High Throughput Mitochondrial Nephrotoxicant Assay

DESCRIPTION (provided by applicant): The kidney is a target of toxicity from drugs, and industrial and environmental chemicals because of its high blood flow, numerous transporters, and reliance on aerobic metabolism. Not surprisingly, mitochondria are a common intracellular target of chemicals in multiple organs, leading to decreased aerobic metabolism and ATP, and cell death. Current in vitro models of nephrotoxicity and mitochondrial damage are inadequate for many of the same reasons: cultured cells are very glycolytic with minimal aerobic metabolism, and there are no moderate or high-throughput real-time metabolomic assays. Consequently, new cellular models and metabolomic methodologies are needed to evaluate nephrotoxicity and mitochondrial damage. We have developed primary cultures of renal proximal tubular cells (RPTC) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions. In conjunction, we have a new technology (Seahorse Extracellular Flux Analyzer) to measure cell metabolism (oxygen consumption and acid extrusion) in real time in 24-well plates. The long-term goal of this proposal is to merge the RPTC model and the Seahorse technology to develop a quantitative high-throughput assay (qHTS) to measure the effects of toxicants on renal mitochondrial function. Phase I of the proposed research has two aims: (1) respiratory measurements for RPTC will be optimized for sensitivity and precision in a 96-well format; (2) the optimized metabolic assay integrated with automated imaging will be tested against a selection of clinically relevant nephrotoxicants and non-nephrotoxicants. Phase II of the research will use these results to develop a 96-well based qHTS format and validate it with 1400 TOXNET compound library. This assay system will identify nephrotoxicants with mechanism- based criteria for assessment of new drugs, consumer products, and environmental agents. PUBLIC HEALTH RELEVANCE: The final results of the proposed research will be a quantitative high-throughput assay that can assess new drugs, consumer products, and environmental agents for their potential to cause kidney damage in humans.