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）：多囊肾病（PKD）的特征是形成肾囊肿、心血管异常和高血压。不同的 PKD 模型已证实存在各种紊乱，包括离子转运异常、细胞极性改变、细胞生长和增殖加速、蛋白质错误定位和细胞外基质组成异常。在常染色体隐性多囊肾病（ARPKD）模型中，纤毛发育或纤毛功能缺陷被认为是一种常见的致病机制。其他工作表明，纤毛的一项功能是介导细胞质钙浓度 [Ca2+]i 的流量依赖性增加。这项工作的中心假设是，纤毛是顶端 Ca2+ 进入的强直调节剂，PKD 中缺乏功能性纤毛会导致 Ca2+ 穿过顶膜的进入增加且不受调节。研究将在对照集合管细胞系和来自 ARPKD (orpk) 橡树岭多囊肾小鼠模型（其中纤毛缺失、纤毛 (-)）或来自具有纤毛突变的 cpk 小鼠的集合管细胞中进行。纤毛蛋白，胱氨酸。第一个目标是确定纤毛通过顶端流动和储存操作的 Ca2+ 通道 (SOCC) 调节顶端 Ca2+ 进入的作用。研究还将确定在没有功能性纤毛的情况下整体顶端 Ca2+ 通透性是否较高。由于瞬时受体电位通道 (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 或封闭抗体将用于确定哪些 TRP 同工型参与流依赖性 Ca2+ 通道和 SOCC 活性以及总体 Ca2+ 通透性。第三个目的是确定纤毛在顶膜 TRP 通道空间组织中的作用。还将讨论纤毛细胞骨架相互作用在调节 TRP 位置和活性中的作用。