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，因为其血流量高、转运蛋白众多且依赖于有氧代谢。毫不奇怪，线粒体是多个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。由于许多相同的原因，目前的肾毒性和线粒体损伤的体外模型是不够的：培养的细胞非常糖酵解，有氧代谢最少，没有中等或高通量的实时代谢组学测定。因此，需要新的细胞模型和代谢组学方法来评价肾毒性和线粒体损伤。我们已经开发了肾近端小管细胞（RPTC）的原代培养物，其表现出体内有氧代谢水平，不糖酵解，并保留较高水平的分化功能。与此同时，我们有一种新技术（海马细胞外通量分析仪），可以在24孔板中真实的测量细胞代谢（耗氧量和酸排出量）。该提案的长期目标是将RPTC模型和Seahorse技术合并，以开发定量高通量测定（qHTS）来测量毒物对肾线粒体功能的影响。拟议研究的第一阶段有两个目标：（1）将在96孔格式中优化RPTC的呼吸测量的灵敏度和精确度;（2）将针对选择的临床相关肾毒性物质和非肾毒性物质测试与自动成像集成的优化代谢测定。研究的第二阶段将使用这些结果开发基于96孔的qHTS格式，并使用1400个TOXNET化合物库对其进行验证。该检测系统将通过基于机制的标准鉴定肾毒性物质，用于评估新药、消费品和环境因子。 公共卫生相关性：拟议研究的最终结果将是一种定量的高通量检测方法，可以评估新药、消费品和环境因子对人类肾脏损伤的潜在影响。

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.