Regulation of podocyte survival by dendrin

树突蛋白对足细胞存活的调节

• 批准号: 8294828
• 负责人: Kirk N Campbell
• 金额: $ 15万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8294828
• 关键词: Actinin; Annexins; Apoptosis; Apoptotic; BAX gene; Binding; Binding Sites; Biological Assay; Bowman' s space; Cell Nucleus; Cells; Cessation of life; Complex; Confocal Microscopy; Data; Dendrites; Development; Diabetic Nephropathy; Disease; Dose; Focal Segmental Glomerulosclerosis; Foot Process; Goals; Health; Immunoglobulin A; Importins; Injury; Kidney; Kidney Diseases; Knockout Mice; Measures; Mediating; Modeling; Molecular; Multiprotein Complexes; Mus; NPHS2 protein; Nuclear; Nuclear Import; Phosphorylation; Point Mutation; Protein Biosynthesis; Proteins; Proteinuria; Publishing; Regulation; Research Personnel; Role; Serine; Severities; Severity of illness; Signal Pathway; Signal Transduction; Staurosporine; Testing; Therapeutic Agents; Threonine; Transfection; Transforming Growth Factor beta; Transgenic Mice; Transgenic Organisms; Work; Yeasts; alpha Actinin; base; dendrin; glomerular filtration; glomerular function; glomerulosclerosis; in vivo; interest; microbial alkaline proteinase inhibitor; molecular sieving; mouse model; nephrin; novel; novel therapeutics; overexpression; podocyte; programs; protein complex; slit diaphragm; urinary; yeast two hybrid system

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.

描述（由申请人提供）： 项目摘要：足细胞足突及其插入的缝隙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关联。足细胞丢失的程度与多种肾脏疾病（包括糖尿病肾病和 IgA 肾病）的疾病严重程度相关 我们最近发表的数据已确定树突蛋白是一种新型狭缝隔膜成分，在转化生长因子β（TGF-β）的影响下，它会重新定位到足细胞核以增强细胞凋亡。我们发现，在局灶性节段性肾小球硬化症小鼠模型中，树突蛋白重新定位到细胞核，其中 TGF-β 水平增加。在本申请中，我们提出了两个具体目标来研究树突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蛋白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-actinin-4 的缺失是否会增加树突蛋白的核表达。我们将通过体内磷酸化测定来测试 TGF-β 是否诱导树突蛋白磷酸化。然后我们将诱导假定结合位点的点突变，以测试14-3-3是否以磷酸化依赖性方式与树突蛋白结合以促进核输入。在具体目标 2 中，我们将确定树突蛋白如何增强 TGF-β 诱导的足细胞凋亡。将通过膜联蛋白结合测定和FACS分析来测量过表达Yes相关蛋白和Nedd4-2对TGF-β诱导的细胞凋亡的影响。随后，我们将树突蛋白缺失小鼠与 CD2AP 缺失和 TGF-β 转基因小鼠进行杂交，以确定树突蛋白缺失是否会改善这两种 TGF-β 表达增加的小鼠模型中肾小球硬化的严重程度。 相关性：拟议的研究旨在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重要机制。 公共健康相关性：这项工作旨在阐明导致泌尿道内壁极其重要的细胞（足细胞）损伤和死亡的重要机制的细节。这些发现可能会对肾脏疾病（如糖尿病肾病、局灶节段性肾小球硬化症和 IgA 肾病）新型治疗药物的开发产生影响，所有疾病都与足细胞数量减少有关。