Assays to Enhance Metabolic Phenotyping in the Kidney.

Abstract

The kidney is highly metabolically active, and injury induces changes in metabolism that can impact repair and fibrosis progression. Changes in expression of metabolism-related genes and proteins provide valuable data, but functional metabolic assays are critical to confirm changes in metabolic activity. Stable isotope metabolomics are the gold standard, but these involve considerable cost and specialized expertise. Both the Seahorse bioflux assays and substrate oxidation assays in tissues ex vivo are two relatively cost-effective assays for interrogating metabolism. Many institutions have access to Seahorse bioflux analyzers, which can easily and quickly generate data, but guidelines to enhance reproducibility are lacking. We investigate how variables (e.g. primary versus immortalized cells, time in culture) impact the data generated by Seahorse bioflux analyzers. In addition, we show the utility of ³H-palmitate, a new approach for assessing fatty acid oxidation in the kidney, in uninjured and injured kidney cortices. The ³H-palmitate substrate oxidation assays also demonstrate significant sex-dependent and strain-dependent differences in rates of fatty acid oxidation. These data should facilitate metabolic interrogation in the kidney field with enhanced reproducibility.