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A High-Throughput Screening Assay to Identify Kidney Toxic Compounds
Susanne Ramm1,2, Melanie Adler1,2, Vishal S Vaidya1,2,3
1Laboratory of Systems Pharmacology, Harvard Program in Therapeutic Sciences, Harvard Medical School, Boston, Massachusetts.
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.
Insights
Developing new methods to detect kidney toxicity early is crucial. This study presents two assays using primary human proximal tubular epithelial cells (HPTECs) to measure heme oxygenase 1 (HO-1) as a biomarker for drug-induced kidney damage.
Area of Science:
- Toxicology
- Cell Biology
- Biochemistry
Background:
- Drug-induced kidney toxicity is a significant clinical and economic problem.
- Current in vitro methods lack the sensitivity and throughput for predictive nephrotoxicity assessment.
- Primary human proximal tubular epithelial cells (HPTECs) offer a relevant in vitro model for kidney toxicity studies.
Purpose of the Study:
- To develop and validate sensitive, high-throughput in vitro assays for predictive nephrotoxicity screening.
- To utilize heme oxygenase 1 (HO-1) protein upregulation as a biomarker for kidney toxicant exposure in HPTECs.
- To establish methods amenable for broader application in toxicological screening.
Main Methods:
- Homogeneous time-resolved fluorescence (HTRF) assay for HO-1 detection.
- Immunofluorescence assay for HO-1 intensity and cell number quantification.
- Utilized primary human proximal tubular epithelial cells (HPTECs) as the model system.
Main Results:
- Two distinct methods (HTRF and immunofluorescence) were established to screen for increased HO-1 expression.
- Immunofluorescence assay demonstrated higher sensitivity by combining HO-1 intensity and cell number readouts.
- The developed assays are adaptable for use with other cell types.
Conclusions:
- The HTRF and immunofluorescence assays provide valuable tools for in vitro nephrotoxicity screening.
- HO-1 upregulation serves as a reliable biomarker for kidney toxicant-induced stress in HPTECs.
- These methods contribute to the development of predictive toxicological assessments, mitigating risks for patients and pharmaceutical development.
