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。我们以前开发的原代培养肾近端小管细胞（RPTC），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。第一阶段提案的目标是将我们的新型相关RPTC模型与Seahorse技术相结合，以开发一种高通量检测方法，从而准确测量肾毒性。除了完成第一阶段目标中描述的目标外，我们还开发了一种化学信息学策略，其中化学相似性用于聚类分子，然后建模以定义三维空间中相似物理化学特征的潜在“毒团”。这些破坏线粒体的化学实体/毒物被预测为肾毒物。我们的阶段目标是使用TOXNET和Toxcast验证我们的综合代谢和成像分析，并使用化学信息学分析开发毒基数据库。该RPTC/Seahorse平台将识别肾毒性物质和线粒体毒性物质，并为上市公司和监管机构提供基于机制和化学的标准，用于评估和预测新药、消费品和环境因子的肾毒性和线粒体毒性，并缩短识别潜在问题化学品的总时间。商业化计划是为制药和联邦机构提供这些筛选和化学信息服务。 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. We previously 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. The goal of the Phase I proposal was to merge our novel and relevant RPTC model and the Seahorse technology to develop a high-throughput assay to accurately measure nephrotoxicity. In addition to completing the objectives described in the Phase I aims, we also developed a cheminformatic strategy in which chemical similarity is used to cluster molecules that are then modeled to define a potential "toxicophore" of similar physicochemical features in 3-dimesional space. These chemical entities/toxicophores that damage mitochondria are predicted to be nephrotoxicants. Our goals for Phase are to validate our integrated metabolic and imaging assay using the TOXNET & Toxcast land to use cheminformatic analyses to develop a toxicophore database. This RPTC/Seahorse platform will identify nephrotoxicants and mitochondrial toxicants and provide public companies and regulatory agencies with mechanism and chemical-based criteria for assessing and predicting nephrotoxicity and mitochondrial toxicity of new drugs, consumer products, and environmental agents, and shorten the overall time to identify potential problem chemicals. The commercialization plan is to offer these screening and cheminformatic services to pharmaceutical & federal agencies. 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.