The exocyst in kidney development and cyst formation

肾脏发育和囊肿形成中的外囊

• 批准号: 7873314
• 负责人: Benjamin C. Fogelgren
• 金额: $ 15.3万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7873314
• 关键词: Affect; American; Apical; Apoptosis; Autosomal Dominant Polycystic Kidney; Award; Calcium; Canis familiaris; Cell Culture Techniques; Cell Line; Cell Polarity; Cell Proliferation; Cell model; Cell physiology; Cells; Cilia; Collagen; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyst; Cystic kidney; Development; Disease; EGF gene; Embryo; Engineering; Ensure; Epithelial; Epithelial Cells; Epithelium; Exons; Gene Targeting; Goals; Growth Factor Receptors; Hereditary Disease; Human; In Vitro; Kidney; Kidney Diseases; Knock-out; Knockout Mice; Laminin; Link; Liquid substance; Locales; Location; MEKs; Measurement; Measures; Membrane Proteins; Mentors; Modeling; Molecular; Morphology; Mus; Organ; Organelles; Pathogenesis; Pathway interactions; Phenotype; Physiological; Physiology; Polycystic Kidney Diseases; Process; Proliferating; Proteins; Regulation; Renal function; Research; Research Personnel; Research Project Grants; Research Proposals; Role; S-Phase Fraction; Secretory Component; Sensory; Signal Pathway; Signal Transduction; Site; Staging; Surface; Testing; Training; Transgenic Mice; Tubular formation; Vesicle; Yeasts; base; cell type; cilium biogenesis; design; improved; in vivo; kidney cell; matrigel; mouse model; nephrogenesis; novel; overexpression; public health relevance; research study; response; trans-Golgi Network; vector

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. PUBLIC HEALTH RELEVANCE: Autosomal dominant polycystic kidney disease (ADPKD), affecting 500,000 Americans, is the most common potentially lethal genetic disease and is characterized by cystic overgrowth that destroys the kidney. A cellular organelle called the primary cilium has been implicated in the pathogenesis of ADPKD and our Preliminary Studies show that the eight-protein exocyst complex is necessary for both cilia and cyst formation. In this application, we propose experiments to analyze the exocyst in kidney development and cyst formation, with successful completion of these experiments laying the groundwork for development of novel therapies for ADPKD.

描述(申请人提供：囊泡是上皮器官的基本“积木”，如肾脏，囊泡发生的异常调节会导致潜在的致命性疾病，如多囊肾病(PKD)。初级纤毛是一种从上皮细胞顶端突起的细胞器，与PKD的发病有关，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态或功能上被破坏时，细胞将其误解为去分化和增殖的信号，导致大的肾囊肿的形成。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，将含有膜蛋白的囊泡运送到靶向亚细胞位置，包括初级纤毛。本研究建议的总体目标是阐明外囊在肾脏发育和囊变中的作用，以便更好地了解致病囊变的形成过程。我们通过对MANINE-Darby犬肾小管上皮细胞系(MDCK)的体外研究表明，外囊对于原发纤毛的形成和正常包囊的形成都是至关重要的。此外，我们还发现，胞外囊泡的中心成分Sec10的沉默导致细胞内钙减少和细胞增殖增加，这两个都是PKD的特征。基于这些发现，我们的总体假设是，外囊对于纤毛发生和囊变是关键的，当在体内被破坏时，将导致类似于其他纤毛疾病的多囊表型。我们将通过以下具体目标来验证这一假说：(1)确定Sec10基因敲除的MDCK细胞是否由于缺陷的管腔融合、空洞或缺陷的凋亡诱导的空化而未能形成包囊。由于肾囊肿的形成可以通过这两种不同的途径完成，我们将使用MDCK细胞的三维培养来识别当没有外囊时囊性发生的特定阶段：极性建立、顶端囊泡交付或细胞凋亡。(2)明确肾上皮细胞受胞外囊调控的增殖途径。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中观察到的类似的分子机制所致。(3)建立肾脏特异的Sec10基因敲除小鼠模型，并将其肾脏表型与已知的常染色体显性遗传性PKD小鼠模型进行比较。由于胚系破坏外囊已导致早期胚胎死亡，我们将使用Cre/lox靶向策略仅在肾小管上皮细胞中敲除Sec10。这将使我们能够测试体内没有外囊是否概括了我们的体外研究结果，即纤毛发生中断和细胞增殖增加，并导致类似PKD的肾脏表型。除了从执行拟议研究中获得的实践培训外，申请人在该奖项期间将接受的指导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。 公共卫生相关性：常染色体显性遗传性多囊肾病(ADPKD)影响着50万美国人，是最常见的潜在致命遗传病，其特征是囊性过度生长，破坏肾脏。一种称为初级纤毛的细胞器与ADPKD的发病机制有关，我们的初步研究表明，8蛋白外囊复合体是纤毛和包囊形成所必需的。在这一应用中，我们提出了分析肾脏发育和包囊形成过程中的胞囊的实验，这些实验的成功完成为开发ADPKD的新疗法奠定了基础。