The role of the Rap guanine nucleotide exchange factor 1 (C3G) in the podocyte and in 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，会导致过滤屏障功能异常。细胞黏附蛋白Nephin通过传递调节肌动蛋白细胞骨架动力学的信号，在发育过程中建立正常的足突结构和连接组成是必需的。编码狭缝横隔膜蛋白Nephin的基因突变的患者无法形成功能性狭缝横隔膜，并患有严重的蛋白尿。Nephin通过细胞骨架适配蛋白Crk1/2和CrkL在足细胞中传递来自裂隙横隔膜的信号，从而在培养中诱导片状脂蛋白的形成(George等，J Clin Invest，2012)。在小鼠体内，Crk1/2或CrkL缺乏保护足细胞免受损伤，而Crk1/2和CrkL缺乏导致足细胞突起异常形成和蛋白尿(George等，肾脏国际，2014)。在足细胞培养中，由于Neferin的激活诱导片状脂血症，我们假设Neferin可能在局部粘连处向整合素发出信号。事实上，在足细胞培养中，肾素的激活会诱导整合素β的激活。初步数据显示，这依赖于Rap鸟嘌呤核苷酸交换因子1(C3G)，它是小GTP酶Rap1的激活剂。利用果蝇肾细胞模型，我们表明敲除Newitin原基棒和结石(SNS)或功能增强会导致整合素的错误定位(Dlugos，Picciotto等人，J am Soc Nephrol，2019)。在后续的授权期，我们将剖析Nephin信号转导整合素β的机制，并表征RAP1及其激活剂C3G在足细胞培养和小鼠中的作用。我们将测试Nephin诱导的整合素激活是否调节足细胞黏附。我们已经在小鼠身上建立了肾脏特异的C3G基因敲除(早期足细胞基因敲除)。小鼠出现蛋白尿和足突消失。我们现在将分析C3G在足细胞维持和损伤反应中的作用。为此，我们将现有的C3G FLOX小鼠与表达由Podocin启动子驱动的Cre重组酶的小鼠杂交，以产生表型特异的足细胞C3G基因敲除小鼠。此外，我们将用足细胞特异的C3G基因敲除小鼠测试C3G在足细胞损伤中是否发挥作用。对准备好的足细胞进行RNA测序将揭示体内C3G信号的机制。整合素活性的改变可能导致足细胞附着在肾小球基底膜(GBM)上的缺陷，并导致足细胞连续丢失进入尿液。足细胞丢失是导致慢性肾小球疾病的重要病理机制。因此，肾素-整合素途径的分子特征可能揭示肾小球疾病的潜在治疗靶点。

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.