Date of Award

2019

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Dr. Monica Valentovic, Committee Chairperson

Second Advisor

Dr. Gary Rankin

Third Advisor

Dr. Richard Egleton

Fourth Advisor

Dr. Travis Salisbury

Fifth Advisor

Dr. Todd Green

Abstract

Radiocontrast media (RCM) are necessary for many diagnostic procedures such as arteriography, percutaneous coronary intervention (PCI), and computed tomography. Contrast-induced acute kidney injury (CI-AKI) is the third most common cause of hospital-associated kidney damage accounting for 10-25% of cases worldwide. The mechanisms of contrast-induced renal impairment are not entirely known, but diminished renal hemodynamics, inflammatory responses, and direct cytotoxicity have been hypothesized. The purpose of this study was to investigate the mechanisms of direct cytotoxicity observed in HK-2 cells following treatment with diatrizoic acid (DA) and to determine the source of this damage. Mitochondrial function, endoplasmic reticulum (ER) stress, oxidative stress, and the role of calcium were examined in response to exposure to DA. HK-2 cells were grown to confluency for 48 hr and exposed to 0-30 mg I/mL of DA for 2, 8, or 24 hr. The vehicle used for all studies was phosphate buffered saline (PBS). Mitochondrial and cell viability were decreased within 2 and 24 hr, respectively, as shown by MTT assays and trypan blue exclusion cell counts. Mitochondrial function was monitored using an Agilent Seahorse analyzer and cell mito stress tests, cell glycolysis stress tests, mito fuel flex tests, and real-time ATP rate assays. Oxidative stress, ER stress, and mitophagy were assessed in whole cell lysate and cell fractions using OxyBlot and Western blot analysis for 4-hydroxynonenol (4-HNE), tumor necrosis factor alpha (TNFα), NADPH oxidase 4 (NOX4), manganese superoxide dismutase (MnSOD), glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), microtubule-associated proteins 1A/1B light chain 3B (LC3B), cytochrome c, and caspases 3, 4, and 12. The role of calcium in mitophagy and apoptosis was determined by pretreating HK-2 cells with various calcium modulators such as BAPTA-AM, EGTA, 2-APB, or calpeptin prior to the addition of DA. Studies conducted using Seahorse XF technology and analysis of LC3BI and II expression determined that DA alters mitochondrial function within 8 hr. MTT and calpain activity assays indicated that disruption of calcium homeostasis plays a role in DA induced cytotoxicity within 8 hr. An increase in oxidative stress and loss of mitochondrial membrane integrity was evident within 24 hr exposure to 18 mg I/mL DA. DA induces apoptosis at 24 hr exposure as shown by Western blot analysis of cytochrome c leakage and activation of caspase 3 and 12. These studies indicate that mitochondrial damage and oxidative stress occur in HK-2 cells treated with DA, and maintaining calcium homeostasis may help prevent DA-induced cytotoxicity.

Subject(s)

Acute renal failure.

Cytokines -- Research.

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