Regulation of podocyte survival by dendrin

描述（由申请人提供）： 项目摘要：足细胞足突及其插入的狭缝隔膜形成了尿蛋白丢失的最终屏障，这解释了足细胞损伤与蛋白尿的典型相关性。足细胞丢失的程度与包括糖尿病肾病和伊加肾病在内的几种肾脏疾病的严重程度相关 我们最近发表的数据已确定dendrin作为一种新的狭缝隔膜组件，重新定位到足细胞核，以增强细胞凋亡的影响下，转化生长因子β（TGF-β）。我们已经发现，在局灶节段性肾小球肾炎小鼠模型中，树突素重新定位到细胞核中，其中TGF-β水平增加。在本申请中，我们提出了两个具体的目标来研究树突状蛋白对足细胞存活的调节。在具体目标1中，我们将阐明调节树突状蛋白从裂孔隔膜迁移到足细胞核的分子机制。我们将利用共聚焦显微镜来测试是否损失α-辅肌动蛋白-4增加树突状蛋白的核表达。我们将通过体内磷酸化测定来测试TGF-β是否诱导树突状蛋白的磷酸化。然后，我们将诱导假定的结合位点的点突变，以测试14-3-3是否以磷酸化依赖的方式结合树突状蛋白，以促进核输入。在具体目标2中，我们将确定树突素如何增强TGF-β诱导的足细胞凋亡。将通过膜联蛋白结合测定和FACS分析测量过表达Yes相关蛋白和Nedd 4 -2对TGF-β诱导的细胞凋亡的影响。随后，我们将使dendrin缺失小鼠与CD 2AP缺失和TGF-β转基因小鼠杂交，以确定dendrin的缺失是否改善了TGF-β表达增加的这两种小鼠模型中肾小球硬化的严重程度。 相关性：所提出的研究试图确定我们感兴趣的蛋白质树突素增强肾脏内脏上皮细胞或足细胞的损伤和程序性死亡的机制。对这些细胞的损伤是蛋白尿肾病进展的重要机制。 公共卫生相关性：这项工作旨在阐明一个重要的机制，导致损伤和死亡的至关重要的细胞（足细胞）内衬的泌尿空间的细节。这些发现可能会对糖尿病肾病、局灶节段性肾小球肾炎和伊加肾病等肾脏疾病的新型治疗药物的开发产生影响，所有疾病均与足细胞数量减少相关。

DESCRIPTION (provided by applicant): Project Summary: Podocyte foot processes and their interposed slit diaphragms form the final barrier to urinary protein loss which explains the typical association of podocyte injury with proteinuria. The degree of podocyte loss correlated with disease severity in several kidney disorders including diabetic nephropathy and IgA nephropathy Our recently published data has identified dendrin as a novel slit diaphragm component that relocates to the podocyte nucleus to enhance apoptosis under the influence of transforming growth factor beta (TGF-beta). We have found that dendrin relocates to the nucleus in a mouse model of focal segmental glomeruloclerosis where TGF-beta levels are increased. In this application we have proposed two specific aims to study the regulation of podocyte survival by dendrin. In Specific Aim 1 we will elucidate the molecular mechanism regulating the relocation of dendrin from the slit diaphragm to the podocyte nucleus. We will utilize confocal microscopy to test whether loss of alpha-actinin-4 increases the nuclear expression of dendrin. We will test whether TGF-beta induces the phosphroylation of dendrin by an in vivo phorphorylation assay. Then we will induce point mutations of putative binding sites to test whether 14-3-3 binds to dendrin in a phosphorylation-dependent manner to promote nuclear import. In Specific Aim 2 we will determine how dendrin enhances TGF-beta induced podocyte apoptosis. The effect of overexpressing Yes associated Protein and Nedd4-2 on TGF-beta induced apoptosis will be measured by an Annexin binding assay and FACS analysis. Subsequently we will cross dendrin null mice with CD2AP null and TGF-beta transgenic to determine whether the loss of dendrin ameliorates the severity of glomerulosclerosis in these two mouse models where TGF-beta expression is increased. Relevance: The proposed studies seek to determine the mechanism whereby our protein of interest, dendrin, enhances injury and programmed death of kidney visceral epithelial cells or podocytes. Injury to these cells is an important mechanism in the progression of proteinuric kidney diseases. PUBLIC HEALTH RELEVANCE: This work seeks to elucidate the details of an important mechanism leading to injury and death of critically important cells (podocytes) lining the urinary space. These findings could have an impact on the development of novel therapeutic agents for kidney diseases such as diabetic nephropathy, focal segmental glomeruloclerosis and IgA nephropathy, all disorders associated with a reduction in podocyte number.