Angiotensin II Type 1 Receptor (AT1R) Changes in Animal Model of Chronic Kidney Disease: Evaluation and Pharmacotherapy By Basma Ismail Thesis submitted to the Faculty of Graduate and Posdoctoral Studies in partial fulfillment of the requirements for a doctoral degree in Cellular and Molecular Medicine. Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa © Basma Ismail, Ottawa, Canada, 2016 Basma Ismail Evaluation of AT1 receptor in chronic kidney disease ABSTRACT Cardiovascular complications represent the leading cause of death in chronic kidney disease (CKD) patients. Significant renal mass reduction induced by 5/6 subtotal nephrectomy (Nx) animal model leads to a chain of events that culminates in hypertension and CKD. The renin angiotensin (Ang) system (RAS) is known to be dysregulated, specifically Ang type 1 receptor (AT1R) plays a major role in development and progression of the disease. However, conflicting results have been reported on intrarenal AT1R levels, and the impact of antihypertensive drugs on RAS signaling is divergent. We hypothesize that PET imaging will be able to quantify kidney AT1R expression reliably in healthy and disease states. The broad objectives of this research project were: (i) to develop a positron emission tomography (PET) probe capable of detecting changes in the AT1R binding in the kidney; (ii) to elucidate the nature/temporal role of renal AT1R in Nx rat model of CKD; and (iii) to explore the predictive value of non-invasive PET imaging of AT1R to guide the use of antihypertensive therapy in preventing the progression of the disease. The novel selective AT1R PET radioligand 18 [ F]FPyKYNE-losartan was successfully used with PET in detecting renal AT1Rs at early and late stages of the CKD. The PET results correlated well with in vitro [125I]- [Sar1, Ile8]Ang II autoradiography. Over the time-course of the study (10-20 weeks), the Nx rats exhibited renal impairment, proteinuria and sustained hypertension. Echocardiography indicated the development of cardiac hypertrophy most likely secondary to the hyperdynamic circulation. These abnormalities were associated with increasing plasma and kidney levels of Ang II, and compensatory downregulation of renal AT1Rs. ACEI enalapril attenuated renal impairment, hypertension and prevented ii Basma Ismail Evaluation of AT1 receptor in chronic kidney disease progression of cardiac hypertrophy in Nx rats. This was successfully accomplished through reduction of systemic and kidney Ang II, and consequent normalization of renal AT1R as measured by PET (and autoradiography). The non-dihydropyridine CCB diltiazem also reduced blood pressure but did not normalize renal AT1R expression. Diltiazem induced elevation in Ang II levels in plasma, kidney and heart, associated with exacerbation of renal and cardiac dysfunction, and no change in AT1R renal expression. This outcome adds value to the use of [18F]FPyKYNE-losartan PET for determination of receptor abnormalities with progression of the disease and monitoring of therapy. iii Basma Ismail Evaluation of AT1 receptor in chronic kidney disease ACKNOWLEDGMENTS I would like to sincerely thank my supervisor Dr. Jean DaSilva for his mentoring, guidance, and continuous faith in my abilities to pursue and accomplish my PhD. To my co-supervisor Dr. Rob Beanlands, I am grateful for your knowledge that has helped in the interpretation of findings and for your encouragement throughout the years of my research project. I would like to extend my thanks to my TAC members Dr. Kevin Burns, Dr. Patrick Burgon and Dr. Richard Hebert for all their experienced feedbacks on my research. I would also like to acknowledge the University of Ottawa and the Ottawa Heart Institute for the funding support. I have had the privilege of working with so many wonderful people. To Drs. Stephanie Thorn and James Thackeray, I will always be grateful for your technical and academic expertise, and the constant support you provided me that greatly contributed to this work. To Marika Kolajova and Dr. Tayebeh Hadizad, thanks for your friendship and all the endless coffee breaks that made it a pleasure to come to the lab every day. The experiments in this thesis could not have been completed without the contributions of the ACVS staff, the radiochemistry lab and the imaging physics group including Keegan Flowers, Dr. Etienne Croteau, Myra Kordos, Christine Archur and Julia Lockwood; and the many members of the PET Biotesting lab over the years. I would like to especially thank Dr. Rob deKemp, for providing his expert assistance with the imaging results. Finally, I dedicate this thesis to my family: I would never have gotten this far without them. To my loving husband Mohamed, for supporting me unconditionally; my parents and sisters, who have never stopped encouraging me; and my precious kids, Ahmed and Anas who mean the world to me. iv Basma Ismail Evaluation of AT1 receptor in chronic kidney disease TABLE OF CONTENTS ABSTRACT.......................................................................................................................ii ACKNOWLEDGEMENTS.............................................................................................iv TABLE OF CONTENTS..................................................................................................v LIST OF TABLES .........................................................................................................viii LIST OF FIGURES ........................................................................................................iix LIST OF ABBREVIATIONS........................................................................................xiv CHAPTER 1: INTRODUCTION ....................................................................................1 1.0 Introduction..................................................................................................................2 1.1. Renin Angiotensin System (RAS)…..........................................................................2 1.1.1 RAS cascade ...............................................................................................2 1.1.2 Angiotensin II .............................................................................................5 1.1.3 Angiotensin II and the AT1R in Disease......................................................6 1.1.4 Angiotensin II Receptors ............................................................................7 1.2 Angiotensin II Type 1 Receptor (AT1R) ...................................................................8 1.2.1 AT1R Signaling ………................................................................................11 1.2.2 AT1R Regulation .........................................................................................14 1.2.3. AT1R and Other Effectors ...........................................................................16 1.3 Tissue RAS ….............................................................................................................16 1.3.1 Myocardial AT1R Distribution .....................................................................17 1.3.2 Renal AT1R Distribution ..............................................................................18 1.4 Role of AT1R in Cardiovascular Diseases................................................................19 1.4.1 AT1R in Myocardial Infarction and Heart Failure ………...........................20 v Basma Ismail Evaluation of AT1 receptor in chronic kidney disease 1.4.2 AT1R in Hypertension ..................................................................................21 1.5 AT1R in Diabetes ......................................................................................................22 1.6 Chronic Kidney Disease (CKD) ...............................................................................23 1.6.1 Epidemiology and Prevalence.......................................................................26 1.6.2. Etiology and Pathophysiology.....................................................................26 1.6.3. Subtotal Nephrectomy Animal Model of CKD...........................................27 1.6.4. Role of AT1R in CKD .................................................................................30 1.7. Antihypertensive Therapy .......................................................................................30 1.7.1. RAS Inhibition ............................................................................................31 1.7.2. Calcium Channel Blockers (CCBs) ............................................................32 1.8. Positron Emission Tomography (PET) ..................................................................34 1.8.1. Small Animal PET ......................................................................................37 1.8.2. Blood Flow PET Imaging............................................................................37 1.8.3. RAS Targets for PET Imaging ....................................................................38 1.8.4. AT1R PET Radioligands .............................................................................39 11 1.8.4.1. [ C]Methyl-EXP3174..............................................................................43 18 1.8.4.2. [ F]FPyKYNE-Losartan .........................................................................43 1.8.4.3. AT1R imaging in Animal Disease Models ...............................................44 1.8.5. PET AT1R Quantification ...........................................................................45