Mechanisms of Glomerular Scarring

肾小球疤痕形成的机制

• 批准号: 7085421
• 负责人: JOHN R. SEDOR
• 金额: $ 28.66万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7085421
• 关键词: biomarker; confocal scanning microscopy; electron microscopy; fluorescence microscopy; gene expression; gene mutation; gene targeting; genetic disorder; genetically modified animals; glomerular filtration; glomerulosclerosis; green fluorescent proteins; immunocytochemistry; in situ hybridization; laboratory mouse; pathologic process; phenotype; polymerase chain reaction; protein transport; regulatory gene; renal glomerulus; transcription factor; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): Glomerular scarring is a hallmark of progressive kidney disease. Identifying molecular mechanisms of rare, familial kidney diseases has provided insights into the pathogenesis of sporadic nephropathy. Mutations in genes encoding podocyte proteins, which comprise the filtration barrier or regulate podocyte phenotype, have been discovered by genetic analyses of families with glomerulosclerosis, suggesting a podocyte phenotype "switch" is a critical mechanism of glomerular scarring. We postulated that regulatory pathways transmit information from filtration barrier to nucleus to regulate podocyte differentiation state in response to environmental signals. WT1, a zinc finger transcription factor, critical for appropriate podocyte differentiation, expressed in mature podocytes and is mutated in familial glomerulosclerosis. WT1 expression is diminished in some human and experimental glomerular diseases, but podocyte expression of WT1 target genes, podocalyxin and nephrin, is diminished even when WT1 levels are unchanged. Using a two-hybrid assay to identify regulators of WT1 activity in a mouse kidney library, we identified a novel protein, WT1 interacting protein (WTIP). WTIP maps within a locus for familial focal sclerosis (FSGS1) on human chromosome 19q13.1 and is part of a family of zyxin-like molecules, which contain 3 LIM domains and shuttle between cytoplasm and nucleus. Our preliminary data demonstrated that WTIP is expressed in podocytes in culture and in vivo. Ectopically expressed WTIP co-localized with CD2-associated protein (CD2AP) in podocyte actin spots, sites of dynamic actin filament reorganization. Full-length WTIP, in the presence of the nuclear export inhibitor leptomycin, was retained in the nucleus, co-localized and coprecipitated with WT1, and inhibited WTl-dependent transcriptional activation of the amphiregulin promoter. Based on these data, we hypothesize the following: (1) In normal glomeruli WTIP is part of a multiprotein complex in podocyte foot process and may link the CD2AP/nephrin/podocin complex to adherens junction proteins by regulating dynamic actin assembly. (2) After injury, WTIP translocates into the nucleus, where it represses WTl-dependent gene expression to dysregulate podocyte phenotype. (3) Loss of WTIP from its cytosolic location also promotes redistribution of slit diaphragm proteins and actin rearrangement characteristic of foot process effacement. In vitro and in vivo experiments will test the validity of our model.

描述（由申请人提供）：肾小球瘢痕形成是进行性肾脏疾病的标志。确定罕见的家族性肾脏疾病的分子机制，提供了对散发性肾病发病机制的见解。通过对肾小球硬化症家族的遗传分析，已经发现编码足细胞蛋白（其构成滤过屏障或调节足细胞表型）的基因突变，这表明足细胞表型“转换”是肾小球瘢痕形成的关键机制。我们推测，调节通路将信息从过滤屏障传递到细胞核，以调节足细胞对环境信号的分化状态。WT 1是一种锌指转录因子，对足细胞的适当分化至关重要，在成熟足细胞中表达，在家族性肾小球硬化症中发生突变。在一些人类和实验性肾小球疾病中，WT 1表达减少，但足细胞表达的WT 1靶基因，足细胞标记蛋白和nephrin，即使WT 1水平不变，也会减少。利用双杂交技术鉴定小鼠肾脏文库中WT 1活性的调节因子，我们鉴定了一种新的蛋白质，WT 1相互作用蛋白（WTIP）。WTIP定位于人类染色体19q13.1上的家族性局灶性硬化症（FSGS 1）的基因座内，并且是zyxin样分子家族的一部分，其包含3个LIM结构域并且在细胞质和细胞核之间穿梭。我们的初步数据表明，WTIP表达在足细胞在培养和在体内。异位表达的WTIP与CD 2相关蛋白（CD 2AP）共定位于足细胞肌动蛋白点，动态肌动蛋白丝重组的网站。全长WTIP，在核输出抑制剂来普霉素的存在下，被保留在细胞核中，与WT 1共定位和共沉淀，并抑制WT 1依赖的双调蛋白启动子的转录激活。基于这些数据，我们假设：（1）在正常肾小球中，WTIP是足细胞足突中多蛋白复合物的一部分，并可能通过调节动态肌动蛋白组装将CD 2AP/nephrin/podocin复合物连接到粘附连接蛋白。(2)损伤后，WTIP易位到细胞核中，在那里它抑制WT 1依赖性基因表达以失调足细胞表型。(3)从其胞质位置的WTIP的损失也促进重新分配的狭缝隔膜蛋白和肌动蛋白重排特征的足突消失。体外和体内实验将检验我们模型的有效性。