Cellular and Mitochondrial Damage by the Antiviral agent Tenofovir in Renal Epithelial Cells

Abstract

Tenofovir disproxil fumarate (TDF) is a highly effective HIV antiviral drug approved for treating Human Immunodeficiency Virus and Hepatitis B. It is one of the first line drugs used to treat HIV and is efficacious in both novel and treatment‐experienced patients. The prodrug is administered orally and deesterified to the active drug TFV. TFV is associated with nephrotoxicity. However, the precise mechanisms for cytotoxicity remain unknown. Our hypothesis is that TFV causes mitochondrial dysfunction including diminished ATP formation and oxidative stress. HK‐2 cells were seeded and grown to confluency for 48h followed by 24‐72h exposure to 0‐28.8uM TFV. The vehicle was phosphate buffered saline (PBS). Cell viability was assessed using the MTT assay. Mitochondrial dysfunction was examined by measuring mitochondrial oxygen consumption, ATP levels, cytochrome c leakage, and by assessing the Cell Energy Phenotype using an Agilent Seahorse system. Oxidative Stress was evaluated by studying protein carbonylation and 4‐hydroxynonenal (4‐HNE) adducts by Western blot. All groups were a minimum of 3 independent studies of different cell passages. Analysis of statistical differences were performed using a One or ANOVA with a 95% confidence interval, p<0.05. TFV reduced HK‐2 cell viability at 24‐72h as shown by the MTT Assay. TFV reduced ATP levels and increased cytochrome c leakage at 72 h compared to PBS‐control. Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) were decreased following 24‐72h exposure to 28.8uM TFV. TFV induced oxidative stress as protein carbonylation and 4‐HNE adduct formation were elevated relative to PBS controls. Protein expression of LC3BI and LC3BII were compared between vehicle and TFV treated groups. TFV mediated oxidative stress, induced mitochondrial damage as indicated by decreased ATP, OCR, and ECAR, and cytochrome c leakage.