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

高糖条件下系膜细胞基质沉积作为肾小球硬化模型

• 批准号: 9052756
• 负责人: Charles Grabel Miller
• 金额: $ 2.3万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2016-05-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9052756
• 关键词: Accounting; Affect; Binding; Binding Sites; Blood Glucose; Cell Culture Techniques; Cell Line; Cells; Cicatrix; Collagen; Defect; Deposition; Detergents; Development; Diabetes Mellitus; Diabetic Nephropathy; Disease; Disease Progression; End stage renal failure; Environment; Event; Exhibits; Extracellular Matrix; Extracellular Matrix Proteins; Feedback; Fibroblasts; Fibronectins; Fibrosis; Filtration; Flow Cytometry; Future; Generations; Glomerular Capillary; Glucose; Human; Immunofluorescence Immunologic; Integrin Binding; Integrins; Kidney; Kinetics; Knock-out; Lead; Measures; Mediating; Mesenchymal; Modeling; Molecular; Nature; Newly Diagnosed; Pathway interactions; Play; Post-Translational Protein Processing; Process; Production; Property; Proteins; Proteinuria; Renal glomerular disease; Role; Structure of glomerular mesangium; Surface; Testing; Therapeutic; Time; Tissues; United States; cell assembly; cell type; density; deoxycholate; dimer; fibrillogenesis; glomerular basement membrane; glomerular filtration; glomerulosclerosis; glycation; glycosylated fibronectin; insight; kidney cell; mesangial cell; neutralizing antibody; new therapeutic target; public health relevance; receptor; research study; sugar

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.

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，其特征在于细胞外基质（ECM）蛋白水平升高，破坏肾小球滤过装置的功能。肾小球系膜细胞是肾小球的主要基质细胞，支持肾小球毛细血管网。纤连蛋白（FN）是全身ECM的主要成分，存在于正常肾小球中，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基质先于并部分决定其他ECM蛋白的沉积。因此，FN基质组装中的扰动对组织适当ECM的整体完整性具有深远影响。细胞通过整合素受体介导FN基质组装，特别是<$5 <$1，与FN二聚体的RGD细胞结合结构域结合。这种结合诱导FN-FN相互作用和短原纤维的形成，然后通过连续加入FN二聚体成熟为稳定的不溶性基质。高葡萄糖条件已显示出增加各种ECM蛋白（包括FN）的系膜细胞表达。高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代表了葡萄糖介导的ECM破坏的另一种模式。糖基化FN，其表现出改变的结合特性，被发现在糖尿病中的水平升高。我们认为，系膜细胞基质组装缺陷代表了肾小球硬化症的最终共同途径，这标志着糖尿病肾病和其他纤维化疾病。首先通过测量脱氧胆酸盐（DOC）洗涤剂不溶性FN（FN基质成熟的既定标志物），在正常和高葡萄糖条件下通过FN蓄积的时间过程确定高葡萄糖对基质组装动力学的影响。将对原纤维密度进行定量，以确定ECM组织中任何葡萄糖介导的变化。将在存在糖化FN的情况下进行类似实验。由于细胞环境的3D性质刺激基质产生，因此将检查3D基质对系膜细胞介导的FN原纤维形成的动力学的影响，以确定是否存在正反馈回路，其中不溶性基质的积累刺激进一步的基质沉积。由于整联蛋白受体启动最终的共同途径，因此将使用中和抗体和敲除细胞检查<$5 <$1和<$v <$3整联蛋白在高糖诱导的基质沉积中的作用。进一步深入了解糖尿病肾病中基质组装增加的机制可能为开发新一代治疗药物铺平道路，这些药物靶向这一过程，希望逆转向ESRD的进展。