Analysis of pathomechanisms of defective podocyte adhesion in FSGS.

FSGS足细胞粘附缺陷的病理机制分析。

• 批准号: 531415133
• 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/531415133?language=en
• 关键词: Analysis; pathomechanisms; defective; podocyte; adhesion

The podocyte is central in the pathogenesis of proteinuric glomerular diseases such as focal segmental glomerulosclerosis (FSGS). FSGS is one of the leading causes of chronic kidney disease. Podocytes form the outer part of the filtration barrier with their intercellular contacts called slit diaphragms. Slit diaphragms are formed by the essential adhesion proteins Nephrin and Neph1. We dissected that Nephrin transmits signals to Integrin β1 at focal adhesions (FA) via the small GTPase Rap1 and its activating factor RAPGEF1 (C3G). This signaling pathway may influence podocyte adhesion and thus podocyte loss in glomerular disease. To analyze molecular mechanisms of Nephrin signaling, we employ the versatile in vivo Drosophila nephrocyte model of podocytes. Nephrocytes exhibit slit diaphragm-like structures consisting of orthologs of mammalian adhesion proteins such as Nephrin. We showed in several mouse models that knockout (KO) of key genes of this pathway in podocytes leads to FSGS. In mass spectrometry analysis of the phospho-proteome of podocytes after activation of Nephrin signaling, we identified over 180 differentially phosphorylated proteins. We will now pursue the overarching hypothesis that Nephrin transmits signals from the slit diaphragm to focal adhesions thereby influencing the composition of the extracellular matrix. In the first work package, we will test whether Nephrin and Integrin α3β1 interact genetically by crossing heterozygous Nephrin KO with podocyte-specific Integrin α3 or β1 mice and analyzing the modulation of the slit diaphragm phenotype. We will dissect the mechanisms by single-nucleus sequencing. In the second part, we will investigate the hypothesis in the Drosophila model that Nephrin affects the composition of the extracellular matrix. In the phospho-proteome analysis after activation of Nephrin signal transduction, we found among others proteins of the Epidermal Growth Factor Receptor (EGFR) signaling pathway enriched phosphorylated. In the third part, we will pursue the hypothesis that Nephrin and EGFR signaling are integrated to affect focal adhesions.

足细胞在蛋白尿肾小球疾病如局灶节段性肾小球硬化（FSGS）的发病机制中起核心作用。FSGS是慢性肾脏疾病的主要原因之一。足细胞形成滤过屏障的外部部分，它们的细胞间接触称为狭缝隔膜。狭缝隔膜由必需的黏附蛋白Nephrin和Neph1形成。我们分析了Nephrin通过小GTPase Rap1及其激活因子RAPGEF1 （C3G）在局灶粘连（FA）处将信号传递给Integrin β1。该信号通路可能影响肾小球疾病中足细胞粘附从而导致足细胞损失。为了分析Nephrin信号传导的分子机制，我们采用了多用途的果蝇肾细胞足细胞模型。肾细胞表现出由哺乳动物黏附蛋白（如Nephrin）的同源物组成的狭缝膈状结构。我们在几种小鼠模型中发现，足细胞中该通路关键基因的敲除（KO）可导致FSGS。在Nephrin信号激活后足细胞磷酸化蛋白质组的质谱分析中，我们鉴定了超过180种不同的磷酸化蛋白。我们现在将继续研究一个重要的假设，即Nephrin将信号从狭缝隔膜传递到局灶粘连，从而影响细胞外基质的组成。在第一个工作包中，我们将通过杂合子Nephrin KO与足细胞特异性整合素α3或β1小鼠杂交，并分析狭缝膈表型的调节，来检验Nephrin和整合素α3β1是否存在遗传相互作用。我们将通过单核测序来剖析其机制。在第二部分，我们将研究果蝇模型中关于Nephrin影响细胞外基质组成的假设。在激活Nephrin信号转导后的磷酸化蛋白质组分析中，我们发现表皮生长因子受体（EGFR）信号通路的其他蛋白富集磷酸化。在第三部分中，我们将探讨Nephrin和EGFR信号共同影响局灶性粘连的假设。