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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Department of Physiology and 2Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322
Submitted 29 December 2003 ; accepted in final form 18 January 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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·cm2. Transepithelial [14C]urea fluxes were measured at 37°C in a HCO3–/CO2 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 [14C]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. urea transporter-A1; arginine vasopressin; collecting duct; Madin-Darby canine kidney cells
The primary method for investigating the rapid regulation of urea transport has been perfusion of rat IMCDs. This method provides physiologically relevant functional data, although it cannot determine which urea transporter isoform is responsible for a specific functional effect, because both UT-A1 and UT-A3 are expressed in this nephron segment. Progress in understanding the cell biology of urea transporters and the functions of the separate isoforms has been hampered by the lack of an appropriate cell culture system. The goal of the present study was to create a polarized epithelial cell line that stably expresses the UT-A1 urea transporter and reproduces many of the functional properties of urea transport in the IMCD.
In early studies of type I, high-resistance Madin-Darby canine kidney (MDCK) cells, it was shown that they responded to arginine vasopressin (AVP, antidiuretic hormone) by increasing the adenylyl cyclase activity, cAMP levels, and the synthesis of prostaglandins (reviewed in Ref. 8). Addition of apical AVP (110 nM = 50 mU/ml) also increased the rate of tracer Na+ efflux from the cells, but the effect was smaller and delayed compared with the increased rate of efflux seen with 1 mM cAMP (8).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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A new cell line of MDCK cells was constructed by using the Flp-In system (Invitrogen). This system creates isogenic cell lines with one or more Flp recombination target (FRT) sites (5, 29). To create the new MDCK-FRT cell line, MDCK cells were first stably transfected with pFRT/lacZeo by using Lipofectamine 2000 (Invitrogen) and were selected for 10 days in 100 µg/ml Zeocin. Single cell colonies were grown, their 
-galactosidase activity was measured, and Southern blot analysis was performed to determine the number of FRT sites inserted into the genome. Since the MDCK-UT-A1 cells are sensitive to Zeocin, all of the FRT sites have UT-A1 inserted into them by homologous recombination.
A clonally selected MDCK-FRT cell line was then cotransfected with pOG44, a vector for transiently expressing the Flp recombinase, and with pcDNA5/FRT/UT-A1, an expression vector that contains the coding region for UT-A1 and possesses an FRT site for homologous recombination. After insertion of the UT-A1 gene into the FRT site, UT-A1 transcription was driven by the human cytomegalovirus immediate-early enhancer/promoter. The latter vector also contains the hygromycin resistance gene under the control of the SV40 promoter to permit selection for recombinant clones in 800 µg/ml hygromycin. The homologous recombination inactivates the lacZ-Zeocin fusion gene. These cells were then grown and passaged in T-75 flasks by using 500 µg/ml hygromycin in DMEM to maintain selection. The final MDCK-UT-A1 cells, which express UT-A1 by functional assay and Western blot analysis, are sensitive to Zeocin, resistant to hygromycin, and lack -galactosidase activity. The cells were grown and passaged in DMEM containing HEPES and bicarbonate buffers by using standard techniques.
Collagen-coated Costar Transwell inserts (1 cm2 growth surface area; Corning) were used to grow an epithelial layer of MDCK cells. After 1-h incubation of the Transwells in DMEM at 37°C, a suspension of selected, trypsinized cells was prepared, and 7.5 x 104 cells were loaded onto each Transwell and fed after 1 h without hygromycin. These cells grew to confluence over 5–7 days. The transmembrane resistance was measured daily by using an epithelial resistance meter (EVOMX-G; World Precision Instruments, Sarasota, FL). Inserts in which there were not orderly increases in the transepithelial electrical resistance from <0.1 k to >1 k were discarded. We used only membranes with 1.5 k or higher resistance for flux measurements. We transferred the membranes from the CO2 incubator to the flux plate in the following manner to minimize changes in ionic conditions. The urea flux medium contained Hanks' balanced salt solution with bicarbonate (HBBS; GIBCO/Invitrogen) supplemented with 20 mM HEPES from a 1-M stock (GIBCO/Invitrogen). Flux medium made of HBBS-HEPES containing 5 mM urea was aliquoted (1.5 ml) into the wells of a 12-well plate and placed with the lid open for 
1 h in the humidified tissue culture incubator (Pco2 = 40 mmHg, 37°C). We replaced the lid before we moved the covered plates to the experimental bench, where we temporarily transferred them to a 37°C water bath and then placed them on top of a thermostated aluminum block.
