Podocyte-Specific VEGF-A Gain of Function Induces Nodular Glomerulosclerosis in eNOS Null Mice

VEGF-A and nitric oxide are essential for glomerular filtration barrier homeostasis and are dysregulated in diabetic nephropathy. Here, we examined the effect of excess podocyte VEGF-A on the renal phenotype of endothelial nitric oxide synthase (eNOS) knockout mice. Podocyte-specific VEGF₁₆₄ gain of function in eNOS^−/− mice resulted in nodular glomerulosclerosis, mesangiolysis, microaneurysms, and arteriolar hyalinosis associated with massive proteinuria and renal failure in the absence of diabetic milieu or hypertension. In contrast, podocyte-specific VEGF₁₆₄ gain of function in wild-type mice resulted in less pronounced albuminuria and increased creatinine clearance. Transmission electron microscopy revealed glomerular basement membrane thickening and podocyte effacement in eNOS^−/− mice with podocyte-specific VEGF₁₆₄ gain of function. Furthermore, glomerular nodules overexpressed collagen IV and laminin extensively. Biotin-switch and proximity ligation assays demonstrated that podocyte-specific VEGF₁₆₄ gain of function decreased glomerular S-nitrosylation of laminin in eNOS^−/− mice. In addition, treatment with VEGF-A decreased S-nitrosylated laminin in cultured podocytes. Collectively, these data indicate that excess glomerular VEGF-A and eNOS deficiency is necessary and sufficient to induce Kimmelstiel-Wilson–like nodular glomerulosclerosis in mice through a process that involves deposition of laminin and collagen IV and de-nitrosylation of laminin.Vascular glomerular endothelial factor-A (VEGF-A) is essential for the development and maintenance of normal glomerular structure and function.¹ Podocytes are the most important source of glomerular VEGF-A.^1–4 Glomerular VEGF-A plays a critical role in the pathogenesis of diabetic nephropathy.^5–7 Transgenic mice with podocyte VEGF₁₆₄ gain of function develop a glomerular phenotype indistinguishable from early diabetic nephropathy, in the context of normal blood glucose and normal systemic VEGF-A.⁵ In the setting of type 1 diabetes, plasma VEGF-A increases but nodular glomerulosclerosis develops only in mice with podocyte VEGF₁₆₄ gain of function, demonstrating that local rather than systemic VEGF excess is critical for the progression of diabetic glomerulopathy to advanced disease.⁶Nitric oxide (NO) is a product of arginine oxidation: L arginine+O₂ → citrulline+NO, catalyzed by NO synthase (NOS). The major source of endogenous NO, NOS isoforms (neuronal NOS, inducible NOS, and endothelial NOS),^8–10 are expressed in the kidney.^11–14 VEGF-A activates endothelial NOS (eNOS), inducing NO generation, which stimulates soluble guanylate cyclase, thereby causing vasodilatation. VEGF-A activates eNOS via phosphatidylinositol-3-kinase/Akt.¹⁵ Signals downstream from VEGF-A and NO stimulate endothelial cell proliferation and migration in human endothelium, regulate endothelial integrity, and contribute to angiogenesis.^16–21 In diabetes, low NO bioavailability is associated with high VEGF-A levels.^7,8,22–25 Nakagawa et al. called this process “uncoupling of VEGF to NO,” connecting mechanistically the advanced nephropathy with the relationship between VEGF and NO in the kidney.²⁶ Experimental diabetes induced in eNOS knockout (KO) mice resulted in severe diabetic nephropathy: nodular glomerulosclerosis, decreased GFR, and hypertension, associated with increased VEGF mRNA renal expression.^26,27 Consistent with these findings, db/db mice treated with l-arginine and sepiapterin had improved albuminuria and glomerular basement membrane (GBM) thickness, associated with reversed eNOS dimerization and phosphorylation, suggesting that improving eNOS activity delays the progression of diabetic nephropathy.²⁸ However, the mechanisms whereby excess VEGF-A and eNOS insufficiency lead to advanced diabetic nephropathy remain unclear.At the cellular level, binding of NO to soluble guanylate cyclase leads to increased cyclic guanosine monophosphate (cGMP) production and activation of protein kinase G, phosphodiesterases, and cGMP-gated ion channels. However, extensive evidence demonstrates that NO exerts multiple biologic functions through cGMP–independent S-nitrosylation of proteins.^29–32 S-Nitrosylation is a reversible, covalent addition of NO to thiol groups on specific cysteine from proteins, forming nitroso-protein (SNO).^30–32 Nitrosylation induces redox-based conformational changes in target proteins that modulate signaling and function.³² Altered protein S-nitrosylation has been demonstrated in pulmonary, hematologic, neurologic, and cardiovascular diseases, as well as in cancer, preeclampsia, and diabetes.^31,33We hypothesized that deficient S-nitrosylation of specific proteins mediates the glomerular phenotype resulting from eNOS deletion and excess VEGF-A in vivo. Here we examined the effects of increased podocyte VEGF₁₆₄ in eNOS KO mice and evaluated whether S-nitrosylation is mechanistically involved in the ensuing glomerular phenotype. Our findings indicate that podocyte VEGF₁₆₄ gain of function in eNOS null mice is sufficient to induce nodular glomerulosclerosis, massive proteinuria, and renal failure in the absence of diabetic milieu. Podocyte VEGF₁₆₄ gain of function decreases glomerular laminin S-nitrosylation in eNOS null mice, linking this post-translational modification to nodular glomerulosclerosis.