NONENZYMATIC GLYCATION OF GLOMERULAR BASEMENT MEMBRANE

导致慢性肾功能衰竭的肾脏疾病是一个严重的公众 健康问题。糖尿病是导致肾脏疾病的最大单一原因 疾病。拟议研究的长期目标是为 在对糖尿病肾病发病机制的认识上 分子水平。在糖尿病中，高血糖会影响许多代谢。 途径和这些变化中的每一个都可能有助于 糖尿病并发症。其中一个主要的变化是非酶 蛋白质的糖基化。该过程分两步进行：第一步， 一个是可逆的(Amadori产物的形成)，另一个是不可逆的 一(交联物的形成)。因为它们的半衰期很长， 基底膜大分子是这一过程的主要靶点。在……里面 糖尿病肾病，最突出的变化之一是 肾小球基底膜增厚、通透性增加。 因此，了解非酶制剂的作用势在必行。 肾小球基底膜及其成分上的糖基化。我们 建议：一、分离小牛肾小球基底膜并孵育 在葡萄糖存在的情况下促进它们的非酶 糖基化；这些孵化将在没有或 氨基胍的存在，这是一种已知的抑制交联的物质 队形。第二，检查葡萄糖的掺入程度和 交联剂的形成和糖基化引起的结构变化 完整的肾小球基底膜，用分光光度法和高 分辨率扫描电子显微镜技术。第三，提取 肾小球基底膜的三种主要大分子成分 (IV型胶原、层粘连蛋白和牙本质蛋白/Nidogen)使用变性剂， 凝胶渗透和离子交换层析，并确定可能的 用凝胶电泳法检测糖基化引起的结构改变， 放射自显影和旋转阴影电子显微镜。第四，到 检查提取的大分子与每个大分子相互作用的能力 其他和定义糖基化引起的缺陷相互作用 浊度法、固相结合分析和旋转跟踪电子 显微镜。这些研究将拓宽我们对这些机制的认识。 肾小球基底区潜在的结构和功能改变 膜在糖尿病中的作用，并有望导致旨在 减少糖尿病并发症。

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.