Urea flux measurements. Immediately before initiating the flux measurements, we removed four epithelial membrane inserts (Costar) from their culture plates and placed them into empty wells of a 12-well plate. We carefully removed the 500–600 µl of culture medium over each epithelial layer and added 400 µl of prewarmed flux medium containing 0.4 µCi of [14C]urea. The four inserts were then transferred to the first row of wells, each containing the 1.5 ml of flux medium to initiate the transepithelial flux. We removed the lid only to add the inserts and then three more times during the flux as the four inserts were moved at designated times from row to row, ending one and initiating the next flux period (Fig. 1). We always measured the [14C]urea flux from the cis solution (insert or apical membrane bathing solution or upper solution) to the trans solution (well or basolateral membrane bathing solution or the lower solution) because it was difficult to rapidly and carefully remove the upper solution without damaging the cells growing on the filter that forms the base of the insert. Forskolin (10 mM stock in DMSO), AVP (10–4 M stock in H2O), phloretin (100 mM stock in ethanol), dimethylurea, and thionicotinamide (Sigma) were certified grade and were added to the trans solution.
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20 µl in the upper chamber above the epithelial layer. There was fluid attached to the lower surface when the insert was moved from well to well. The residual that was inadvertently carried over from well to well on the underside of the insert during the flux measurements was minimized by dipping the insert three times and tipping it at an angle as it was removed and transferred to the next well. The carried-over counts did not alter the calculated flux more than a few percent, since in most cases the amount carried forward from the last well was not too different from the amount carried forward to the next well. Having consistent residual volumes transferred was a matter of practice and of consistency of tilting and dipping of the insert. We took all of the lower well solution to be counted. In control experiments, there was 27 µl residual in the well from a total of 1,500 µl. Triplicate 10-µl samples of the upper insert solution supplemented with 1.5 ml nonradioactive flux solution, to give equal volumes, were each counted with 3 ml of OptiFluor (Packard) in a Packard Tricarb liquid scintillation counter. Each time period was an independent flux measurement. The total counts transferred across the epithelial layer into the well divided by the specific activity of the upper solution and the time interval was the calculated flux. A near-saturated solution of thionicotinamide (5 mM) was prepared in HBBS-HEPES. Thionicotinamide color quenches the [14C]urea counting rate when present in the scintillation fluid. We corrected the counts (using a quench curve derived from data obtained at a fixed [14C]urea amount and variable thionicotinamide concentrations) when thionicotinamide was present in the trans well.
During a long series of flux measurements, the specific activity in the cis (apical) compartment decreases. We calculated this by using the total counts that were transported to the preceding trans compartments, and we used this corrected specific activity to calculate the flux. The validity of this calculation was determined by directly measuring the specific activity of the cis compartment at the beginning and end of the flux measurements. When necessary, we therefore calculated the change in specific activity by interpolation between initial and final measurements and corrected the flux calculations accordingly.
Western blot and phosphorylation methods. We radiolabeled cells using our previously published methods (13, 14). Briefly, we washed confluent cell layers with phosphate-free DMEM and then incubated them with 1 ml of phosphate-free DMEM containing 0.1 mCi/ml of [32P]orthophosphate for 3 h, 37°C, 5% CO2, and 100% humidity. At the end of the 3-h loading period, inhibitors or activators were added for further incubation. We then washed and solubilized the cells in RIPA buffer. All cellular material was collected. Each phosphorylation immunoprecipitation sample contained the contents of two wells from a six-well plate. We sheared the cells in the samples with a 26-gauge needle, centrifuged the samples for 15 min at 14,000 g, and removed the top 80% of each supernatant fraction to a fresh tube containing 10 µl of antibody. We removed an additional 50 µl, which we added to an equal volume of Laemmli sample buffer and boiled before using it as a preimmunoprecipitation control sample in Western blots.
