Sirt3 attenuates LPS-induced mitochondrial fission in renal tubular epithelial cells through the AMPK-Drp1 pathway.

Sepsis-associated acute kidney injury (S-AKI) is characterized by profound tubular epithelial cell injury and mitochondrial dysfunction, yet the mechanisms linking mitochondrial dynamics to renal damage remain incompletely defined. This study investigated the role of sirtuin 3 (Sirt3) in regulating mitochondrial dynamics during endotoxin-induced renal injury. Reanalysis of the kidney subset of GSE40180 identified Sirt3 downregulation and AMPK-related transcriptional changes in cecal ligation and puncture (CLP)-injured mouse kidneys. In LPS-induced AKI, Sirt3 deficiency aggravated renal dysfunction, tubular injury, oxidant-associated fluorescence, mitochondrial fragmentation, and Drp1 Ser616 phosphorylation; these findings were independently supported in a CLP model. In HK-2 cells, Sirt3 overexpression preserved mitochondrial morphology, oxygen consumption, and ATP content, whereas Sirt3 silencing exacerbated LPS-induced injury. Re-expression of Sirt3-WT, but not the catalytically inactive H248Y mutant, restored these protective effects. AMPK silencing attenuated the effects of Sirt3 overexpression on Drp1 Ser616 phosphorylation, mitochondrial morphology, respiration, and ATP content, supporting AMPK involvement in the Sirt3-associated mitochondrial phenotype. LPS increased LKB1 acetylation, whereas Sirt3 overexpression reduced it, and co-immunoprecipitation demonstrated an association between Sirt3 and LKB1. Under LPS exposure, Sirt3 silencing further increased LKB1 acetylation, and this increase was reversed by re-expression of Sirt3-WT but not by the catalytically inactive H248Y mutant. AMPK silencing strongly attenuated but did not abolish the Sirt3-associated improvements, indicating that the pathway is not strictly linear. Together, these findings support a Sirt3-dependent pathway involving LKB1 and AMPK in the regulation of Drp1 Ser616 signaling and mitochondrial integrity in experimental endotoxin-induced AKI.