A Podocyte-Based Automated Screening Assay Identifies Protective Small Molecules
A Podocyte-Based Automated Screening Assay Identifies Protective Small Molecules
复制标题
DOI：
10.1681/asn.2014090859
复制
发表时间：
2015-11-01
影响因子：
13.6
通讯作者：
Gupta, Vineet
中科院分区：
文献类型：
--
作者：
Lee, Ha Won;Khan, Samia Q.;Gupta, Vineet
足细胞损伤和丢失标志着各种肾小球疾病发病机制的早期阶段，使这些细胞成为治疗的绝佳靶标。然而，目前缺乏用于合理开发足细胞导向疗法的基于细胞的高通量筛选分析。在这里，我们描述了一种新型的基于高内涵筛选的表型测定，该测定在 96 孔板中的每种测定条件下分析数千个足细胞，以定量测量多种细胞特征的剂量依赖性变化。我们的测定始终产生 >0.44 的 Z' 值，使其适合化合物筛选。通过筛选超过 2100 种药理活性药物，我们鉴定出 24 种小分子可以在体外保护足细胞免受损伤（命中率 1%）。在已确定的命中中，我们确认了 β1-整合素激动剂 Pyrintegrin 作为足细胞保护剂。 Pyrintegrin 治疗可防止损伤引起的 F-肌动蛋白应力纤维、粘着斑和培养细胞中活性 β1-整合素水平的降低。在体内，给予pyrintegrin可以保护小鼠免受LPS诱导的足细胞足突消失和蛋白尿的影响。对小鼠肾小球的分析表明，LPS 给药降低了足细胞中活性整合素的水平，而与 Pyrintegrin 共同处理可以防止这种情况发生。在大鼠中，pyrintegrin 可降低由嘌呤霉素氨基核苷诱发的肾病引起的蛋白尿峰值。我们的研究结果将pyrintegrin确定为潜在的治疗候选者，并展示了基于足细胞的筛选测定法用于识别蛋白尿性肾病的新疗法的用途。
Podocyte injury and loss mark an early step in the pathogenesis of various glomerular diseases, making these cells excellent targets for therapeutics. However, cell-based high-throughput screening assays for the rational development of podocyte-directed therapeutics are currently lacking. Here, we describe a novel high-content screening-based phenotypic assay that analyzes thousands of podocytes per assay condition in 96-well plates to quantitatively measure dose-dependent changes in multiple cellular features. Our assay consistently produced a Z' value >0.44, making it suitable for compound screening. On screening with >2100 pharmacologically active agents, we identified 24 small molecules that protected podocytes against injury in vitro (1% hit rate). Among the identified hits, we confirmed an beta 1-integrin agonist, pyrintegrin, as a podocyte-protective agent. Treatment with pyrintegrin prevented damage-induced decreases in F-actin stress fibers, focal adhesions, and active beta 1-integrin levels in cultured cells. In vivo, administration of pyrintegrin protected mice from LPS-induced podocyte foot process effacement and proteinuria. Analysis of the murine glomeruli showed that LPS administration reduced the levels of active integrin in the podocytes, which was prevented by cotreatment with pyrintegrin. In rats, pyrintegrin reduced peak proteinuria caused by puromycin aminonucleoside-induced nephropathy. Our findings identify pyrintegrin as a potential therapeutic candidate and show the use of podocyte-based screening assays for identifying novel therapeutics for proteinuric kidney diseases.