For immunoprecipitation, we incubated samples with UT-A1 antibody at 4°C overnight with gentle agitation. Protein A-agarose beads (25 µl) were added, and cold incubation continued for a further 2 h. We pelleted the beads in a microcentrifuge and then washed the pellet seven times with RIPA buffer. We verified the completeness of the washing protocol by counting all of the washes as well as the supernatant and sample. On the basis of the counts present on the protein A-agarose beads, we added an amount of Laemmli buffer to the beads and boiled the samples for 1–3 min.
For electrophoretic analysis, proteins were size separated by SDS-PAGE on Laemmli gels and then either stained with Coomassie blue and dried for autoradiography or electroblotted to polyvinylidene difluoride membranes for Western blot analysis as described previously (12–15, 32). Western blots were incubated with our antibody to the COOH terminus of UT-A1 (5% milk, Tris-buffered saline, 0.05% Tween-20) overnight at 4°C (20). The secondary antibody we used depended on the method of analysis. For enhanced chemiluminescence detection, we further incubated the blot with horseradish peroxidase-linked goat anti-rabbit IgG at a dilution of 1:5,000 (2 h, room temperature) and then washed and analyzed it. For infrared detection, we incubated the blots with goat anti-rabbit IgG fluorescently labeled with Alexa 680 (1:4,000, 2 h, room temperature), and then we washed and visualized the blot by using the LI-COR Odyssey gel scanning system (LI-COR Biotechnology, Lincoln, NE). We stained gels in parallel with Coomassie blue to verify the uniformity of gel loading.
UT-A1 protein was immunoprecipitated from equal amounts of the whole cell lysates by using our previously described method (13, 14, 32). Proteins were size separated on two identical SDS-polyacrylamide gels containing an equal portion of the total immunoprecipitated protein per lane. The proteins on one gel were transferred to a polyvinylidene difluoride membrane, and the amount of immunoprecipitated UT-A1 was assayed by Western blot. The other gel was dried, and 32P incorporation into UT-A1 was analyzed by autoradiography.
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RESULTS |
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20–30 min and then appeared to reach a plateau value. When after 42 min of stimulation the forskolin was removed from two of the inserts, the flux in the absence of forskolin remained stimulated for 20 min and then declined 40% over the next 35 min before 5 mM thionicotinamide was included on the basolateral side to inhibit the flux. In the continued presence of forskolin, the urea flux remained stimulated for a significantly longer period and then decayed more slowly. The control fluxes in the absence of forskolin were nearly constant (average 2.6 ± 0.3 nmol·min–1·cm–2) over the same time period.
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DISCUSSION |
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Our Western blot analysis of urea transporter protein showed no evidence for intrinsic urea transporters in the parental untransfected MDCK cells (Fig. 2). A negative Western blot could be obtained if the antibody, which was raised against a COOH-terminal peptide from rat UT-A1, does not recognize the corresponding canine peptide sequence. However, our flux experiments in untransfected cells also showed no change in basal transport after treatment with AVP or forskolin, both of which produced a strong stimulation in transfected cells. Since expression of UT-A1 without hormonal activation did not increase the [14C]urea flux above the native flux rate, all of the hormonal activation can be ascribed to the expressed rat UT-A1. It therefore appears that the MDCK cells did not express a native canine urea transporter or, if they did, that it did not react with the antibody and was not activated by forskolin or AVP.
The MDCK-UT-A1 cells expressed significant amounts of UT-A1 protein as shown by Western blot analysis (Fig. 2), and our transport experiments showed that it was functionally active. In the renal tubule in vivo, the UT-A1 protein was present in two different glycosylation states, with apparent molecular weights of 97 kDa and 117 kDa (1), which differ in their degree of glycosylation and whose relative abundance depended on physiological and pathological conditions (1, 3, 11, 16). In comparison, in the MDCK-UT-A1 cells, the UT-A1 protein was primarily present in the 97-kDa form. However, this did not appear to impair the functional status of the protein, because the MDCK-UT-A1 cells exhibited a robust urea permeability after stimulation with forskolin.
