Diabetes is a major public health problem in the United States. The disease is often a progressive and is characterized by several secondary complications that include kidney failure (diabetic nephropathy). The pathogenic mechanism(s) linking hyperglycemia to the diverse complications is unknown, but circumstantial evidence has been reported which implicates the chemical attachment of glucose (glycation) to certain proteins as a causal event. In the case of diabetic nephropathy, the probable target proteins are constituents of the glomerular basement membrane (GBM), an extracellular matrix that plays a crucial role in the ultrafiltration function of the kidney. The central thrust of this proposal is to examine the role of glycation in the pathogenesis of diabetic nephropathy by investigating the effect of glycation on the physicochemical properties of the major constituent of GBM, type IV collagen. The central hypothesis to be tested is: that glycation and AGE-products of the type IV collagen constituent of GBM causes; 1) alterations in collagen-collagen binding and the binding of other GBM constituents and plasma proteins to the collagen scaffold; and 2) an alteration in the susceptibility of collagen IV to proteolytic degradation. The overall experimental approach is: 1) to investigate the in vitro glycation of collagen IV domains (7S, NC1, triple-helical, and E1 fragment) with respect to a) major sites of glycation and AGE-product formation, b) effect on binding of laminin, fibronectin, and plasma proteins, and c) susceptibility to proteolytic degradation; and 2) to investigate the in vivo glycation of these same domains from GBM of age-matched controls and streptozotocin-diabetic rats with respect to the same parameters. The methodology involves a broad spectrum of technologies. These include an animal model system, light and electron microscopy, immunochemical and protein chemistry techniques to include: electrophoresis, Western blotting, HPLC, amino acid analyses, peptide sequencing, ELISAs, fluorescence and UV spectroscopy.
