NONENZYMATIC GLYCATION IN DIABETIC KIDNEY DISEASES

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A major pathology associated with long standing diabetes mellitus is the loss of kidney function. The molecular, cellular, biochemical and physiological mechanisms which result in kidney failure have not been determined. However, almost all diabetologists will agree that strict control of blood glucose levels will slow the onset of glucose related pathologies including kidney dysfunction. The mechanism behind the toxicity of elevated glucose concentrations on cell and organ function are also poorly understood. Due to the central role of hyperglycemia in the onset of kidney disease this research project will evaluate how glucose alters a major function of the kidney, namely the filtration of serum proteins. The biochemical process known as nonenzymatic glycation will be studied using serum proteins and basement membrane components as targets for the addition of carbohydrates. We hypothesize that the increased incidence of nonenzymatic glycation of proteins in poorly controlled diabetics results in increased diffusion or flux of proteins across the glomerular basement membrane. To study the diffusion of serum proteins in basement membranes we have developed an in vitro fluorescence assay based on fluorescence recovery after photobleaching or FRAP. FRAP permits the direct measurement of protein diffusion in basement membranes derived from reconstituted matrix proteins, from isolated glomerular basement membranes as well as basement membranes synthesized by cells derived from isolated glomeruli. We will nonenzymatically glycate both serum proteins and proteins present in the basement membrane and evaluate the effect on solute diffusion. The effect of long standing diabetes will be modelled by exposing cells and proteins to elevated concentrations of glucose and other reactive monosaccharides for extended periods of time. Solute flux in these extensively glycated basement membranes will then be quantified. The proposed studies will provide insight into the mechanism of albuminuria which is observed rapidly after the onset of diabetes.