The exocyst in kidney development and cyst formation

描述（由申请人提供）：囊肿是上皮器官（如肾脏）的基本“构建块”，囊肿发生的异常调节导致潜在的致命疾病，如多囊肾病（PKD）。初级纤毛是一种从上皮细胞顶面突出的细胞器，与PKD的发病机制有关，被认为是细胞的感觉天线。当肾小管上皮细胞的初级纤毛在形式或功能上被破坏时，细胞将其误解为去分化和增殖的信号，导致形成大肾囊肿。高度保守的八蛋白外囊复合物是分泌途径的重要组成部分，穿梭含有膜蛋白的囊泡到靶向亚细胞区域，包括初级纤毛。本研究的总体目标是阐明外囊在肾脏发育和囊肿形成中的作用，以便更好地了解致病性囊肿形成的过程。我们已经表明，通过体外研究的Manine-Darby犬肾（MDCK）肾小管上皮细胞系，外囊是至关重要的初级纤毛和正常的囊肿的形成。此外，我们发现Sec 10（外囊的中心成分）的沉默导致细胞内钙离子减少和细胞增殖增加，这两个都是PKD的标志。基于这些发现，我们的总体假设是外囊对于纤毛发生和囊形成是至关重要的，并且当在体内被破坏时，将导致类似于在其他纤毛病中所见的多囊表型。我们将通过以下具体目的来检验这一假设：（1）确定Sec 10敲低的MDCK细胞是否由于有缺陷的管腔聚结中空化或有缺陷的细胞分裂诱导的空化而不能形成包囊。由于肾囊肿的形成可以通过这两种替代途径完成，我们将使用MDCK细胞的三维培养物来识别当不存在外囊时被破坏的囊形成的特定阶段（或多个阶段）：极性建立、顶端囊泡递送或细胞凋亡。(2)确定肾上皮细胞中由外囊调节的增殖途径。为此，我们将确定Sec 10沉默MDCK细胞中测得的增殖增加是否是由于在PKD中观察到的类似分子机制所致。(3)生成Sec 10的肾脏特异性小鼠敲除，并将肾脏表型与已知的常染色体显性PKD小鼠模型进行比较。由于外囊的生殖系破坏导致早期胚胎死亡，我们将使用Cre/lox靶向策略仅在肾小管上皮细胞中敲除Sec 10。这将使我们能够测试体内外囊的缺乏是否重现了我们在体外发现的纤毛发生中断和细胞增殖增加，并导致类似于PKD的肾表型。除了从执行拟议的研究中获得的实践培训外，申请人在此奖项期间将获得的教学和指导将确保从博士后实习生成功过渡到专注于了解肾脏疾病分子基础的独立研究人员。

DESCRIPTION (provided by applicant: Cysts are basic "building blocks" for epithelial organs, such as the kidney, and abnormal regulation of cystogenesis results in potentially lethal disorders such as polycystic kidney disease (PKD). The primary cilium, an organelle that projects from the apical surface of epithelial cells and has been implicated in the pathogenesis of PKD, is thought to act as a sensory antenna for the cell. When the primary cilia of renal tubular epithelial cells are disrupted in form or function, the cells misinterpret this as a signal to dedifferentiate and proliferate, resulting in the formation of large renal cysts. The highly conserved eight-protein exocyst complex is a critical component of the secretory pathway, shuttling vesicles containing membrane proteins to targeted subcellular locales, including the primary cilium. The overall goal of this research proposal is to elucidate the role of the exocyst in kidney development and cystogenesis, in order to better understand the process of pathogenic cyst formation. We have shown, through in vitro studies of the Manine-Darby Canine Kidney (MDCK) tubular epithelial cell line, that the exocyst is critical both for the formation of primary cilia and normal cysts. Additionally, we showed the silencing of Sec10, a central component of the exocyst, resulted in decreased intracellular calcium and increased cellular proliferation, both hallmarks of PKD. Based on these findings, our overall hypothesis is that the exocyst is critical for ciliogenesis and cystogenesis, and when disrupted in vivo, will result in a polycystic phenotype similar to that seen in other ciliopathies. We will test this hypothesis through the following Specific Aims: (1) Determine if Sec10-knockdown MDCK cells fail to form cysts due to defective lumen-coalescing hollowing or defective apoptosis-induced cavitation. Since renal cyst formation can be accomplished through these two alternative pathways, we will use three-dimensional cultures of MDCK cells to identify the specific stage (or stages) of cystogenesis that is disrupted when the exocyst is absent: polarity establishment, apical vesicle delivery, or apoptosis. (2) Identify the proliferative pathways regulated by the exocyst in renal epithelial cells. In this aim, we will determine if the increased proliferation measured in Sec10-silenced MDCK cells is due to similar molecular mechanisms that have been observed in PKD. (3) Generate a kidney-specific murine knockout of Sec10 and compare the renal phenotype with a known mouse model of autosomal dominant PKD. Since germline disruption of the exocyst has led to early embryonic lethality, we will use a Cre/lox targeting strategy to knockout Sec10 only in renal tubular epithelial cells. This will allow us to test if the absence of the exocyst in vivo recapitulates our in vitro findings of disruption of ciliogenesis and increased cellular proliferation, and leads to a renal phenotype similar to PKD. In addition to the practical training received from performing the proposed research, the didactic and mentoring that the applicant will receive during this award will ensure the successful transition from a postdoctoral trainee to an independent researcher focused on understanding the molecular basis of renal diseases.