The exocyst in kidney development and cyst formation

描述（由申请人提供：囊肿是上皮器官（例如肾脏）的基本“构件”，囊肿发生的异常调节会导致潜在的致命性疾病，例如多囊肾病（PKD）。初级纤毛是一种从上皮细胞顶端表面突出的细胞器，与 PKD 的发病机制有关，被认为充当细胞的感觉天线。当肾小管上皮细胞的初级纤毛当形态或功能受到破坏时，细胞将其误解为去分化和增殖的信号，导致形成大型肾囊肿。高度保守的八蛋白外囊复合体是分泌途径的关键组成部分，它将含有膜蛋白的囊泡运送到目标亚细胞部位，包括初级纤毛。该研究计划的总体目标是阐明外囊在肾脏发育和囊肿发生中的作用。通过对 Manine-Darby 犬肾 (MDCK) 肾小管上皮细胞系的体外研究，我们发现外囊对于初级纤毛和正常囊肿的形成至关重要。此外，我们发现外囊的核心成分 Sec10 的沉默会导致细胞内钙的减少和细胞增殖的增加，这都是 PKD 的标志。外囊对于纤毛发生和囊肿发生至关重要，当在体内被破坏时，将导致与其他纤毛病中所见的多囊表型相似，我们将通过以下具体目标来检验这一假设：（1）确定 Sec10 敲低的 MDCK 细胞是否由于有缺陷的管腔聚结空化或有缺陷的细胞凋亡诱导的空化而无法形成囊肿。我们将使用 MDCK 细胞的三维培养来识别当外囊不存在时被破坏的囊肿发生的特定阶段：极性建立、顶端囊泡递送或细胞凋亡 (2) 识别肾上皮细胞中外囊调节的增殖途径。为此，我们将确定在 Sec10 沉默的 MDCK 细胞中测量到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 Sec10 的肾脏特异性小鼠敲除模型，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。由于外囊的种系破坏导致早期胚胎致死，我们将使用 Cre/lox 靶向策略仅在肾小管上皮细胞中敲除 Sec10，这将使我们能够测试体内外囊的缺失是否重现了纤毛发生破坏的体外发现。细胞增殖增加，并导致类似于 PKD 的肾脏表型 除了从执行拟议研究中获得的实践培训之外，申请人在该奖项期间将接受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的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.