Differential Alteration of Branched-Chain Amino Acid Catabolism in Mouse Primary Proximal Tubules

Background: Acute kidney injury (AKI) disrupts key metabolic pathways in proximal tubule (PT) cells, including fatty acid oxidation (FAO) and branched-chain amino acid (BCAA) catabolism, both essential for energy production. Our previous results showed pharmacological activation of BCAA catabolism could protect against nephrotoxic AKI in male mice. Females are more resistant to AKI than males, and RNA-seq analysis of human AKI patients revealed sex-specific differences in BCAA catabolic enzyme expression. Our aim was to investigate the alteration of BCAA catabolism in nephrotoxin-treated primary PT cells from male and female mice in vitro. Methods: Mouse primary PT cells were isolated from wild-type C57Bl/6 littermates and treated with aristolochic acid I (AAI) to induce injury, with or without the BCAA catabolism activator BT2. Injury markers and BCAA genes and transporters were analyzed by qRT-PCR, mitochondrial respiration by Seahorse assay, intracellular BCAA levels by BCAA-Glo, and mTORC1 signaling by Western blot. Leucine treatment effects on injury markers and mTORC1 activation were also tested. Results: AAI induced injury in both sexes, with slightly higher Krt20 expression in males. Cell cycle arrest markers Cdkn1a and Ccng1 increased in both sexes but were more elevated in females. BCAA catabolic genes were downregulated after AAI, with baseline expression of known sex-dependent genes Oxct1, Mut, Ivd, Auh and Acadm higher in females. BCAA transporter genes were downregulated to similar levels in both sexes after AAI treatments. AAI reduced mitochondrial respiration similarly in both sexes, but BT2 improved respiration in males only. In male cells, AAI resulted in BCAA accumulation and mTORC1 activation. In female cells, BCAA levels were lower at baseline and did not accumulate after AAI, and mTORC1 signaling was not activated. Leucine treatments increased mTORC1 signaling pathway in male cells but not female cells. Conclusion: These findings suggest a sex-specific role of BCAA catabolism in the pathophysiology of AKI. The differential impact of BCAA catabolism on cellular respiration, mTORC1 signaling, BCAA accumulation and response to therapeutic intervention with BT2 highlights the importance of considering sex as a biological variable in the study and treatment of AKI. Funding: NIDDK Support, Private Foundation Support