Functional and Phenotypic Characterization of a New FSGS Gene

描述（由申请人提供）：局灶节段性肾小球硬化（FSGS）是肾衰竭的常见原因，是肾小球滤过屏障（GFB）功能完整性改变的致病性改变的结果。家族性FSGS病例的研究指出足细胞在其发病机制中的核心作用。我们的长期目标是通过确定对维持GFB功能完整性至关重要的途径来了解FSGS的分子发病机制，并确定FSGS的新治疗靶点。本应用程序的总体目标是研究f -肌动蛋白结合细胞周期基因ANLN突变导致FSGS的机制。我们的方法是可行的，因为我们最近发现ANLN的f -肌动蛋白结合域R431C突变是家族性FSGS的一个原因。我们发现，在FSGS塌陷的人和小鼠的肾脏活检中，苯胺素上调。表达R431C突变的足细胞细胞系表现出与关键足细胞蛋白CD2AP结合缺陷、AKT活化和细胞运动异常。此外，斑马鱼胚胎中苯胺素的下调会破坏GFB。我们的总体假设是，在苯胺的f -肌动蛋白结合域的突变影响f -肌动蛋白细胞骨架聚合和导致异常足细胞增殖，凋亡和迁移；足细胞稳态的破坏破坏了正常的GFB功能并导致FSGS的发病机制。我们将通过以下具体目标来探索这一假设：1)通过表征a) R431C突变激活的信号级联，确定ANLN R431C破坏足细胞稳态的机制；b)确定R431C突变对细胞凋亡、细胞增殖和细胞迁移的功能影响；c) PI3K/AKT偶联信号通路的药物抑制剂对突变诱导的表型的影响。2)通过体内互补实验确定R431C突变对斑马鱼胚胎GFB的功能影响，以确定R431C ANLN突变的等位基因致病性，并使用肾小球滤过作为生理学相关读数评估药理学拯救。3)通过对FSGS患者的ANLN外显子测序，比较突变组和非突变组的疾病表型，分析FSGS患者队列中ANLN基因的突变情况。创新：这一建议代表了第一个旨在定义氨酰胺变异体引起FSGS机制的研究。意义：揭示ANLN突变导致FSGS的机制可能会发现对维持足细胞细胞骨架功能完整性很重要的途径。此外，通过探索氨苄素在细胞增殖，凋亡和运动中的作用，我们将

DESCRIPTION (provided by applicant): Focal segmental glomerulosclerosis (FSGS), a common cause of kidney failure, is the result of pathogenic changes that alter the functional integrity of the glomerular filtration barrier (GFB). The study of familial FSGS cases points to a central role of the podocyte in its pathogenesis. Our long term goals are to understand the molecular pathogenesis of FSGS by identifying pathways that are critical for the maintenance of the functional integrity of the GFB and identify novel therapeutic targets for FSGS. The overall objective of this application is to study the mechanisms by which mutations in an F-actin binding cell cycle gene, ANLN, cause FSGS. Our approach is feasible because we recently identified a mutation in F-actin binding domain of ANLN, R431C, as a cause of familial FSGS. We showed that anillin is upregulated in kidney biopsies of humans and mice with collapsing FSGS. Podocyte cell lines expressing the R431C mutation demonstrate defective binding to CD2AP a key podocyte protein, activation of AKT and aberrant cell motility. Also, knockdown of anillin in zebrafish embryos disrupts the GFB. Our overarching hypothesis is that mutations in the F-actin binding domain of anillin affect F-actin cytoskeleton polymerization and lead to aberrant podocyte proliferation, apoptosis and migration; disruption of podocyte homeostasis then disrupts normal GFB function and leads to the pathogenesis of FSGS. We will explore this hypothesis through the following specific aims: 1) Determine the mechanisms by which podocyte homeostasis is disrupted by ANLN R431C by characterizing a) the signaling cascades activated by the R431C mutation b) determine the functional effect of the R431C mutation on apoptosis, cell proliferation and cell migration, and c) the effect of pharmacologic inhibitors of the PI3K/AKT coupled signaling pathways on the phenotype induced by the mutation. 2) Determine the functional effect of the R431C mutation on the GFB of zebrafish embryos using an in vivo complementation assay to determine allele pathogenicity of R431C ANLN mutation and assess pharmacologic rescue using glomerular filtration as a physiologically relevant readout. 3) Analyze mutations of the ANLN gene in a cohort of patients with FSGS by sequencing the exons of ANLN in FSGS patients and comparing the disease phenotype in subjects with and without mutations. Innovation: This proposal represents the first study designed to define the mechanisms by which anillin variants cause FSGS. Significance: Unraveling the mechanisms by which mutations in ANLN cause FSGS may identify pathways that are important for maintaining the functional integrity of the podocyte cytoskeleton. Furthermore, by probing the role of anillin in cell proliferation, apoptosis, and motility, we will provide insight into the mechanisms of podocyte renewal in health and disease. Our genetic and mechanistic approaches will advance our understanding of the molecular pathogenesis of podocyte phenotype changes in FSGS and lead to identification of novel therapeutic targets and less toxic pharmacologic approaches.