The role of the primary cilium in glomerular podocyte cells

描述（由申请人提供）： 纤毛是由一个专门的质膜包围微管为基础的轴丝。纤毛起源于与中心粒相同的基体.有三种类型的纤毛：运动的，非运动的（初级），或结节。哺乳动物体内几乎每一个细胞上都有纤毛。初级纤毛被认为是具有化学或机械感知能力的感觉细胞器。在肾脏中，初级纤毛是沿着肾单位的流体流动的机械传感器。纤毛异常与许多肾囊性疾病有关。肾小球足细胞具有从细胞体向外延伸并进入Bowman间隙的纤毛。这项研究计划的主要目的是确定足细胞纤毛的功能。足突表达了一种收缩系统，但其目的尚不清楚。我们对足细胞纤毛和这种记录的收缩系统的观察使我们假设纤毛激活导致收缩，从而调节狭缝隔膜尺寸和过滤质量。我们的目的是纤毛和裂膈之间的沟通缺陷导致足细胞功能障碍和疾病。主要假设被整合为三个具体目标。在第一个目标中，将通过体外和体内纤毛表征研究来评估足细胞的流量传感能力。此外，我们将通过监测以下变化来评估足细胞流动反应：1）水平流体流动刺激后的细胞溶质钙，2）垂直流动刺激后的葡聚糖通量和3）通过肌动蛋白和微管蛋白的免疫荧光标记的细胞骨架重组。第二个目标将确定纤毛激活后负责流动刺激钙反应的分子组分。将使用RNA干扰技术评估离子通道的亲和力，以降低候选蛋白质表达和流动刺激后读数的后续变化。在第三个目标中，足细胞系和转基因小鼠模型将用于评估细胞骨架及其在狭缝隔膜处的结合伴侣在纤毛介导的足细胞流动反应中的贡献。这一目标将比较正常和肾小球疾病患者足细胞纤毛的结构差异，以表明异常流量传感作为蛋白尿性肾病进展的一个特征。将使用来自正常和转基因小鼠模型的分离的灌注肾小球来测试足细胞纤毛对流动反应的贡献。这些研究将增加我们对基本肾功能的理解，并为新发现的足细胞器提供全面的功能评估，因为它涉及基于足细胞的肾小球疾病。

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.