Urea flux across MDCK-mUT-A2 monolayers is acutely sensitive to AVP, cAMP, and [ - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:41 |只看该作者 |倒序浏览 |打印

作者：Elizabeth A. Potter, Gavin Stewart, and Craig P. Smith作者单位：Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom



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      【摘要】
   In this study, we engineered a Madin-Darby canine kidney (MDCK) type I cell line to stably express the mouse urea transporter UT-A2. Monolayers of MDCK-mUT-A2 cells had a basal phloretin-inhibitable urea permeability of 8.4 x 10 -6 ± 0.3 cm/s. Treatment of MDCK-mUT-A2 monolayers with AVP led to a rapid dose-dependent increase in trans -monolayer phloretin-inhibitable urea flux. The temporal pattern of response was markedly different from that observed for MDCK cells expressing rat UT-A1. Exposure of MDCK-mUT-A2 cells to either 10 µM forskolin or 250 µM 8-bromo cAMP also increased urea flux rate. Inclusion of the PKA inhibitor H89 (10 µM) had no effect on the forskolin-stimulated increase in urea flux across MDCK-mUT-A2 monolayers. Treatment with either 10 µM CPA or 1 mM ATP also caused an increase in UT-A2-mediated urea flux, although these responses where transient compared with those induced by AVP or elevated cAMP. Taken together, these results show for the first time that UT-A2 is acutely sensitive to AVP, cAMP, or increased intracellular calcium. : O4 H6 r6 f9 d! J* a& \
      【关键词】 arginine vasopressin intracellular calcium urine concentration urea transporter
   UREA TRANSPORTERS REGULATE the passive movement of urea across plasma membranes. In mammals, they are the product of two genes: UT-A ( Slc14a2 ) and UT-B ( Slc14a1 ) ( 16 ). Five protein isoforms have been characterized to date that originate from the UT-A gene and a single protein species derived from the UT-B gene has been characterized ( 13, 15 ). Products of both genes play important roles in the kidney, where they serve to maintain a high inner medullary solute concentration as part of the urinary concentrating mechanism ( 6, 21, 28 ).

In the mouse and rat kidney, isoforms UT-A1, UT-A2, and UT-A3 are the most abundant. UT-A1 is the largest UT-A isoform with 97- and 117-kDa glycosylated protein forms ( 8, 19 ). UT-A3 consists of the NH 2 -terminal 460 amino acids of UT-A1 and occurs as 44- and 67-kDa glycosylated proteins ( 7, 8, 11 ). Both UT-A1 and UT-A3 localize to the inner medullary collecting duct (IMCD) of the kidney where they regulate urea reabsorption into the interstitium under the acute control of arginine vasopressin (AVP) ( 6, 14, 24, 25 ). In rat isolated perfused IMCD, treatment with AVP is known to lead to rapid increases in intracellular calcium ([Ca 2  ] i ) and cAMP concentrations and transepithelial phloretin-sensitive urea transport ( 23 ). Urea transport mediated by UT-A1 or UT-A3 expressed in Xenopus laevis oocytes is acutely increased by cAMP ( 8, 19 ). Treatment of IMCD suspensions with AVP or cAMP has been found to elicit an increased phosphorylation of UT-A1, indicating that these stimulants may directly activate UT-A1 possibly via protein kinase A ( 30 ).
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UT-A2 consists of the COOH-terminal 397 amino acids of UT-A1 and exists as 43- to 55-kDa glycosylated proteins ( 20, 27, 29 ). This isoform has been localized to type 1 and type 3 thin descending limbs (tDL) of the loop of Henle ( 7, 17 ). In these nephron segments, it is proposed to participate in intrarenal recycling of urea between the IMCD and loop of Henle ( 8, 26, 27 ). Its expression is significantly upregulated during chronic dehydration and following prolonged treatment with 1-deamino 8- D -arginine vasopressin (DDAVP), possibly indicating a role in the urinary concentrating mechanism ( 2, 18, 20, 27 ). UT-A2 function has been previously observed to be unresponsive to cAMP when expressed in X. laevis oocytes ( 8, 19, 29 ) and also when expressed in human embryonic kidney cells (HEK-293) ( 11 ).

