Aquaporin-2 transfection of Madin-Darby canine kidney cells reconstitutes vasopressin-regulated transcellular osmotic water transport.

Water transport across the mammalian collecting tubule is regulated by vasopressin-dependent aquaporin -2 in­ sertion into and retrieval from the apical cell membrane. To establish a cell line that properly expresses aquaporin -2 and its hormone-dependent shuttling, Madin-Darby canine kidney cells were stably transfected with an aquaporin-2 expression construct. Cells of a representative clone (wild-type 10 (WT- 10)) were grown on semipermeable supports, and transcellular osmotic water permeability (Pf; in ijlm/s ± SEM) was mea­ sured. The basal Pf of WT-10 cells, which was lowered with indomethacin, increased from 10.6 ± 0.8 to 35.7 ± 1.2 upon incubation with l-desamino-8-D-a rginine vasopressin (dDAVP). This increase coincided with the translocation of aquaporin-2 from an intracellular compartment to the apical membrane. The Pf of untransfected cells (6.5 ± 0.8) was Since the identification of the first mammalian water channel, the channel-forming integral membrane protein of 28 kD (CHIP28), or aquaporin-1 (AQP-1), the aquaporin family in mammals has been expanded to six members and is likely to grow (reviewed in references 1 and 2). In general, aquaporins are intrinsic membrane proteins of approximately 30 kD that form channels and facilitate selective water transport across cell membranes. For most aquaporins, this water permeability can be blocked by sulfhydryl reagents, which is due to binding of mercury to a cysteine amino acid present in an extracellular loop, thought to be near the mouth of the water pore (3,4). Although mammalian aquaporins are very similar in primary structure, they differ greatly in tissue distribution, subcellular localization, and regulation of expression (1,2). In the kidney, four aquaporins (AQP-1, AQP-2, AQP-3, and AQP-4) are expressed. AQP-1 is constitutively and abundantly expressed in the apical and basolateral membranes of epithelial cells lining renal proximal tubules and descending limbs of Henle