Vasopressin Increases Plasma Membrane Accumulation of Urea Transporter UT-A1 in Rat Inner Medullary Collecting Ducts
Janet D. Klein*,
Otto Fröhlich,
Mitsi A. Blount*,
Christopher F. Martin*,
Tekla D. Smith* and
Jeff M. Sands*,
* Renal Division, Department of Medicine; and Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
Address correspondence to: Dr. Janet D. Klein, Emory University School of Medicine, Renal Division, 1639 Pierce Drive NE, WMB Room 3319B, Atlanta, GA 30322. Phone: 404-727-9933; Fax: 404-727-3425; E-mail: janet.klein{at}emory.edu
Received for publication March 20, 2006.
Accepted for publication July 25, 2006.
Urea transport, mediated by the urea transporter A1 (UT-A1)and/or UT-A3, is important for the production of concentratedurine. Vasopressin rapidly increases urea transport in rat terminalinner medullary collecting ducts (IMCD). A previous study showedthat one mechanism for rapid regulation of urea transport isa vasopressin-induced increase in UT-A1 phosphorylation. Thisstudy tests whether vasopressin or directly activating adenylylcyclase with forskolin also increases UT-A1 accumulation inthe plasma membrane of rat IMCD. Inner medullas were harvestedfrom rats 45 min after injection with vasopressin or vehicle.UT-A1 abundance in the plasma membrane was significantly increasedin the membrane fraction after differential centrifugation andin the biotinylated protein population. Vasopressin and forskolineach increased the amount of biotinylated UT-A1 in rat IMCDsuspensions that were treated ex vivo. The observed changesin the plasma membrane are specific, as the amount of biotinylatedUT-A1 but not the calcium-sensing receptor was increased byforskolin. Next, whether forskolin or the V2-selective agonistdDAVP would increase apical membrane expression of UT-A1 inMDCK cells that were stably transfected with UT-A1 (UT-A1-MDCKcells) was tested. Forskolin and dDAVP significantly increasedUT-A1 abundance in the apical membrane in UT-A1-MDCK cells.It is concluded that vasopressin and forskolin increase UT-A1accumulation in the plasma membrane in rat IMCD and in the apicalplasma membrane of UT-A1-MDCK cells. These findings suggestthat vasopressin regulates urea transport by increasing UT-A1accumulation in the plasma membrane and/or UT-A1 phosphorylation.
Urea plays a critical role in the urinary concentrating mechanismand hence in the regulation of water homeostasis. Urea’simportance to the generation of concentrated urine has beenappreciated since at least 1934 (1). Several studies have shownthat maximal urine-concentrating ability is decreased in protein-deprivedanimals and humans and is restored by urea infusion (reviewedin references [2,3]). Urea is transported by the products ofmammalian genes: UT-A (Slc14a2) and UT-B (Slc14a1). The UT-Afamily of urea transporters currently consists of six proteinisoforms, four of which (UT-A1, UT-A2, UT-A3, and UT-A4) areexpressed in kidney medulla (reviewed in references [2,3]).UT-A1 is the largest protein and is expressed in the apicalplasma membrane of the inner medullary collecting duct (IMCD)(4–6). UT-A3 also is expressed in the IMCD (reviewed inreferences [2,3]). A UT-A1/UT-A3 knockout mouse (7,8) lacksurea transport across its IMCD and has a severe urine-concentratingdefect, indicating that urea transport, mediated by UT-A1 and/orUT-A3, is important for the production of concentrated urine.
Adding arginine vasopressin (AVP; also known as antidiuretichormone) to the bath of a perfused rat terminal IMCD resultsin binding to V2 receptors, stimulating adenylyl cyclase andcAMP production and increasing urea transport (9–12).We showed that one mechanism for rapid regulation is that vasopressinincreases UT-A1 phosphorylation (13). Vasopressin (and forskolin)also increases urea flux and UT-A1 phosphorylation in MDCK cellsthat we stably transfected with UT-A1 (UT-A1-MDCK cells) (14).
Another mechanism by which vasopressin could rapidly increaseurea transport is by increasing UT-A1 accumulation in the apicalplasma membrane. Vasopressin-regulated trafficking of aquaporin-2(AQP2) between subapical vesicles and the apical plasma membraneis the major mechanism for acute regulation of water absorptionby vasopressin in the collecting duct (reviewed in reference[15]). One previous study tested whether vasopressin regulatesthe trafficking of UT-A1 in the rat IMCD but did not find evidencefor regulated trafficking in Brattleboro rats, which lack endogenousvasopressin (16). The collecting duct of Brattleboro rats ischronically conditioned (17) and may not be representative ofcollecting duct of normal rats. In this study, we reexaminedthis question by studying normal (basal), vasopressin-repleteSprague-Dawley rats. We found that exogenous vasopressin doesincrease UT-A1 accumulation in the plasma membrane of the IMCDfrom basal rats. In addition, forskolin, which directly activatesthe catalytic subunit of adenylyl cyclase (18), increases UT-A1accumulation in the plasma membrane of the IMCD from basal ratsand from rats that were made water diuretic for 2 wk and inour stably transfected UT-A1-MDCK cells (14).
Animal Preparation
All animal protocols were approved by the Emory University InstitutionalAnimal Care and Use Committee. Male Sprague-Dawley rats (CharlesRiver Laboratories, Wilmington, MA) that weighed 125 to 200g received free access to water and standard rat chow (TestDiet 5001; Purina Mills, Brentwood, MO) that contained 23% protein.Some rats were made water diuretic for 2 wk by feeding them10% glucose water (19).
