Immunolocalization of the mercurial - insensitive water channel and glycerol intrinsic protein in epithelial cell plasma membranes ( water transport / aquaporin / kidney collecting duct / airways / brain ) ANTONIO FRIGERI

Two water channel homologs were cloned recently from rat kidney, mercurial-insensitive water channel (MIWC) and glycerol intrinsic protein (GLIP). Polyclonal antibodies were raised against synthetic C-terminal peptides and purified by affinity chromatography. MIWC and GLIP antibodies recognized proteins in rat kidney with an apparent molecular mass of 30 and 27 kDa, respectively, and did not cross-react. By immunofluorescence, MIWC and GLIP were expressed together on the basolateral plasma membrane of collecting duct principal cells in kidney. By immunohistochemistry, MIWC and GLIP were expressed on tracheal epithelial cells with greater expression of GLIP on the basal plasma membrane and MIWC on the lateral membrane; only MIWC was expressed in bronchial epithelia. In eye, GLIP was expressed in conjunctival epithelium, whereas MIWC was found in iris, ciliary body, and neural cell layers in retina. MIWC and GLIP colocalized on the basolateral membrane of villus epithelial cells in colon and brain ependymal cells. Expressiopn of MIWC and GLIP was not detected in small intestine, liver, spleen, endothelia, and cells that express water channels CHIP28 or WCH-CD. These studies suggest water/solute transporting roles for MIWC and GLIP in the urinary concentrating mechanism, cerebrospinal fluid absorption, ocular fluid balance, fecal dehydration, and airway humidification. The unexpected membrane colocalization of MIWC and GLIP in several tissues suggests an interaction at the molecular and/or functional levels. Several mammalian water-selective transporting proteins (aquaporins) with homology to members of the MIP (major intrinsic protein of lens) protein family (1) have been cloned recently. Channel-forming integral protein (CHIP28) is expressed in erythrocytes, kidney proximal tubule, thin descending limb of Henle, and epithelial and/or endothelial cells in choroid plexus, alveolus, colonic crypts, ciliary body, and other fluid-transporting tissues (2-7). WCH-CD (AQP2) is the vasopressin-regulated water channel expressed in intracellular vesicles and the apical membrane of principal cells in kidney collecting duct (8, 9). Humans with mutations in CHIP28 have been found recently to have no obvious clinical abnormalities (10), whereas mutations in WCH-CD are associated with hereditary nephrogenic diabetes insipidus (11). To identify other water channel homologs expressed in mammalian tissues with high water permeability that do not express CHIP28 and WCH-CD, the application of an homology cloning strategy yielded several more MIP family members (12-14). A mercurial insensitive water channel (MIWC) was cloned from a rat lung cDNA library and functioned as a water-selective channel when expressed in Xenopus oocytes; in situ hybridization indicated MIWC transcript expression in kidney medulla, brain surface, and retina (13). An unusual The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4328 feature of MIWC expression was the presence of full-length mRNA encoding a functional water channel and a spliced mRNA of unclear significance; the relative expression of full-length transcript to spliced transcript was high in kidney and lung but low in liver and salivary gland. A second homologous protein, glycerol intrinsic protein (GLIP), was cloned from rat kidney independently by Ma et al (14) and Ishibashi et aL (AQP3; ref. 15). Both groups reported increased glycerol uptake when GLIP was expressed in Xenopus oocytes, whereas Ishibashi et at (15) also reported increased osmotic water permeability. Preliminary immunolocalization studies indicated strong expression of GLIP protein in basolateral membrane of kidney collecting duct, and Northern and PCR Southern blot analyses indicated a wide tissue distribution of the GLIP transcript (14, 15). The purpose of this study was to determine the tissue distribution and membrane localization of MIWC and GLIP proteins by immunohistochemistry and to determine whether the spliced MIWC transcript was translated. Purified polyclonal antibodies localized MIWC and GLIP in basolateral membrane of principal cells in kidney collecting duct, selected epithelial cells in trachea, colon, and eye, and ependymal cells in brain. Cellular expression of the spliced MIWC transcript was not associated with expression of immunoreactive protein. An interesting result was the colocalization of MIWC and GLIP in certain cell membranes that do not express CHIP28 and WCH-CD.