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Phosphodiesterase 1 (Pde1) at the Crossroads of Calcium And

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Phosphodiesterase 1 (Pde1) at the Crossroads of Calcium And PHOSPHODIESTERASE 1 (PDE1) AT THE CROSSROADS OF CALCIUM AND CYCLIC NUCLEOTIDE SIGNALING IN DIABETIC NEPHROPATHY by Asim Bikash Dey A Dissertation Submitted to the Faculty of Purdue University In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy Department of Biology Indianapolis, Indiana May 2020 THE PURDUE UNIVERSITY GRADUATE SCHOOL STATEMENT OF COMMITTEE APPROVAL Dr. Simon J. Atkinson Department of Biology Dr. Mark C. Kowala Eli Lilly and Company Dr Ruben C. Aguilar Purdue University Dr. Guoli Dai Department of Biology Dr. Anthony J. Baucum II Department of Biology Approved by: Dr. Theodore R. Cummins Head of the Graduate Program 2 Dedicated to my family 3 ACKNOWLEDGMENTS I greatly appreciate Dr. Simon Atkinson at IUPUI and Dr. Mark Kowala at Eli Lilly and Company, my supervisors, for their support and encouragement throughout my graduate work in parallel to my day job. Their unrelenting support and profound belief in me have been instrumental in completing this work. Special thanks to Dr. Kowala for his flexibility that made it easier to juggle graduate work and the demands of a full-time job. I’m deeply indebted to Dr. Mark Rekhter whose unconditional support at all stages of my thesis made this a reality. Your insightful comments and input have helped me develop my critical analytical skills and help me become a better scientist. Completion of my experiments would not have been possible without your critical input, technical assistance and constant support over the last several years. I also would like to thank my committee members, Dr. Guoli Dai, Dr. Claudio Aguilar, and Dr. AJ Baucum for their invaluable comments and suggestions during this process. Special thanks to Dr Baucum for graciously agreeing to serve as a member at the last minute and for a very thorough review of the writing. Thanks to Laura Flak and Anita Sale at the Graduate school for helping me with administrative and technical issues through my time at IUPUI. I thank Leah Porras, Fang Li, Wayne Blosser, my co-workers at Lilly who has been very helpful in executing experiments. Big thanks to Dr Tao Wei, at Eli Lilly for his insightful bioinformatics help. My computational biology part would not been possible without his help. I learned a lot from him. This arduous process of earning a doctorate and writing a dissertation would not be possible without the support of my family specially my wife my daughters who put up with a husband and a father missing in action all the time during this process. Without their constant support and understanding it would not have been possible to achieve my educational goals. 4 TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................................ 8 LIST OF TABLES ........................................................................................................................ 11 ABBREVIATIONS ...................................................................................................................... 12 ABSTRACT .................................................................................................................................. 14 DIABETIC KIDNEY DISEASE: CHALLENGES AND POTENTIAL NOVEL MECHANISM …………………………………………………………………………………..15 1.1 Introduction ....................................................................................................................... 15 1.2 Risk factors for diabetic kidney disease............................................................................ 16 1.2.1 Hemodynamic factors ................................................................................................ 16 1.2.2 Metabolic factors ....................................................................................................... 18 1.3 Structural changes in diabetic kidney disease ................................................................... 19 1.4 Second messengers in chronic kidney disease .................................................................. 20 1.4.1 Cyclic nucleotide signaling in kidney ........................................................................ 21 1.4.2 PDE family ................................................................................................................ 22 1.4.3 Intracellular calcium and kidney disease ................................................................... 26 1.5 Use of computational biology in deciphering DKD ......................................................... 29 1.6 Conclusion ........................................................................................................................ 31 ROLE OF PHOSPHODIESTERASE 1 IN SYSTEMIC HEMODYNAMICS AND POTENTIAL IN DIABETIC KIDNEY DISEASE ............................................................ 33 2.1 Introduction ....................................................................................................................... 33 2.2 Methods and materials ...................................................................................................... 35 2.2.1 Animals ...................................................................................................................... 35 2.2.2 Generation of PDE proteins ....................................................................................... 36 2.2.3 Phosphodiesterase enzyme assays ............................................................................. 37 2.2.4 IC50 calculation ......................................................................................................... 37 2.2.5 Calcium-calmodulin dependent PDE enzyme assays ................................................ 38 2.2.6 PDE enzyme assays using [3H]cAMP as substrate ................................................... 38 2.2.7 PDE enzyme assays using [3H]cGMP as substrate ................................................... 38 5 2.2.8 ReninAAV db/db uNx model for renal failure .......................................................... 39 2.2.9 Rat telemetry studies .................................................................................................. 39 2.2.10 Rat Ear temperature ................................................................................................. 40 2.2.11 Histopathologic Evaluation ..................................................................................... 42 2.2.12 Isolation of rat glomeruli ......................................................................................... 42 2.2.13 Enzyme-Linked Immunosorbent Assay (ELISA) ................................................... 43 2.2.14 Urinary cAMP and cGMP measurement in rats ...................................................... 44 2.2.15 Measurement of apoptosis using Caspase-3/7 Green Detection Reagent ............... 44 2.3 Results ............................................................................................................................... 45 2.3.1 Effect of LY1 on PDE1 activity ................................................................................ 45 2.3.2 Development of rat model of vasodilation ................................................................ 45 2.3.3 LY1 lowered blood pressure in normal and spontaneously hypertensive rats .......... 45 2.3.4 LY1 attenuated disease progression in the mouse model of DKD ............................ 50 2.3.5 LY1 improved histopathology in DKD mouse model ............................................... 52 2.3.6 LY1 reduced fibrotic and inflammatory gene expression ......................................... 54 2.3.7 LY1 reduced urinary bio markers of kidney injury ................................................... 54 2.3.8 Acute effect of LY1 on cyclic nucleotides in the rat urine ........................................ 57 2.3.9 Expression analysis of major calcium channels in patients with CKD ..................... 59 2.3.10 LY1 attenuated renal cell apoptosis induced by in vitro activation of TRPC6 ....... 59 2.4 Discussion ......................................................................................................................... 67 2.4.1 Hemodynamic effects of PDE1 inhibition ................................................................. 67 2.4.2 Renal benefits of PDE1 inhibition in diabetic nephropathy ...................................... 72 2.4.3 Mechanism of PDE1 activation and reno-protective effects of PDE1 inhibition ...... 77 ROLE OF TRPC6 AND PDE1 IN THE CROSSTALK OF CALCIUM AND CYCLIC NUCLEOTIDE SIGNALING ....................................................................................... 80 3.1 Introduction ....................................................................................................................... 80 3.2 Methods and materials ...................................................................................................... 83 3.2.1 Reagents ..................................................................................................................... 83 3.2.2 Cell Culture ................................................................................................................ 83 3.2.3 Construction of recombinant plasmid and generation of TRPC6 stable cell line...... 83 3.2.4 Adenoviral construction of GCamp2 Ca sensor with hTRPC6 ................................. 84 6 3.2.5 Membrane potential assay ......................................................................................... 84 3.2.6 Measurement of Calcium
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