A Madin-Darby canine kidney (MDCK) type I cell line has recently been engineered that stably expresses rat UT-A1 protein. This cell line, when grown on permeable supports, has successfully been used to study acute regulation of UT-A1-mediated urea transport ( 9 ). This system allows urea transporter function to be examined via radiolabeled urea flux across a cultured cell monolayer and serves as a model of a polarized epithelia. The aim of the current study was to establish a cell line stably expressing the mouse urea transporter UT-A2 and to define the characteristics of the UT-A2 response to AVP and its established second messengers cAMP and Ca 2 .
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METHODS
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Establishment of MDCK-mUT-A2 cells. MDCK cells stably transfected with pFRT/ lac Zeo (termed "MDCK-FLZ" cells, Invitrogen) and MDCK-FLZ cells stably expressing rat UT-A1 (termed MDCK-rUT-A1) were the kind gift of Prof. R. Gunn (Emory University, Atlanta, GA). Cells were cultured as previously described ( 9 ). MDCK-FLZ cells stably expressing mouse UT-A2 (termed MDCK-mUT-A2) were derived using the Flp-In system (Invitrogen) as follows: sequence encoding a c- myc tag was incorporated into the open reading frame of mUT-A2 cDNA immediately 5' to the stop codon by PCR/plasmid subcloning. This incorporated sequence encoding 10 amino acids (EQKLISEEDL), a stop codon and a Spe 1 restriction site. Correct incorporation was verified by restriction digest and nucleotide sequencing. Treatment of X. laevis oocytes expressing either wild-type or c- myc -tagged UT-A2 protein with 8-Br-cAMP (250 µM) for 1 h caused a significant twofold increase in urea permeability compared with controls not exposed to 8-Br-cAMP ( P 9 v- J6 |( n- F( |

The UT-A2 c- myc construct was subcloned into pcDNA5/FRT (Invitrogen) and cotransfected with pOG44 into the MDCK-FLZ cells using an Amaxa nucleofector (Amaxa, Cologne, Germany) according to the manufacturer's instructions. Cells were selected with 300 µg/ml hygromycin after 24 h and individual clones were isolated following 2 wk. Five clonal cell lines were isolated (termed EP10-EP15) and protein expression was assessed using semiquantitative immunoblotting as follows: cells were harvested using 0.05% trypsin-EDTA (GIBCO) and washed twice in PBS. Cell protein was homogenized, spun at 1,000 g at 4°C for 5 min, and the pelleted cellular debris was removed. Samples were then spun at 20,000 g at 4°C for 20 min and the pelleted membrane fraction was retained. Ten micrograms of protein were run on a 12% SDS-polyacrylamide gel, and protein was transferred electrophoretically to a nitrocellulose membrane. Membranes were blocked with 10% milk and probed with antisera: ML446 (1:500) ( 24 ), ML194 (1:500) ( 8 ), or anti-c- myc monoclonal mouse IgG1 (1:5,000) (Sigma). Immunoblotting methods are essentially as previously described ( 24 ).
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Epithelial monolayers were cultured on semipermeable polyester supports (0.4-µm pore, Transwell, Corning, UK) as described ( 9 ). Monolayers were fed on day 2 following seeding then daily until use. The resistance of each monolayer was measured using an EVOM resistance meter (World Precision Instruments). After becoming confluent, membranes developed a transmembrane resistance 1.5 k. Only membranes that showed sequential increases in transmembrane resistance to 1.5 k were used for flux experiments.