Basal or water diuretic rats were given a midscapular subcutaneousinjection of vasopressin (5 nmol in 100 µl of normal saline;American Regent, Shirley, NY) or vehicle, then after 45 minwere killed by decapitation. The kidneys were removed and placedon ice immediately, and the inner medullas were separated. Theinner medullary tissue was placed into ice-cold isolation buffer(10 mM triethanolamine, 250 mM sucrose [pH 7.6], 1 µg/mlleupeptin, and 2 mg/ml PMSF) and homogenized, then SDS was addedto a final concentration of 1% for Western analysis of the totalcell lysate (19–21). Total protein in each sample wasmeasured by a modified Lowry method (DC protein assay reagent;Bio-Rad, Richmond, CA). In some cases, inner medullary tissuelysate was subjected to differential centrifugation to obtaina 17,000 x g (low-speed, high-density) plasma membrane fractionand a 200,000 x g (high-speed, low-density) intracellular vesiclefraction as described previously (16,22–26).
Fresh suspensions of rat IMCD (from rats that were not pretreatedwith vasopressin) were prepared as described previously (13,27).Briefly, rat inner medullas were minced in 1 ml of suspensionbuffer (in mM: 118 NaCl, 5 KCl, 25 NaHCO3, 1.2 MgSO4, 2 CaCl2,5.5 glucose, 5 Na-acetate, and 4 Na2PO4) that contained 2 mg/mlcollagenase and 0.65 mg/ml hyaluronidase, then incubated for30 min at 37°C. Next, DNAse (5 µl of 1 mg/ml) wasadded and incubated for another 30 min. Finally, the IMCD werewashed free of enzymes by centrifugation three times with exchangesof suspension buffer, then resuspended in 1 ml of this buffer.Rat IMCD suspensions were pretreated with 10 nM vasopressinor 10 µM forskolin for 15 min at 37°C, then vasopressinor forskolin was left resident during the biotinylation incubation(described next).
UT-A1-MDCK Cells
Construction and selection of the UT-A1-MDCK cells were describedpreviously (14). We plated 2 x 105 cells per Costar Transwellinsert (Corning, Acton, MA; 1 cm2 growth surface area) and studiedconfluent high-resistance (>800 cm2) polarized epithelialmonolayers 4 d later. Transmembrane resistance was measureddaily using an epithelial resistance meter (EVOMX-G; World PrecisionInstruments, Sarasota, FL). Inserts in which there was not anorderly increase in the transepithelial electrical resistancefrom <100 to >800 cm2 were discarded (14).
Transepithelial tracer urea fluxes at 37°C were measuredas described previously (14,28). The measurements consistedof adding tracer urea to the medium in the Transwell insert(apical side, containing 5 mM cold urea) to start the flux experiment.The insert then was moved in 3-min intervals from one well ofa 12-well culture plate to the next, and the radioactivity thataccumulated during this interval in the basolateral medium wasused to calculate the rate of urea flux. At the end of the fluxexperiment, the cells were washed in flux medium to remove thetracer, 400 µl of 100 mM HCl was added, and the cellswere scraped off the filter with a rubber spatula. The cellsuspension was centrifuged, and the supernatant cell extractwas assayed for its cAMP concentration using a cAMP ELISA kit(Assay Designs, Ann Arbor, MI).
Western Analysis
Western analysis was performed as described previously (14,25,29–31).Briefly, rat inner medullary tissue, IMCD suspensions, or UT-A1-MDCKcells were homogenized in ice-cold isolation buffer and broughtto 1% SDS for Western analysis of total cell lysate (25,29,32).Proteins were size-separated by SDS-PAGE on Laemmli gels, electroblottedto polyvinylidene difluoride membranes, and incubated with primaryantibody overnight at 4°C. After washing, the blot was incubatedwith fluorescently labeled secondary antibody and visualizedwith the Odyssey infrared imaging scanner. Parallel gels werestained with Coomassie blue to verify uniformity of gel loading(LICOR, Lincoln, NE). Laser densitometry was used to quantifythe intensity of the resulting bands on Western blot. Antibodiesto anti–UT-A1 and anti-AQP2 were prepared by our laboratory;anti–calcium-sensing receptor (33,34) was a gift of Dr.Tyler Miller (Case Western Reserve University, Cleveland, OH).
