title: A High-Throughput Screening Assay to Identify Kidney Toxic Compounds.
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.
authors.affiliation: Laboratory of Systems Pharmacology, Harvard Program in Therapeutic Sciences, Harvard Medical School, Boston, Massachusetts.|Renal Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
authors.forename: Susanne
authors.initials: S
authors.lastname: Ramm
authors.affiliation: Laboratory of Systems Pharmacology, Harvard Program in Therapeutic Sciences, Harvard Medical School, Boston, Massachusetts.|Renal Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
authors.forename: Melanie
authors.initials: M
authors.lastname: Adler
authors.affiliation: Laboratory of Systems Pharmacology, Harvard Program in Therapeutic Sciences, Harvard Medical School, Boston, Massachusetts.|Renal Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.|Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts.
authors.forename: Vishal S
authors.initials: VS
authors.lastname: Vaidya
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D015925:Cryopreservation; D004790:Enzyme Induction / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D015200:Fluoroimmunoassay; D051547:Heme Oxygenase-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D018384:Oxidative Stress / Q000187:drug effects; D023382:Toxicity Tests, Acute / Q000295:instrumentation / Q000379:methods*; D015262:Xenobiotics / Q000633:toxicity*
chemical_list: D015415:Biomarkers; D015262:Xenobiotics; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1
keywords: HTRF; biomarker; heme oxygenase 1; high-throughput; in vitro; primary human proximal tubular epithelial cells; screening