SS-31 Attenuates Lipopolysaccharide-Induced Injury in HK-2 Renal Tubular Epithelial Cells via SIRT3-Mediated Regulation of Mitochondrial Metabolism

Background: The mitochondrial-targeting peptide Elamipretide (SS-31) has shown protective effects against sepsis-associated acute kidney injury (S-AKI). Therefore, we investigated the mechanism underlying its effects on lipopolysaccharide (LPS)-induced injury in renal tubular epithelial cells, with a focus on mitochondrial lipid metabolism and the SIRT3-CPT2 signaling pathway.Methods: Human renal proximal tubular epithelial (HK-2) cells were pretreated with different concentrations of SS-31, followed by exposure to LPS to establish an in vitro model of LPS-induced renal tubular injury. Cell viability and inflammatory cytokine levels were quantified by Cell Counting Kit-8 (CCK-8) and enzyme-linked immunosorbent assay (ELISA), respectively. Oxidative stress was determined through quantification of reactive oxygen species (ROS), malondialdehyde (MDA), and antioxidant enzyme activities. Mitochondrial membrane potential was detected using JC-1 staining, while intracellular lipid accumulation was assessed with BODIPY 493/503 staining. Fatty acid oxidation (FAO)-associated mitochondrial respiration was analyzed using a Seahorse XF Analyzer. Cell apoptosis was quantified by flow cytometry, and SIRT3 and CPT2 protein expressions were determined by Western blotting. Short hairpin RNA-mediated SIRT3 knockdown was performed to verify the mechanistic role of SIRT3.Results: SS-31 increased LPS-treated HK-2 cell viability, and the effect was more significant at a concentration of 100 μM (p < 0.01). SS-31 intervention markedly downregulated inflammatory cytokines, ROS, and MDA while restoring antioxidant defenses (p < 0.05). In addition, SS-31 reduced intracellular lipid accumulation, altered mitochondrial oxygen consumption under FAO assay conditions, and decreased apoptosis (p < 0.01). Mechanistically, SS-31 markedly upregulated SIRT3 and CPT2 (p < 0.05). The knockdown of SIRT3 attenuated the protective effects of SS-31 and reduced the expression of CPT2 (p < 0.05), suggesting that the SIRT3-dependent regulation may be involved in the protective mechanism of SS-31.Conclusion: SS-31 protects renal tubular epithelial cells against LPS-induced damage by modulating lipid metabolism and mitochondrial function through a SIRT3-dependent mechanism involving CPT2 regulation. These findings suggest its potential preventive utility and provide a mechanistic basis for further investigation in the context of S-AKI.