Biotinylation of UT-A1
Rat inner medullary tissue, IMCD suspensions, or UT-A1-MDCKcells were biotinylated using a modification of the method describedpreviously (16). Suspensions were prepared by enzyme digestas described previously (13). In the studies in which the ratreceived a subcutaneous injection of vasopressin, the innermedullary tissue was not treated with vasopressin ex vivo. Inthe studies of fresh suspensions of rat IMCD (from rats thatwere not pretreated with vasopressin) that were subsequentlytreated ex vivo or in the UT-A1-MDCK cell studies, the treatments(vasopressin, forskolin, or dDAVP) were added for 30 min at37°C, then samples were washed free of excess solution twicewith PBS and three times with biotinylation buffer without biotin(215 mM NaCl, 4 mM KCl, 1.2 mM MgSO4, 2 mM CaCl2, 5.5 mM glucose,10 mM triethanolamine, and 2.5 mM Na2HPO4). Treatments wereadded back during the incubation with biotinylation buffer thatcontained 3 mg/ml biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimideester (cat. no. B1022; Sigma, St. Louis, MO) for 60 min at 4°C(16). This incubation procedure results in the biotinylationof both apical and basolateral plasma membrane proteins in ratIMCD suspensions but only apical plasma membrane proteins inUT-A1-MDCK cells (Figure 1). Cells then were washed free ofunattached biotin by three washes with biotin quenching buffer(0.1 mM CaCl2, 1 mM MgCl2, and 260 mM glycine in PBS) with thelast wash incubated for 20 min at 4°C. Next, samples werewashed three times with lysis buffer without detergent, andthe cells were solubilized for 1 h in lysis buffer that contained1% NP-40 (150 mM NaCl, 5 mM EDTA, and 50 mM Tris). After centrifugation(14,000 x g, 10 min, 4°C) to remove insoluble particulates,streptavidin beads were added to the supernatant fractions andallowed to absorb biotinylated proteins overnight at 4°C.After washing with high-salt and no-salt buffers, Laemmli SDS-PAGEsample buffer was added directly to the pellets, samples wereboiled for 1 min, and the pool of biotinylated proteins wasanalyzed by Western blot.
Figure 1. Confocal micrographs of biotinylated proteins that were probed with Alexa 488–streptavidin. Isolated tubules (A and B) or UT-A1 MDCK cells that were grown on semipermeable supports (C and D) were biotinylated as described in the Materials and Methods section, then fixed with paraformaldehyde, permeabilized with NP40, labeled with fluorescence-tagged streptavidin (light), mounted on glass slides, and visualized with a Zeiss LSM 510 confocal microscope using a x63 oil objective. Z stacks were collected. Shown are various cut planes from those stacks as follows: Isolated tubule xz sliced with luminal stain apparent (A), center slice 50% into the Z plane illustrating staining both apical and basolateral but with no staining inside the cell (B), UT-A1 MDCK cells at 33% Z cut from the top showing no evidence of interior biotin (C), and UT-A1-MDCK cells XZ cut plane view (D). Arrows mark the location of the supporting semipermeable membrane.
Statistical Analyses
All data are presented as mean ± SEM. To test for statisticalsignificance between two groups, we used a t test. To test forstatistical significance among three or more groups, we usedANOVA followed by Fisher least significant difference. The criterionfor statistical significance was P < 0.05.
Vasopressin Increases Plasma Membrane Expression of UT-A1 in Rat Inner Medulla
Vasopressin, administered to the rat, significantly increasesUT-A1 protein abundance (densitometry in arbitrary units) ina membrane fraction that is enriched in plasma membranes (22,23,35)from basal rats (from 22 ± 1 to 26 ± 2; n = 4,P < 0.03; Figure 2). Vasopressin did not change UT-A1 abundancein a membrane fraction that is enriched in plasma membranesfrom rats that were made water diuretic for 2 wk (hydrated)to suppress endogenous vasopressin levels (from 20 ±1 to 19 ± 2; n = 4; NS). The latter result is consistentwith a previous study in which dDAVP (desmopressin, a V2-selectiveagonist) did not increase UT-A1 abundance in the membrane fractionthat is enriched in plasma membranes from Brattleboro rats (whichlack endogenous vasopressin) (16). There was no significantchange in UT-A1 abundance in the vesicle fraction for eitherbasal or hydrated rats (data not shown).
Figure 2. Acute vasopressin (AVP) administration increases UT-A1 abundance in the membrane fraction of rat inner medullas. Rats were given AVP (5 nmol subcutaneously) 45 minutes before being killed; inner medullary membranes were isolated and probed with anti–UT-A1. Left four lanes show UT-A1 in inner medullas from basal rats (duplicate samples shown). Right four lanes show UT-A1 in the inner medulla membranes from diuretic rats (water loaded for 2 wk). Arrows indicate the two prominent UT-A1 glycoprotein forms at 117 and 97 kD. Shown are two of four rats from each group.
Vasopressin significantly increases UT-A1 biotinylation (densitometryin arbitrary units) in inner medullas from basal rats (62 ±3 to 71 ± 4; n = 5; P < 0.05; Figure 3). Figure 3also shows that in the pool of biotinylated proteins from thesecells, our antibody detects only bands that are consistent withthe size of biotinylated UT-A1 (36).
Figure 3. Biotinylation of inner medullary (IM) proteins reveal that AVP treatment increases the abundance of biotinylated UT-A1. Rats were given AVP (5 nmol subcutaneously) 45 minutes before being killed, and an IM collecting duct (IMCD) suspension was prepared. The suspended IMCD were biotinylated, and the biotinylated protein pool was analyzed by Western blot for the presence of UT-A1. Left two lanes show UT-A1 abundance in the absence of vasopressin (duplicate samples shown). Right two lanes show UT-A1 in the membranes from rats that were treated with vasopressin. Arrows indicate the two prominent glycoprotein forms at 117 and 97 kD. Shown are two of five rats from each group.
Vasopressin and Forskolin Increase Plasma Membrane Expression of UT-A1 in Rat IMCD Suspensions
The preceding studies were performed by injecting vasopressininto rats and establish the physiologic response in the intactrat. In the following experiments, we isolated IMCD suspensionsand treated them ex vivo with vasopressin or forskolin.
