先前的研究表明,小RhoGTPases,肌动蛋白细胞骨架的调节剂,是肾小球疾病中足细胞足突消失的驱动因素,比如FSGS。然而,缺乏对足细胞中小型RhoGTPases调控网络的全面了解。
我们对RhoGTPases的足细胞转录组和蛋白质组数据集进行了分析;在体内定位,足细胞特异性RhoGTPase亲和网络;并检查了靶向Srgap1的条件性敲除小鼠和小鼠疾病模型。为了评估足细胞足突形态,我们使用超分辨率显微镜和电子显微镜;原位邻近连接测定用于确定小GTP酶激活蛋白SRGAP1的亚细胞定位.我们在二维和三维培养和定量相互作用蛋白质组学中对CRISPR/Cas9产生的SRGAP1敲除足细胞进行了功能分析。
我们证明了SRGAP1在体内定位到足细胞足突和在体外定位到细胞突起。Srgap1fl/fl*Six2Cre而不是Srgap1fl/fl*hNPHS2Cre敲除小鼠在成年期发展出FSGS样表型。hNPHS2Cre对Srgap1的足细胞特异性缺失导致对多柔比星诱导的肾病的易感性增加。详细的分析表明足细胞足突明显消失。此外,SRGAP1敲除的足细胞在体外显示出过度的突起形成和小RhoGTP酶机制的抑制。对SRGAP1依赖性相互作用组的评估显示SRGAP1参与了突出和收缩肌动蛋白网络。肾小球活检标本的分析通过在FSGS中显示SRGAP1的明显重新分布,将这些发现转化为人类疾病。
SRGAP1,一种足细胞特异性RhoGAP,通过限制突出的活动来控制足细胞足过程结构,分支肌动蛋白网络。因此,阐明复杂的调控小RhoGTP酶亲和网络为肾小球疾病的潜在精确干预提供了新的靶标。
Previous research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking.
We conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped in vivo, podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting
Srgap1. To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy; in situ proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated
SRGAP1 knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics.
We demonstrated
SRGAP1 localization to podocyte foot processes in vivo and to cellular protrusions in vitro. Srgap1fl/fl*Six2Cre but not Srgap1fl/fl*hNPHS2Cre knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of Srgap1 by hNPHS2Cre resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore,
SRGAP1-knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery in vitro. Evaluation of a
SRGAP1-dependent interactome revealed the involvement of
SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS.
SRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases.