High glucose promotes O-GlcNAcylation of ACSL4 to induce ferroptosis of renal tubular epithelial cell

O-linked N-acetylglucosamine (O-GlcNAc) glycosylation represents a prevalent post-translational modification of proteins. Accumulating evidence indicates that dysregulated O-GlcNAcylation can induce glucose toxicity and plays critical roles in the pathogenesis of diabetic nephropathy (DN). Human kidney proximal tubular epithelial HK-2 cells were exposed to high glucose (HG) conditions to establish DN cellular models. Co-immunoprecipitation (Co-IP) combined with western blot analysis was employed to assess the expression levels of O-GlcNAc, O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), ACSL4, and ferroptosis-associated proteins. Cell viability was evaluated using the CCK-8 assay, while cell death was analyzed through DAPI/propidium iodide (PI) double staining. Ferroptosis was assessed by measuring intracellular iron accumulation, glutathione (GSH) content, and reactive oxygen species (ROS) levels. Additionally, <i>in vivo</i> experiments were conducted using male C57BL/6J mice divided into four groups: control, DN, DN+shOGT, and DN+shNC. DN was induced by a high-fat diet (HFD) followed by streptozotocin (STZ) injection. OGT was specifically knocked down in the kidneys using AAV-shOGT. Kidney tissues were analyzed for pathological changes, O-GlcNAcylation levels, and ferroptosis markers. Our findings demonstrated that O-GlcNAc and OGT expressions were significantly upregulated in HG-treated HK-2 cells. OGT knockdown effectively attenuated HG-induced ferroptosis. Importantly, ACSL4 protein levels exhibited strong positive correlation with OGT expression. Subsequent investigation revealed direct interaction between ACSL4 and OGT. O-GlcNAcylation modification was found to enhance ACSL4 protein stability. Moreover, overexpression of ACSL4 counteracted the protective effects of OGT knockdown against ferroptosis. Additionally, OGT knockdown reversed the high iron concentration, ROS, and MDA levels, and restored GSH and SOD levels in DN mice. O-GlcNAc, OGT, and ACSL4 levels, which were markedly increased in the DN group, were downregulated by OGT knockdown treatment <i>in vivo</i>. Collectively, these results demonstrate that high glucose promotes OGT-mediated O-GlcNAcylation of ACSL4, thereby stabilizing this enzyme and facilitating ferroptosis progression in DN.