The role of the Rap guanine nucleotide exchange factor 1 (C3G) in the podocyte and in glomerular disease

Rap 鸟嘌呤核苷酸交换因子 1 (C3G) 在足细胞和肾小球疾病中的作用

• 批准号: 242796563
• 负责人: Professorin Dr. Britta George
• 金额: --
• 依托单位: Klinik für Nephrologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/242796563?language=en
• 关键词: role; Rap; guanine; nucleotide; exchange

In most forms of glomerular diseases, abnormal filter barrier function results when podocytes undergo foot process spreading and retraction by remodeling their cytoskeletal architecture and intercellular junctions. The cell adhesion protein Nephrin is necessary for establishing regular foot process architecture and junction composition in development by transducing signals that regulate actin cytoskeletal dynamics. Patients with mutations in the gene encoding the slit diaphragm protein Nephrin fail to develop functional slit diaphragms and suffer from severe proteinuria. Nephrin transmits signals from the slit-diaphragm in podocytes via the cytoskeletal adapter proteins Crk1/2 and CrkL to induce lamellipodia formation in culture (George et al, J Clin Invest, 2012). In vivo in mice, Crk1/2 or CrkL deficiency protected from podocyte injury while Crk1/2 and CrkL deficiency resulted in abnormal podocyte process formation and proteinuria (George et al, Kidney International, 2014). As Nephrin activation induces lamellipodia in podocyte culture, we hypothesized that Nephrin may signal to Integrin at focal adhesions. Indeed, in podocyte culture Nephrin activation induces Integrin β activation. Preliminary data showed that this is dependent on the Rap guanine nucleotide exchange factor 1 (C3G) which is an activator of the small GTPase Rap1. Employing the Drosophila nephrocyte model, we showed that knockdown of the Nephrin ortholog Sticks-and stones (Sns) or gain-of-function results in mistargeting of Integrin (Dlugos, Picciotto et al, J Am Soc Nephrol, 2019). In the sequential grant period, we will dissect the mechanisms of Nephrin signaling to Integrin β and characterize the role of Rap1 and its activator C3G in podocyte culture and mice. We will test whether Nephrin-induced Integrin activation modulates podocyte adhesion. We already established a nephron-specific C3G knockout in mice (early podocyte knockout). Mice exhibit proteinuria and foot process effacement. We will now analyze the role of C3G in podocyte maintenance and reaction to injury. For this, we will cross available C3G flox mice with mice that express Cre recombinase driven by the Podocin promoter to generate and phenotype podocyte-specific C3G knockout mice. Additionally, we will test with podocyte-specific C3G knockout mice whether C3G plays a role during podocyte injury. RNA sequencing of prepared podocytes will uncover mechanisms of C3G signaling in vivo. Changes in Integrin activation may result in defective podocyte attachment to the glomerular basement membrane (GBM) and consecutive loss of podocytes into the urine. Podocyte loss is an important pathomechanism that results in chronic glomerular disease. Thus, molecular characterization of the Nephrin-Integrin pathway may expose potential therapeutic targets for glomerular disease.

在大多数形式的肾小球疾病中，当足细胞通过重塑其细胞骨架结构和细胞间连接而经历足突伸展和收缩时，导致异常的滤过屏障功能。细胞粘附蛋白Nephrin通过转导调节肌动蛋白细胞骨架动力学的信号，在发育中建立规则的足突结构和连接组成是必需的。编码裂孔隔膜蛋白Nephrin的基因突变的患者不能发育功能性裂孔隔膜，并患有严重的蛋白尿。Nephrin通过细胞骨架衔接蛋白Crk 1/2和CrkL从足细胞中的裂隔膜传递信号，以诱导培养物中的板状伪足形成（乔治et al，J Clin Invest，2012）。在小鼠体内，Crk 1/2或CrkL缺乏可保护足细胞免受损伤，而Crk 1/2和CrkL缺乏导致异常足细胞突起形成和蛋白尿（乔治et al，Kidney International，2014）。由于在足细胞培养中，肾配蛋白激活诱导板状伪足，我们假设肾配蛋白可能在粘着斑处向整联蛋白发出信号。事实上，在足细胞培养物中，Nephrin活化诱导整联蛋白β活化。初步数据表明，这是依赖于Rap鸟嘌呤核苷酸交换因子1（C3 G），这是一个小的GTP酶Rap 1的激活剂。采用果蝇肾细胞模型，我们发现，Nephrin直系同源物Sticks-and-stones（Sns）或功能获得性的敲低导致整联蛋白的错误定位（Dlugos，Picciotto et al，J Am Soc Nephrol，2019）。在后续研究中，我们将分析Nephrin信号传导至Integrin β的机制，并描述Rap 1及其激活剂C3 G在足细胞培养和小鼠中的作用。我们将测试肾蛋白诱导的整合素激活是否调节足细胞粘附。我们已经在小鼠中建立了肾单位特异性C3 G敲除（早期足细胞敲除）。小鼠表现出蛋白尿和足突消失。我们现在将分析C3 G在足细胞维持和对损伤的反应中的作用。为此，我们将使可用的C3 G flox小鼠与表达由Podocin启动子驱动的Cre重组酶的小鼠杂交，以产生足细胞特异性C3 G敲除小鼠的表型。此外，我们将用足细胞特异性C3 G基因敲除小鼠测试C3 G是否在足细胞损伤过程中起作用。制备的足细胞的RNA测序将揭示体内C3 G信号传导的机制。整合素活化的变化可能导致足细胞与肾小球基底膜（GBM）的附着缺陷和足细胞进入尿液的连续损失。足细胞丢失是导致慢性肾小球疾病的重要病理机制。因此，肾蛋白-整合素途径的分子表征可能揭示肾小球疾病的潜在治疗靶点。