title: A Quantitative Approach to Screen for Nephrotoxic Compounds In Vitro.
abstract: Nephrotoxicity due to drugs and environmental chemicals accounts for significant patient mortality and morbidity, but there is no high throughput in vitro method for predictive nephrotoxicity assessment. We show that primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells rendering them desirable to use in such in vitro systems. To identify a reliable biomarker of nephrotoxicity, we conducted multiplexed gene expression profiling of HPTECs after exposure to six different concentrations of nine human nephrotoxicants. Only overexpression of the gene encoding heme oxygenase-1 (HO-1) significantly correlated with increasing dose for six of the compounds, and significant HO-1 protein deregulation was confirmed with each of the nine nephrotoxicants. Translatability of HO-1 increase across species and platforms was demonstrated by computationally mining two large rat toxicogenomic databases for kidney tubular toxicity and by observing a significant increase in HO-1 after toxicity using an ex vivo three-dimensional microphysiologic system (kidney-on-a-chip). The predictive potential of HO-1 was tested using an additional panel of 39 mechanistically distinct nephrotoxic compounds. Although HO-1 performed better (area under the curve receiver-operator characteristic curve [AUC-ROC]=0.89) than traditional endpoints of cell viability (AUC-ROC for ATP=0.78; AUC-ROC for cell count=0.88), the combination of HO-1 and cell count further improved the predictive ability (AUC-ROC=0.92). We also developed and optimized a homogenous time-resolved fluorescence assay to allow high throughput quantitative screening of nephrotoxic compounds using HO-1 as a sensitive biomarker. This cell-based approach may facilitate rapid assessment of potential nephrotoxic therapeutics and environmental chemicals.
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;
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;
authors.forename: Marc
authors.initials: M
authors.lastname: Hafner
authors.affiliation: Laboratory of Systems Pharmacology, Harvard Program in Therapeutic Sciences, Harvard Medical School, Boston, Massachusetts;
authors.forename: Jeremy L
authors.initials: JL
authors.lastname: Muhlich
authors.affiliation: Laboratory of Systems Pharmacology, Harvard Program in Therapeutic Sciences, Harvard Medical School, Boston, Massachusetts;
authors.forename: Esther Maria
authors.initials: EM
authors.lastname: Gottwald
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington;
authors.forename: Elijah
authors.initials: E
authors.lastname: Weber
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington;
authors.forename: Alenka
authors.initials: A
authors.lastname: Jaklic
authors.affiliation: Renal Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts;
authors.forename: Amrendra Kumar
authors.initials: AK
authors.lastname: Ajay
authors.affiliation: Social and Scientific Systems, Durham, North Carolina;
authors.forename: Daniel
authors.initials: D
authors.lastname: Svoboda
authors.affiliation: National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina;
authors.forename: Scott
authors.initials: S
authors.lastname: Auerbach
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington;
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Kelly
authors.affiliation: Kidney Research Institute, Department of Medicine, University of Washington, Seattle, Washington; and.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Himmelfarb
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 vvaidya@bwh.harvard.edu.
authors.forename: Vishal S
authors.initials: VS
authors.lastname: Vaidya
mesh_terms: D015415:Biomarkers / Q000032:analysis; D002478:Cells, Cultured; D051547:Heme Oxygenase-1 / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007674:Kidney Diseases / Q000139:chemically induced* / Q000201:enzymology / Q000235:genetics; D007687:Kidney Tubules, Proximal / Q000166:cytology; D018675:Toxicity Tests / Q000379:methods
chemical_list: D015415:Biomarkers; D051547:Heme Oxygenase-1
keywords: acute renal failure; nephrotoxicity; tubule cells