Carnosine alleviates high glucose-induced renal tubular cell pyroptosis by activating the AMPK/SIRT3/SOD2 pathway

Diabetic nephropathy (DN) is a major complication driven by inflammation and oxidative stress (OS); mitochondrial reactive oxygen species (mtROS)-activated NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome-induced pyroptosis is a key mechanism. Carnosine, notably an endogenous dipeptide with antioxidant and anti-glycation effects, has renoprotective potential but its mechanism remains unclear. High glucose (HG)-treated HK-2 cells were used as an <i>in vitro</i> model. We assessed cell viability, mtROS, and the expression of AMP-activated protein kinase (AMPK)/sirtuin 3 (SIRT3)/superoxide dismutase 2 (SOD2) and NLRP3 pathway proteins using Western blot and quantitative real-time PCR (qPCR). Pyroptotic cell death was confirmed by measuring the cleavage of gasdermin D (GSDMD) and lactate dehydrogenase (LDH) release. The roles of SIRT3 and AMPK were validated using small interfering RNA (siRNA) and a pharmacological inhibitor. Cellular adenosine triphosphate (ATP) levels were measured to assess the bioenergetic status. Carnosine reversed HG-induced decreases in cell viability and increases in mtROS. HG conditions also led to a significant depletion of cellular ATP, which was partially restored by carnosine. Mechanistically, carnosine activated the AMPK/SIRT3 axis, promoting the deacetylation and activation of SOD2. This suppressed NLRP3 inflammasome activation, evidenced by reduced levels of NLRP3, ASC, cleaved caspase-1, as well as reduced cleavage of GSDMD into its N-terminal fragment (GSDMD-N), reduced LDH release, and downstream cytokines. These protective effects were dependent on both AMPK and SIRT3. Carnosine protects renal tubular cells from HG-induced injury by alleviating mitochondrial OS and subsequent NLRP3 inflammasome-mediated pyroptosis through the activation of the AMPK/SIRT3/SOD2 signaling pathway. This activation is likely mediated by carnosine's ability to restore cellular bioenergetics.