title: Urea transport in MDCK cells that are stably transfected with UT-A1.
abstract: Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was >1,500 Omega.cm(2). Transepithelial [(14)C]urea fluxes were measured at 37 degrees C in a HCO(3)(-)/CO(2) buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-A1-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-A1-transfected cells was UT-A1 protein expressed (as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-A1. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [(14)C]urea fluxes in the UT-A1-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial cell line stably expresses UT-A1 and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts.
authors.affiliation: Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Otto
authors.initials: O
authors.lastname: Fröhlich
authors.forename: Janet D
authors.initials: JD
authors.lastname: Klein
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Smith
authors.forename: Jeff M
authors.initials: JM
authors.lastname: Sands
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gunn
mesh_terms: D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D001692:Biological Transport / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002999:Clone Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D010693:Phloretin / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D014508:Urea / Q000378:metabolism*; D014882:Water-Electrolyte Balance / Q000187:drug effects / Q000502:physiology
chemical_list: D026901:Membrane Transport Proteins; D001127:Arginine Vasopressin; D005576:Colforsin; D009536:Niacinamide; C070820:thionicotinamide; D014508:Urea; D010693:Phloretin