AQUAPORIN-3 WATER CHANNEL LOCALIZATION AND REGULATION IN RAT-KIDNEY

The aquaporins are a family of water channels expressed in several wat er-transporting tissues, including the kidney. We have used a peptide- derived, affinity-purified polyclonal antibody to aquaporin-3 (AQP-3) to investigate its localization and regulation in the kidney. Immunobl otting experiments showed expression in both renal cortex and medulla, with greatest expression in the base of the inner medulla. Subcellula r fractionation of membranes, using progressively higher centrifugatio n speeds, revealed that AQP-3 is present predominantly in the 4,000 an d 17,000 g pellets and, in contrast to AQP-2, is virtually absent in t he high-speed (200,000 g) pellet that contains small intracellular ves icles. Immunocytochemistry and immunofluorescence studies revealed tha t labeling is restricted to the cortical, outer medullary, and inner m edullary collecting ducts. Within the collecting duct, principal cells were labeled, whereas intercalated cells were unlabeled. Consistent w ith previous immunofluorescence studies (K. Ishibashi, S. Sasaki, K. F ushimi, S. Uchida, M. Kuwahara, H. Saito, T. Furukawa, K. Nakajima, Y. Yamaguchi, T. Gojobori, and F. Marumo. Proc. Natl. Acad. Sci. USA 91: 6269-6273, 1994; T. Ma, A. Frigeri, H. Hasegawa, and A. S. Verkman. J . Biol. Chem. 269: 21845-21849, 1994), the labeling was confined to th e basolateral domain. Immunoelectron microscopy, using the immunogold technique in ultrathin cryosections, demonstrated a predominant labeli ng of the basolateral plasma membranes. In contrast to previous findin gs with AQP-2, there was only limited AQP-3 labeling of intracellular vesicles, suggesting that this water channel is not regulated acutely through vesicular trafficking. Immunoblotting studies revealed that th irsting of rats for 48 h approximately doubled the amount of AQP-3 pro tein in the inner medulla. These studies are consistent with a role fo r AQP-3 in osmotically driven water absorption across the collecting d uct epithelium and suggest that the expression of AQP-3 is regulated o n a long-term basis.