Flux measurements. Urea flux experiments were performed as described by Frohlich and colleagues ( 9 ) at 37 ± 0.2°C in an apical-to-basolateral direction using 14 C urea (0.8 µCi/apical well) as the radiolabeled tracer. The basolateral solution was collected at 3-min intervals. At the beginning of an experiment, consecutive baseline collections were made, thereafter AVP or test compounds were added to the basolateral chamber. All test compounds were of certified grade and were made up either in sterile dH 2 O or anhydrous DMSO. Stocks were given: AVP; 100 µM/dH 2 O, forskolin; 50 µM/DMSO, cyclopiazonic acid (CPA); 2.5 mM/DMSO, N -[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, 2 HCl (H89); 10 mM DMSO, 8-bromo-cAMP (8-BrcAMP); 50 mM/dH 2 O, phloretin; 100 µM in DMSO. Stocks were then diluted in Hanks' balanced salt solution (GIBCO) containing 20 mM HEPES before use. In parallel, control solutions were made by adding the corresponding solvent minus the test compound.) d2 B$ F: H& U2 l7 G$ J9 a8 U

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Semiquantitative immunoblotting of MDCK-mUT-A2 cell lines. Following transfection of MDCK-FLZ cells with the pcDNA5/FRT/mUT-A2 construct, a hygromycin-resistant clone, termed EP10, was selected and expanded. Western analysis of this cell line and the MDCK-rUT-A1 cell line ( 9 ) using a previously characterized UT-A1/UT-A2-selective antiserum (ML194) ( 8 ) detected UT-A1 bands in the control MDCK-rUT-A1 cells and a 43- to 50-kDa smear in the cell line transfected with pcDNA5/FRT/mUT-A2 ( Fig. 1 ). Protein from untransfected MDCK-FLZ cells showed no ML194 signal ( Fig. 1 ). Antiserum ML446, raised to the common NH 2 terminus of UT-A1 and UT-A3, only detected UT-A1 in the MDCK-rUT-A1 cell line ( Fig. 1 ). Immunoblotting analysis of EP10 protein using an anti-c- myc monoclonal antibody identified a predominant protein signal at 43-50 kDa. This was in agreement with the protein signal identified by ML194 and we conclude represents the UT-A2 c- myc fusion protein expressed in the MDCK type I cell line." R- Z8 R$ z, q, C/ T) m1 @- l

Fig. 1. Characterization by immunoblotting of the newly derived Madin-Darby canine kidney (MDCK)-mUT-A2 cell line (EP10). Three sets of identical 10-µg whole cell protein samples from UT-A1, EP10 (MDCK-mUT-A2), and MDCK-FLZ (untransfected) cell lines were compared simultaneously by Western blotting. Membranes were incubated with ML446, ML194 antisera, or anti-c- myc monoclonal mouse IgG1 (Sigma) as indicated. ML194 labeled a 97-kDa protein in the UT-A1 cell line and 43- to 50-kDa protein in the EP10 cell line. No signal was apparent in the EP10 cell line when ML446 was the primary antiserum, whereas a 43- to 50-kDa protein was detected by the c- myc monoclonal antibody. No signals were observed in the MDCK-FLZ cell line with any of the antisera.

Functional analysis of MDCK-mUT-A2 cell monolayers. The mean urea permeability of the untransfected MDCK-FLZ cell monolayers was 2.1 x 10 -6 ± 0.5 cm/s ( n = 6). This was comparable to the MDCK-rUT-A1 monolayer with a permeability of 2.3 x 10 -6 ± 0.4 ( n = 6), whereas the basal urea permeability of the MDCK-mUT-A2 was significantly higher at 8.4 x 10 -6 ± 0.3 cm/s ( n = 91).
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As previously reported, the addition of 10 -8 M AVP to the basolateral chamber of MDCK-rUT-A1 monolayers caused an increase in urea flux rate ( 9 ) ( Fig. 2 A ). Urea flux rate increased within the first 3 min of AVP exposure and leveled to a plateau after 9 min. It then increased to a peak of 6.9 ± 3 nmol·min -1 ·cm -2 ( n = 4) after 42 min of AVP exposure. Removal of AVP resulted in a rapid decrease in urea flux to 1.5 ± 0.3 nmol·min -1 ·cm -2 ( n = 4). Urea flux did not return to the basal (pre-AVP) rate despite 60 min of washout. This response of rapid increase followed by a small plateau (or shoulder) then a further increase in flux rate was as previously reported by Frohlich and colleagues ( 9 ).; ^5 V! z8 ?7 E2 r7 p7 Y% l

