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问题 健康问题。 糖尿病是肾病的最大单一原因 疾病。 拟议研究的长期目标是贡献 深入了解糖尿病肾病的发病机制 分子水平。 在糖尿病中，高血糖会影响许多代谢 路径和这些变化中的每一个都可能有助于发展 糖尿病并发症。 主要改变之一是非酶促 蛋白质的糖基化。 这个过程分两步进行：首先， 可逆的一种（Amadori 产品的形成）和第二种不可逆的 一（形成交联产物）。 由于其半衰期长， 基底膜大分子是该过程的主要目标。 在 糖尿病肾病最显着的改变之一是 肾小球基底膜增厚和通透性增加。 因此，有必要了解非酶促作用的作用。 肾小球基底膜及其成分上的糖基化。 我们 建议：首先分离小牛肾小球基底膜并孵育 它们在葡萄糖存在下以促进其非酶促作用 糖基化；这些孵化将在缺席或 存在氨基胍，一种已知能抑制交联的物质 形成。 其次，检查葡萄糖掺入的程度和 交联形成和糖基化诱导的结构改变 完整的肾小球基底膜，使用分光光度法和高 分辨率扫描电子显微技术。 三、提取 肾小球基底膜的三大分子成分 （IV 型胶原蛋白、层粘连蛋白和巢蛋白/巢蛋白）使用变性剂， 凝胶渗透和离子交换色谱法并确定可能的 使用凝胶电泳进行糖基化诱导的结构改变， 放射自显影和旋转阴影电子显微镜。 四、至 检查提取的大分子与每个大分子相互作用的能力 其他并使用定义糖基化诱导的缺陷相互作用 比浊法、固相结合分析和旋转阴影电子 显微镜。 这些研究将拓宽我们对机制的认识 肾小球基底的潜在结构和功能改变 膜在糖尿病中的应用，希望能提出旨在 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.