CALCIUM ENTRY REGULATED BY CILIA IN POLYCYSTIC DISEASE

多囊病中纤毛调节的钙进入

• 批准号: 7030207
• 负责人: Phillip Darwin Bell
• 金额: $ 2.94万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-15 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7030207
• 关键词: apical membrane; calcium channel; calcium ion; cilium; confocal scanning microscopy; cytoskeleton; epithelium; immunofluorescence technique; membrane activity; polycystic kidney; polymerase chain reaction; protein isoforms; western blottings

DESCRIPTION (provided by applicant): Polycystic kidney disease (PKD) is characterized by the formation of renal cysts, cardiovascular abnormalities and hypertension. Various derangements including abnormal ion transport, altered cell polarity, accelerated cell growth and proliferation, protein mis-localization, and abnormal extracellular matrix composition have been demonstrated in various models of PKD. In models of autosomal recessive polycystic kidney disease (ARPKD), defects in cilia development or ciliary function have been proposed as a common pathogenic mechanism. Other work has shown that one function of cilia is to mediate flowdependent increases in cytosolic calcium concentration [Ca2+]i. The central hypothesis of this work is that cilia are tonic regulators of apical Ca2+ entry and that the absence of a functioning cilia in PKD leads to elevated and unregulated Ca2+ entry across the apical membrane. Studies will be performed in control collecting duct cell lines and in collecting duct cells from either the Oak Ridge Polycystic Kidney mouse model of ARPKD (orpk) in which cilia are absent, cilia(-), or from the cpk mouse which has a mutation in the ciliary protein, cystin. The first aim will be to determine the role of cilia regulated apical Ca2+ entry by apical flow and store operated Ca2+ channels (SOCC). Studies will also determine if overall apical Ca2+ permeability is higher in the absence of a functioning cilia. Since transient receptor potential channels (TRP) are likely candidates for these two pathways, the second aim will identify which of the TRP channels are expressed in these cells and determine if there are differences in message and proteins levels in cilia+/- cells. siRNA or blocking antibodies will be used to establish which TRP isoforms are involved in flowdependent Ca2+ channels and SOCC activity and in overall Ca2+ permeability. The third aim is to define the role of cilia in the spatial organization of TRP channels at the apical membrane. The role of cilia cytoskeletal interactions in the regulating TRP location and activity will also be addressed.

描述（由申请人提供）：多囊性肾脏疾病（PKD）的特征是形成肾脏囊肿，心血管异常和高血压。在PKD的各种模型中已经证明了各种危险，包括异常离子转运，细胞极性改变，加速的细胞生长和增殖，蛋白质错误定位以及异常细胞外基质组成。在常染色体隐性多囊肾脏疾病（ARPKD）的模型中，已经提出了纤毛发育或睫状功能的缺陷作为一种常见的致病机制。其他工作表明，纤毛的一种功能是介导胞质钙浓度的流动依赖性增加[Ca2+] i。这项工作的核心假设是纤毛是顶端Ca2+进入的补品调节剂，而在PKD中缺乏纤毛的缺乏会导致整个顶端膜上升高和不受管制的Ca2+进入。研究将在对照收集管道细胞系和从不存在纤毛的ARPKD（ORPK）的橡树岭多囊肾脏小鼠模型中收集管道细胞的对照中进行研究，或者是从纤毛蛋白，ciliary蛋白中具有突变的CPK小鼠（ - ）。第一个目的是确定纤毛调节的顶端Ca2+通过顶部流动和存储操作的Ca2+通道（SOCC）的作用。研究还将确定在没有功能纤毛的情况下，总体顶端Ca2+渗透率是否更高。由于瞬态受体电位通道（TRP）可能是这两种途径的候选者，因此第二个目标将确定哪些TRP通道在这些细胞中表达，并确定纤毛+/-细胞中的信息和蛋白质水平是否存在差异。 siRNA或阻断抗体将用于确定哪些TRP同工型与流动依赖性CA2+通道和SOCC活性以及总体Ca2+渗透性有关。第三个目的是定义纤毛在顶部膜上TRP通道的空间组织中的作用。还将解决纤毛细胞骨架相互作用在调节TRP位置和活动中的作用。