with a diminished response to dDAVP. Figure 3 Immunoblot Analyses of AQP2 from Mouse Kidneys (A) Western blot analyses of total kidney membranes from littermate mice. An intermediate form of AQP2 at 31 kDa was identified in kidney membranes from a mutant mouse (Mut) and partially in a heterozygous mouse (Het). (B) Total kidney membranes were subjected to endoglycosidase H treatment (Endo H) prior to Western blotting. High-mannose (h.m.) glycosylated proteins that have not exited the ER are sensitive to endoglycosidase H digestion. In humans, recessive alleles of Aqp2 are postulated to cause NDI because they do not properly translocate to the apical cell surface in response to AVP. This postulate comes solely from in vitro studies in which mutant Aqp2 cDNAs corresponding to human disease mutations are transfected into kidney cell lines. In general, such recessive alleles, when visualized immunocytochemically, fail to localize to AVP-responsive vesicles. Rather, they get trapped in the endoplasmic reticulum (ER). Our mouse model of NDI affords the first opportunity to test this hypothesis in a mature animal. As shown in Figure 4A (top row of photomicrographs), AQP2 (stained red) normally localized to the subapical region of collecting duct cells in kidneys of wild-type mice. Upon stimulation with dDAVP, AQP2 translocated to or near the cell surface (Figure 4A, second row). In kidneys taken from mutant animals, however, AQP2 was distributed randomly throughout the cell in the basal state (Figure 4A, third row), while AQP3 (green) appropriately localized to the basolateral surface [23]. Furthermore, upon dDAVP stimulation, AQP2-F204V failed to translocate to the cell surface (Figure 4A, bottom row). To confirm these findings, the staini