Date of Award

2017

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Monica Valentovic

Second Advisor

Gary Rankin

Third Advisor

Richard Egleton

Fourth Advisor

Travis Salisbury

Fifth Advisor

Todd Green

Abstract

Tenofovir (TFV) is a reverse transcriptase inhibitor that is approved by the United States Food and Drug Administration (FDA) to treat HIV and chronic Hepatitis B. It has a long half-life, allowing for once a day dosing and is effective in treatment of both naive and experienced patients. It is administered orally as tenofovir disoproxil fumarate (TDF) and is deesterified in plasma to the active drug TFV. However, renal impairment is associated with its use; TFV can induce decreased glomerular filtration rate (GFR) and free calcitriol, renal failure, and Fanconi Syndrome. The exact mechanism of toxicity currently remains unknown, largely due to limited experimental models. The purpose of this study was to investigate the mechanisms of cytotoxicity and oxidative damage observed in HK-2 cells following treatment with TFV and to determine if managing oxidative damage mitigates toxicity. TFV is the active form of TDF and was used for all studies. HK-2 cells were grown to confluency for 48 h and then exposed to 0- 28.8 µM TFV for 24, 48, or 72 h. The vehicle used for all studies was phosphate buffered saline (PBS). TFV induces a loss of cell viability compared to the control within 24 h as shown by an MTT assay, Trypan Blue Exclusion cell counts, and lactate dehydrogenase (LDH) leakage. Oxidative stress and mitochondrial damage were assessed in whole cell lysate and different cell fractions using OxyBlot and western blot for 4-hydroxynonenol (4-HNE), tumor necrosis factor alpha (TNFα), caspase 3, 8, and 9, MnSOD, ATP Synthase, and cytochrome c and showed an increase in protein carbonylation and loss of mitochondrial membrane integrity following 72 h exposure to 28.8 µM TFV. TFV induces apoptosis at 72 h exposure as shown by western blot analysis of cytochrome c leakage and activation of caspase 3 and 9. Studies conducted using Seahorse XFp technology determined that TFV alters mitochondrial function. Studies were conducted using a 1 h pretreatment with antioxidants resveratrol, N-acetyl-L-cysteine, or ascorbic acid, and results showed protection of cell viability following 24 h exposure to 3 and 14.5 μM TFV. These studies suggest that mitochondrial damage and oxidative stress occur in HK-2 cells treated with TFV and that controlling oxidative damage may help prevent toxicity from developing. Additional knowledge of subcellular events associated with tenofovir nephrotoxicity can be used to develop clinical methods to mitigate toxicity.

Subject(s)

Reverse transcriptase -- Inhibitors -- Therapeutic use.

Drugs -- Side effects -- Research.

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