Fig. 2. AVP increased urea flux across UT-A2-expressing monolayers. 14 C urea flux was measured in an apical-to-basolateral direction at 3-min intervals across MDCK monolayers grown on permeable supports. Basal flux rates were established by 3 consecutive measurements before addition of 1 x 10 -8 M AVP to the basolateral chamber. AVP was then removed from the basolateral chamber (washout). A : as previously described, the urea flux across UT-A1-expressing MDCK cells was increased by AVP ( ). The flux rate increased gradually during the first 9 min of AVP treatment, then leveled out before increasing to a peak after 42 min of AVP treatment. Removal of AVP caused a rapid decrease in the urea flux rate. After 63 min of washout, the rate of flux had not returned to the pretreatment basal rate. In comparison, untreated MDCK-rUT-A1 membranes ( ) showed no response to AVP. B : urea flux across MDCK-mUT-A2 cells ( ) was also increased by 1 x 10 -8 M AVP. The profile of response differed from that observed for UT-A1. Within 9 min of AVP treatment, the rate had reached a peak and thereafter declined. Removal of AVP caused the urea flux rate to rapidly decrease to the pretreatment rate after 15 min of washout. In comparison, untreated MDCK-mUT-A2 membranes ( ) showed no response. Traces are representative of 4 independent experiments.- x! j' ^; K; C: s8 f
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The addition of 10 -8 M AVP to the basolateral chamber of MDCK-mUT-A2 monolayers resulted in a rapid increase in urea flux that peaked at 6.8 ± 1 nmol·min -1 ·cm -2 ( n = 4) within 9 min followed by a slow rate of decline that leveled off after 30 min of AVP exposure ( Fig. 2 B ). This rate of flux persisted until the end of the AVP period. Stimulation with AVP was reversed on washout, and urea flux rate returned to basal values within 24 min of AVP removal. These data suggest that transfection of MDCK-FLZ cells with pcDNA5/FRT/mUT-A2 instilled an AVP-sensitive urea pathway that had a pattern of activation by AVP that was distinctly different to that observed for MDCK-rUT-A1.
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Treatment of MDCK-mUT-A2 monolayers with 300 µM phloretin, an established inhibitor of UT-A-mediated urea transport ( 22 ), caused a decrease in urea flux rate, indicating that the basal urea flux across MDCK-mUT-A2 monolayers is, in part, due to a phloretin-sensitive pathway ( Fig. 3 ). Following pretreatment with phloretin, addition of 10 -8 M AVP to MDCK-mUT-A2 monolayers had no effect on urea flux rate, although the rate was increased in time-matched controls not pretreated with phloretin ( Fig. 3 ). This shows that the AVP-stimulated flux across MDCK-mUT-A2 monolayers is sensitive to phloretin. From these data together with our immunoblotting data ( Fig. 1 ), we conclude that the EP10 MDCK-mUT-A2 cell line expresses functional UT-A2. Interestingly, and for the first time, these data show that urea transport mediated by UT-A2 is acutely responsive to AVP.8 t/ F/ V: F0 g" b* R

Fig. 3. AVP-stimulated urea flux across MDCK-mUT-A2 monolayers is inhibited by 300 µM phloretin. Following 4 collections to establish basal urea flux rates, 300 µM phloretin was added to the basolateral chamber of 3 monolayers (open points). This caused a decrease in urea flux rate compared with monolayers not exposed to phloretin (filled points, n = 3). Addition of 1 x 10 -8 M AVP to the basolateral chamber of all monolayers caused an increase in urea flux in monolayers unexposed to phloretin but had no effect on urea flux rate in cells incubated with phloretin.

