Leucine-Based Catabolic Disorder of Branched Chain Amino Acids Accelerates the Progression of AKI

Background: The disorder of branched chain amino acid (BCAAs) after kidney injury has been previously reported. However, the characteristics of this metabolic abnormality and its relationship with the progression of acute kidney injury (AKI) remain unexplored. Methods: Using a bilateral renal ischemia-reperfusion (BIR) injury model in male C57BL/6 mice, we analyzed the expression of BCAAs catabolic enzymes and their transmembrane transporters through single-cell RNA sequencing, and measured BCAAs levels in primary tubular epithelial cells (pTECs) using LC-MS/MS. We also tested the effects of administration of BT2 (a BCAAs catabolism promoter), BCAAs compound and independent components (leucine, isoleucine, valine, respectively) of BCAAs on BIR-induced renal tubule injury. Alkyne-labeled leucine was used to track leucine localization and targets in pTECs. Additionally, activity-based protein profiling (ABPP) was used to identify leucine-interacting proteins in pTECs, followed by in vitro experiments to explore the potential mechanisms. Results: Our study revealed that the downregulated genes in the kidney after BIR injury were notably involved in oxidative phosphorylation, carbon metabolism, and the valine, leucine and isoleucine degradation. BCAAs catabolic enzymes and transmembrane transporters were primarily expressed in tubular epithelial cells (TECs), with significantly reduced expression after BIR injury. Besides, BCAAs levels in pTECs increased after AKI. The BCAAs catabolism promoter, BT2, alleviated BIR-induced renal tubular injury. The administration of BCAAs compound and leucine respectively worsened AKI, while the supplement of isoleucine or valine has little effect on BIR-induced renal tubular injury. Alkyne-labeled leucine primarily localized in cytoplasm of pTECs and colocalized with mitochondria. ABPP revealed leucine interacts with RHOT1, a mitochondrial autophagy-related protein. In vitro experiments showed high leucine concentrations suppressed mitophagy in pTECs. Conclusion: The downregulation of BCAAs catabolic enzymes and transmembrane transporters results in abnormal BCAAs accumulation in TECs after BIR injury. Only excessive leucine results in abnormal mitophagy via interaction with Rhot1, which aggravates the progression of AKI, suggesting the Leu-Rhot1 axis is a potential target for prevention of AKI.