Linking polarity signaling to mechanotransduction in glomerular podocytes

肾滤过屏障疾病是影响全球5亿多人的慢性肾脏疾病的主要原因。与潜在原因无关，肾小球损伤的程度总是由肾小球上皮细胞-足细胞的损伤来定义。后者是高度特化的肾小球上皮细胞，与有窗口的内皮细胞和肾小球基底膜一起构成实际的肾滤过屏障。足细胞表现出独特的形态，形成初级和次级足突，完全包裹肾小球毛细血管。损伤后，这种特殊的功能形态发生改变，足细胞类似于经典的柱状上皮细胞。直接的结果是，足细胞的细胞形状发生了变化，细胞消失在初级尿液中。考虑到足细胞的有丝分裂后性质，任何细胞的损失都不能通过邻近细胞的增殖来弥补，留下空白的毛细血管，并导致蛋白质大量损失到初级尿液中。肌动蛋白-细胞骨架在这一去分化过程中起着重要作用，但目前对其调控途径的研究还不够深入。损伤小鼠原代足细胞的初步MS/MS数据显示，损伤后假定的机械传感器蛋白Filamin B显著增加。在果蝇肾细胞中极性信号转导机制存在缺陷的果蝇肾细胞中，其同源基因Cher的表达也显著增加，这不仅表明机械转导过程和细胞极性信号通路在足细胞中的高度重要性，而且表明这两条通路都具有潜在的遗传依赖性。因此，在这个项目提案中，我们的目标是研究机械转导和细胞极性信号对足细胞生物学的影响。我们将利用果蝇肾细胞模型进一步揭示机械感受器蛋白丝蛋白的功能作用。为此，我们将产生不同的转基因果蝇，并表征不同的丝氨酸结构域对肾细胞形态和功能的贡献。在第二个独立的方法中，我们将在体内研究细胞极性异常信号在果蝇肾细胞机械转导过程中的影响。为了进一步研究这两条通路之间的相互联系，我们还将分析机械感觉受扰对肾细胞细胞极性的影响。在这项建议中，我们的目标不仅是解开丝胺在体内的功能作用，还旨在表征体内肾细胞中机械转导和细胞极性信号之间的潜在相互联系。这一建议的发现将清楚地有助于理解足细胞在损伤时形态变化的潜在机制。基于所获得的知识，未来可以开发治疗肾小球疾病的新疗法。

Diseases of the kidney filtration barrier are a major cause of chronic kidney disease affecting more than 500 million people worldwide. Independent of the underlying cause, the degree of glomerular injury is always defined by the damage to the epithelial cells of the glomerulus, the podocytes. The latter are highly specialized epithelial cells of the glomerulus, which build the actual kidney filtration barrier together with fenestrated endothelial cells and the glomerular basement membrane. Podocytes exhibit a unique morphology and form primary and secondary foot processes, which enwrap the glomerular capillaries completely. Upon injury this specialized and functional morphology alters and podocytes resemble classical columnar epithelial cells. As a direct consequence, the podocytes’ cell shape changes and the cells are lost into the primary urine. Given the post mitotic nature of podocytes any cellular loss cannot be compensate by proliferation of neighboring cells leaving the capillaries blank and causing immense loss of protein into the primary urine. The actin-cytoskeleton plays an important role during this dedifferentiation process, but the regulatory pathways are either not identified so far or only characterized insufficiently. Preliminary MS/MS data of injured primary mouse podocytes revealed a significant increase of the putative mechanosensor protein Filamin B upon injury. Its Drosophila homolog Cher was also significantly increased in Drosophila nephrocytes with a defective polarity signaling machinery, suggesting not only a high importance of mechanotransduction processes and cell polarity signaling pathways in podocytes, but also a potential genetic dependency of both pathways. Therefore, within this project proposal we aim to investigate the impact of mechanotransduction and cell polarity signaling on podocyte biology. We will make use of the Drosophila nephrocyte model to further unravel the functional role of the mechanosensor protein Filamin. To this end, we will generate different transgenic flies and characterize the contribution of the different Filamin domains for nephrocyte morphology and function. In a second and independent approach, we will study the impact of aberrant cell polarity signaling on mechanotransduction process in Drosophila nephrocytes in vivo. To further examine the interconnection between the two pathways, we will also analyze effects of disturbed mechanosensation on cell polarity in nephrocytes. Within this proposal, we not only aim to unravel the functional role of Filamin in vivo, but also aim to characterize the potential interconnection between mechanotransduction and cell polarity signaling in nephrocytes in vivo. The findings of this proposal will clearly help to understand the underlying mechanisms regulating morphological changes of podocytes upon injury. Based on this acquired knowledge novel therapies treating glomerular diseases can be developed in the future.