The increase in urea flux rate induced by AVP was found to be dependent on the dose of AVP ( Fig. 4 ). Concentrations 5 p6 s* d" \! h' ~# l  M  y5 w
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Fig. 4. AVP stimulation of MDCK-mUT-A2-mediated urea flux is dose dependent. Different doses of AVP were applied after the initial control period for a duration of 45 min to 5 separate membranes expressing UT-A2., 3 x 10 -8 M;, 1 x 10 -9 M;, 3 x 10 -10 M;, 1 x 10 -10 M., UT-A2 monolayer in absence of AVP. A dose-dependent activation of UT-A2-mediated flux was observed between 1 x 10 -10 M and 1 x 10 -9 M AVP.

Classically, AVP acting via V 2 vasopressin receptors triggers an increase in intracellular cAMP via activation of adenylate cyclase. We assessed the effect on MDCK-mUT-A2 monolayers of raising intracellular cAMP by treating cells with forskolin, an adenylate cyclase activator. Ten micromolar forskolin had no detectable effect on MDCK-FLZ monolayers (data not shown), but rapidly stimulated urea flux when applied to MDCK-mUT-A2 monolayers ( Fig. 5 ). This increase peaked after 9 min and was followed by a gradual decrease that did not return to basal levels following washout. As previously reported, forskolin caused an increase in urea flux across MDCK-rUT-A1 monolayers, although the profile of response distinctly differed from that observed for MDCK-mUT-A2 ( Fig. 5 ). As observed with AVP, addition of forskolin to MDCK-rUT-A1 cells caused an increase in urea flux to a small peak at 9-12 min followed by a plateau then a second larger peak at 39 min.
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Fig. 5. Activation of UT-A2-mediated urea flux by forskolin. The apical-to-basolateral radiolabeled urea flux across MDCK-mUT-A2 (triangles) or MDCK-rUT-A1 (diamonds) cell monolayers was measured before, during, and after the basolateral addition of 10 µM forskolin. Forskolin was applied for 45 min (filled points) followed by a 45-min washout period during which persistence of activation was observed. Open points represent time-matched controls expressing UT-A2 or UT-A1. Traces shown are representative of 4 experiments. The activation profile of 10 µM forskolin compound was similar to that of AVP.& V3 I2 ?( J/ P2 L  |! S0 A
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To corroborate our studies with forskolin, we performed parallel studies using 8-BrcAMP (10 and 250 µM). Basolateral application of 10 µM 8-BrcAMP to MDCK-UT-A2 monolayers did not markedly increase UT-A2-mediated urea flux. In comparison, 250 µM 8-BrcAMP induced a well-defined increase in urea flux that reached a peak after 24 min and thereafter decreased until the agonist was removed. Stimulation was rapidly reversed following washout, retuning to basal values within 15 min (data not shown).
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To test whether cAMP was acting through PKA, we used the cAMP-dependent protein kinase antagonist H89. Application of 10 µM H89 to MDCK-rUT-A1-expressing membranes had no discernible affect on the initial peak/plateau but caused a decrease in urea flux at later time points with the result that the tertiary stage of the response was decreased ( Fig. 6 ). In contrast, H89 had no apparent effect on forskolin-stimulated increase in MDCK-mUT-A2 urea flux ( Fig. 6 ). This suggests that activation of UT-A2 is not via PKA.
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Fig. 6. Lack of effect of H89 on forskolin-activated urea flux across MDCK-mUT-A2-expressing cell monolayers; 10 µM forskolin was applied basolaterally to UT-A1 and UT-A2-expressing membranes for 36 min. In some monolayers, 10 µM H89 was also applied basolaterally. Following this, membranes were subject to washout conditions for 9 min, and then 300 µM phloretin was basolaterally applied. Traces are representative of 3 experiments. In comparison, H89 partially reduced forskolin-stimulated UT-A1-mediated urea flux ( ). In contrast, exposure to H89 had no discernable effect on MDCK-mUT-A2-mediated urea flux ( ). Open symbols represent membranes not exposed to H89.0 v$ e" Y; n$ {1 U1 B! @! P

