EXPRESSION AND FUNCTIONAL-ANALYSIS OF WATER CHANNELS IN A STABLY AQP2-TRANSFECTED HUMAN COLLECTING DUCT CELL-LINE

In this study, we describe the establishment of a stably transfected e pithelial cell line with the cDNA for the rat aquaporin 2 (AQP2). To t his end, we used a human cell line (HCD) derived from the cortical col lecting duct and having characteristics of principal cells (Prie, D., Friedlander, G., Coureau, C., Vandewalle, A., Cassigena, R., and Ronco , P. M. (1995) Kidney Int. 47, 1310-1318). The HCD cells were first sc reened for the constitutive expression of AQPs. By Western blot analys is, we found a low expression of immunoreactive AQP2 and AQP4 proteins . In contrast, transfected cells (clone CD8) probed with AQP2 antiseru m expressed an intense 29-kDa protein on immunoblot in addition to a b road band between 35-45 kDa corresponding to the glycosylated form of the protein, indicating that full maturity of the protein is attained in transfected cells. Immunofluorescence demonstrated that AQP2 was lo cated in intracellular vesicles. After vasopressin stimulation, the st aining redistributed from an intracellular site to the apical pole of the cells, an effect similar to that described on collecting duct prin cipal cells in vivo (Sabolic, I., Katsura, T., Verbavatz, J. M., and B rown, D. (1995) J. Membr. Biol. 143, 165-175) and in perfused tubules (Nielsen, S., Chou, C. L., Marples, D., Christensen, E. I., Kishore, B . K., and Knepper, M. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10 13-1017). The redistribution of AQP2 in CD8 cells was accompanied by a n approximately B-fold increase in osmotic water permeability coeffici ent (P-f), which was inhibited by 0.3 mM HgCl2. These data indicate th at functional vasopressin-sensitive water channels are expressed in tr ansfected cells. The stably transfected cells represent a suitable mod el to unravel by direct experimental approach the intracellular signal s involved in the translocation of AQP2 to the apical plasma membrane in the presence of vasopressin.