Glycosaminoglycans and Podocyte Behavior in the Renal Glomerulus

描述（由申请人提供）：先前的研究表明硫酸肝素糖胺聚糖（HS-GAG）在肾小球超滤过程中起着不可或缺的作用，表明去除HS-GAG会损害超滤屏障的透选率。随后的研究表明，在糖尿病等疾病中，肾小球毛细血管基底膜（GBM） HS-GAG的丢失与蛋白尿的发生和肾小球早衰改变的开始有关。虽然GBM的渗透性可能是HS-GAG的一个功能，但我们实验室的数据表明，GBM HS-GAG更关键的作用可能是控制足细胞的行为。利用Cre-lox技术选择性删除编码蛋白聚糖核心蛋白上HS-GAG链延伸的第一个酶的EXT1基因，我们消除了足细胞产生的所有HS-GAG的组装。传统观点认为，以这种方式进行基因操作的动物会因严重的急性蛋白尿而导致围产期死亡。然而，PEXTKO（足细胞EXT1敲除）小鼠不会发生明显的蛋白尿。年轻成年PEXTKO动物的常规光学显微镜研究显示在形态学上没有明显的差异。然而，在超微结构水平上，肾小球发生了显著的变化，如足细胞足突消失，GBM合成和转换增强。免疫组织化学研究表明，足细胞从这些动物的肾小球中分离和丢失。随着动物年龄的增长，会出现轻度蛋白尿。基于上述数据，我们的假设是HS-GAG通过介导足细胞- gbm相互作用直接影响正常组织中足细胞的行为，而这些相互作用的缺失表现在导致HS-GAG缺失的疾病中。为了验证这一假设，我们提出了以下具体目标：1。在发育和成年野生型小鼠和PEXTKO小鼠中，研究hs - gag在调节足细胞- gbm相互作用和最终调节肾小球功能中的作用；2. 利用永生化的EXT1 fl/fl和EXT1 fl/+足细胞株系研究HS-GAG影响足细胞行为的机制。

DESCRIPTION (provided by applicant): Previous studies have demonstrated that the heparan sulfate glycosaminoglycans (HS-GAG) have an integral role in the process of glomerular ultrafiltration, showing that removal of HS-GAG compromised the permeaselectivity of the ultrafiltration barrier. Subsequent studies have shown that in diseases, such as diabetes mellitus, the loss of HS-GAG from the glomerular capillary basement membrane (GBM) is associated with the onset of proteinuria and the beginning of prosclerotic changes in the renal glomerulus. Although permeaselectivity of the GBM may be one function of the HS-GAG, data from our laboratory suggests perhaps an even more critical role for GBM HS-GAGs is the control of podocyte behavior. Using Cre-lox technology to selectively delete the EXT1 gene, which encodes for the first enzyme in HS-GAG chain elongation on proteoglycan core proteins, we eliminated the assembly of all HS-GAGs made by podocytes. Conventional wisdom would predict that animals genetically manipulated in this manner should suffer perinatal mortality due to severe, acute proteinuria. However, PEXTKO (Podocyte EXT1 knockout) mice do not develop significant proteinuria. Routine light microscopy studies of young adult PEXTKO animals show no distinct differences in morphology. However at the ultrastructural level, there are significant changes in the glomerulus such as podocyte foot process effacement and enhanced GBM synthesis and turnover. Immunohistochemistry studies indicate that podocytes detach and are lost from the glomeruli of these animals. As the animals age, mild proteinuria does develop. Because of the above data, our hypothesis is that HS-GAGs directly influence podocyte behavior in normal tissues by mediating podocyte-GBM interactions and the loss of these interactions are manifested in diseases that cause the loss of HS-GAG. To test the hypothesis we propose the following specific aims: 1. To investigate the role of HS-GAGs in modulating podocyte-GBM interactions and ultimately glomerular function in developing and adult wild-type mice and PEXTKO mice; 2. Investigate the mechanism by which HS-GAG influences podocyte cell behavior using immortalized EXT1 fl/fl and EXT1 fl/+ podocyte cell lines. PUBLIC HEALTH RELEVANCE Diseases,such as diabetes mellitus, have profound effects on the filtration apparatus of the kidney. The primary manifestation of these diseases is gradual scarring of the filtration apparatus of the kidney, which results in dysfunction and eventual demise of the filtration apparatus. Using a novel animal model which manifests several changes seen in renal diseases we will investigate the mechanism by which glomerular cells interact with the filtration surface.