An increase in [Ca 2  ] i has previously been reported to accompany stimulation by AVP in IMCD cells ( 23 ). To test the possibility that UT-A2 may respond to changes in cellular calcium, we employed CPA, a cell-permeable, reversible inhibitor of the endoplasmic reticulum Ca 2  -ATPase. Treatment of cells with CPA causes a gradual increase in [Ca 2  ] i due to a decrease in Ca 2  reuptake into intracellular pools ( 4, 12 ). The application of 10 µM CPA to MDCK-mUT-A2 monolayers caused a fairly rapid increase in urea flux rate (within 12 min) followed by a gradual decline back to the basal urea flux rate after 40 min of CPA exposure ( Fig. 7 A ). At the end of the treatment period, the urea flux rate remained at basal levels despite the presence of CPA. Removal of CPA caused the urea flux rate to decrease below the basal rate. This decrease was sustained for the remainder of the experiment. The application of CPA to MDCK-rUT-A1 monolayers also caused a rapid and transient increase in urea flux ( Fig. 8 A ). In MDCK-mUT-A2 to discern whether the changes brought about by CPA were due to increased UT-A2-mediated transport, we pretreated MDCK-mUT-A2 monolayers with phloretin. This manoeuvre abolished the response to CPA confirming that the response was mediated by UT-A2 ( Fig. 7 B ).
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Fig. 7. Effect of 10 µM cyclopiazonic acid (CPA) on MDCK-mUT-A2 monolayers. A : 10 µM CPA was applied basolaterally to MDCK-mUT-A2-expressing membranes for 45 min followed by 45-min washout period. Traces are representative of 6 experiments. CPA elicited a transitory increase in UT-A2-mediated urea flux, compared with time-matched MDCK-mUT-A2 controls ( ), that returned to basal rates after 42 min. Following washout, the urea flux rate of CPA-exposed monolayers decreased to below baseline and this persisted until the end of the experiment. B : following 4 collections to establish basal urea flux rates, 300 µM phloretin was added to the basolateral chamber of 3 monolayers (open points). This caused a decrease in urea flux rate compared with monolayers not exposed to phloretin (filled points, n = 3). The addition of 10 µM CPA to the basolateral chamber of all monolayers caused an increase in urea flux in monolayers unexposed to phloretin, but this effect was not apparent in cells incubated with phloretin., v. u, z# S0 b# F4 k, t, q" o

