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J Vis Exp. 2018 Jun 20;(136). doi: 10.3791/57718.

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells.

Author information

1
Department of Molecular and Cellular Pharmacology, University of Miami Leonard M. Miller School of Medicine; Interdisciplinary Stem Cell Institute, University of Miami Leonard M. Miller School of Medicine.
2
Interdisciplinary Stem Cell Institute, University of Miami Leonard M. Miller School of Medicine; Department of Medicine, Division of Cardiology, University of Miami Leonard M. Miller School of Medicine; Vascular Biology Institute, University of Miami Leonard M. Miller School of Medicine; Peggy and Harold Katz Family Drug Discovery Center, University of Miami Leonard M. Miller School of Medicine; lshehadeh@med.miami.edu.

Abstract

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

PMID:
29985358
PMCID:
PMC6101965
DOI:
10.3791/57718
[Indexed for MEDLINE]
Free PMC Article

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