The role of the primary cilium in glomerular podocyte cells

描述(由申请人提供)： 纤毛是以微管为基础的轴丝，被特殊的质膜包裹。纤毛起源于基底体，其结构与母中心粒相同。纤毛有三种类型：运动型、非运动型(初生型)或节状型。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传感能力的感觉细胞器。在肾脏中，初级纤毛是液体沿肾单位流动的机械传感器。纤毛异常与许多肾囊性疾病有关。肾小球足细胞有纤毛，从细胞体向外延伸，进入鲍曼间隙。这项研究的主要目的是确定足细胞纤毛的功能。Podoctyes表达了一种收缩系统，但其目的不明。我们对足细胞纤毛和这一记录在案的收缩系统的观察，使我们假设纤毛激活导致收缩，从而调节裂隙横隔膜的尺寸和滤过的质量。我们的目的是纤毛和裂孔隔膜之间的沟通障碍会导致足细胞功能障碍和疾病。这一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特性研究来评估足细胞的血流传感能力。此外，我们将通过监测1)水平流动刺激后细胞内钙的变化，2)垂直流动刺激后的葡聚糖流量，以及3)通过免疫荧光标记肌动蛋白和微管蛋白对细胞骨架的重组来评估足细胞流动的反应。第二个目标是确定纤毛激活后引起流动刺激的钙反应的分子成分。离子通道的候选能力将使用RNA干扰技术进行评估，以降低候选蛋白的表达和流刺激后读数的后续变化。在第三个目标中，将使用足细胞系和转基因小鼠模型来评估细胞骨架及其在裂隙隔膜的结合伙伴在纤毛介导的足细胞流动反应中的作用。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，以表明异常的血流感觉是基于蛋白尿的肾脏疾病进展的一个特征。足细胞纤毛对血流反应的贡献将使用正常和转基因小鼠模型的分离灌流肾小球来测试。这些研究将增加我们对基本肾功能的了解，并为新发现的足细胞细胞器提供全面的功能评估，因为它与基于足细胞的肾小球疾病有关。

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.