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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.
Current Protocols in Toxicology
|August 2, 2016
Summary
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.
