The aquaporins are a family of water channels expressed in several water-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. Immunoblotting experiments showed expression in both renal cortex and medulla, with greatest expression in the base of the inner medulla. Subcellular fractionation of membranes, using progressively higher centrifugation speeds, revealed that AQP-3 is present predominantly in the 4,000 and 17,000 g pellets and, in contrast to AQP-2, is virtually absent in the high-speed (200,000 g) pellet that contains small intracellular vesicles. Immunocytochemistry and immunofluorescence studies revealed that labeling is restricted to the cortical, outer medullary, and inner medullary collecting ducts. Within the collecting duct, principal cells were labeled, whereas intercalated cells were unlabeled. Consistent with previous immunofluorescence studies (K. Ishibashi, S. Sasaki, K. Fushimi, 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 the basolateral domain. Immunoelectron microscopy, using the immunogold technique in ultrathin cryosections, demonstrated a predominant labeling of the basolateral plasma membranes. In contrast to previous findings 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 thirsting of rats for 48 h approximately doubled the amount of AQP-3 protein in the inner medulla. These studies are consistent with a role for AQP-3 in osmotically driven water absorption across the collecting duct epithelium and suggest that the expression of AQP-3 is regulated on a long-term basis.

The einal part of the inner medullary cleing duct exhibits a high degree ofwater permeability that is independent of increased intracellular cAMP and not accounted for by the activity of the known renal epithelial water channels CH1P28

Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC50 = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.