your persistence to improve my spice tolerance! Finally, thanks to Robo who provided a fine balance of banter and moral support as well as an invaluable 6th sense for locating any item in the lab. I am also grateful to friends beyond the lab who have shown encouragement and understanding, thanks to Lizzi for her ‘progress presents’ to shine some light on the long weekends spent writing up. Thank you to my parents, Mum and Derry, and Dad and Denise for their consistent support, and taking pride in my ventures even if it is unfamiliar territory to them. To quote mum, ‘I’m very impressed you’ve written so much gobbledey-gook!’ A special thanks to the newlyweds, team A&A, who kindly welcomed me into their home during the write up. It was much appreciated, if short lived, as a quick turn of events took them to lands-a-far. They are an inspiration to embrace the challenges and opportunities life presents. 4 Abbreviations 19-nor-1,25(OH)2D2 19-nor-1α, 25 dihydroxy vitamin D2 (paricalcitol) 1OHD3 1α hydroxyvitamin D3 (alfacalcidol) 1,25(OH)2D 1α, 25 dihydroxyvitamin D (calcitriol) 25OHD 25 hydroxyvitamin D ALP Alkaline phosphatase AMP Adenosine monophosphate ATP Adenosine triphosphate BMD Bone mineral disorder BMP-2 Bone morphogenetic protein 2 BSA Bovine serum albumin BSP Bone sialoprotein / Osteopontin BGP Bone gla protein / Osteocalcin Ca Calcium CaSR Calcium sensing receptor CKD Chronic kidney disease cMGP Carboxylated MGP DEPC Diethylpyrocarbonate DMSO Dimethyl sulfoxide Dp-ucMGP Dephosphorylated uncarboxylated MGP ESKD End stage kidney disease FBS Fetal bovine serum FGF23 Fibroblast growth factor 23 FGFR Fibroblast growth factor receptor GFR Glomerular filtration rate H and E Haematoxylin and eosin HBSS Ca free Hanks balanced saline solution HCl Hydrochloric acid HD Haemodialysis HGPS Hutchinson Guilford progeria syndrome MACS Myeloid angiogenic cells MGP Matrix gla protein P Phosphorous PBS Phosphate buffer saline Pi Inorganic phosphate 5 PD Peritoneal dialysis PFA Paraformaldehyde P-Np Para-nitrophenol P-Npp Para-nitrophenol phosphate PSG Penicillin, streptomycin and glutamate PTH Parathyroid Hormone RANKL Receptor activator of nuclear factor kappa-B ligand RXR Retinoid X receptor SE Standard error SLE Systemic lupus erythematosus SMA Smooth muscle α actin SMC Smooth muscle cell SMMHC Smooth muscle myosin heavy chain TNF-α Tumour necrosis factor alpha TRPV6 Transient receptor potential cation channel, subfamily V, member 6, uMGP Uncarboxylated MGP VDR Vitamin D receptor VDRA Vitamin D receptor activator VDRE Vitamin D response element VDR-A Vitamin D receptor A isoform VDR-B Vitamin D receptor B isoform VSMC Vascular smooth muscle cell 6 Table of Contents Declaration............................................................................................................ 2 Abstract ................................................................................................................. 3 Acknowledgements .............................................................................................. 4 Abbreviations ....................................................................................................... 5 Table of Contents ................................................................................................. 7 List of Figures ..................................................................................................... 13 List of Tables ...................................................................................................... 16 Chapter 1 Introduction ....................................................................................... 17 1.1 Chronic Kidney Disease is associated with increased cardiovascular risk. 17 1.2 Mineral metabolism .................................................................................... 17 1.2.1 The role of the kidney, FGF23 and PTH in Ca and P homeostasis. ............17 1.2.2 The impact of CKD on FGF-23, PTH and Ca and P homeostasis. .............22 1.3 Vascular Calcification ................................................................................. 23 1.3.1 What is vascular calcification?....................................................................23 1.3.2 Characteristics of arteriosclerosis are distinct from atherosclerosis. ..........23 1.4 The highly regulated process of vascular calcification and its risk factors in CKD. 25 1.4.1 Apoptosis of VSMCs ..................................................................................25 1.4.2 Matrix vesicle release. .................................................................................25 1.4.3 Perturbation in the level of physiological calcification inhibitors ..............26 1.4.4 Osteo / chondrocytic differentiation............................................................28 1.5 Vitamin D .................................................................................................... 31 1.5.1 Vitamin D and the systemic regulation of Ca .............................................31 1.5.2 Vitamin D metabolism ................................................................................32 1.5.3 Vitamin D Receptor (VDR): Mechanism of action and different VDR isoforms ..................................................................................................................35 1.5.4 Vitamin D plethora! ....................................................................................39 1.5.5 CKD patients are deficient in Vitamin D ....................................................40 1.5.6 Vitamin D receptor activators .....................................................................44 1.6 Direct effects of 1,25(OH)2D on VSMCs ................................................... 46 1.6.1 Vitamin D: Protective