Glycation of basement membrane collagen IV has been implicated as a major pathogenetic process leading to diabetic microvascular complications. To evaluate the relevance of carbohydrate-induced modifications on collagen IV in diabetic nephropathy, we isolated the cross-linking domains 7S and NC1 from the glomerular basement membrane (GBM) of patients with diabetes mellitus. Modifications characteristic for glycated proteins were identified when the domains from diabetic kidney were compared with the same domains from human placenta as an unmodified control. In both domains a marked formation of inter-and intramolecular cross links could be demonstrated by SDS-PAGE. Furthermore circular dichroism studies showed a decrease in helicity of the 7S domain from human diabetic kidneys of 13%, indicating denaturation already at room temperature. Thermal transition profiles, showing a shift of the denaturation temperature towards a lower temperature, with loss of a distinct second melting point, confirmed this observation. Our data provide further evidence for a possible role of protein-modification by glycoxidative reactions in the onset of diabetic nephropathy in vivo.