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.
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|Steele, Stacy L; Wu, Yongren; Kolb, Robert J et al. (2010) Telomerase immortalization of principal cells from mouse collecting duct. Am J Physiol Renal Physiol 299:F1507-14|