Renal disease leading to chronic renal failure represents a severe public health problem. Diabetes mellitus is the largest single cause of renal disease. The long term objective of the proposed studies is to contribute in the understanding of the pathogenesis of diabetic kidney disease at the molecular level. In diabetes, hyperglycemia affects many metabolic pathways and each of these changes may contribute to the development of diabetic complications. One of the major alterations is the nonenzymatic glucosylation of proteins. This process occurs in two steps: a first, reversible one (formation of Amadori products) and a second, irreversible one (formation of crosslinked products). Because of their long half-life, basement membrane macromolecules are primary targets of this process. In diabetic kidney disease, one of the most prominent alterations is the thickening and increased permeability of the glomerular basement membrane. Therefore, it is imperative to understand the effect of nonenzymatic glucosylation on the glomerular basement membrane and its components. We propose: First, to isolate calf glomerular basement membranes and incubate them in the presence of glucose in order to promote their nonenzymatic glucosylation; these incubations will be performed in the absence or the presence of aminoguanidine, a substance known to inhibit crosslink formation. Second, to examine the extent of glucose incorporation and crosslink formation and the glucosylation-induced structural alterations of the intact glomerular basement membrane, using spectrophotometric and high resolution scanning electron microscopic techniques. Third, to extract three major macromolecular components of the glomerular basement membrane (type IV collagen, laminin, and entactin/nidogen) using denaturing agents, gel permeation and ion-exchange chromatography and to determine possible glucosylation-induced structural alterations using gel electrophoresis, autoradiography and rotary shadowing electron microscopy. Fourth, to examine the ability of the extracted macromolecules to interact with each other and define glucosylation-induced defective interactions using turbidimetry, solid phase binding assays, and rotary shadowing electron microscopy. These studies will broaden our knowledge on the mechanisms underlying structural and functional alterations of the glomerular basement membrane in diabetes and hopefully will lead to proposals aiming at reducing diabetic complications.
|Hasegawa, G; Hunter, A J; Charonis, A S (1995) Matrix nonenzymatic glycosylation leads to altered cellular phenotype and intracellular tyrosine phosphorylation. J Biol Chem 270:3278-83|
|Nelson, T R; Charonis, A S; McIvor, R S et al. (1995) Identification of a cDNA encoding tubulointerstitial nephritis antigen. J Biol Chem 270:16265-70|
|Tsopanoglou, N E; Zioudrou, C; Tsilibary, E C et al. (1995) Putrescine: a novel inhibitor of glycosylation-induced cross-links in laminin. Microcirculation 2:283-7|
|Charonis, A; Kalfa, T; Butkowski, R et al. (1994) Interactions between tubulointerstitial nephritis antigen and laminin or type IV collagen. Contrib Nephrol 107:180-7|
|Kalfa, T A; Thull, J D; Butkowski, R J et al. (1994) Tubulointerstitial nephritis antigen interacts with laminin and type IV collagen and promotes cell adhesion. J Biol Chem 269:1654-9|
|Haitoglou, C S; Tsilibary, E C; Brownlee, M et al. (1992) Altered cellular interactions between endothelial cells and nonenzymatically glucosylated laminin/type IV collagen. J Biol Chem 267:12404-7|
|Charonis, A S; Tsilbary, E C (1992) Structural and functional changes of laminin and type IV collagen after nonenzymatic glycation. Diabetes 41 Suppl 2:49-51|