Hyperglycemia in combination with excess fat intake promotes renal pyroptosis and fibrosis through Gα ₁₂ -dependent endoplasmic reticulum stress

<b>Background:</b> Chronic exposure to free fatty acids (FFAs) and glucose may disrupt metabolic homeostasis and initiate pathological processes. This study investigated the effects of hyperglycemia and fat overload on renal endoplasmic reticulum (ER) stress, pyroptosis and fibrogenesis in mice and the underlying basis. We hypothesized that the combined insult would more severely induce Gα₁₂-dependent ER stress and renal complications. <b>Methods:</b> Mice were subjected to either high fat diet (HFD)+streptozotocin (STZ), or STZ treatment, and AZ2 was used as an anti-diabetic agent. Blood sera were used for blood biochemistry, and tissues were employed for RNA sequencing, immunoblottings, TEM, histology and immunohistochemistry. HEK293 and other cells were used for high glucose (HG) and palmitate treatment, or Gα₁₂ or siGα₁₂ transfection. <b>Results:</b> The combined HFD and STZ treatment, showing enrichment of genes related to GPCR signaling, inflammasome, ER stress, and pyroptosis in the RNA-sequencing analysis, upregulated Gα₁₂ in the kidney, alongside increased PGC1α and PPARα. IRE1α and ATF6 were elevated without an increase in GRP78. This was accompanied by elevated blood glucose, creatinine, and BUN levels. We also found increases of pro-IL-1β, IL-1β, caspase-1, and NLRP3, demonstrating pyroptosis. Immunoassays revealed increased fibrosis markers. AZ2 reversed these changes. STZ treatment alone exhibited mild complications in the absence of Gα₁₂ induction despite severe hyperglycemia. In cell-based assays, HG+palmitate elicited IRE1 activation along with Gα₁₂ overexpression although HG alone had a minimal effect. Overexpression of Gα₁₂ facilitated the effect of HG+palmitate on ER stress, pyroptosis, and fibrosis, whereas Gα₁₂ knockdown had the opposite effect, as corroborated by the outcomes obtained using STZ-treated Gα₁₂-/-, Gα₁₂+/-, and Gα₁₃ liver-specific KO mice. <b>Conclusion:</b> These findings support the role of HG and lipid overload combination in driving renal pyroptosis and fibrogenesis through Gα₁₂-mediated ER stress and inflammasome, delineating the mechanism underlying the conditions of diabetic renal complications and pharmacological intervention.