A High-Throughput Screening Assay to Identify Kidney Toxic Compounds

Curr Protoc Toxicol. 2016 Aug 1;69:9.10.1-9.10.26. doi: 10.1002/cptx.12.

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.

Keywords: HTRF; biomarker; heme oxygenase 1; high-throughput; in vitro; primary human proximal tubular epithelial cells; screening.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Biomarkers / metabolism
  • Cells, Cultured
  • Cryopreservation
  • Enzyme Induction / drug effects
  • Fluorescence Resonance Energy Transfer
  • Fluoroimmunoassay
  • Heme Oxygenase-1 / chemistry
  • Heme Oxygenase-1 / genetics
  • Heme Oxygenase-1 / metabolism
  • High-Throughput Screening Assays*
  • Humans
  • Kidney Tubules, Proximal / cytology
  • Kidney Tubules, Proximal / drug effects*
  • Kidney Tubules, Proximal / immunology
  • Kidney Tubules, Proximal / metabolism
  • Kinetics
  • Oxidative Stress / drug effects
  • Toxicity Tests, Acute / instrumentation
  • Toxicity Tests, Acute / methods*
  • Xenobiotics / toxicity*

Substances

  • Biomarkers
  • Xenobiotics
  • HMOX1 protein, human
  • Heme Oxygenase-1