Regulation of podocyte survival by dendrin

描述(由申请人提供)： 项目概述：足细胞足突及其插入的裂隙横隔膜构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联系。在包括糖尿病肾病和IgA肾病在内的几种肾脏疾病中，足细胞丢失的程度与疾病严重程度相关 我们最近发表的数据表明，树突状蛋白是一种新的裂隙横隔膜成分，在转化生长因子-β(TGF-β)的影响下，它重新定位到足细胞核，促进细胞凋亡。我们发现，在局灶性节段性肾小球硬化的小鼠模型中，树突状蛋白重新定位到细胞核，其中转化生长因子-β水平增加。在这一应用中，我们提出了两个特定的目标来研究树突蛋白对足细胞存活的调节。在特定的目标1中，我们将阐明树突从裂隙隔膜到足细胞核重新定位的分子机制。我们将利用共聚焦显微镜来测试α-肌动蛋白-4的缺失是否会增加树突状蛋白的核表达。我们将通过体内磷酸化实验来测试转化生长因子-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部位的点突变，以测试14-3-3是否以磷酸化依赖的方式与树突结合，以促进核进口。在特定的目标2中，我们将确定树突蛋白如何促进转化生长因子-β诱导的足细胞凋亡。Annexin结合实验和流式细胞仪分析将检测过表达Yes相关蛋白和Nedd4-2对转化生长因子-β诱导的细胞凋亡的影响。随后，我们将与CD2AP缺失和转化生长因子-β转基因的树突状蛋白缺失的小鼠杂交，以确定在这两种转化生长因子-β表达增加的小鼠模型中，树突状蛋白的缺失是否改善了肾小球硬化的严重程度。 相关性：拟议的研究试图确定我们感兴趣的蛋白质树突蛋白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。这些细胞的损伤是蛋白尿肾病进展的一个重要机制。 公共卫生相关性：这项工作试图阐明导致尿腔内至关重要的细胞(足细胞)损伤和死亡的重要机制的细节。这些发现可能会对糖尿病肾病、局灶性节段性肾小球硬化和IgA肾病等肾脏疾病的新治疗药物的开发产生影响，所有这些疾病都与足细胞数量减少有关。

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.