Glycosaminoglycans and Podocyte Behavior in the Renal Glomerulus

肾小球中的糖胺聚糖和足细胞行为

• 批准号: 8109885
• 负责人: KEVIN John MCCARTHY
• 金额: $ 30.51万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-10 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8109885
• 关键词: Acute; Adhesions; Adult; Affect; Age; Age-Months; Albuminuria; Animal Model; Animals; Apoptosis; Basement membrane; Behavior; Binding; Blood capillaries; Cell Line; Cell Proliferation; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Charge; Chronic Disease; Cicatrix; Control Animal; Data; Development; Diabetes Mellitus; Disease; Dystroglycan; EXT1 gene; Enzymes; Event; Excision; Fibroblasts; Filtration; Focal Adhesions; Foot Process; Functional disorder; Glomerular Capillary; Glycosaminoglycans; Glypican; Heparitin Sulfate; Immunohistochemistry; In Vitro; Integrins; Investigation; Kidney; Kidney Diseases; Kidney Glomerulus; Knock-out; Knockout Mice; Laboratories; Lead; Mediating; Modeling; Molecular; Morphology; Normal tissue morphology; Pattern; Perinatal mortality demographics; Process; Protein Isoforms; Proteinuria; Proteoglycan; Role; Staging; Structure; Surface; System; Technology; Testing; Time; Ultrafiltration; Wild Type Mouse; capillary; cell behavior; density; glomerular function; knockout animal; light microscopy; membrane synthesis; mouse model; novel; podocyte; polysulfated glycosaminoglycan; protein expression; proteoglycan core protein; public health relevance; receptor; slit diaphragm; syndecan; young adult

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.

描述（由申请方提供）：既往研究已证明硫酸乙酰肝素糖胺聚糖（HS-GAG）在肾小球超滤过程中具有不可或缺的作用，表明去除HS-GAG会损害超滤屏障的渗透选择性。随后的研究表明，在疾病如糖尿病中，HS-GAG从肾小球毛细血管基底膜（GBM）的损失与蛋白尿的发作和肾小球中前列腺变化的开始相关。虽然GBM的选择性渗透性可能是HS-GAG的功能之一，但来自我们实验室的数据表明，GBM HS-GAG甚至更关键的作用可能是控制足细胞行为。使用Cre-lox技术选择性地删除EXT 1基因，该基因编码蛋白聚糖核心蛋白上HS-GAG链延伸中的第一种酶，我们消除了足细胞产生的所有HS-GAG的组装。传统的智慧会预测，以这种方式进行基因操作的动物应该由于严重的急性蛋白尿而遭受围产期死亡。然而，PEXTKO（足细胞EXT 1敲除）小鼠不发生显著的蛋白尿。年轻成年PEXTKO动物的常规光学显微镜研究显示形态学无明显差异。然而，在超微结构水平上，肾小球中存在显著变化，例如足细胞足突消失和GBM合成和周转增强。免疫组织化学研究表明，足细胞脱离，并从这些动物的肾小球丢失。随着动物年龄的增长，会出现轻度蛋白尿。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，并且这些相互作用的丧失在导致HS-GAG丧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在发育和成年野生型小鼠和PEXTKO小鼠中调节足细胞-GBM相互作用和最终肾小球功能的作用; 2.使用永生化EXT 1 fl/fl和EXT 1 fl/+足细胞系研究HS-GAG影响足细胞行为的机制。 公共卫生相关性 疾病，如糖尿病，对肾脏的过滤器官有深远的影响。这些疾病的主要表现是肾脏的过滤装置逐渐结疤，这导致过滤装置功能障碍和最终死亡。使用一种新的动物模型，表现出几个变化，在肾脏疾病，我们将研究肾小球细胞与滤过表面相互作用的机制。