Macrophage ACOX3-Associated Fatty Acid Oxidation Dysfunction Promotes Ferroptosis-Related Tubular Injury in Renal Ischemia-Reperfusion Injury

Renal ischemia–reperfusion injury (RIRI) is a major cause of acute kidney injury and impaired graft recovery after kidney transplantation. Although tubular epithelial injury is central to RIRI, the contribution of immune-cell metabolic remodeling to ferroptosis-related tissue damage remains incompletely understood. Integrated spatial and single-cell transcriptomic analyses revealed broad, cell type–biased remodeling of fatty acid metabolism during early RIRI and, together with network-based prioritization, highlighted marked ACOX3 downregulation in macrophages. In hypoxia/reoxygenation (HR)-treated RAW264.7 macrophages, ACOX3 overexpression preserved fatty acid oxidation, limited lipid accumulation and lipid peroxidation, maintained mitochondrial function, and supported SLC7A11/GPX4 expression, whereas ACOX3 silencing produced the opposite phenotype. Ferrostatin-1 partially rescued several injury-related changes, supporting the involvement of ferroptosis-related mechanisms. ACADM knockdown partially weakened the protective effects associated with ACOX3 overexpression, indicating downstream metabolic involvement. Transwell co-culture and macrophage-conditioned medium experiments further showed that macrophage ACOX3 status modulated ferroptosis-related injury in HK-2 cells through non-contact paracrine signaling. Virtual screening identified Isoginkgetin as a candidate ACOX3-binding compound, and CETSA and SPR supported target engagement and direct binding. Isoginkgetin attenuated ferroptosis-related stress in HR-treated macrophages and reduced renal dysfunction, tubular injury, and ferroptosis-related oxidative stress in a mouse RIRI model. Collectively, these findings identify macrophage ACOX3-associated fatty acid oxidation dysfunction as a contributor to RIRI and support Isoginkgetin as a candidate intervention requiring further target-specific and genetic validation.