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

Abstract

Water transport across the mammalian collecting tubule is regulated by vasopressin-dependent aquaporin-2 insertion 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 microm/s +/- SEM) was measured. 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 1-desamino-8-D-arginine 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 unchanged by dDAVP. Kinetic studies with WT-10 cells revealed that maximal Pf was obtained within 30 min after dDAVP addition, which remained elevated for at least 90 min. Intracellular cAMP levels peaked within 5 min after dDAVP admission and decreased to basal levels within 45 min. After preincubation with dDAVP, the Pf decreased within 15 min after dDAVP washout and returned to basal levels within 75 min. In conclusion, the WT-10 cells mimic the vasopressin-regulated transcellular water transport and aquaporin-2 translocation as found in collecting duct cells to a great extent, and therefore constitute an in vitro cell model that can be used to study the regulation of transcellular water transport in detail and provide a simplified test system for screening putative aquaporin-2 blockers.