PODOCYTE CELL LINEAGE IN GENITOURINARY DEVELOPMENT

说明(改编自应用程序) 申请者研究计划的长期目标将检验以下几点 两个假设：1)足细胞(肾小球内脏上皮细胞)是 在发育过程中肾小球血管生成所必需的，以及2) 足细胞在慢性肾功能障碍的进展中起关键作用 各种潜在原因，包括糖尿病、衰老和炎症。这个 足细胞特异性基因neparin的启动子已经被克隆。一种转基因 包含1.25kb的人类neparin基因5‘侧翼区 扼要介绍了内源性neparin mRNA转录本的表达情况，并 仅在毛细血管环和成熟期肾小球的足细胞中表达 发育中的小鼠肾脏。拟议的研究将使用这一启动子来 产生新的试剂来描述足细胞在肾脏中的作用 发展和疾病。本申请的主要目的是 建立小鼠模型系统，在体外操纵足细胞基因表达并 在活体内。具体目标包括：1.生成特定于足细胞的 Cre-重组酶小鼠品系中基因的缺失和过度表达 肾脏发育过程中的细胞特异性方式。2.生成鼠标创建者 可用于以可诱导的方式修改基因活性的品系 足细胞。3.分离表达A蛋白的新的小鼠足细胞系 “活的”分化的标志。4.进一步刻画顺位角色 赤霉菌newitin基因1.25kb足细胞特异性启动子内的元件 以确定负责的交易因素 特定于足细胞的表达式。5.生成特定于足细胞的敲除 血管内皮生长因子(VEGF)。 在本申请中产生的试剂将允许时间和 特定于足细胞的基因活性的空间修饰。会是 可能操纵参与肾小球血管生成的候选基因和 确定足细胞在设置肾小球滤过中的作用 障碍。在未来的研究中，这些试剂将有助于确定其作用 足细胞在肾小球硬化中的作用，并最终确定治疗 慢性肾脏疾病的目标。

DESCRIPTION (adapted from the application) The long-term goals of the applicant's research program will test the following two hypotheses: 1) that the podocyte (glomerular visceral epithelial cell) is required for glomerular angiogenesis during development and 2) that the podocyte is pivotal in progression of chronic renal dysfunction due to a variety of underlying causes that include diabetes, aging and inflammation. The promoter of a podocyte-specific gene, nephrin, has been cloned. A transgene containing 1.25-kb of the 5' flanking region of the human nephrin gene recapitulates the endogenous expression of the nephrin mRNA transcript, and is only expressed in podocytes in capillary-loop and mature stage glomeruli of the developing murine kidney. The proposed studies will use this promoter to generate novel reagents to delineate the role of the podocyte in kidney development and disease. The main objective of the present application is to develop mouse model systems to manipulate podocyte gene expression in vitro and in vivo. The Specific Aims include: 1. To generate a podocyte-specific Cre-recombinase mouse line in order to delete and over-express genes in a cell-specific manner during renal development. 2. To generate mouse founder lines that can be used to modify gene activity in an inducible manner in podocytes. 3. To isolate novel murine podocyte cell lines that expresses a "living" marker of differentiation. 4. To further characterize the cis-acting elements within the 1.25-kb podocyte-specific promoter of the nephrin gene in order to identify trans-acting factors that are responsible for podocyte-specific expression. 5. To generate a podocyte-specific knockout of vascular endothelial growth factor (VEGF). The reagents generated in the present application will allow both temporal and spatial modification of gene activity specifically in podocytes. It will be possible to manipulate candidate genes involved in glomerular angiogenesis and to determine the role of the podocyte in setting up the glomerular filtration barrier. In future studies, these reagents will be useful to determine the role of the podocyte in glomerulosclerosis and ultimately, to identify therapeutic targets for chronic renal disease.