Deciphering interaction and collaborative function of basement membrane and slit diaphragm in Drosophila.

破译果蝇基底膜和裂隙隔膜的相互作用和协作功能。

• 批准号: 539635640
• 负责人: Privatdozent Dr. Tobias Hermle
• 金额: --
• 依托单位: Klinik für Innere Medizin IV - Nephrologie und Allgemeinmedizin
• 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/539635640?language=en
• 关键词: Deciphering; interaction; collaborative; function; basement

Glomerular disease underlies the majority of chronic kidney failure. Targeted therapies are frequently lacking due to limited understanding of the complex pathogenesis. The critical layers of the filtration barrier are the glomerular basement membrane and the slit diaphragm. Both structures also represent signaling platforms. However, the collaborative function and exchange of signaling cues between both layers remain poorly understood. Approximately 10% of chronic kidney disease has a genetic cause. Recent studies suggest that variants in the three type IV collagens of the basement membrane account for about 30% of these, even in adult cohorts. Surprisingly, these DNA variants frequently manifest as focal segmental glomerulosclerosis, i.e. as a podocytopathy. This highlights the relevance of the interaction of podocytes with the extracellular matrix. Slit diaphragms and basement membranes are lacking in cultured cells and mouse models are hampered by low throughput and difficult accessibility of the filtration barrier. The Drosophila nephrocyte combines a functional, molecularly conserved slit diaphragm and a basement membrane in a genetically tractable model organism. Nephrocytes are therefore ideal to explore the dynamic interaction between the slit diaphragm and the extracellular matrix. We first want to establish the nephrocytes as model for the glomerular basement membrane (Aim 1) by identification of the matrix components, their localization, origin and loss-of-function phenotypes. Fluorescence Recovery After Photobleaching (FRAP) analysis will reveal the dynamics of the basement membrane. Then, we want to unravel the signaling cues exchanged between basement membrane and slit diaphragm (Aim 2). We will study the role of the basement membrane for nephrin trafficking and slit diaphragm establishment, identity matrix receptors, and examine a putative role for excessive autophagy and defective lysosomal degradation. We further will compare transcriptional profiles using RNA sequencing using fine dissection of nephrocytes to obtain RNA exclusively from this cell type for an unbiased trancriptomic analysis. To establish a personalized platform for the glomerular collagen IV genes, we will employ overexpression of the human protein in comparison to the mutant for rescue and gain-of-function. Taken together, the proposed work is suitable to elucidate the collaborative function of slit diaphragm and the glomerular basement membrane.

肾小球疾病是大多数慢性肾衰竭的基础。由于对复杂发病机制的了解有限，经常缺乏靶向治疗。滤过屏障的关键层是肾小球基底膜和裂隙隔膜。这两种结构也代表信号平台。然而，两层之间的协作功能和信号线索交换仍然知之甚少。大约 10% 的慢性肾病有遗传原因。最近的研究表明，基底膜的三种 IV 型胶原蛋白的变异约占其中的 30%，即使在成人群体中也是如此。令人惊讶的是，这些 DNA 变异经常表现为局灶性节段性肾小球硬化症，即足细胞病。这凸显了足细胞与细胞外基质相互作用的相关性。培养细胞中缺乏狭缝隔膜和基底膜，并且小鼠模型因通量低和过滤屏障难以接近而受到阻碍。果蝇肾细胞在遗传上易处理的模型生物体中结合了功能性的、分子保守的狭缝隔膜和基底膜。因此，肾细胞是探索狭缝隔膜和细胞外基质之间动态相互作用的理想选择。我们首先希望通过鉴定基质成分、其定位、起源和功能丧失表型来建立肾细胞作为肾小球基底膜的模型（目标 1）。光漂白后荧光恢复 (FRAP) 分析将揭示基底膜的动态。然后，我们想要解开基底膜和裂隙隔膜之间交换的信号线索（目标 2）。我们将研究基底膜在去氧肾上腺素运输和裂隙隔膜建立、同一基质受体中的作用，并检查过度自噬和有缺陷的溶酶体降解的假定作用。我们将进一步使用 RNA 测序来比较转录谱，使用肾细胞的精细解剖来专门从这种细胞类型中获取 RNA，以进行无偏转录组分析。为了建立肾小球胶原蛋白 IV 基因的个性化平台，我们将采用与突变体相比的人类蛋白的过度表达来拯救和获得功能。综上所述，所提出的工作适合阐明裂隙隔膜和肾小球基底膜的协同功能。