A High-Throughput Screening Assay to Identify Kidney Toxic Compounds

Current protocols in toxicology Pub Date : 2018-08-06 DOI:10.1002/cptx.12

Susanne Ramm, Melanie Adler, Vishal S. Vaidya

{"title":"A High-Throughput Screening Assay to Identify Kidney Toxic Compounds","authors":"Susanne Ramm, Melanie Adler, Vishal S. Vaidya","doi":"10.1002/cptx.12","DOIUrl":null,"url":null,"abstract":"<p>Kidney toxicity due to drugs and chemicals poses a significant health burden for patients and a financial risk for pharmaceutical companies. However, currently no sensitive and high-throughput in vitro method exists for predictive nephrotoxicity assessment. Primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells, making them a desirable model to use in in vitro screening systems. Additionally, heme oxygenase 1 (HO-1) protein expression is upregulated as a protective mechanism during kidney toxicant-induced oxidative stress or inflammation in HPTECs and can therefore be used as a biomarker for nephrotoxicity. In this article, we describe two different methods to screen for HO-1 increase: A homogeneous time resolved fluorescence (HTRF) assay and an immunofluorescence assay. The latter provides lower throughput but higher sensitivity due to the combination of two readouts, HO-1 intensity and cell number. The methods described in the protocol are amendable for other cell types as well. © 2016 by John Wiley & Sons, Inc.</p>","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"69 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2018-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cptx.12","citationCount":"17","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols in toxicology","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cptx.12","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}

引用次数: 17

Abstract

Kidney toxicity due to drugs and chemicals poses a significant health burden for patients and a financial risk for pharmaceutical companies. However, currently no sensitive and high-throughput in vitro method exists for predictive nephrotoxicity assessment. Primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells, making them a desirable model to use in in vitro screening systems. Additionally, heme oxygenase 1 (HO-1) protein expression is upregulated as a protective mechanism during kidney toxicant-induced oxidative stress or inflammation in HPTECs and can therefore be used as a biomarker for nephrotoxicity. In this article, we describe two different methods to screen for HO-1 increase: A homogeneous time resolved fluorescence (HTRF) assay and an immunofluorescence assay. The latter provides lower throughput but higher sensitivity due to the combination of two readouts, HO-1 intensity and cell number. The methods described in the protocol are amendable for other cell types as well. © 2016 by John Wiley & Sons, Inc.

Abstract Image

查看原文本刊更多论文

鉴别肾毒性化合物的高通量筛选试验

药物和化学品引起的肾毒性给患者带来了巨大的健康负担，也给制药公司带来了财务风险。然而，目前还没有一种灵敏、高通量的体外肾毒性预测评估方法。原代人近端小管上皮细胞(HPTECs)具有分化上皮细胞的特征，使其成为体外筛选系统中理想的模型。此外，血红素加氧酶1 (HO-1)蛋白表达上调是肾毒物诱导的氧化应激或炎症过程中的保护机制，因此可以用作肾毒性的生物标志物。在本文中，我们描述了筛选HO-1增加的两种不同方法:均匀时间分辨荧光(htf)测定和免疫荧光测定。后者提供较低的吞吐量，但由于两个读数，HO-1强度和细胞数量的组合，灵敏度更高。协议中描述的方法也适用于其他细胞类型。©2016 by John Wiley &儿子,Inc。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Current protocols in toxicology

自引率

0.00%

发文量