THE EXOCYST, ADPCKD, AND RENAL ORGANOGENESIS

描述（改编自应用程序） 上皮囊肿和小管的形成是复杂的， 这些过程对器官发育至关重要， 损伤和疾病状态，如常染色体显性多囊肾病 （ADPCKD）。在体外模型系统中，Madin-Darby犬肾（MDCK）细胞 在胶原基质中生长时形成囊肿， 肝细胞生长因子，也称为分散因子（HGF/SF）。期间 当小管发生细胞侵入胶原蛋白时，它们暂时失去极性， 当新的小管形成时重新获得极性。调节这种调节的因素 极性基本上是未知的。一个候选因素是外囊复合体， 其是芽殖酵母中极化分泌的决定因素。 哺乳动物的外囊是高度保守的，并参与极化膜 上皮细胞的运输。初步数据显示外囊 复合物主要参与囊肿和小管的形成。外囊复合体 定位于极化MDCK细胞的紧密连接， 沿着生长的小管重新定位， 随着极性的变化。当外囊的关键成分hSec 10， 在MDCK细胞中过表达， 在用以下物质刺激后，发生沿着小管形成的极大增加 HGF/SF。 在NIH Grant K 08 DKO 2509的R 03补充申请中， 在肾抑制剂控制下过表达hSec 10的转基因小鼠 细胞特异性启动子，以评估在体内的作用， hSec 10在肾小管发生中的作用。外囊复合体成员的表达模式， 还将研究发育中的肾脏和ADPCKD组织中的细胞。一个可能 将显性阴性形式的hSec 10克隆到腺病毒载体中， 用于感染对照细胞和MDCK细胞，这些细胞过表达全长hSec 10， 为更广泛的研究提供初步数据， 疗法最后，将为小鼠的产生奠定基础， hSec 10基因的靶向破坏。 总之，外囊复合物以前没有涉及到 囊肿和小管形成的过程。我们的初步数据显示 参与体外模型系统中的囊形成和管形成。我 将检验hSec 10组分的过表达是否会导致细胞凋亡的假设。 外囊复合体增加小鼠体内的囊形成和小管形成 模型和来自ADPCKD患者的组织样品中，因此， 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.