High Throughput Mitochondrial Nephrotoxicant Assay

描述(申请人提供)：肾脏是药物、工业和环境化学品的毒性目标，因为它的高血流量、大量的转运体和对有氧代谢的依赖。不足为奇的是，线粒体是多个器官中常见的细胞内化学物质的靶标，导致有氧代谢和ATP减少，并导致细胞死亡。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充分的：培养的细胞非常糖酵解，有氧代谢最少，并且没有中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。我们已经发展了肾近端小管细胞(RPTC)的原代培养，这种细胞在体内表现出有氧代谢水平，不发生糖酵解，并保持较高水平的分化功能。同时，我们拥有一项新技术(海马氏胞外通量分析仪)，可以在24孔板中实时测量细胞代谢(氧气消耗和酸排出)。这项建议的长期目标是将RPTC模型和SeaHorse技术结合起来，开发一种定量高通量分析(QHTS)来衡量毒物对肾脏线粒体功能的影响。拟议研究的第一阶段有两个目标：(1)RPTC的呼吸测量将以96孔的形式进行灵敏度和精确度的优化；(2)与自动化成像相结合的优化代谢分析将与一些临床相关的肾毒性药物和非肾毒性药物进行测试。第二阶段的研究将利用这些结果来开发一个基于96井的qHTS格式，并用1400个TOXNET化合物文库进行验证。这一检测系统将用基于机制的标准来识别肾毒物，以评估新药、消费品和环境制剂。 与公共健康相关：拟议研究的最终结果将是一种定量的高通量分析，可以评估新药、消费品和环境制剂是否有可能对人类造成肾脏损害。

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.