CELL SURFACE & CYTOSKELETAL PROTEOGLYCANS OF GLOMERULUS

细胞表面

基本信息

批准号：
3239778
负责人：
DAVID J KLEIN
金额：
$ 11.58万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-09-30 至 1992-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3239778
关键词：
basement membrane cellular pathology cytoskeleton diabetes mellitus electron microscopy glycosylation high performance liquid chromatography human tissue immunofluorescence technique laboratory rat membrane proteins monoclonal antibody nephrosclerosis proteoglycan radiotracer renal glomerulus tissue /cell culture

项目摘要

Proteoglycans (PG) are strategically located along the glomerular basement membrane (GBM) where they are in part responsible for the charge barrier to plasma ultrafiltration. GBM from both humans and rats with diabetes mellitus contains a decreased content of heparan sulfate (HS) PG. The pathogenesis of this abnormality is poorly understood. Decreased PG synthesis, synthesis of a biochemically abnormal molecule, or abnormal interaction of PG with other GBM molecules in diabetes could explain a decreased GBM HS PG content. We have previously shown that glomerular PG synthesis and hydrodynamic size (both HS and chondroitin/dermatan sulfate (CS/DS) PGs and glycosaminoglycan (GAG) side chains) were similar in normal and streptozotocin-induced diabetic rats. However, GBM from diabetics were missing a rapidly synthesized and released pool of HS, pointing to the possibility that PG interaction with GBM may be perturbed by diabetes. Indeed, isolated glomeruli from diabetic rats had a decreased proportion of a unique heparin- displaced cell surface HS PG. Thus, abnormal interaction of cell surface PGs with GBM may be involved in the pathogenesis of the diabetic nephropathy. PGs on cell surfaces may be important links between the tissue and the intracellular cytoskeleton, through interaction with tissue glycoproteins such as fibronectin, laminin, and type IV collagen, all molecules to which PG binding has been demonstrated. We hypothesize that PG-matrix interaction is abnormal in diabetes and perturbs binding of HS to GBM with a breakdown in the charge barrier to plasma ultrafiltration. In addition, altered interactions between cell surface PGs and extracellular matrix macromolecules may alter cellular functions communicated by PGs to the intracellular environment through the cellular cytoskeleton. We propose to study the association between glomerular cell surfaces and PGs using visceral epithelial and mesangial cells in culture isolated from human kidneys. We will compare the biochemical characteristics of cell surface PGs with those present in cytoskeletal preparations from these cells. We will attempt to show that PGs associated with cell surfaces interact with extracellular matrix glycoproteins causing changes in cell behavior (adhesion, spreading) and that these interactions may be altered by glycosylation. Antibodies will be produced to human PGs from the cells in culture and used to characterize the glomerular PGs and to co-localize PGs and the cytoskeleton by immunofluorescent and electron microscopy. The morphologic effects upon PG localization of agents which disturb the cytoskeletal architecture will also be examined.

蛋白多糖（PG）战略性地位于肾小球上基底膜（GBM），它们部分负责血浆超滤的电荷势垒。来自两者的 GBM 患有糖尿病的人类和大鼠含有减少的硫酸乙酰肝素（HS）PG的含量。本病的发病机制对异常现象了解甚少。 PG合成减少，生化异常分子的合成，或异常 PG 与糖尿病中其他 GBM 分子的相互作用可能解释 GBM HS PG 含量减少。我们之前有过表明肾小球 PG 合成和流体动力学尺寸（两者 HS 和软骨素/硫酸皮肤素 (CS/DS) PG 和糖胺聚糖（GAG）侧链）在正常和链脲佐菌素诱导的糖尿病大鼠。然而，GBM 糖尿病患者缺少快速合成和释放的 HS，指出 PG 与 GBM 相互作用的可能性受到糖尿病的困扰。事实上，分离的肾小球糖尿病大鼠的独特肝素比例下降移位的细胞表面 HS PG。因此，细胞之间的异常相互作用 GBM 表面 PG 可能参与了 GBM 的发病机制糖尿病肾病。细胞表面的 PG 可能很重要组织和细胞内细胞骨架之间的联系，通过与组织糖蛋白（如纤连蛋白）相互作用，层粘连蛋白和 IV 型胶原蛋白，所有与 PG 结合的分子已被证明。我们假设 PG 矩阵糖尿病中相互作用异常，并扰乱 HS 与 GBM 等离子体电荷势垒被击穿超滤。此外，细胞之间的相互作用也发生了改变。表面PG和细胞外基质大分子可能会改变 PGs 向细胞内传递细胞功能通过细胞骨架调节环境。我们建议研究肾小球细胞之间的关联使用内脏上皮细胞和系膜细胞的表面和PG 从人类肾脏中分离出的培养物。我们将比较细胞表面 PG 的生化特征及其存在在这些细胞的细胞骨架制剂中。我们将尝试表明与细胞表面相关的 PG 相互作用细胞外基质糖蛋白引起细胞变化行为（粘附、扩散），并且这些相互作用可能是被糖基化改变。将会产生针对人类的抗体来自培养细胞的 PG，用于表征肾小球 PG 并通过以下方式将 PG 和细胞骨架共定位免疫荧光和电子显微镜。形态学干扰剂对 PG 定位的影响还将检查细胞骨架结构。