Vasopressin significantly increases biotinylated UT-A1 by 99± 29% (n = 7; P < 0.02; Figure 4A). Forskolin alsosignificantly increases biotinylated UT-A1 by 49 ± 10%(n = 6; P < 0.01). As a positive control, we measured AQP2and found that vasopressin significantly increases biotinylatedAQP2 by 34 ± 7% (n = 7; P < 0.01; Figure 4B). As acontrol to ensure that our biotinylation protocol did not resultin biotinylation of a non–plasma membrane protein, weprobed the same samples using a mAb to the nuclear envelopeproteins lamins A and C. We did not detect any biotinylatedlamin in the biotinylated IMCD suspensions (data not shown).As an additional control to ensure that the biotinylation reagentwas not entering the cells and labeling the total cellular proteinpool, we probed the total cell lysate (pre-streptavidin beadprotein sample) for UT-A1. There was no difference in UT-A1abundance among control, forskolin-, and vasopressin-treatedIMCD suspensions (Figure 4C) or in AQP2 abundance between controland vasopressin-treated IMCD suspensions (Figure 4D). Thesecontrols indicate that our biotinylation protocol is technicallycorrect and that the biotinylation reagent is not entering thecells.
Figure 4. Biotinylation reveals increased UT-A1 in IMCD suspensions that were treated with either 10 µM forskolin or 10 nM AVP for 30 minutes. (A) Western blot of UT-A1 in the biotinylated protein population from duplicate samples of control (left), forskolin-treated (center), or vasopressin-treated (right) rat IMCD suspensions. Shown are two to three of six to seven rats from each group. (B) Western blot of aquaporin-2 (AQP2) in the biotinylated protein population from duplicate samples of control (left) or vasopressin-treated (right) rat IMCD suspensions. Shown are three of seven rats from each group. (C) Western blot of the total cell lysate (pre-streptavidin bead) protein samples that were probed for UT-A1. There was no difference in UT-A1 abundance among control, forskolin-treated, and vasopressin-treated IMCD suspensions. (D) Western blot of the total cell lysate protein samples probed for AQP2. There was no difference in AQP2 abundance between control and vasopressin-treated IMCD suspensions.
Next, we tested whether direct activation of adenylyl cyclasewith forskolin (18) would stimulate an increase in plasma membraneaccumulation of UT-A1 in IMCD suspensions from hydrated rats.Forskolin significantly increases biotinylated UT-A1 in IMCDsuspensions from hydrated rats (from 37 ± 11 to 62 ±4; n = 3; P < 0.05; Figure 5A) and from additional basalrats (from 57 ± 8 to 90 ± 5; n = 3; P < 0.05)that were analyzed in parallel. Forskolin also increases biotinylatedAQP2 in the same samples in both hydrated rats (from 55 ±3 to 64 ± 2; n = 3; P < 0.05; Figure 5B) and frombasal rats (from 78 ± 2 to 97 ± 9; n = 3; P <0.05). As a control to ensure that the increases in UT-A1 andAQP2 were not part of a generalized and nonspecific responseto forskolin, the same samples were probed for calcium-sensingreceptor, an apical plasma membrane protein in the rat IMCD(37). The abundance of biotinylated calcium-sensing receptordid not change with forskolin in basal rats (Figure 5C).
Figure 5. Forskolin increases biotinylated UT-A1 (A) and AQP2 (B) but not calcium-sensing receptor (CaR; C) in both basal and 2-wk hydrated rats. Basal and hydrated rats were killed, then IMCD suspensions were prepared and treated with 10 µM forskolin or vehicle. The biotinylated (membrane) population of proteins was examined by Western blot for UT-A1 (top), AQP2 (middle), and CaR (bottom). (–) and (+), absence or presence of forskolin treatment, respectively. Shown are duplicate samples from a total of three samples per condition. Arrows indicate the molecular weights of UT-A1, AQP2, and CaR.
Forskolin and dDAVP Increase UT-A1 in the Apical Plasma Membrane of UT-A1-MDCK Cells
Forskolin (10 µM, 55 min) significantly increases ureaflux, consistent with our previous findings (14,28), and cAMPproduction in UT-A1-MDCK cells. Forskolin (10 µM, 20 min)significantly increases UT-A1 biotinylation (by 173 ±15%; n = 6; P < 0.005) and hence UT-A1 protein abundancein the apical plasma membrane of the UT-A1-MDCK cells (Figure 6).dDAVP (10 nM, 15 min) also significantly increases UT-A1 biotinylationby 59% (from 35 ± 3 to 55 ± 12; n = 3; P <0.05) in the apical plasma membrane of the UT-A1-MDCK cells(data not shown).
Figure 6. Forskolin (10 µM; and , duplicate determinations) significantly increases urea flux (A) over control () levels. As the final portion of the flux experiment, we add dimethyl urea (DMU) to inhibit carrier-mediated urea transport, and it reduced urea flux to the basal level. The cAMP production (B) in parallel wells (n = 4 duplicate determinations/group) of our UT-A1-MDCK cells was increased by a comparable 60-min treatment with forskolin. (C) Forskolin-induced increase in UT-A1. The cells that were grown on six-well cluster dishes were treated for 30 min with 10 µM forskolin, in parallel with control cells. This Western blot shows duplicate samples of both control (left) and forskolin-treated (right) cells that were probed with anti–UT-A1. Arrows indicate UT-A1. Shown are two of six wells of cells for each condition.
