Abstract Body: Introduction:
Type 2 diabetic kidney disease (T2DKD) is a major cause of end-stage renal disease and exhibits marked sex differences in onset and progression. NO is a key regulator of renal function and blood pressure, yet its specific effects on renal epithelial cells, particularly glomerular podocytes, and its interactions with mitochondrial bioenergetics remain underexplored. Our studies uncover a novel mechanistic link between nitric oxide synthase (NOS) uncoupling, oxidative stress, and mitochondrial dysfunction in a sex-specific context of T2DKD.
Methods:
We used the T2DKD and age-matched Wistar male and female rats and an immortalized podocyte cell culture to perform real-time confocal imaging, Seahorse bioenergetics assay, Western blot, and electron microscopy. ANOVA with post-hoc was used for comparisons (p<0.05). Targeted metabolomics analysis was performed to identify metabolic 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. MetaboAnalyst 6.0 was used for statistical analysis (cutoff fold change 0.5, p<0.05).
Results:
We demonstrated that angiotensin II type 2 receptor (AT2R) activation promotes NO production in podocytes, protecting against Ca²⁺ overload and cytoskeletal instability. Under HG conditions, NOS isoforms undergo pathological redistribution, leading to an increase in NOS2 activity (n ≥ 20 for each group, p < 0.001) and reduced NO bioavailability (n ≥ 16 for each group, p < 0.001). Seahorse analysis of renal cortex in T2DKD rats revealed significant sex differences in mitochondrial respiration, with females showing higher respiratory and spare capacity compared to T2DN males and Wistar controls (n ≥ 46, p < 0.001). Comprehensive metabolomic profiling further identified profound sex-dependent alterations in renal and serum metabolic pathways. Notably, arginine metabolism and biosynthesis, key pathways for NO production, were significantly disrupted in both sexes (n = 6, p<0.05), underscoring the central role of NO deficiency in the progression of T2DKD.
Conclusions:
Together, our findings identify NOS uncoupling as a driver of mitochondrial damage and highlight distinct redox and metabolic vulnerabilities in male and female diabetic kidneys. Our data support the development of sex-specific therapeutic strategies aimed at restoring NO bioavailability and mitochondrial health to slow T2DKD progression.