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

肾小球疾病是大多数慢性肾功能衰竭的基础。由于对其复杂的发病机制的了解有限，靶向治疗常常是缺乏的。滤过屏障的关键层是肾小球基底膜和裂隙隔膜。这两个结构也代表了信号平台。然而，两层之间的信号线索的协作功能和交换仍然知之甚少。大约10%的慢性肾脏疾病是由基因引起的。最近的研究表明，基底膜的三种IV型胶原的变异约占这些变异的30%，即使在成人队列中也是如此。令人惊讶的是，这些DNA变异体经常表现为局灶性节段性肾小球硬化，即足细胞病变。这突出了足细胞与细胞外基质相互作用的相关性。培养的细胞缺乏缝隙隔膜和基底膜，小鼠模型受到低通量和难以进入过滤屏障的阻碍。果蝇肾细胞结合了一个功能性的、分子上保守的裂隙横隔膜和一个基底膜，这是一个遗传上容易处理的模式生物。因此，肾细胞是研究裂隙横隔膜和细胞外基质之间动态相互作用的理想细胞。我们首先希望通过鉴定肾小球基底膜的基质成分、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变化。然后，我们想要解开基底膜和裂隙横隔膜之间交换的信号线索(目标2)。我们将研究基底膜在neparin运输和裂隙横隔膜建立中的作用，身份基质受体，并研究过度自噬和缺陷溶酶体降解的推定作用。我们将进一步通过对肾细胞的精细解剖，使用RNA测序来比较转录谱，以获得仅从这种细胞类型中获得的RNA，用于无偏见的转录本分析。为了建立一个针对肾小球IV型胶原基因的个性化平台，我们将利用人类蛋白的过度表达来拯救和获得功能。综上所述，所提出的工作适合于阐明裂隙隔和肾小球基底膜的协同功能。

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.