The major findings in this study are that (1) vasopressin increasesUT-A1 accumulation in the plasma membrane of the IMCD from basalrats; (2) forskolin increases UT-A1 accumulation in the plasmamembrane of the IMCD, both in basal and in hydrated rats; and(3) forskolin and dDAVP increase UT-A1 accumulation in the apicalplasma membrane of UT-A1-MDCK cells. Two urea transporters,UT-A1 and UT-A3, are expressed in the IMCD (5,38,39). However,because we transfected only UT-A1 into the UT-A1-MDCK cells,we can conclude that the forskolin- and dDAVP-mediated increasesin UT-A1 accumulation in the apical plasma membrane do not requirethe presence of UT-A3 in the UT-A1-MDCK cells; we cannot excludethe possibility that UT-A3 is involved in the rat IMCD. A vasopressin-stimulatedincrease in UT-A1 accumulation in the plasma membrane may bea mechanism that contributes to the vasopressin-stimulated increasein urea transport that occurs in the perfused rat terminal IMCD(9).
The only previous study to examine whether vasopressin-regulatedtrafficking of UT-A1 occurs concluded that it did not (16).However, this study examined Brattleboro rats, which lack endogenousvasopressin. Although that study was well performed, its findingshave been generalized, resulting in the incorrect conclusionthat vasopressin-regulated trafficking of UT-A1 to the plasmamembrane does not occur (16). Our study suggests that vasopressindoes regulate the accumulation of UT-A1 in the plasma membranein basal Sprague-Dawley rats (which have endogenous vasopressinpresent). In addition, when forskolin is used to stimulate directlythe catalytic subunit of adenylyl cyclase (18), it significantlyincreases UT-A1 accumulation in the plasma membrane in IMCDsuspensions from either basal or hydrated rats. Because collectingducts in general are known to be conditioned in polyuric ratsand their cAMP response to vasopressin is subdued (17,40,41),we speculate that the failure to detect an increase in UT-A1accumulation after vasopressin administration in the Brattlebororat (16) or our hydrated Sprague-Dawley rats may have resultedfrom an insufficient cAMP response. However, in Sprague-Dawleyrats with a presumably normal cAMP response to vasopressin orwhen forskolin is used as the agonist, increases in both UT-A1and AQP2 accumulation in the plasma membrane are clearly detected.These increases seem to be specific because biotinylated calcium-sensingreceptor is unchanged.
Vasopressin also regulates the trafficking of AQP2 between subapicalvesicles and the apical plasma membrane in rat collecting ducts,provided that at least one AQP2 molecule within a tetramer isphosphorylated at serine-256 (reviewed in reference [15]). Vasopressin-regulatedtrafficking of AQP2 occurs in both Sprague-Dawley and Brattlebororats (reviewed in reference [15]). We speculate that this differencebetween vasopressin regulation of UT-A1 and AQP2 suggests thatless cAMP stimulation may be needed to stimulate the plasmamembrane accumulation of AQP2 than of UT-A1. Vasopressin alsoincreases the apical plasma membrane accumulation of AQP2 inseveral cell culture systems (42–45).
In the whole-animal experiments (those in which the rat receiveda subcutaneous injection of vasopressin), the percentage changesare somewhat modest, although they are statistically significant(Figures 2 and 3). These data are important for establishingthe physiologic response in the intact rat kidney. Because themagnitude of the change may have been limited by variable absorptionof the injected vasopressin and/or delivery to the kidney collectingduct, we proceeded to study IMCD suspensions and treated themex vivo with vasopressin or forskolin. A limitation to the IMCDsuspensions is that the plasma membrane of an occasional IMCDcell may be damaged during the process of preparing them, therebyallowing the biotin reagent to enter the cytoplasm of the damagedcell (Figure 1). If this occurs, then it would be equally likelyin control and in vasopressin- or forskolin-treated IMCD suspensions,thereby increasing the noise of the measurement and potentiallyunderestimating the percentage increase in UT-A1 in the plasmamembrane in the treated IMCD suspensions. UT-A1 accumulationin the plasma membrane (biotinylated UT-A1) consistently increasesby 50 to 100% in the IMCD suspensions (Figures 4 and 5). However,even a 100% increase in UT-A1 in the plasma membrane may notaccount for the six- to eight-fold increase in urea permeabilitythat was stimulated by vasopressin in the perfused tubule. Wepreviously showed that vasopressin increases UT-A1 phosphorylationin rat inner medulla, rat IMCD suspensions, and UT-A1-MDCK cells(13,14), with a time course that is similar to vasopressin-inducedstimulation of urea transport in the perfused rat terminal IMCD(10,12,13,46). Thus, vasopressin may increase urea permeabilitythrough increases in both UT-A1 accumulation in the plasma membraneand UT-A1 phosphorylation.
Our study shows that vasopressin increases UT-A1 accumulationin the plasma membrane in basal Sprague-Dawley rats, and forskolinincreases it in both basal and hydrated rats. In addition, ourstudy further establishes our UT-A1-MDCK cells as a model systemthat reproduces the physiologic properties that we find in ratinner medulla and IMCD suspensions.
Acknowledgments
This work was supported by National Institutes of Health grantsR01-DK41707, R01-DK63657, R01-DK62081, and P01-DK61521 and AmericanHeart Association Grant-in-Aid 0655280B.
