<b>O-GlcNAcylation Stabilizes NEK7 to Drive Podocyte Pyroptosis in Diabetic Kidney Disease</b>

<p dir="ltr">Diabetic kidney disease (DKD) progression involves NEK7-driven podocyte pyroptosis, with hyperglycemia-induced O-GlcNAcylation as a key post-translational regulator. This study elucidates how O-GlcNAc modification governs NEK7 stability and its pathological role. We employed clinical DKD specimens, high-glucose-stimulated podocytes, and streptozotocin-induced diabetic mice to first examine NEK7, O-GlcNAc, OGT, and GFPT1 expression, confirming NEK7's pyroptosis role via siRNA knockdown. Bioinformatics analysis (YinOYang 1.2) predicted O-GlcNAcylation motifs, validated by T302A mutagenesis and co-immunoprecipitation. Protein stability was assessed using cycloheximide chase and ubiquitination assays. Therapeutic efficacy of GFPT1 inhibitor DON was evaluated in vitro and in vivo through biochemical parameters, histopathology, and pyroptosis markers. Chronic hyperglycemia activated the hexosamine biosynthetic pathway (HBP), elevating pathology-associated O-GlcNAc modifications that promoted NEK7 accumulation via post-translational stabilization. This was accompanied by upregulated O-GlcNAc, OGT, and GFPT1 in DKD glomeruli and high-glucose podocytes. Crucially, threonine 302 (T302) was identified as NEK7's primary O-GlcNAcylation site. This modification reduced proteasomal degradation, extended NEK7 half-life, and enhanced NLRP3 inflammasome activation and interleukin release. Pharmacological HBP inhibition using DON normalized O-GlcNAcylation, suppressed pyroptosis, and mitigated renal injury. We pioneer the discovery of the glucose-O-GlcNAc-NEK7-NLRP3 axis driving podocyte pyroptosis in DKD, nominating Thr302 as a potential therapeutic target. These findings establish a novel post-translational modification mechanism and propose a dual-target therapeutic strategy for DKD management.</p>