S-nitrosylation of extracellular matrix proteins in diabetic nephropathy
Diabetic nephropathy (ON) is the leading cause of end-stage renal disease worldwide. The factors that induce ON in -30% of diabetic patients and the molecular mechanisms leading to ON glomerular damage and renal failure are not fully understood. Protein S-nitrosylation is a redox-dependent, reversible post-translational modification that plays important roles in physiology, insulin resistance and cardiovascular disease. However, the role of protein S-nitrosylation in ON is largely unknown. We recently identified laminin S-nitrosylation in glomeruli and podocytes, and its regulation by nitric oxide (NO) and vascular endothelial growth factor A (VEGF-A). We find that laminin de-nitrosylation associates with laminin accumulation in glomerular nodules, suggesting a role in prototypical ON lesions. Excessive TGF131 and CTGF signaling are responsible for mesangial. cell proliferation and extracellular matrix expansion in human and rodent ON. We have identified potential nitrosylation sites in active TGF131 and CTGF. We propose to examine S-nitrosylation of the mature GBM laminin 521. TGFB1 and CTGF, which are key contributors to extracellular matrix accumulation and nodular glomerulosclerosis in ON. Aim 1 will evaluate laminin 521 S-nitrosylation: We will assess S-nitrosylation of each (a5, 132 and yl ) laminin chain by biotin-switch test (BST) and proximity link assay (PLA), identify nitrosylated Cys residues by mutagenesis, BST and LC/MS/MS, and evaluate a5, 132 and y1 laminin-SNO effect on laminin secretion and degradation by podocytes, and on glomerular nodule formation in advanced ON mouse models. Aim 2 will examine TFGpl and CTGF S-nitrosylation. will determine whether endogenous TGFP1 and CTGF are S-nitrosylated in mesangial cells and mouse kidneys using BSTI' and PLA, and assess S-nitrosylation influence on TGFP1 and CTGF activity, measured by mesangial cell proliferation, SMAO phosphorylation and collagen IV production. ,

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