THE EXOCYST, ADPCKD, AND RENAL ORGANOGENESIS

说明(改编自应用程序) 上皮性囊肿和小管的形成是复杂的，知之甚少。 对器官发育、再生至关重要的过程 损伤和疾病状态，如常染色体显性遗传性多囊肾病 (ADPCKD)。在体外模型系统中，Madin-Darby犬肾(MDCK)细胞 在胶原基质和管状物中生长时会形成包囊 肝细胞生长因子，又称散射因子(HGF/SF)。在.期间 小管生成细胞在侵入胶原蛋白时暂时失去极性 随着新的小管的形成，重新获得极性。调节这种调节的因素 极性在很大程度上是未知的。一个候选因素是胞囊复合体， 这是发芽酵母分泌极化的决定因素。 哺乳动物的外囊高度保守，并参与极化的膜。 上皮细胞的流量。初步数据显示，这个排泄物 复合体主要参与囊和小管的形成。外囊复合体 定位于极化的MDCK细胞的紧密连接，并在 小管发生，沿生长的小管以一致的模式重新定位 随着极性的变化。当胞囊的关键成分hSec10， 在MDCK细胞中过表达，更有效和更快地形成包囊 在刺激时，随着小管形成的显著增加而发生 HGF/SF。 在NIH Grant K08DKO2509的R03补充申请中，创建了 肾脏控制下过表达hSec10的转基因小鼠 细胞特异性启动子的提出是为了评价在体内的作用。 HSec10在肾小管发生中的作用。卵泡外囊复合体成员的表达模式 还将对肾脏发育和ADPCKD组织进行研究。一个可能的 显性阴性形式的hSec10将被克隆入腺病毒载体，并 用于感染高表达全长hSec10的对照和MDCK细胞 以便为涉及基因的更广泛的研究产生初步数据 心理治疗。最后，将为小鼠的产生奠定基础。 HSec10基因的靶向干扰。 综上所述，胞囊复合体以前并未与 包囊和小管的形成过程。我们的初步数据显示，这个复杂的 在体外模型系统中参与了囊泡发生和小管发生。我 将检验这样一种假设，即hSec10成分的过度表达 胞外包囊复合体增加小鼠体内的囊性和小管生成 模型和ADPCKD患者的组织样本中，因此是一种 新的治疗靶点。

DESCRIPTION (adapted from the application) Epithelial cyst and tubule formation represent complex, poorly understood processes that are crucial for organ development, regeneration following injury, and disease states such as autosomal dominant polycystic kidney disease (ADPCKD). In an in vitro model system, Madin-Darby canine kidney (MDCK) cells form cysts when grown in a collagen matrix and tubulate in response to hepatocyte growth factor, also known as scatter factor (HGF/SF). During tubulogenesis cells transiently lose polarity as they invade the collagen and regain polarity as new tubules form. Factors regulating this modulation of polarity are largely unknown. One candidate factor is the exocyst complex, which is a determinant of polarized secretion in budding yeast. The mammalian exocyst is highly conserved and is involved in polarized membrane traffic in epithelial cells. Preliminary data indicates that the exocyst complex is centrally involved in cyst and tubule formation. The exocyst complex localized to the tight junction of polarized MDCK cells and, during tubulogenesis, relocalized along the growing tubules in a pattern consistent with the changes in polarity. When a critical component of the exocyst, hSec10, was overexpressed in MDCK cells, more efficient and rapid cyst formation occurred along with greatly increased tubule formation upon stimulation with HGF/SF. In this R03 supplement application for NIH Grant K08DKO2509, the creation of transgenic mice overexpressing hSec10 under the control of a renal cell-specific promoter is proposed in order to evaluate the in vivo role of hSec10 in tubulogenesis. The expression pattern of exocyst complex members in developing kidneys and in ADPCKD tissue will also be studied. A probable dominant negative form of hSec10 will be cloned into an adenovirus vector and used to infect control and MDCK cells overexpressing full-length hSec10 in order to generate preliminary data for a more extensive study involving gene therapy. Finally, the groundwork will be laid for the generation of mice with targeted disruptions in the hSec10 gene. In summary, the exocyst complex has not previously been implicated in the process of cyst and tubule formation. Our preliminary data shows this complex is involved in cystogenesis and tubulogenesis in an in vitro model system. I will test the hypothesis that overexpression of the hSec10 component of the exocyst complex increases cystogenesis and tubulogenesis in vivo in a murine model and in tissue samples from patients with ADPCKD and is, therefore, a novel therapeutic target.