Mesangial cell matrix deposition in high glucose as a model of glomerulosclerosis

描述（由申请人提供）：糖尿病肾病是糖尿病最具破坏性的长期后果之一，目前是美国终末期肾病（ESRD）的主要原因。该疾病的影响主要集中在肾小球，其特征是细胞外基质（ECM）蛋白水平升高，破坏肾小球滤过器的功能。系膜细胞支持肾小球毛细血管网络，是肾小球主要的基质生成细胞。纤维连接蛋白（FN）是全身ECM的主要成分，在正常肾小球中发现，在糖尿病肾病中水平显著升高。FN是细胞-基质相互作用的重要参与者，FN基质先于其他ECM蛋白的沉积，并在一定程度上决定了其沉积。因此，FN矩阵组装中的扰动对组织适宜的ECM的整体完整性有深远的影响。细胞通过整合素受体介导FN基质的组装，特别是与FN二聚体的RGD细胞结合结构域的结合。这种结合诱导FN-FN相互作用并形成短原纤维，然后通过继续添加FN二聚体成熟为稳定的不溶性基质。高糖条件已被证明可增加系膜细胞各种ECM蛋白的表达，包括FN。高葡萄糖对系膜细胞介导的成熟基质组装的影响尚未确定。基质蛋白的非酶糖基化是葡萄糖介导的ECM破坏的另一种模式。糖化FN表现出改变的结合特性，在糖尿病中发现其水平升高。我们认为系膜细胞基质组装缺陷是肾小球硬化的最终共同途径，肾小球硬化是糖尿病肾病和其他纤维化疾病的标志。高葡萄糖对基质组装动力学的影响将首先通过测量脱氧胆酸（DOC）洗涤剂不溶性FN （FN基质成熟度的既定标志）在正常和高葡萄糖条件下FN积累的时间过程来确定。纤维密度将被量化，以确定任何葡萄糖介导的ECM组织变化。类似的实验将在糖基化FN存在的情况下进行。由于细胞环境的3D性质刺激了基质的产生，因此将研究3D基质对系膜细胞介导的FN纤维形成动力学的影响，以确定是否存在正反馈回路，其中不溶性基质的积累刺激了进一步的基质沉积。由于整合素受体启动了最终的共同途径，因此将使用中和抗体和敲除细胞来检查整合素在高糖诱导的基质沉积中的作用。进一步了解糖尿病肾病中基质组装增加的机制可能为新一代治疗方法的开发铺平道路，这些治疗方法以这一过程为目标，希望逆转ESRD的进展。

DESCRIPTION (provided by applicant): Diabetic nephropathy is among the most devastating long-term consequences of diabetes mellitus and is now the leading cause of end-stage renal disease (ESRD) in the United States. The effects of the disease are predominantly focused in the glomerulus and are characterized by increased levels of extracellular matrix (ECM) proteins that disrupt the function of the glomerular filtration apparatus. Mesangial cells, which support th glomerular capillary network, are the primary matrix-producing cells of the glomerulus. Fibronectin (FN), a major component of the ECM throughout the body, is found in the normal glomerulus and at significantly elevated levels in diabetic nephropathy. FN is an important player in cell-matrix interactions and FN matrix precedes and in part dictates the deposition of other ECM proteins. Consequently, perturbations in FN matrix assembly have profound effects on the overall integrity of tissue-appropriate ECM. Cells mediate FN matrix assembly via integrin receptors, notably ¿5¿1, binding to RGD cell-binding domains of FN dimers. This binding induces FN-FN interactions and formation of short fibrils that then mature into a stable insoluble matrix through continued addition of FN dimers. High glucose conditions have been shown to increase mesangial cell expression of various ECM proteins, including FN. The effects of high glucose on mesangial cell-mediated assembly of a mature matrix are as yet undetermined. Non-enzymatic glycation of matrix proteins represents another mode of glucose-mediated ECM disruption. Glycated FN, which exhibits altered binding properties, is found at elevated levels in diabetes. We propose that defects in mesangial cell matrix assembly represent a final common pathway in glomerulosclerosis, which marks diabetic nephropathy and other fibrotic diseases. The influence of high glucose on the kinetics of matrix assembly will first be determined by a time course of FN accumulation under normal and high glucose conditions by measuring deoxycholate (DOC) detergent-insoluble FN, an established marker for FN matrix maturity. Fibril density will be quantified to determine any glucose- mediated changes in ECM organization. Similar experiments will be performed in the presence of glycated FN. Since the 3D nature of a cell's environment stimulates matrix production, the effects of 3D matrix on the dynamics of mesangial cell-mediated FN fibrillogenesis will be examined to determine if a positive feedback loop exists, in which accumulation of insoluble matrix stimulates further matrix deposition. Since integrin receptors initiate the final common pathway, the roles of ¿5¿1 and ¿v¿3 integrins in high glucose-induced matrix deposition will be examined using neutralizing antibodies and knockout cells. Further insight into the mechanisms of increased matrix assembly in diabetic nephropathy may pave the way for the development of a new generation of therapeutics, which target this process in hopes of reversing the progression towards ESRD. PUBLIC HEALTH RELEVANCE: Chronically high blood sugar levels in diabetes mellitus can have devastating effects on the kidney's filtration apparatus. This apparatus depends on proper organization of a network of proteins called the extracellular matrix, which accumulate in excess and block filtration when exposed to high sugar levels. Using a kidney cell culture model, we plan to clarify the effects of high sugar levels on the filtration matrix in hopes of revealing new targets for future therapies aimed at reversing the progression of this increasingly common and potentially deadly disease.