Mechanisms of Glomerular Scarring

肾小球疤痕形成的机制

• 批准号: 6670833
• 负责人: JOHN R. SEDOR
• 金额: $ 36.17万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6670833
• 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）：肾小球疤痕是进行性肾病的标志。鉴定罕见的家族性肾病的分子机制为了解散发性肾病的发病机制提供了见解。通过对肾小球硬化家族的遗传分析发现了编码足细胞蛋白的基因突变，这些蛋白构成了过滤屏障或调节足细胞表型，这表明足细胞表型“转换”是肾小球疤痕形成的关键机制。我们假设调节途径将信息从过滤屏障传递到细胞核，以响应环境信号调节足细胞的分化状态。 WT1 是一种锌指转录因子，对于足细胞的适当分化至关重要，在成熟足细胞中表达，并在家族性肾小球硬化症中发生突变。 WT1 表达在一些人类和实验性肾小球疾病中减少，但即使 WT1 水平不变，WT1 靶基因、足萼蛋白和去氧肾上腺素的足细胞表达也会减少。使用双杂交测定来鉴定小鼠肾脏文库中 WT1 活性的调节因子，我们鉴定了一种新的蛋白质，WT1 相互作用蛋白 (WTIP)。 WTIP 位于人类染色体 19q13.1 上的家族性局灶性硬化症 (FSGS1) 基因座内，属于 zyxin 样分子家族的一部分，该分子包含 3 个 LIM 结构域并在细胞质和细胞核之间穿梭。我们的初步数据表明 WTIP 在培养物和体内的足细胞中表达。异位表达的 WTIP 与 CD2 相关蛋白 (CD2AP) 共定位于足细胞肌动蛋白点（动态肌动蛋白丝重组位点）。在核输出抑制剂瘦霉素存在下，全长WTIP保留在细胞核中，与WT1共定位并共沉淀，并抑制双调蛋白启动子的WT1依赖性转录激活。基于这些数据，我们假设如下：（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，并且可能通过调节动态肌动蛋白组装将CD2AP/去氧肾上腺素/podocin复合物与粘附连接蛋白连接起来。 (2)损伤后，WTIP易位到细胞核中，在细胞核中抑制WT1依赖性基因表达，从而失调足细胞表型。 (3) WTIP 从胞质位置的丢失也会促进缝隙隔膜蛋白的重新分布和足突消失特征的肌动蛋白重排。体外和体内实验将测试我们模型的有效性。