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Mechanistic Study of Diatrizoic Acid Induced Proximal Tubule Cytotoxicity Dakota Blake Ward

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Mechanistic Study of Diatrizoic Acid Induced Proximal Tubule Cytotoxicity Dakota Blake Ward Marshall University Marshall Digital Scholar Theses, Dissertations and Capstones 2019 Mechanistic Study of Diatrizoic Acid Induced Proximal Tubule Cytotoxicity Dakota Blake Ward Follow this and additional works at: https://mds.marshall.edu/etd Part of the Medical Cell Biology Commons, Medical Toxicology Commons, and the Reproductive and Urinary Physiology Commons MECHANISTIC STUDY OF DIATRIZOIC ACID INDUCED PROXIMAL TUBULE CYTOTOXICITY A dissertation submitted to the Graduate College of Marshall University In partial fulfillment of the requirements for the degree of Doctor of Physiology In Biomedical Sciences by Dakota Blake Ward Approved by Dr. Monica Valentovic, Committee Chairperson Dr. Gary Rankin Dr. Richard Egleton Dr. Travis Salisbury Dr. Todd Green Marshall University July 2019 APPROVAL OF THESIS We, the faculty supervising the work of Dakota Blake Ward, affirm that the dissertation, Mechanistic Study of Diatrizoic Acid Induced Proximal Tubule Cytotoxicity, meets the high academic standards for original scholarship and creative work established by the Biomedical Sciences Program and the Graduate College of Marshall University. This work also conforms to the editorial standards of our discipline and the Graduate College of Marshall University. With our signatures, we approve the manuscript for publication. ii © 2019 Dakota Blake Ward ALL RIGHTS RESERVED iii DEDICATION I dedicate this work to my incredible wife Paige. It is impossible to say where I would be without your support, understanding, and love, but it is safe to say it would not be writing a dedication page. Although it is no secret how difficult and time consuming getting a doctorate is, no one explains the burdens it puts on the people you love. I am beyond thankful for you being there for me throughout this chapter in our lives. I cannot wait to build the life we have always dreamed of together. I love you, Das. iv ACKNOWLEDGMENTS First and foremost, I want to acknowledge my advisor and mentor, Dr. Monica Valentovic. Our relationship has had its fair share of ups and downs, however, my respect and admiration for her has never wavered. I always joke and say, “if I knew half of what she has forgotten, I’d be set for life.” Dr. Valentovic sees things in people that they cannot see in themselves and I am no exception. Her recognition has driven me to be the scientist and person I am today. Dr. Valentovic is the type of person that you can always count on and I could not be happier with an advisor. Thank you, Dr. V. Also, thanks for all the snacks. I would like to extend a special thanks to Dr. Gary Rankin. My first experiences in research began in Dr. Rankin’s laboratory. Through working in his lab, I realized that research is the career path that I wanted to follow. I was a lost kid with an interest in science prior to meeting Dr. Rankin and I will be forever grateful for the opportunity. To the rest of my committee, Dr. Egleton, Dr. Salisbury, and Dr. Green, I want to thank you all individually. Dr. Egleton, thank you for being a source of great conversation and the occasional beer. Your stream of consciousness is unrivalled. Every time I eat a piece of ginger candy, I will think of you. Dr. Salisbury, thank you for being an absolute machine of a human being. Your drive and determination inspire me daily. I cannot think of a day where I was at the lab that I did not see you, late nights and weekends included. Dr. Green, a more helpful person does not exist, and I definitely cannot think of a person happier to help. You always seem to have a smile on and you never fail to put a smile on my face. Thank you all. Quickly, I want to thank Jamie Friedman. You started as a peer and colleague. Then you became a friend. Now, you’re a lifer. v Finally, I want to thank Katie Brown, better known as BJ. I owe the vast majority of the work in this dissertation to you. All the problems that I was having in the lab seemed to vanish when you became a part of our lab. I truly wish I could bring you with me on all of my future endeavors. To the countless people that impacted my life positively or negatively to lead me to where I am today, I cannot thank you enough. vi TABLE OF CONTENTS Approval of Thesis .......................................................................................................................... ii Dedication ...................................................................................................................................... iv Acknowledgments........................................................................................................................... v List of Tables .................................................................................................................................. x List of Figures ................................................................................................................................ xi Abstract ........................................................................................................................................ xiii Chapter 1: Contrast Induced Acute Kidney Injury and Direct Cytotoxicity of Iodinated Radiocontrast Media on Renal Proximal Tubule Cells................................................................... 1 Abstract ............................................................................................................................... 2 Introduction ......................................................................................................................... 2 Radiocontrast Media ........................................................................................................... 3 Renal Pathogenesis ............................................................................................................. 7 Risk Factors ...................................................................................................................... 10 Cytotoxicity....................................................................................................................... 15 Interventions for Cytotoxicity by Hydration, Drugs, and Natural Products ..................... 24 Conclusions ....................................................................................................................... 29 Chapter 2: General Methods, Materials, and Statement of Hypothesis ........................................ 30 The HK-2 Cell Model ....................................................................................................... 30 Experimental Protocol ...................................................................................................... 30 General Protocol for Western Blotting and OxyBlot® ..................................................... 32 Seahorse XFe Assays ........................................................................................................ 33 Cell and Protein Normalization ........................................................................................ 36 vii Statistical Analysis ............................................................................................................ 36 Statement of Hypothesis ................................................................................................... 37 Chapter 3: Radiocontrast Agent Diatrizoic Acid Induces Mitophagy and Oxidative Stress Via Calcium Dysregulation ................................................................................................................. 39 Abstract ............................................................................................................................. 40 Introduction ....................................................................................................................... 40 Methods............................................................................................................................. 42 Chemicals and Reagents ....................................................................................... 42 Cell Line and Diatrizoic Acid (DA) Treatment .................................................... 43 Mitochondrial and Cell Viability .......................................................................... 43 Mitochondrial Isolation ......................................................................................... 44 Oxyblot and Western Blot .................................................................................... 45 Seahorse XF Assays .............................................................................................. 46 TNFα in Cell Media and Cell Lysate .................................................................... 47 SOD Activity Assay .............................................................................................. 48 Calcium Assays ..................................................................................................... 48 Statistical Analysis ................................................................................................ 49 Results ............................................................................................................................... 49 DA Effects on Mitochondrial and Cell Viability .................................................. 49 DA Effects on Mitochondrial Function and Energy Utilization ........................... 51 DA Effects on Mitophagy ..................................................................................... 56 DA Effects on Endoplasmic Reticulum Stress ....................................................
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