Caseinolytic Protease P-Mediated Degradation of Unfolded/Misfolded TUFM Restores Mitochondrial Proteostasis and Attenuates AKI

Background: Acute kidney injury (AKI) is characterized by mitochondrial dysfunction in renal tubular epithelial cells, which are particularly vulnerable to metabolic stress. The mitochondrial unfolded protein response (UPRmt) serves as a critical adaptive mechanism; however, the role of the caseinolytic protease P (ClpP) in AKI remains unclear. We hypothesized that ClpP can mitigate AKI by degrading unfolded and misfolded proteins, particularly phosphorylated mitochondrial elongation factor Tu (p-TUFM), thereby restoring mitochondrial homeostasis. Methods: In vivo, we generated an AKI mouse model through ischemia-reperfusion injury (IRI) using Clpp knockout mice (CRISPR/Cas9) and validated findings with ClpP-specific agonists and inhibitors. In vitro, primary renal tubular epithelial cells were subjected to hypoxia/reoxygenation (HR). TPE-MI fluorescence was used to detect unfolded and misfolded proteins. Results: Unfolded and misfolded proteins accumulated significantly in the mitochondria of renal tubular epithelial cells, activating the UPRmt pathway during AKI. ClpP expression in these cells was notably upregulated, and this protease effectively degraded unfolded and misfolded proteins in the mitochondria. Functional gain- and loss-of-function experiments demonstrated that Clpp gene knockdown or pharmacological inhibition exacerbated IRI, while ClpP agonist intervention significantly improved kidney injury. Mechanistic studies revealed that TUFM is a critical substrate of ClpP. When its guanosine triphosphate (GTP)-binding domain is truncated, TUFM adopted an unfolded state and accumulates abnormally in the mitochondria, impairing mitochondrial protein translation. ClpP could bind and degrade this unfolded/misfolded TUFM, thereby restoring mitochondrial protein translation capacity. Notably, p-TUFM was the primary form that determines TUFM binding and degradation by ClpP. Conclusion: During the AKI process, ClpP plays a crucial role in UPRmt by specifically degrading accumulated unfolded and misfolded proteins in the mitochondria. By degrading p-TUFM, ClpP restores mitochondrial translation function and alleviates AKI. Funding: Government Support – Non-U.S.