title: Loss of primary cilia results in deregulated and unabated apical calcium entry in ARPKD collecting duct cells.
abstract: Recent genetic analysis has identified a pivotal role of primary cilia in the pathogenesis of polycystic kidney disease (PKD). However, little is known regarding how cilia loss/dysfunction contributes to cyst development. In epithelial cells, changes in apical fluid flow induce cilia-mediated Ca2+ entry via polycystin-2 (PC2), a cation channel. The Oak Ridge Polycystic Kidney (orpk) mouse contains a mutated Tg737 gene that disrupts expression of polaris, a protein required for ciliogenesis. These studies examine the effect of cilia malformation on Ca2+ entry in orpk cilia(-) collecting duct principal cells, and in orpk cells in which wild-type Tg737 was reintroduced, orpk cilia(+). [Ca2+]i was monitored in confluent cell monolayers using fluorescence microscopy. Intrinsic apical Ca2+ entry was measured by Mn2+ quenching and Ca2+ depletion/readdition under flow conditions below the threshold for stimulation. We found that unstimulated apical Ca2+ entry was markedly increased in cilia(-) cells and was sensitive to Gd3+, an inhibitor of PC2. Electrophysiological measurements demonstrate increased abundance of an apical channel, consistent with PC2, in cilia(-) cells. Immunofluorescence studies revealed that PC2, normally expressed on and at the base of cilia in orpk cilia(+) cells, was observed throughout the apical membrane in cilia(-) cells. Furthermore, cilia(-) cells displayed elevated subapical Ca2+ levels measured with the near-membrane Ca2+ indicator FFP-18. We propose that cilia exert a tonic regulatory influence on apical Ca2+ entry, and absence of cilia results in loss of spatial organization of PC2, causing unregulated Ca2+ entry and elevations in subapical [Ca2+], a factor which may contribute to cyst formation.
authors.affiliation: Department of Physiology, Univ. of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Siroky
authors.forename: William B
authors.initials: WB
authors.lastname: Ferguson
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Fuson
authors.forename: Yi
authors.initials: Y
authors.lastname: Xie
authors.forename: Attila
authors.initials: A
authors.lastname: Fintha
authors.forename: Peter
authors.initials: P
authors.lastname: Komlosi
authors.forename: Bradley K
authors.initials: BK
authors.lastname: Yoder
authors.forename: Erik M
authors.initials: EM
authors.lastname: Schwiebert
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Guay-Woodford
authors.forename: P Darwin
authors.initials: PD
authors.lastname: Bell
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002923:Cilia / Q000473:pathology* / Q000502:physiology; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D005682:Gadolinium / Q000494:pharmacology; D007685:Kidney Tubules, Collecting / Q000378:metabolism / Q000648:ultrastructure*; D008345:Manganese / Q000378:metabolism; D051379:Mice; D016891:Polycystic Kidney, Autosomal Dominant / Q000235:genetics / Q000473:pathology*; D050396:TRPP Cation Channels / Q000037:antagonists & inhibitors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics
chemical_list: D002118:Calcium; D005456:Fluorescent Dyes; D016257:Fura-2; D005682:Gadolinium; D008345:Manganese; D050396:TRPP Cation Channels; D025521:Tumor Suppressor Proteins; C490898:Tg737Rpw protein, mouse; C098105:fura-FFP18