Variation of medium osmolalities induces changes in cilia related signalling pathways in primary cultured renal inner medullary collecting duct cells

摘要

Compared to other organs the cells of the renal innermedulla are challenged with an environment of highlyvariable osmolality. Cellular adaption to changing osmolalityis associated with changes in gene expression. To identifynovel genes and pathways that are affected by highosmolality we performed microarray experiments usingprimary cultured rat inner medullary collecting duct cellscultivated at 300, 600, or 900 mosmol/kg for six days.Compared to 300 mosmol/kg more than 2000 genesshowed significant changes in expression at 600 or 900mosmol/kg. Pathway analysis revealed that the WNT/betacateninpathway was also affected. Western blot analysisconfirmed the down regulation of beta-catenin and ofphosphorylated beta-catenin protein. Immunofluorescenceanalysis indicated massive changes in intracellular localizationof beta-catenin. At 300 mosmol/kg beta-catenin wasdistributed diffusively within the cells. At higher osmolalitiesbeta-catenin was concentrated at the cell to cell contactsand in the nucleus. Similar effects were observed forphosphorylated beta-catenin. We also observed strikingchanges in cell morphology. While at 300 mosmol/kg theactin filaments were diffusively distributed the cultivationat 600 mosmol/kg was associated with massive reorganisationand enrichment of the actin filaments at the cell tocell contacts. Staining of the microtubules with betatubulinclearly showed the primary cilia in cells cultivatedat 300 mosmol/kg. Surprisingly the length of the cilia wasdrastically reduced in cells cultivated at 600 mosmol/kg.The analysis of the underlying physiological mechanismsand the consequences for cilia related signalling atdifferent osmolalities could help to add new aspects tocilia function.