Abstract
Primary cilia are hair-like cellular extensions that sense microenvironmental signals surrounding cells. The role of adenylyl cyclases in ciliary function has been of interest because the product of adenylyl cyclase activity, cAMP, is relevant to cilia-related diseases. In the present study, we show that vasopressin receptor type-2 (V2R) is localized to cilia in kidney epithelial cells. Pharmacologic inhibition of V2R with tolvaptan increases ciliary length and mechanosensory function. Genetic knockdown of V2R, however, does not have any effect on ciliary length, although the effect of tolvaptan on ciliary length is dampened. Our study reveals that tolvaptan may have a cilia-specific effect independent of V2R or verapamil-sensitive calcium channels. Live-imaging of single cilia shows that V2R activation increases cilioplasmic and cytoplasmic cAMP levels, whereas tolvaptan mediates cAMP changes only in a cilia-specific manner. Furthermore, fluid-shear stress decreases cilioplasmic, but not cytoplasmic cAMP levels. Our data indicate that cilioplasmic and cytoplasmic cAMP levels are differentially modulated. We propose that the cilium is a critical sensor acting as a responsive cAMP microcompartment during physiologically relevant stimuli.
Highlights
Primary cilia are hair-like cellular extensions that sense microenvironmental signals surrounding cells
In vascular endothelial cells, which generally have shorter cilia than epithelial cells, ciliary length increased from 3.67 ± 0.06 μm to 7.56 ± 0.14 μm after treatment with tolvaptan
CAMP-dependent protein kinase (PKA) and other downstream signaling molecules are activated[52,53]. This leads to the insertion of aquaporin 2 (AQP2) into the apical membrane[54], increasing water reabsorption in the kidney collecting duct and enhancing urinary concentration in the thick ascending limb
Summary
Primary cilia are hair-like cellular extensions that sense microenvironmental signals surrounding cells. Functional defects of the cilium, such as dysfunctional polycystins proteins (PC1 or PC2), cause polycystic kidneys[2,3,4,11,12] Both PC1 and PC2 form a mechanosensory complex in the cilium, which upon bending modulates calcium fluxes[13,14]. Studies have shown that increasing cAMP levels in cystic epithelial cells, via either adenylyl cyclase (AC) activation or addition of membrane-permeable cAMP analogs, enhances cyst formation and/or cyst enlargement. Polycystic Kidney (PCK) rats develop progressive cystic enlargement of the kidneys and hepatic histologic abnormalities that resemble human autosomal dominant PKD26 Using this model, Wang et al evaluated renal cyst development in the PCK rats without circulating vasopressin. Previous studies have demonstrated that increasing intracellular cAMP levels, through the addition of forskolin or a cell-permeable cAMP analog, causes an increase in ciliary length of renal epithelia[34,35]. Given the potential treatment of tolvaptan and the role of cilia in PKD, we sought to characterize the effect of tolvaptan on ciliary length and ciliary cAMP signaling
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