Portions of this work have been published in abstract form (FASEBJ 20: A1218, 2006; J Am Soc Nephrol 2006, in press) and presentedat Experimental Biology; April 1 through 5, 2006; San Francisco,CA.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Gamble JL, McKhann CF, Butler AM, Tuthill E: An economy of water in renal function referable to urea.
Am J Physiol 109
: 139
–154, 1934[Free Full Text]
Bagnasco SM, Peng T, Janech MG, Karakashian A, Sands JM: Cloning and characterization of the human urea transporter UT-A1 and mapping of the human Slc14a2 gene.
Am J Physiol Renal Physiol 281
: F400
–F406, 2001[Abstract/Free Full Text]
Nielsen S, Terris J, Smith CP, Hediger MA, Ecelbarger CA, Knepper MA: Cellular and subcellular localization of the vasopressin-regulated urea transporter in rat kidney.
Proc Natl Acad Sci U S A 93
: 5495
–5500, 1996[Abstract/Free Full Text]
Kim Y-H, Kim D-U, Han K-H, Jung J-Y, Sands JM, Knepper MA, Madsen KM, Kim J: Expression of urea transporters in the developing rat kidney.
Am J Physiol Renal Physiol 282
: F530
–F540, 2002[Abstract/Free Full Text]
Fenton RA, Chou C-L, Stewart GS, Smith CP, Knepper MA: Urinary concentrating defect in mice with selective deletion of phloretin-sensitive urea transporters in the renal collecting duct.
Proc Natl Acad Sci U S A 101
: 7469
–7474, 2004[Abstract/Free Full Text]
Fenton RA, Flynn A, Shodeinde A, Smith CP, Schnermann J, Knepper MA: Renal phenotype of UT-A urea transporter knockout mice.
J Am Soc Nephrol 16
: 1583
–1592, 2005[Abstract/Free Full Text]
Sands JM, Nonoguchi H, Knepper MA: Vasopressin effects on urea and H2O transport in inner medullary collecting duct subsegments.
Am J Physiol Renal Physiol 253
: F823
–F832, 1987[Abstract/Free Full Text]
Star RA, Nonoguchi H, Balaban R, Knepper MA: Calcium and cyclic adenosine monophosphate as second messengers for vasopressin in the rat inner medullary collecting duct.
J Clin Invest 81
: 1879
–1888, 1988[Medline]
Sands JM, Schrader DC: An independent effect of osmolality on urea transport in rat terminal IMCDs.
J Clin Invest 88
: 137
–142, 1991[Medline]
Nielsen S, Knepper MA: Vasopressin activates collecting duct urea transporters and water channels by distinct physical processes.
Am J Physiol Renal Physiol 265
: F204
–F213, 1993[Abstract/Free Full Text]
Zhang C, Sands JM, 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[Abstract/Free Full Text]
Frohlich O, Klein JD, Smith PM, Sands JM, Gunn RB: Urea transport in MDCK cells that are stably transfected with UT-A1.
Am J Physiol Cell Physiol 286
: C1264
–C1270, 2004[Abstract/Free Full Text]
Nielsen S, Frokiaer J, Marples D, Kwon ED, Agre P, Knepper M: Aquaporins in the kidney: From molecules to medicine.
Physiol Rev 82
: 205
–244, 2002[Abstract/Free Full Text]
Inoue T, Terris J, Ecelbarger CA, Chou C-L, Nielsen S, Knepper MA: Vasopressin regulates apical targeting of aquaporin-2 but not of UT1 urea transporter in renal collecting duct.
Am J Physiol Renal Physiol 276
: F559
–F566, 1999[Abstract/Free Full Text]
Cornett LE, Breckinridge SM, Koike TI: Induction of V2 receptors in renal medulla of homozygous Brattleboro rats by arginine vasopressin.
Peptides 10
: 985
–991, 1989[CrossRef][Medline]
Seamon KB, Padgett W, Daly JW: Forskolin: Unique diterpene activator of adenylate cyclase in membranes and intact cells.
Proc Natl Acad Sci U S A 87
: 3363
–3367, 1981
Kim D-U, Sands JM, Klein JD: Role of vasopressin in diabetes mellitus-induced changes in medullary transport proteins involved in urine concentration in Brattleboro rats.
Am J Physiol Renal Physiol 286
: F760
–F766, 2004[Abstract/Free Full Text]
Kim D-U, Sands JM, Klein JD: Changes in renal medullary transport proteins during uncontrolled diabetes mellitus in rats.
Am J Physiol Renal Physiol 285
: F303
–F309, 2003[Abstract/Free Full Text]
Kim D-U, Klein JD, Racine S, Murrell BP, Sands JM: Urea may regulate urea transporter protein abundance during osmotic diuresis.
Am J Physiol Renal Physiol 288
: F188
–F197, 2005[Abstract/Free Full Text]
Ecelbarger CA, Terris J, Frindt G, Echevarria M, Marples D, Nielsen S, Knepper MA: Aquaporin-3 water channel localization and regulation in rat kidney.
Am J Physiol Renal Physiol 269
: F663
–F672, 1995[Abstract/Free Full Text]
Marples D, Knepper MA, Christensen EI, Nielsen S: Redistribution of aquaporin-2 water channels induced by vasopressin in rat kidney inner medullary collecting duct.