Fig. 8. Effect of 10 µM CPA or 1 mM ATP on urea flux across MDCK-rUT-A1 monolayers. A : 10 µM CPA was applied basolaterally to MDCK-rUT-A1-expressing membranes for 36 min followed by 18-min washout period. CPA elicited a transitory increase in UT-A1-mediated urea flux that returned to basal rates after 18 min. The trace is representative of 3 experiments. B : 1 mM ATP applied to the basolateral chamber of MDCK-rUT-A1-expressing monolayers for 36 min caused a rapid increased in urea flux that peaked after 9 min. Thereafter, the rate decreased but remained above the unstimulated flux rate for the remainder of the 36-min ATP period. Thereafter, addition of 300 µM phloretin to the basolateral chamber caused a decrease in urea flux rate. The trace is representative of 3 experiments.
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In addition to experiments using CPA, we also made use of the fact that extracellular ATP rapidly increases [Ca 2  ] i in IMCD and MDCK cells via membrane-bound purinoceptors ( 5, 10 ). Basolateral addition of 1 mM ATP elicited a very rapid stimulation of urea flux across UT-A2 monolayers with the peak response seen within 6 min ( Fig. 9 A ). This was followed by a decline to basal rates. ATP did not have a detectable effect on urea flux rate in untransfected MDCK monolayers (data not shown). As we found with both AVP and CPA, pretreatment of MDCK-mUT-A2 monolayers with phloretin abolished the response to ATP ( Fig. 9 B ), indicating that the increased urea transport was mediated by UT-A2. Application of ATP to MDCK-rUT-A1 monolayers elicited a rapid increase in urea flux that peaked after 9 min and then decreased. Thereafter, the flux rate remained elevated above the pretreatment value until addition of 300 µM phloretin caused a rapid decrease in urea flux rate to the pretreatment rate ( Fig. 8 B ). Taken together, these results and those from our experiments using CPA suggest that increasing [Ca 2  ] i in UT-A2 or UT-A1 expressing MDCK monolayers elicits an increase in urea flux via UT-A2 or UT-A1, respectively. In the case of CPA, this effect is transient and urea transport returns to basal levels in the presence of the activator. In comparison, the response to ATP is more sustained and in the case of UT-A1 persists for at least 36 min in the presence of ATP.
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Fig. 9. Effect of 1 mM ATP on urea flux across MDCK-mUT-A2 monolayers. A : 1 mM ATP was applied to the basolateral chamber of MDCK-mUT-A2 ( )- expressing monolayers for 36 min. The membranes were then subject to control conditions for 9 min. ATP elicited a rapid increase in urea flux across UT-A2-expressing monolayers, but had no effect on MDCK-FLZ monolayers (data not shown). Washout of ATP caused a return to baseline flux rates. Untreated time-matched control MDCK-mUT-A2 monolayers maintained the basal flux rate throughout the experiment ( ). B : following 4 collections to establish basal urea flux rates, 300 µM phloretin was added to the basolateral chamber of 3 monolayers (open symbols). This caused a decrease in urea flux rate compared with monolayers not exposed to phloretin (filled symbols, n = 3). Addition of 1 mM ATP to the basolateral chamber of all monolayers caused an increase in urea flux in monolayers not exposed to phloretin but had no effect on urea flux rate in cells incubated with phloretin.
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DISCUSSION
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The function of the three major renal urea transporters, UT-A1, UT-A2, and UT-A3, and regulation of these proteins by the central antidiuretic hormone AVP are of key importance to the urinary concentrating mechanism and consequently water balance. Recently, studies utilizing MDCK cells stably expressing UT-A1 have shed new light on urea transporter regulation. To further our understanding, we aimed in this study to derive a renal cell line heterologously expressing mouse UT-A2 and to study acute regulation of UT-A2 function by AVP and its known second messengers cAMP and Ca 2 . With respect to our first aim, immunoblotting of clonally selected mUT-A2 cotransfected MDCK-FLZ cells confirmed that the newly derived cell line EP10 expressed significant amounts of UT-A2 protein and that UT-A2 expression persisted following subsequent cell passage. These data indicated that we successfully engineered a MDCK cell line to stably express mUT-A2. This is the first renal epithelial cell line to stably express heterologously UT-A2. 1.5 k /cm 2 ) tight epithelial monolayers that under basal nonstimulated conditions have a fourfold higher urea permeability than untransfected MDCK-FLZ monolayer. This indicates that UT-A2 when expressed in MDCK cells increases epithelial urea permeability and may imply that UT-A2 is partially activated under basal conditions. The latter suggestion is supported by the finding that treatment of MDCK-mUT-A2 monolayers with phloretin cased a substantial decrease in the urea flux rate.
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The finding that UT-A2-mediated urea flux was functionally upregulated by AVP came as a surprise and is of considerable interest. In the kidney, UT-A2 has been localized to type 1 and type 3 tDL ( 8, 17 ) where it is proposed to mediate recycling of urea into the thin limbs from the medullary interstitium as part of the urinary concentrating mechanism. The V 1a vasopressin receptor has been localized to the outer medullary tDL of short loop nephrons in the rat ( 1 ) and therefore may couple to UT-A2. The role of the V 1a receptor has however not been determined in this tissue, but in other tissues is known to mobilize intracellular calcium stores and activate PKC ( 3 ). Potentially then, AVP could stimulate UT-A2 activity in thin tDL by triggering an increase in [Ca 2  ] i. Certainly under conditions of thirsting, UT-A2 mRNA in thin limbs significantly increases indicating that upregulation or activation of UT-A2 may somehow potentiate the urinary concentrating mechanism ( 20 ). However, the recent report that mice deficient in UT-A2 were able to concentrate their urine to the same extent as wild-type mice argues that under normal circumstance, this role is not essential for concentration of urine ( 26 ).+ L- S) F7 w7 K" y% p7 {  O2 S# S
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The stimulation profile of UT-A2 by AVP differs markedly from that observed for UT-A1. The application of AVP to UT-A2 monolayers caused a rapid increase in urea permeability that peaked in 9 min. This was followed by a decrease and then leveling-off of the decrease. In contrast, the profile of UT-A1-mediated increase in urea permeability observed by Frohlich and colleagues ( 9 ) and by us consisted of a rapid increase in urea flux rate within the first 9 min of exposure to AVP. This was followed by a brief leveling off and then a sustained increase in flux rate that peaked at 42 min. Interestingly, the timing of the first phase UT-A2 activation peak coincides with the first phase of UT-A1 stimulation.

