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

Content Provider	Europe PMC
Author	Ding, Wen Yousefi, Keyvan Shehadeh, Lina A.
Copyright Year	2018
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.
Journal	Journal of Visualized Experiments : JoVE
PubMed Central reference number	PMC6101965
Issue Number	136
PubMed reference number	29985358
e-ISSN	1940087X
DOI	10.3791/57718
Language	English
Publisher	MyJove Corporation
Publisher Date	2018-06-20
Access Restriction	Open
Rights License	This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License. To view a copy of this license, visithttp://creativecommons.org/licenses/by/3.0/us/ Copyright © 2018, Journal of Visualized Experiments
Subject Keyword	Biology Issue 136 Primary cell isolation primary cell characterization tubular epithelial cells kidney bioenergetics mitochondria respiration high throughput extracellular flux analysis fatty acid oxidation chronic kidney disease
Content Type	Text
Resource Type	Article
Subject	Immunology and Microbiology Chemical Engineering Neuroscience Biochemistry, Genetics and Molecular Biology