Rôle du complexe protéique NPHP1/NPHP4/RPGRIP1L impliqué dans la néphronophtise et les ciliopathies associées, dans la morphogenèse épithéliale, la polarité cellulaire et la ciliogenèse

Abstract : Nephronophthisis, a hereditary nephropathy characterized by interstitial fibrosis and cyst formation, is caused by mutations in NPHP genes encoding the ciliary proteins called nephrocystins. We investigate the function of nephrocystin-1, -4 and -8, in vitro and in vivo in mammalian kidney cells and in zebrafish respectively. Depletion of either NPHP1 (N1-KD), NPHP4 (N4-KD) or RPGRIP1L (RPGRIP1L-KD) by shRNA-mediated knockdown in MDCK cells led to abnormal ciliogenesis, delay in tight junction formation and disorganized structures in 3D culture. Moreover NPHP4 modulates the Wnt pathways during morphogenesis of the zebrafish pronephros and in mammalian kidney cells in which NPHP4 interacts with inversin and dishevelled, regulating its stability and its subcellular localization. Rpgrip1l is required for dishevelled stabilization at the cilium base and is necessary for polarized positioning of motile cilia of the zebrafish floor plate and sensory hair cells of the mouse cochlea. In either N1-KD or N4-KD cells, we also showed an over activation of Cdc42 and RhoA, downstream targets of dishevelled. This was accompanied by actin cytoskeletal disorganization, enhanced spreading on collagen, over-activation of proteins that regulate focal adhesion structures i.e p130cas-Pyk2 and increased cell migration. Interestingly, the stable expression of dominant negative form of Cdc42 in knockdown cells rescued the migration and the 3D phenotypes. In parallel, we observed that loss of Nphp4 in mice caused cystic tubular dilatation after subtotal nephrectomy correlated with alteration of ciliogenesis and over activation of Cdc42 and RhoA. Our data show a role of nephrocystins in epithelial cell organization and kidney morphogenesis in particular in regulation of focal adhesion, tight junction, ciliogenesis via dishevelled stability.
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Helori-Mael Gaudé. Rôle du complexe protéique NPHP1/NPHP4/RPGRIP1L impliqué dans la néphronophtise et les ciliopathies associées, dans la morphogenèse épithéliale, la polarité cellulaire et la ciliogenèse. Sciences agricoles. Université René Descartes - Paris V, 2012. Français. ⟨NNT : 2012PA05T048⟩. ⟨tel-00839849⟩

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