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

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+]iwas 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.