Loss of primary cilia results in deregulated and unabated apical calcium entry in ARPKD collecting duct cells.

Academic Article

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.
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    Keywords

  • Animals, Calcium, Cell Membrane Permeability, Cells, Cultured, Cilia, Fluorescent Antibody Technique, Fluorescent Dyes, Fura-2, Gadolinium, Kidney Tubules, Collecting, Manganese, Mice, Polycystic Kidney, Autosomal Dominant, TRPP Cation Channels, Tumor Suppressor Proteins
  • Digital Object Identifier (doi)

    Authorlist

  • Siroky BJ; Ferguson WB; Fuson AL; Xie Y; Fintha A; Komlosi P; Yoder BK; Schwiebert EM; Guay-Woodford LM; Bell PD
  • Start Page

  • F1320
  • End Page

  • F1328
  • Volume

  • 290
  • Issue

  • 6