Am J Physiol Cell Physiol 269
: C655
–C664, 1995[Abstract/Free Full Text]
Sands JM, Naruse M, Jacobs JD, Wilcox JN, Klein JD: Changes in aquaporin-2 protein contribute to the urine concentrating defect in rats fed a low-protein diet.
J Clin Invest 97
: 2807
–2814, 1996[Medline]
Naruse M, Klein JD, Ashkar ZM, Jacobs JD, Sands JM: Glucocorticoids downregulate the rat vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts.
J Am Soc Nephrol 8
: 517
–523, 1997[Abstract]
Nielsen S, Marples D, Birn H, Mohtashami M, Dalby NO, Trimble W, Knepper M: Expression of VAMP2-like protein in kidney collecting duct intracellular vesicles. Colocalization with aquaporin-2 water channels.
J Clin Invest 96
: 1834
–1844, 1995[Medline]
Klein JD, Gunn RB, Roberts BR, Sands JM: Down-regulation of urea transporters in the renal inner medulla of lithium-fed rats.
Kidney Int 61
: 995
–1002, 2002[CrossRef][Medline]
Frohlich O, Klein JD, Smith PM, Sands JM, Gunn RB: Regulation of UT-A1-mediated transepithelial urea flux in MDCK cells.
Am J Physiol Cell Physiol 2006
, in press
Klein JD, Price SR, Bailey JL, Jacobs JD, Sands JM: Glucocorticoids mediate a decrease in the AVP-regulated urea transporter in diabetic rat inner medulla.
Am J Physiol Renal Physiol 273
: F949
–F953, 1997[Abstract/Free Full Text]
Klein JD, Timmer RT, Rouillard P, Bailey JL, Sands JM: UT-A urea transporter protein expressed in liver: Upregulation by uremia.
J Am Soc Nephrol 10
: 2076
–2083, 1999[Abstract/Free Full Text]
Kato A, Klein JD, Zhang C, Sands JM: Angiotensin II increases vasopressin-stimulated facilitated urea permeability in rat terminal IMCDs.
Am J Physiol Renal Physiol 279
: F835
–F840, 2000[Abstract/Free Full Text]
Timmer RT, Klein JD, Bagnasco SM, Doran JJ, Verlander JW, Gunn RB, Sands JM: Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues.
Am J Physiol Cell Physiol 281
: C1318
–C1325, 2001[Abstract/Free Full Text]
Awata H, Huang C, Handlogten ME, Miller RT: Interaction of the calcium-sensing receptor and filamin, a potential scaffolding protein.
J Biol Chem 276
: 34871
–34879, 2001[Abstract/Free Full Text]
Huang C, Handlogten ME, Miller RT: Parallel activation of phosphatidylinositol 4-kinase and phospholipase C by the extracellular calcium-sensing receptor.
J Biol Chem 277
: 20293
–20300, 2002[Abstract/Free Full Text]
Mandon B, Chou CL, Nielsen S, Knepper MA: Syntaxin-4 is localized to the apical plasma membrane of rat renal collecting duct cells: Possible role in aquaporin-2 trafficking.
J Clin Invest 98
: 906
–913, 1996[Medline]
Bradford AD, Terris J, Ecelbarger CA, Klein JD, Sands JM, Chou C-L, Knepper MA: 97 and 117 kDa forms of the collecting duct urea transporter UT-A1 are due to different states of glycosylation.
Am J Physiol Renal Physiol 281
: F133
–F143, 2001[Abstract/Free Full Text]
Sands JM, Naruse M, Baum M, Jo I, Hebert SC, Brown EM, Harris HW: An apical extracellular calcium/polyvalent cation-sensing receptor regulates vasopressin-elicited water permeability in rat kidney inner medullary collecting duct.
J Clin Invest 99
: 1399
–1405, 1997[Medline]
Stewart GS, Fenton RA, Wang W, Kwon TH, White SJ, Collins VM, Cooper G, Nielsen S, Smith CP: The basolateral expression of mUT-A3 in the mouse kidney.
Am J Physiol Renal Physiol 286
: F979
–F987, 2004[Abstract/Free Full Text]
Terris JM, Knepper MA, Wade JB: UT-A3: Localization and characterization of an additional urea transporter isoform in the IMCD.
Am J Physiol Renal Physiol 280
: F325
–F332, 2001[Abstract/Free Full Text]
Shen TS, Suzuki Y, Poyard M, Miyamoto N, Defer N, Hanoune J: Expression of adenylyl cyclase mRNAs in the adult, in developing, and in the Brattleboro rat kidney.
Am J Physiol Cell Physiol 273
: C323
–C330, 1997[Abstract/Free Full Text]
Homma S, Gapstur SM, Coffey A, Valtin H, Dousa TP: Role of cAMP-phosphodiesterase isozymes in pathogenesis of murine nephrogenic diabetes insipidus.
Am J Physiol Renal Physiol 261
: F345
–F353, 1991[Abstract/Free Full Text]
Van Balkom BWM, Savelkoul PJM, Markovich D, Hofman E, Nielsen S, Van der Sluijs P, Deen PMT: The role of putative phosphorylation sites in the targeting and shuttling of the aquaporin-2 water channel.
J Biol Chem 277
: 41473
–41479, 2002[Abstract/Free Full Text]
Lu H, Sun TX, Bouley R, Blackburn K, McLaughlin M, Brown D: Inhibition of endocytosis causes phosphorylation (S256)-independent plasma membrane accumulation of AQP2.
