BIOCHEMICAL AND BIOPHYSICAL ALTERATIONS OF THE 7S AND NC1 DOMAIN OF COLLAGEN-IV FROM HUMAN DIABETIC KIDNEYS

Glycation of basement membrane collagen IV has been implicated as a ma jor pathogenetic process leading to diabetic microvascular complicatio ns. To evaluate the relevance of carbohydrate-induced modifications on collagen IV in diabetic nephropathy, we isolated the cross-linking do mains 7S and NCI from the glomerular basement membrane (GBM) of patien ts with diabetes mellitus. Modifications characteristic for glycated p roteins were identified when the domains from diabetic kidney were com pared with the same domains from human placenta as an unmodified contr ol. In both domains a marked formation of inter-and intramolecular cro ss links could be demonstrated by SDS-PAGE. Furthermore circular dichr oism studies showed a decrease in helicity of the 78 domain from human diabetic kidneys of 13%, indicating denaturation already at room temp erature. Thermal transition profiles, showing a shift of the denaturat ion temperature towards a lower temperature, with loss of a distinct s econd melting point, confirmed this observation. Our data provide furt her evidence for a possible role of protein-modification by glycoxidat ive reactions in the onset of diabetic nephropathy in vivo.