The role of the primary cilium in glomerular podocyte cells

初级纤毛在肾小球足细胞中的作用

• 批准号: 7903302
• 负责人: ROBERT John KOLB
• 金额: $ 13.03万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-05 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7903302
• 关键词: Actins; Binding; Binding Proteins; Bowman' s space; Calcium; Cell Adhesion; Cell Line; Cell membrane; Cells; Centrioles; Cilia; Cilium Microtubule; Communication; Contractile System; Coupling; Cystic Kidney Diseases; Cytoskeletal Modeling; Cytoskeleton; Data; Dextrans; Dimensions; Disease; Filtration; Functional disorder; In Vitro; Ion Channel; Kidney; Kidney Diseases; Label; Lead; Link; Mediating; Molecular; Monitor; Mothers; Nephrons; Nodal; Organelles; Patients; Permeability; Predisposition; Process; RNA Interference; Renal function; Renal glomerular disease; Research; Research Proposals; Role; Sensory; Stimulus; Structure; System; Technology; Testing; Transgenic Mice; Tubulin; base; design; dextran; fluid flow; glomerular filtration; in vivo; kinetosome; mouse model; neuronal cell body; podocyte; protein expression; response; sensor; shear stress; slit diaphragm

DESCRIPTION (provided by applicant): Cilia are microtubule-based axonemes enclosed by a specialized plasma membrane. Cilia originate from the basal body a structure identical to the mother centriole. There are three types of cilia: motile, non-motile (primary), or nodal. Cilia are on nearly every cell in the mammalian body. Primary cilia are considered sensory organelles with either chemo or mechanosensing abilities. In the kidney, primary cilia are mechanosensors of fluid flow along the nephron. Cilium abnormalities are linked to numerous renal cystic diseases. Glomerular podocytes have a cilium that extends outward from the cell body and into Bowman's space. The broad objective of this research proposal is to determine the function of the podocyte cilium. Podoctyes express a contractile system but its purpose in unknown. Our observation of the podocyte cilium and this documented contractile system, led us to hypothesize that cilium activation results in a contraction that modulates slit diaphragm dimensions and the quality of filtration. We purpose that defective communication between cilia and slit diaphragm leads to podocyte dysfunction and diseases. The major hypothesis is integrated into three specific aims. In the first aim, podocyte flow sensing ability will be assessed through in vitro and in vivo cilium characterization studies. In addition, we will assess the podocyte flow response by monitoring changes in 1) cytosolic calcium after a horizontal fluid flow stimulus, 2) dextran flux after vertical flow stimulus and 3) reorganization of the cytoskeleton by immunofluorescent labeling of actin and tubulin. The second aim will determine molecular components responsible for the flow stimulated calcium response after cilium activation. Candidacy of the ion channels will be assessed using RNA interference technology to lower candidate protein expression and subsequent changes in the readout after flow stimulation. In the third aim, podocyte cell lines and transgenic mouse models will be used to assess the contribution the cytoskeleton, and its binding partners at the slit diaphragm, have in ciliary mediated podocyte flow responses. This aim will compare structural differences in podocyte cilia from normal and glomerular diseased patients to indicate abnormal flow sensing as a feature in the progression of proteinuric-based kidney disease. The contribution of podocyte cilia to the flow response will be tested using isolated perfused glomeruli from normal and transgenic mouse models. These studies will increase our understanding of basic kidney functions and provide a thorough functional assessment for a newly discovered podocyte organelle, as it relates podocyte based glomerular diseases.

描述（由申请人提供）： 纤毛是基于微管的轴突，由专门的质膜包围。纤毛源自基体的一个与母中心相同的结构。纤毛有三种类型：运动，非运动（原发性）或淋巴结。纤毛几乎在哺乳动物体内的每个细胞上。原发性纤毛被认为具有化学或机械感应能力的感觉细胞器。在肾脏中，原发性纤毛是沿肾单位流体流动的机械传感器。纤毛异常与许多肾囊性疾病有关。肾小球足细胞的纤毛从细胞体向外延伸到鲍曼的空间。该研究建议的广泛目标是确定足细胞纤毛的功能。 Podoctyes表示收缩系统，但其目的是未知。我们对足细胞纤毛的观察和该记录的收缩系统，使我们假设纤毛激活导致收缩，从而调节缝隙膜片尺寸和过滤质量。我们的目的是纤毛和缝隙隔膜之间有缺陷的通信会导致足细胞功能障碍和疾病。主要的假设纳入了三个特定目标。在第一个目标中，将通过体外和体内纤毛表征研究评估足细胞流动感应能力。此外，我们将通过监测1）水平流体流动刺激后的胞质钙的变化来评估足细胞流动响应，2）垂直流动刺激后的右旋量磁通量和3）通过肌动蛋白和tubulin的免疫荧光标记对细胞骨架的重组。第二个目的将确定纤毛激活后负责流动的钙反应的分子成分。将使用RNA干扰技术评估离子通道的候选性能，以降低候选蛋白的表达，并在流动刺激后随后的读数变化。在第三个目的中，足细胞细胞系和转基因小鼠模型将用于评估细胞骨架的贡献，并且其在裂缝隔膜处的结合伴侣具有纤毛介导的Podocyte流动反应的贡献。该目标将比较正常和肾小球患者的足细胞纤毛的结构差异，以表明流动感应异常，作为基于蛋白尿的肾脏疾病进展的特征。足细胞纤毛对流动响应的贡献将使用正常和转基因小鼠模型的孤立灌注肾小球进行测试。这些研究将增加我们对基本肾脏功能的理解，并为新发现的足细胞细胞器提供彻底的功能评估，因为它与基于足细胞的肾小球疾病有关。