NONENZYMATIC GLYCATION IN DIABETIC KIDNEY DISEASES

与长期存在的糖尿病相关的主要病理是 肾功能丧失。 分子、细胞、生物化学和 导致肾衰竭的生理机制尚未被阐明 测定 然而，几乎所有的糖尿病学家都会同意，严格的 控制血糖水平将减缓与血糖相关的 病理学包括肾功能障碍。 背后的机制 葡萄糖浓度升高对细胞和器官功能的毒性 也是知之甚少。 由于高血糖症的核心作用， 该研究项目将评估肾脏疾病的发病 葡萄糖改变了肾脏的主要功能，即对 血清蛋白 被称为非酶糖化的生化过程 将使用血清蛋白和基底膜成分作为 添加碳水化合物的目标。 我们假设 不良反应中蛋白质非酶糖化的发生率增加 控制糖尿病导致蛋白质的扩散或流动增加 穿过肾小球基底膜 研究血清的扩散 我们已经开发了一种体外荧光 基于光漂白或FRAP后荧光恢复的测定。 FRAP 允许直接测量基底膜中的蛋白质扩散 来源于重组基质蛋白，来自分离的肾小球 基底膜以及细胞合成的基底膜 来源于分离的肾小球。 我们将非酶糖化 血清蛋白和蛋白质存在于基底膜，并评估 对溶质扩散的影响。 长期糖尿病的影响 将通过将细胞和蛋白质暴露于升高的 葡萄糖和其他活性单糖的浓度， 一段时间。 这些广泛糖化的基底中的溶质通量 然后对膜进行定量。 拟议的研究将提供 了解蛋白尿的机制， 糖尿病的发病

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.