NONENZYMATIC GLYCATION IN DIABETIC KIDNEY DISEASES

与长期存在的糖尿病相关的一个主要病理是 肾功能丧失。分子、细胞、生化和 导致肾衰竭的生理机制尚未得到证实 下定决心。然而，几乎所有的糖尿病专家都会同意这种严格的 控制血糖水平会延缓血糖相关疾病的发病 包括肾功能障碍在内的病理改变。其背后的机制是 葡萄糖浓度升高对细胞和器官功能的毒性作用 也是知之甚少。由于高血糖在心脏疾病中的中心作用 这项研究项目将评估肾脏疾病的发病情况 葡萄糖改变了肾脏的一项主要功能，即滤过 血清蛋白质。被称为非酶糖基化的生化过程 将使用血清蛋白和基底膜成分作为研究对象 添加碳水化合物的目标。我们假设 不良患者蛋白质非酶糖基化的发生率增加 受控糖尿病患者导致蛋白质扩散或流量增加 穿过肾小球基底膜。研究血清中的扩散 基底膜中的蛋白质我们已经开发出一种体外荧光 基于光漂白或FRAP后的荧光恢复的分析。FRAP 允许直接测量蛋白质在基底膜中的扩散 来自重组的基质蛋白，来自分离的肾小球 基底膜和细胞合成的基底膜 源自孤立的肾小球。我们将非酶糖化这两种物质 血清蛋白和基底膜蛋白的存在及评价 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在升高的 延长的葡萄糖和其他反应性单糖的浓度 一段时间。这些广泛糖化基底物中的溶质通量 然后将对膜进行量化。拟议的研究将提供 对蛋白尿机制的洞察，在治疗后迅速观察 糖尿病的发作。

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.