In the MDCK-mUT-A2 cell line, we found that UT-A2-mediated urea flux was strongly stimulated by the adenylate cyclase activator forskolin and also 8-bromo cAMP. This shows that a cAMP signaling cascade is capable of eliciting a functional response. The common downstream effector of increased intracellular cAMP is the cAMP-dependent protein kinase (PKA). However, blockade of this molecule by H89 did not affect cAMP activation in MDCK-mUT-A2 cells, suggesting that PKA is not involved in UT-A2 stimulation.; \% N# k" j' n

In this study, we also tested the effect of raising [Ca 2  ] i in MDCK-mUT-A2 monolayers on transepithelial urea flux. We found that CPA or application of exogenous ATP, both known to induce an increase in intracellular [Ca 2  ] i in MDCK cells ( 10, 12 ), stimulated UT-A2-mediated urea flux. The pattern of stimulation was more transient than observed with AVP or cAMP and probably reflects the transient, but distinct, nature of the increase in [Ca 2  ] i induced by CPA ( 10 ) or ATP ( 12 ).9 E# O$ v* y, ~5 O, {0 l. G
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Taken together, our data show that transport of urea across MDCK-mUT-A2 monolayers is increased by treatment with cAMP or by raising intracellular Ca 2 . One interpretation of these data is that an increase in [Ca 2  ] i and cAMP both participate in the AVP-induced increase in UT-A2-mediated urea flux across MDCK-mUT-A2 monolayers. In addition, it is possible that other as yet untested second messengers are also triggered by AVP and contribute to the observed response. Furthermore, we do not know the nature of the downstream effectors and importantly it remains to be determined whether the increases in urea flux induced by [Ca 2  ] i or cAMP are due to direct activation of UT-A2, possibly as a result of phosphorylation, as possibly is the case for UT-A1 ( 30 ), or due to shuttling of UT-A2 moieties to the plasma membrane. Future experiments aim to address these important questions.
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In conclusion, we engineered for the first time an MDCK cell line that heterologously expresses mUT-A2. When expressed in MDCK cell, urea transport by mUT-A2 is acutely sensitive to AVP and increased intracellular cAMP or Ca 2 . These findings have implications for the renal handling of urea and the urinary concentrating mechanism.6 R$ {5 J: t' j# B; {/ i0 W5 u

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This work was supported in part by the Biotechnology and Biological Science Research Council and Kidney Research UK.
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ACKNOWLEDGMENTS
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The authors thank Dr. O. Frohlich and Dr. J. Bruce for expert advice.6 p' R" X  ~0 ^$ W1 F* {
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