Figure 4  AQP2 Subcellular Localization and Translocation in Mouse Kidney Collecting Ducts and MDCK Cell Lines
(A) Immunohistochemistry on collecting ducts in kidney sections from an AQP2-F204V mutant (Mut) mouse and an age-sex matched wild-type (WT) littermate. Mice were injected intraperitoneally with PBS (NT) or dDAVP before sacrificing and fixation of the kidneys. Kidneys sections were immunostained for AQP2 (red) and the basolateral marker AQP3 (green). The images were merged and an area of the cytoplasm was magnified (zoom). Note that mutant AQP2 is not properly localized to the subapical compartment, nor does it respond to dDAVP.
(B) MDCK cell lines, stably transfected with constructs encoding mouse WT or AQP2-F204V, were treated with and without 150 μM forskolin for 90 min, after which cells were fixed, permeabilized, and subjected to immunocytochemistry. AQP2 is shown in green, and the basolateral marker Na+/K+-ATPase is shown in red, alongside the nuclear stain DAPI. The z-profile images were reconstructed from multiple z-sections, along the dotted line. Mutant AQP2 fails to localize to the cell surface upon forskolin stimulation. Rather, the perinuclear staining is consistent with an ER localization of mutant AQP2.
(C) The MDCK cell line expressing AQP2-F204V was grown on fibronectin-coated coverslips until tight junctions formed, at which point the cells were treated with 150 μM forskolin for 90 min. Cells were fixed, permeabilized, and sequentially immunoblotted for AQP2 (green) and calnexin (red), an ER marker. The merged image shows that AQP2-F204V colocalizes with the endoplasmic reticulum marker. Scale bar refers to 10 μm.