High Throughput Mitochondrial Nephrotoxicant Assay

描述（由申请人提供）：肾脏是药物、工业和环境化学品毒性的靶点，因为其高血流量、大量转运蛋白和依赖有氧代谢。毫不奇怪，线粒体是多种器官中化学物质的常见细胞内靶点，导致有氧代谢和ATP减少，以及细胞死亡。目前的体外肾毒性和线粒体损伤模型是不充分的，原因有很多：培养的细胞糖酵解性很强，有氧代谢很少，而且没有中等或高通量的实时代谢组学分析。因此，需要新的细胞模型和代谢组学方法来评估肾毒性和线粒体损伤。我们开发了肾近端小管细胞（RPTC）的原代培养物，这些细胞在体内表现出有氧代谢水平，不进行糖酵解，并保留了更高水平的分化功能。同时，我们有一项新技术（海马细胞外通量分析仪），可以在24孔板上实时测量细胞代谢（耗氧量和酸挤压）。本提案的长期目标是合并RPTC模型和海马技术，以开发定量高通量测定（qHTS）来测量毒物对肾脏线粒体功能的影响。第一阶段的研究有两个目标：(1)在96孔格式下优化RPTC呼吸测量的灵敏度和精度；(2)与自动成像相结合的优化代谢分析将针对临床相关的肾毒性药物和非肾毒性药物进行测试。研究的第二阶段将利用这些结果开发基于96口井的qHTS格式，并使用1400 TOXNET化合物库对其进行验证。该检测系统将识别肾毒性与机制为基础的标准，评估新药，消费品，和环境因素。

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.