Mechanisms of Glomerular Scarring

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。鉴定罕见的家族性肾脏疾病的分子机制为散发性肾病的发病机制提供了新的见解。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，这些基因包括过滤屏障或调节足细胞表型，这表明足细胞表型“开关”是肾小球瘢痕形成的关键机制。我们假设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。WT1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，并在家族性肾小球硬化中发生突变。在一些人类和实验性肾小球疾病中，WT1表达减少，但即使在WT1水平不变的情况下，足细胞中WT1靶基因podocalyxin和nephrin的表达也减少。利用双杂交实验鉴定小鼠肾库中WT1活性的调节因子，我们鉴定出一种新的蛋白，WT1相互作用蛋白（WTIP）。WTIP位于人类染色体19q13.1的家族性局灶性硬化症（FSGS1）位点，是zyxin样分子家族的一部分，它包含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在培养和体内足细胞中均有表达。异位表达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。全长WTIP在核输出抑制剂leptomycin存在下保留在细胞核中，与WT1共定位共沉淀，抑制wtl依赖性双调节蛋白启动子的转录激活。基于这些数据，我们提出以下假设：(1)在正常肾小球中，WTIP是足细胞足突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CD2AP/nephrin/podocin复合物与粘附连接蛋白连接起来。(2)损伤后，WTIP易位进入细胞核，抑制wtl依赖基因的表达，导致足细胞表型失调。(3) WTIP从细胞质位置的丢失也促进了狭缝隔膜蛋白的重新分布和足突消失特征的肌动蛋白重排。体外和体内实验将检验我们模型的有效性。

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.