In MDCK-UT-A1 cells, AVP and forskolin increased UT-A1 phosphorylation (Figs. 4 and 6) and both agonists activated [14C]urea fluxes (Figs. 3 and 5). The activated urea fluxes were inhibited by three urea transport inhibitors: thionicotinamide, dimethylurea, and phloretin (Figs. 8–10). Since the MDCK-UT-A1 cells increased their phosphorylation of UT-A1 and increased their flux in response to low concentrations of AVP or forskolin, which activates protein kinase A, these cells appeared to have functional V2 receptors for AVP. To our knowledge, this is the first epithelial cell model to stably express UT-A1, the urea transporter that is expressed in the IMCD.
The time courses of phosphorylation and flux activation of UT-A1 by forskolin were different. The phosphorylation was rapid (2–5 min; Fig. 6) compared with the flux activation (10–30 min; Fig. 5). There are many possible explanations for a delay in flux activation. This may be due to phosphorylation of UT-A1 at multiple sites, many of which are unrelated to activation or many of which must be phosphorylated before the one critical activating site is phosphorylated. Alternatively, phosphorylation of UT-A1 may be a parallel phenomenon unrelated directly to its activation that may result from the phosphorylation of another protein, or linked chain of proteins, which then activate UT-A1 already in the plasma membrane. The delay in MDCK cells may be due to the slow insertion of UT-A1-containing vesicles into the plasma membrane, although this mechanism has been disproved for AVP activation of urea transport in the renal IMCD of Brattleboro rats (which lack AVP) (10, 22).
AVP increased urea permeability in perfused rat terminal IMCDs (28) and increased UT-A1 phosphorylation in IMCD suspensions (32). Forskolin also increased urea permeability in perfused rat terminal IMCDs (9). Phloretin inhibited AVP-stimulated urea transport in perfused rat terminal IMCDs (4). Although neither dimethylurea nor thionicotinamide has been tested in the perfused IMCD, methylurea and acetamide did inhibit urea transport in the rat terminal IMCD (4). Thus the properties of our stably transfected MDCK-UT-A1 cells reproduced the properties of urea transport in the rat terminal IMCD. This suggests that our transfected cells will be a useful model system for studying the cell biology and signaling pathways that regulate urea transport by each of the urea transporter isoforms.
A potential advantage of the approach that we used to create this cell line is that we initially cloned an FRT site into the genome of the parental MDCK cells and created a stable line of MDCK-FRT cells. By cloning UT-A1 into the MDCK-FRT cells, the UT-A1 cDNA can only incorporate into the FRT site that we introduced into the genome of the parental MDCK cells. In future studies, we will be able to clone other urea transporter isoforms into the MDCK-FRT cells, and these UT-A cDNAs should only be incorporated into the same FRT sites. Thus any difference in function between MDCK cell lines that are stably transfected with different UT-A cDNAs should result from differences in the UT-A protein that is expressed and not from where the transgene is incorporated into the MDCK cell genome.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Barker G and Simmons NL. Identification of two strains of cultured canine renal epithelial cells (MDCK cells) which display entirely different physiological properties. Q J Exper Physiol 66: 61–72, 1981.
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4. Chou CL and Knepper MA. Inhibition of urea transport in inner medullary collecting duct by phloretin and urea analogues. Am J Physiol Renal Fluid Electrolyte Physiol 257: F359–F365, 1989.
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7. Fenton RA, Stewart GS, Carpenter B, Howorth A, Potter EA, Cooper GJ, and Smith CP. Characterization of the mouse urea transporters UT-A1 and UT-A2. Am J Physiol Renal Physiol 283: F817–F825, 2002.
