Rodent cholangiocytes express 6 of the 11 known channel proteins called aquaporins (AQPs) that are involved in transcellular water transport in mammals. However, clarifying the role of AQPs in mediating water transport in biliary epithelia has been limited in part because of the absence of physiologically relevant experimental models. In this study, we established a novel AQP4-transfected polarized mouse cholangiocyte cell line suitable for functional studies of transepithelial water transport, and, using this model, we define the importance of this AQP in water transport across biliary epithelia. Polarized normal mouse cholangiocytes (NMCs) lacking endogenous AQP4 were transfected stably with functional AQP4 or cotransfected with functional AQP4 and a transport-deficient AQP4 dominant negative mutant using a retroviral delivery system. In transfected NMCs, AQP4 is expressed on both the mRNA and protein levels and is localized at both the apical and basolateral membranes. In nontransfected NMCs, the transcellular water flow, Pf, value was relatively high (i.e., 16.4 ± 3.2 μm/sec) and likely was a reflection of endogenous expression of AQP1 and AQP8. In NMCs transfected with AQP4, P f increased to 75.7 ± 1.4 μm/sec, that is, by 4.6-fold, indicating the contribution of AQP4 in channel-mediated water transport across MNCs monolayer. In cotransfected NMCs, AQP4 dominant negative reduced P f twofold; no changes in Pf were observed in NMCs transfected with the empty vector. In conclusion, we developed a novel polarized mouse cholangiocyte monolayer model, allowing direct study of AQP4-mediated water transport by biliary epithelia and generated data providing additional support for the importance of AQP4 in cholangiocyte water transport.
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