Disruption of Branched-Chain Amino Acid (BCAA) Catabolism in Proximal Tubule Cells Impairs Mitochondrial Function and Induces Injury

Background: Branched-chain amino acids (BCAAs) catabolism is increasingly acknowledged as a key factor in the metabolic disturbances associated with kidney diseases. Targeting BCAA catabolism through the branched-chain alpha-ketoacid dehydrogenase complex (BCKDH), the enzyme responsible for the rate-limiting step in this pathway, represents a valuable strategy for understanding the BCAAs metabolic dysfunction underlying kidney pathologies, including acute kidney injury (AKI), and addresses an important gap in the field. Methods: A mouse model with floxed Bckdhb (a key subunit of BCKDH) on a C57BL/6J background was generated using CRISPR/Cas9-mediated genome editing. Primary proximal tubule cells were isolated from 3.5- to 4-week-old male pups and cultured until 85-95% confluency. Cells were then transduced with adenovirus-Cre (50 MOI) to induce Bckdhb knockdown, with adenovirus-GFP (50 MOI) serving as a control. Mitochondrial function was evaluated using Seahorse MitoStress test. In parallel, quantitative PCR was performed to assess the expression of injury and inflammation markers associated with Bckdhb knockdown. Results: Quantitative PCR of RNA from adenovirus-Cre–treated cells confirmed successful knockdown of the Bckdhb gene without compensatory changes in Bckdha levels. Functionally, Bckdhb knockdown impaired mitochondrial respiration, with significant reductions observed in ATP-linked respiration, basal respiration, and spare respiratory capacity. In parallel, expression of injury markers, including connective tissue growth factor (Ctgf), vimentin (Vim), and kidney injury molecule-1 (Havcr1), were significantly elevated. Additional markers, such as Cdkn1a (p21), Krt20 (Cytokeratin-20), and Tgfb, also trended upward. Notably, expression of pro-inflammatory cytokines Tnfa and Il6 remained unchanged compared to controls. Conclusion: Proximal tubule-specific knockdown of Bckdhb disrupts mitochondrial bioenergetics and induces injury-associated gene expression without triggering a strong inflammatory response. These findings highlight the critical role of BCAA catabolism in maintaining renal mitochondrial function and epithelial cell health, and suggest that dysregulation of this pathway may contribute to the development or progression of kidney injury. Funding: NIDDK Support - NIDDK Support, NIDDK Support