Glycosaminoglycans and Podocyte Behavior in the Renal Glomerulus

描述(申请人提供)：先前的研究表明，硫酸乙酰肝素糖胺聚糖(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会损害超滤屏障的渗透选择性。后来的研究表明，在糖尿病等疾病中，肾小球毛细血管基底膜(GBM)上HS-GAG的丢失与蛋白尿的开始和肾小球硬化性改变的开始有关。尽管GBM的渗透选择性可能是HS-GAG的一个功能，但我们实验室的数据表明，GBM HS-GAG的一个更关键的作用可能是控制足细胞的行为。利用Cre-lox技术选择性地删除了编码蛋白多糖核心蛋白HS-GAG链延长第一个酶的EXT1基因，消除了足细胞产生的HS-GAG的组装。传统智慧会预测，以这种方式进行基因操作的动物应该会因为严重的急性蛋白尿而遭受围产期死亡。然而，PEXTKO(足细胞EXT1基因敲除)小鼠不会出现明显的蛋白尿。对幼年成年PEXTKO动物的常规光学显微镜研究显示，在形态上没有明显的差异。但在超微结构水平上，肾小球有明显的变化，如足细胞足突消失，GBM合成和周转增强。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，轻度蛋白尿确实会出现。由于上述数据，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的足细胞行为，这些相互作用的丧失在导致HS-GAG丢失的疾病中表现出来。为了验证这一假设，我们提出了以下具体目标：1.研究HS-GAG在发育中和成年野生型小鼠和PEXTKO小鼠中调节足细胞-GBM相互作用并最终调节肾小球功能的作用；2.使用永生化的EXT1 fl/fl和EXT1 fl/+足细胞系来研究HS-GAG影响足细胞行为的机制。 公共卫生相关性 糖尿病等疾病对肾脏的过滤装置有深远的影响。这些疾病的主要表现是肾脏滤过装置逐渐形成疤痕，导致滤过装置功能障碍和最终死亡。利用一种新的动物模型，我们将研究肾小球细胞与滤过表面相互作用的机制，该模型显示了肾脏疾病中的几种变化。

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.