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The contribution of endothelial nitric oxide synthase (eNOS) to podocyte integrity remains unclear. This study therefore examined podocytes and mitochondrial abnormalities in eNOS deficient mice. Absence of eNOS caused glomerular hypertrophy, along with occasional glomerular sclerosis and mesangiolysis. While many glomeruli did not have such advanced lesions, ultrastructural analysis showed cellular hypertrophy, vacuolization, lysosomal enlargement, and microvillus formation in podocytes of eNOS knockout (KO) mice. Increased oxidative stress was associated with mitochondrial abnormalities, including an increase in number, coupled with a reduction in size, of mitochondria in podocytes of eNOS-KO mice. While the levels of expression of several mitochondrial proteins were not altered, the d -17 mutation in mitochondrial DNA was significantly associated with the eNOS deficiency. Renal ATP level in the renal cortex and mitochondrial respiration in the primary podocytes were significantly lower in eNOS-KO mice, suggesting that renal mitochondria may be functionally impaired. Podocytes cultured with endothelial conditioned medium lacking NO consistently showed a greater degree of mitochondrial fragmentation and an increase in mitochondrial oxidative stress, with these mitochondrial alterations rescued by an NO donor. In conclusion, eNOS may be necessary to maintain podocyte integrity, especially mitochondrial function. [ABSTRACT FROM AUTHOR]

Background: We have already reported Gas6 is involved in glomerular hypertrophy observed in diabetic nephropathy. However, the molecular mechanisms involved in glomerular hypertrophy are still unknown, especially in vivo.Methods: In vivo, diabetes was induced in rats and mice by streptozotocin (STZ) and the activation of the Akt/mTOR pathway in glomeruli was examined. In vitro, mesangial hypertrophy was assessed by [(3)H]leucine incorporation and measuring cell areas.Results: Akt, p70 S6 kinase, and 4E-BP-1 were induced and phosphorylated in rat glomerular lysates after 12 weeks of STZ injection when mesangial and glomerular hypertrophy was observed. We then examined the role of Gas6 by treating STZ-rats with warfarin, and found that warfarin treatment inhibited the phosphorylation of these molecules as well as the hypertrophy. We next examined whether high glucose stimulation can induce the expression of Gas6/Axl in mesangial cells. Stimulation of the cells with 25 mmol/L of glucose increased the expression of Gas6/Axl and mesangial cell size compared with that with 5.6 mmol/L of glucose. This hypertrophic effect was abolished in mesangial cells derived from Gas6 knockout mice. We also found that LY294002 and rapamycin blocked Gas6-induced activation of the Akt/mTOR pathway and mesangial hypertrophy. Furthermore, less phosphorylated Akt-positive or 4E-BP-1-positive areas were found in STZ-treated Gas6 knockout mice than in STZ-treated wild-type mice.Conclusion: Our study indicates that the Akt/mTOR pathway is a key signaling cascade in Gas6-mediated mesangial and glomerular hypertrophy and revealed a crucial role of Gas6/Axl and the Akt/mTOR pathway in the development of diabetic nephropathy. [ABSTRACT FROM AUTHOR]