High‐Throughput Imaging of Kidney Cell Function to Elucidate Unknown Mechanisms of Antiretroviral Drug Toxicity
Authors: Pearson Adam , Haenni Dominik , Hall Andrew M. 
Published: April 2020

Globally, the World Health Organization estimates that 38 million people live with human immunodeficiency virus (HIV) infection and 257 million with hepatitis B virus (HBV). Toxicity from antiretroviral drugs is a major cause of kidney disease in these individuals. Tenofovir disoproxil fumarate (TDF) is a first line therapy for both HIV and HBV. TDF induces functional defects in the kidney proximal tubule (PT) for reasons that are unknown, partly due to a lack of appropriate experimental models to investigate this. TDF nephrotoxicity is characterized by two major clinical phenotypes: approximately 25% of patients develop defects in PT solute transport (Labarga et al., 2009); whilst 1% develop severe damage characterized by the appearance of enlarged and dysmorphic mitochondria in the PT (Hall et al., 2011). The aim of our study was to establish realistic in vitro models of TDF toxicity, to further investigate the underlying metabolic phenotypes.Experiments were performed on human‐derived PT cells (RPTEC/TERT1) grown to confluence. A high‐throughput imaging pipeline was established using automated microscopy and machine learning based image analysis. Solute transport was assessed by measuring the number/size of domes formed in the monolayer. Metabolism was assessed by antibody staining for mitochondrial morphology and a marker of autophagy (LC3).We screened a number of different treatment regimes, and identified two that produced phenotypes matching those reported in patients. Treatment with 300 μM TDF for 24 hours induced a decrease in solute transport, but without changing cell number or producing mitochondrial defects. In contrast, treatment with 500 μM TDF for 24 hours caused mitochondrial enlargement and activated autophagy. Further experiments were then performed with these treatment protocols to investigate the underlying metabolic phenotypes. TDF produced a dose dependent decrease in ATP, despite an increase in glycolysis. Baseline oxygen consumption rate (OCR) was also decreased, with a blunted response to oligomycin. However, a substantial increase in OCR occurred in response to the uncoupler FCCP. Moreover, metabolomics analysis revealed no evidence of major defects in the citric acid cycle, beta‐oxidation or urea cycle.Taken together, these findings are consistent with a partial inhibition of the mitochondrial ATP synthase (complex V). Of note, TDF is phosphorylated within cells to produce Tenofovir diphosphate (TFVpp), a structural analogue of ATP. In subsequent experiments using an established in vitro assay of complex V activity, we observed a dose dependent inhibition with TFVpp.In summary, we have developed a high‐throughput imaging pipeline of human‐derived PT cells to generate realistic in vitro models of functional TDF toxicity. Metabolic characterization of these models revealed a phenotype consistent with complex V inhibition, which might explain toxicity observed in patients, since PT solute transport in vivo is almost entirely dependent on mitochondrial ATP production.Support or Funding InformationSwiss National Science Foundation and Swiss National Centre of Competence in Research (NCCR) Kidney Control of Homeostasis.