Am J Physiol Renal Physiol 286
: F233
–F243, 2004[Abstract/Free Full Text]
Gustafson CE, Katsura T, McKee M, Bouley R, Casanova JE, Brown D: Recycling of AQP2 occurs through a temperature- and bafilomycin-sensitive
trans-Golgi-associated compartment. Am J Physiol Renal Physiol 278
: F317
–F326, 2000
Zelenina M, Christensen BM, Palmer J, Nairn AC, Nielsen S, Aperia A: Prostaglandin E2 interaction with AVP: Effects on AQP2 phosphorylation and distribution.
Am J Physiol Renal Physiol 278
: F388
–F394, 2000[Abstract/Free Full Text]
Wall SM, Suk Han J, Chou C-L, Knepper MA: Kinetics of urea and water permeability activation by vasopressin in rat terminal IMCD.
Am J Physiol Renal Physiol 262
: F989
–F998, 1992[Abstract/Free Full Text]
This article has been cited by other articles:
A. R. Mehta Why does the plasma urea concentration increase in acute dehydration?
Advan Physiol Educ,
December 1, 2008;
32(4):
336 - 336.
[Full Text][PDF]
N. W. Blessing, M. A. Blount, J. M. Sands, C. F. Martin, and J. D. Klein Urea transporters UT-A1 and UT-A3 accumulate in the plasma membrane in response to increased hypertonicity
Am J Physiol Renal Physiol,
November 1, 2008;
295(5):
F1336 - F1341.
[Abstract][Full Text][PDF]
G. Chen, H. Huang, O. Frohlich, Y. Yang, J. D. Klein, S. R. Price, and J. M. Sands MDM2 E3 ubiquitin ligase mediates UT-A1 urea transporter ubiquitination and degradation
Am J Physiol Renal Physiol,
November 1, 2008;
295(5):
F1528 - F1534.
[Abstract][Full Text][PDF]
X. Wang, P. C. Harris, S. Somlo, D. Batlle, and V. E. Torres Effect of calcium-sensing receptor activation in models of autosomal recessive or dominant polycystic kidney disease
Nephrol. Dial. Transplant.,
September 30, 2008;
(2008)
gfn527v1.
[Abstract][Full Text][PDF]
G. S. Stewart, J. H. O'Brien, and C. P. Smith Ubiquitination regulates the plasma membrane expression of renal UT-A urea transporters
Am J Physiol Cell Physiol,
July 1, 2008;
295(1):
C121 - C129.
[Abstract][Full Text][PDF]
M. A. Blount, A. C. Mistry, O. Frohlich, S. R. Price, G. Chen, J. M. Sands, and J. D. Klein Phosphorylation of UT-A1 urea transporter at serines 486 and 499 is important for vasopressin-regulated activity and membrane accumulation
Am J Physiol Renal Physiol,
July 1, 2008;
295(1):
F295 - F299.
[Abstract][Full Text][PDF]
O. Frohlich, D. Aggarwal, J. D. Klein, K. J. Kent, Y. Yang, R. B. Gunn, and J. M. Sands Stimulation of UT-A1-mediated transepithelial urea flux in MDCK cells by lithium
Am J Physiol Renal Physiol,
March 1, 2008;
294(3):
F518 - F524.
[Abstract][Full Text][PDF]
P. Uawithya, T. Pisitkun, B. E. Ruttenberg, and M. A. Knepper Transcriptional profiling of native inner medullary collecting duct cells from rat kidney
Physiol Genomics,
January 17, 2008;
32(2):
229 - 253.
[Abstract][Full Text][PDF]
A. C. Mistry, R. Mallick, O. Frohlich, J. D. Klein, A. Rehm, G. Chen, and J. M. Sands The UT-A1 Urea Transporter Interacts with Snapin, a SNARE-associated Protein
J. Biol. Chem.,
October 12, 2007;
282(41):
30097 - 30106.
[Abstract][Full Text][PDF]
M. A. Blount, J. D. Klein, C. F. Martin, D. Tchapyjnikov, and J. M. Sands Forskolin stimulates phosphorylation and membrane accumulation of UT-A3
Am J Physiol Renal Physiol,
October 1, 2007;
293(4):
F1308 - F1313.
[Abstract][Full Text][PDF]
Top
Abstract
Introduction
cm2) polarized epithelial monolayers 4 d later. Transmembrane resistance was measured daily using an epithelial resistance meter (EVOMX-G; World Precision Instruments, Sarasota, FL). Inserts in which there was not an orderly increase in the transepithelial electrical resistance from <100 to >800 
and 
, duplicate determinations) significantly increases urea flux (A) over control (
) levels. As the final portion of the flux experiment, we add dimethyl urea (DMU) to inhibit carrier-mediated urea transport, and it reduced urea flux to the basal level. The cAMP production (B) in parallel wells (n = 4 duplicate determinations/group) of our UT-A1-MDCK cells was increased by a comparable 60-min treatment with forskolin. (C) Forskolin-induced increase in UT-A1. The cells that were grown on six-well cluster dishes were treated for 30 min with 10 µM forskolin, in parallel with control cells. This Western blot shows duplicate samples of both control (left) and forskolin-treated (right) cells that were probed with anti–UT-A1. Arrows indicate UT-A1. Shown are two of six wells of cells for each condition.