8. Fernandez-Castelo S, Bolivar JJ, Lopez-Vancell R, Beaty G, and Cereijido M. Ion transport in MDCK cells. In: Tissue Culture of Epithelial Cells, edited by Taub M. New York: Plenum Press, 1985, 37–50.
9. Gillin AG and Sands JM. Characteristics of osmolarity-stimulated urea transport in rat IMCD. Am J Physiol Renal Fluid Electrolyte Physiol 262: F1061–F1067, 1992.
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11. Kim DU, Sands JM, and Klein JD. Changes in renal medullary transport proteins during uncontrolled diabetes mellitus in rats. Am J Physiol Renal Physiol 285: F303–F309, 2003.
12. Klein JD, Gunn RB, Roberts BR, and Sands JM. Down-regulation of urea transporters in the renal inner medulla of lithium-fed rats. Kidney Int 61: 995–1002, 2002.[CrossRef][Web of Science][Medline]
13. Klein JD, Lamitina T, and O'Neill WC. JNK is a volume-sensitive kinase that phosphorylates the Na-K-2Cl cotransporter in vitro. Am J Physiol Cell Physiol 277: C425–C431, 1999.
14. Klein JD and O'Neill WC. Volume-sensitive myosin phosphorylation in vascular endothelial cells: correlation with Na-K-2Cl cotransport. Am J Physiol Cell Physiol 269: C1524–C1531, 1995.
15. Klein JD, Quach DL, Cole JM, Disher K, Mongiu AK, Wang X, Bernstein KE, and Sands JM. Impaired urine concentration and the absence of tissue ACE: the involvement of medullary transport proteins. Am J Physiol Renal Physiol 283: F517–F524, 2002.
16. Klein JD, Rouillard P, Roberts BR, and Sands JM. Acidosis mediates the upregulation of UT-A protein in livers from uremic rats. J Am Soc Nephrol 13: 581–587, 2002.
17. Knauf PA, Gasbjerg PK, and Brahm J. The asymmetry of chloride transport at 30°C in human red blood cell membranes. J Gen Physiol 108: 577–589, 1996.
18. Lavelle JP, Negrete HO, Poland PA, Kinlough CL, Meyers SD, Hughey RP, and Zeidel ML. Low permeabilities of MDCK cell monolayers: a model barrier epithelium. Am J Physiol Renal Physiol 273: F67–F75, 1997.
19. Morris RG and Schafer JA. cAMP increases density of ENaC subunits in the apical membrane of MDCK cells in direct proportion to amiloride-sensitive Na+ transport. J Gen Physiol 120: 71–85, 2002.
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23. Nielsen S, Terris J, Smith CP, Hediger MA, Ecelbarger CA, and Knepper MA. Cellular and subcellular localization of the vasopressin-regulated urea transporter in rat kidney. Proc Natl Acad Sci USA 93: 5495–5500, 1996.
24. Promeneur D, Rousselet G, Bankir L, Bailly P, Cartron JP, Ripoche P, and Trinh-Trang-Tan MM. Evidence for distinct vascular and tubular urea transporters in the rat kidney. J Am Soc Nephrol 7: 852–860, 1996.[Abstract]
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27. Sands JM. Molecular mechanisms of urea transport. J Membrane Biol 191: 149–163, 2003.[CrossRef][Web of Science][Medline]
28. Sands JM, Nonoguchi H, and Knepper MA. Vasopressin effects on urea and H2O transport in inner medullary collecting duct subsegments. Am J Physiol Renal Fluid Electrolyte Physiol 253: F823–F832, 1987.
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32. Zhang C, Sands JM, and Klein JD. Vasopressin rapidly increases the phosphorylation of the UT-A1 urea transporter activity in rat IMCDs through PKA. Am J Physiol Renal Physiol 282: F85–F90, 2002.
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), 3 x 10–10 M (
), 10–9 M (
), 3 x 10–9 M (
), and 10–8 M (+). Stimulation was evident at a physiological 10–10 M AVP and was maximal at 3 x 10–9 M. All of the AVP-stimulated flux was thionicotinamide inhibitable.
