Impaired mitophagy is a core driver of acute kidney injury (AKI), yet effective pharmacological strategies to restore this mitochondrial quality control mechanism remain limited. Paeoniflorin (Pae), a natural monoterpene glycoside, has shown renoprotective potential, but whether and how it regulates mitophagy remain unclear.. This study investigated the protective effects of Pae in mice subjected to bilateral renal ischaemia-reperfusion injury (IRI) and in cisplatin-treated TCMK-1 cells, with renal tubular-specific Prdx5 conditional knockout (Prdx5-CKO) mice used to determine the requirement for PRDX5. Pae treatment dose-dependently ameliorated renal tubular injury, reduced serum creatinine and BUN levels and inflammatory cytokine expression, and restored mitochondrial DNA copy number.Mechanistically, Pae upregulated PRDX5, which interacted with and negatively regulated RACK1. PRDX5 deficiency abolished the protective effects of Pae, aggravated renal injury, and further suppressed PINK1/Parkin expression. Conversely, RACK1 overexpression suppressed PINK1/Parkin and induced cellular injury, while RACK1 knockdown restored mitophagy and alleviated cisplatin-induced damage. Critically, the renoprotective effect of Pae was strictly PRDX5-dependent, as RACK1 knockdown rescued Pae efficacy in PRDX5-deficient cells. These findings demonstrate that Pae alleviates AKI by activating PINK1/Parkin-mediated mitophagy through the PRDX5/RACK1 signalling axis, identifying PRDX5 as an indispensable therapeutic target and revealing a novel mechanism for maintaining mitochondrial homeostasis in AKI.
