GLYCATION DEPENDENT ALTERATION OF BASEMENT MEMBRANES

Chronic renal failure, characterized by proteinuria and alterations of glomerular filtration rate, is a major complication of longstanding diabetes mellitus. In this disorder, the glomerular basement membrane (GBM) filtration barrier becomes increasingly permeable to large and anionic proteins and, as both GBM and mesangial matrix become thicker, filtration surface decreases. Three biochemical changes have been identified in diabetic basement membranes (BMs): [1] an increase in the extent of non-enzymatic glycosylation of component proteins, [2] an increase in the covalent crosslinking of type IV collagen, and [3] a decrease in the amount of heparan sulfate proteoglycan (HSPG) within the GBM. While protein crosslinking may result in both sieving changes and account for GBM thickening due to decreased degradation, the decrease in HSPG is thought to explain the loss of the polyanionic sieving barrier. We propose to analyze the structure of diabetic BMs and explore the hypothesis that accelerated non-enzymatic glycosylation (glycation), a direct consequence of chronic hyperglycemia in which ketoamine and crosslinks form in proteins, plays a major role in the alteration of the molecular architecture and dependent sieving function of BMs in diabetes. These studies will be conducted in the context of a newly developed model for BM structure and function from our laboratory. In particular we will focus on the supromolecular organization of BM collagen, laminin and glycosaminoglycan. (A) We will compare normal, diabetic and experimentally glycated collagen structure in GBM: here we will employ electron microscopy of high resolution platinum/carbon replicas and X-ray diffraction. (B) We will evaluate the ketoamines and crosslinks in diabetic and experimentally glycated GBM by a variety of biochemical and biophysical techniques (proteolytic solubilization, ketoamine and fluorescence crosslink characterization, fragment size analysis, and protein domain identification). (C) We will evaluate the consequences of glycation on the polymerization of laminin and its interaction with proteoglycan using assays we have developed for the study of normal BM assembly. With this approach we expect to be able to construct models for the alterations which occur in BM molecular architecture.