Sex-Specific Differences in Branched Chain Amino Acid (BCAA) Catabolism and Mitochondrial Respiration in Healthy and Injured Kidneys

Background: Proximal tubule (PT) cells are highly susceptible to acute kidney injury (AKI) and undergo significant changes in cellular metabolism, directly contributing to injury. PT cells use fatty acid oxidation (FAO) and branched chain amino acid (BCAA) catabolism to generate ATP, however during AKI, both pathways are downregulated. Women and female mice are protected from AKI versus men and male mice. Our aim was to investigate BCAA catabolism in male and female human subjects and mice PT cells. Methods: Publicly available RNA-sequencing data from kidney cortex of males and female subjects with and without AKI (three per group) were used for differential gene expression and pathway enrichment analysis using Seurat and EnrichR. Primary PT cells from Bckdhbfl/fl male and female mice were infected with adenoviral GFP (control) or CRE to excise Bckdhb, which encodes a crucial subunit of BCKDH, the rate-limiting enzyme in BCAA catabolism, followed by qRT-PCR. Wildtype (WT) primary PT cells were treated with aristolochic acid I to induce injury, and BT2 to activate BCKDH and increase BCAA catabolism. All primary PT cells underwent live cell metabolic assays using a Seahorse bioanalyzer. Results: The BCAA catabolism pathway was significantly downregulated in males and females with AKI compared to healthy controls. However, more genes in the pathway were downregulated in males than females, and the downregulated genes were different between males and females. For example, BCKDHB was significantly downregulated in males but not females. To determine the specific role of Bckdhb, CRE recombination was undertaken in Bckdhbfl/fl primary PT cells, resulting in Bckdhb mRNA knockdown of 60% and 80% in male and female cells, respectively. Seahorse assays showed significant downregulation of basal respiration and mitochondrial ATP production rate in male cells but not in female cells upon Bckdhb knockdown, with no significant differences in glycolytic ATP production rate. BT2 treatment protected mitochondrial function in WT male primary PT cells treated with AAI but not female primary PT cells. Conclusion: These findings suggest that BCKDH may play a more important role in males versus females, both in healthy and injured kidneys, which may have implications for targeted therapeutic strategies for AKI. Funding: NIDDK Support - NIDDK Support, NIDDK Support