Abstract Body: Background: Type 2 diabetic kidney disease (T2DKD) is a leading cause of end-stage renal disease, marked by metabolic dysregulation and mitochondrial dysfunction. While emerging evidence implicates sex differences in T2DKD pathophysiology, the specific mitochondrial pathways underlying disease progression remain poorly understood. We hypothesized that mitochondria-related metabolic mechanisms drive renal sex-specific disparities and female renoprotection in T2DKD.

Methods: Male and female T2DKD rat model (T2DN) at 14-15 weeks (early stage of T2DKD), 48-52 weeks (late stage of T2DKD), and age-matched Wistar controls were used (N = 6 for each group). Targeted metabolomic analysis of mitochondria-related metabolic pathways of kidney cortex and urine, electron microscopy (FIJI) and Western blot analysis for the proteins of interest were performed. A semi-automatic approach was employed for the analysis of pathway-specific alterations. Metabolic profiles were generated using UHPLC-HRMS; metabolites were identified in El-MAVEN using exact mass and retention time with an in-house library. OriginPro and MetaboAnalyst 6.0 were used for statistical analysis (cutoff fold change 0.5, p<0.05; 2-way ANOVA).

Results: Early-stage T2DKD males exhibited dysregulation in nucleotide, pyruvate, and sulfur amino acid metabolism, while early-stage T2DKD females showed broader changes involving amino acid biosynthesis, glycolysis/gluconeogenesis, and redox pathways (p<0.05) compared to Wistars. Late-stage T2DKD males had widespread disruptions in nucleotide, energy, redox, and amino acid metabolism, whereas late-stage T2DKD females demonstrated alterations in glycolysis, lipid metabolism, and select amino acid pathways (p<0.05) compared to age-matched Wistars. Unbiased EM analysis of late-stage T2DKD renal cortex proximal tubule samples showed enlarged less circular mitochondria in female T2DN vs all groups (p<0.05). Western blot analyses of late-stage T2DKD renal cortex confirmed changes in mitochondrial morphology (higher Pink1, decreased Opa1, Parkin and PGC1a in female T2DN vs all other groups), and antioxidant systems (decreased Sod1 and Gpx4 in T2DN rats vs Wistars, higher Sod2 in female T2DN vs all groups, p<0.05).

Conclusions: Our findings demonstrate that T2DKD progression is accompanied by distinct, sex-specific metabolic mitochondrial adaptations, underscoring the need for tailored therapeutic strategies targeting mitochondrial dysfunction.