CELL SURFACE & CYTOSKELETAL PROTEOGLYCANS OF GLOMERULUS

细胞表面

基本信息

批准号：
3239779
负责人：
DAVID J KLEIN
金额：
$ 11.84万
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/3239779
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.

蛋白聚糖（PG）沿肾小球策略性地位于地下室膜（GBM）部分负责血浆超滤的电荷障碍。来自两者的GBM 糖尿病的人和大鼠含有减少硫酸乙酰肝素（HS）pg的含量。这是发病机理异常知之甚少。 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含量降低。我们以前有表明肾小球PG合成和流体动力学大小 HS和软骨素/皮肤硫酸盐（CS/DS）PGS和糖胺聚糖（GAG）侧链在正常情况下相似，并且链蛋白酶诱导的糖尿病大鼠。但是，GBM来自糖尿病患者缺少迅速合成并释放的池 HS，指出PG与GBM相互作用的可能性可能受到糖尿病的干扰。确实，孤立的glomeruli 糖尿病大鼠的独特肝素 - 位移的细胞表面HS PG。因此，细胞异常相互作用具有GBM的表面PG可能参与糖尿病性肾病。细胞表面上的PG可能很重要组织与细胞内细胞骨架之间的联系，通过与组织糖蛋白（例如纤连蛋白）相互作用，层粘连蛋白和IV型胶原蛋白，所有分子与Pg结合已被证明。我们假设PG-Matrix 相互作用在糖尿病和HS结合的糖尿病异常 GBM，在等离子体的电荷屏障中分解超滤。另外，细胞之间的相互作用改变了表面PG和细胞外基质大分子可能会改变 PGS传播到细胞内的细胞功能通过细胞细胞骨架的环境。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肾小球细胞的表面和PG 从人类肾脏孤立的文化。我们将比较细胞表面PG的生化特征与存在的生化特征在这些细胞的细胞骨架制剂中。我们将尝试证明与细胞表面相关的PG与细胞外基质糖蛋白导致细胞的变化行为（粘附，扩散），这些相互作用可能是通过糖基化改变。将生产抗体来自培养细胞的PG，用于表征肾小球PGS并通过共定位PG和细胞骨架。免疫荧光和电子显微镜。形态学对PG定位的影响，这些药物的定位会干扰还将检查细胞骨架架构。