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Am J Physiol Renal Physiol 293: F1308-F1313, 2007. First published August 8, 2007; doi:10.1152/ajprenal.00197.2007
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Forskolin stimulates phosphorylation and membrane accumulation of UT-A3

Mitsi A. Blount, Janet D. Klein, Christopher F. Martin, Dmitry Tchapyjnikov, and Jeff M. Sands

Renal Division, Emory University School of Medicine, Atlanta, Georgia

Submitted 24 April 2007 ; accepted in final form 2 August 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
UT-A1 is regulated by vasopressin and is localized to the apical membrane and intracellular compartment of inner medullary collecting duct (IMCD) cells. UT-A3 is also expressed in the IMCD and is regulated by forskolin in heterologous systems. The goal of the present study is to investigate mechanisms by which vasopressin regulates UT-A3 in rat IMCD. In fresh suspensions of rat IMCD, forskolin increases the phosphorylation of UT-A3, similar to UT-A1. Biotinylation studies indicate that UT-A3 is located in the plasma membrane. Forskolin treatment increases the abundance of UT-A3 in the plasma membrane similar to UT-A1. However, these two transporters do not form a complex through a protein-protein interaction, suggesting that transporter function is unique to each protein. While immunohistochemistry localized UT-A3 to the basal and lateral membranes, a majority of the staining was cytosolic. Immunohistochemistry of vasopressin-treated rat kidney sections also localized UT-A3 primarily to the cytosol with basal and lateral membrane staining but also showed some apical membrane staining in some IMCD cells. This suggests that under normal conditions, UT-A3 functions as the basolateral transporter but in a high cAMP environment, the transporter may move from the cytosol to all plasma membranes to increase urea flux in the IMCD. In summary, this study confirms that UT-A3 is located in the inner medullary tip where it is expressed in the basolateral membrane, shows that UT-A3 is a phosphoprotein in rat IMCD that can be trafficked to the plasma membrane independent of UT-A1, and suggests that vasopressin may induce UT-A3 expression in the apical plasma membrane of IMCD.

cAMP; vasopressin; concentrating mechanism; urea transport

UNDERSTANDING UREA MOVEMENT is important for determining proper kidney function. The renal handling of urea has been greatly clarified by the molecular identification of distinct epithelial urea transporters (17, 18, 28). Urea transporters are derived from the UT-A (Slc14a2) and UT-B (Slc14a1) genes. The UT-B gene, expressed in red blood cells and in descending vasa recta endothelial cells, transcribes two protein isoforms (1, 22, 26, 27). To date, the UT-A family of urea transporters consists of six protein isoforms. UT-A1, UT-A2, UT-A3, and UT-A4 isoforms are located in the kidney (6, 10, 17, 18, 28), whereas UT-A5 is expressed in mouse testis (5) and UT-A6 is expressed in human colon (19). UT-A1 is the largest protein in the UT-A family and is expressed in the apical membrane of the inner medullary collecting duct (IMCD) (2, 11, 15). UT-A2 consists of the COOH-terminal half of UT-A1 and is expressed in thin descending limbs (16, 25). UT-A3 is the NH2-terminal half of UT-A1 and is also expressed in IMCDs (21, 24). UT-A4 contains the NH2-terminal quarter of UT-A1 spliced to the COOH-terminal quarter (9, 10).

Genetically engineered mice lacking both UT-A1 and UT-A3 have a severe urine concentrating defect indicating that these two transporters are major contributors to the urinary concentrating mechanism (4). The apical membrane urea flux is primarily controlled by UT-A1. This transporter is specifically localized to the apical membrane and subapical intracellular vesicles of the inner medulla (IM) (15, 16). Vasopressin (AVP) increases both UT-A1 phosphorylation and apical membrane accumulation in IMCDs (12).

UT-A3 is also located in the terminal region of the IMCD, although the exact function and cellular location of this transporter remain controversial (14, 21, 23, 24). Urea flux is increased in transfected cells or oocytes containing UT-A3 after addition of forskolin or AVP (6, 10, 23) indicating that UT-A3 function is regulated by cAMP, similar to UT-A1. Recent studies show that activation of PKA by AVP stimulates urea flux through UT-A3 (23). However, deletion of two conserved PKA sites in UT-A3 did not alter urea flux (19) suggesting that UT-A3 is phosphorylated by PKA at a nonclassical site, phosphorylated by another kinase (3), or is not phosphorylated and is indifferent to the phosphorylation event. To date, it is unknown whether UT-A3 is directly phosphorylated in rat IMCD. In this manuscript, we will demonstrate in rat IM that UT-A3 is a phosphoprotein that can be directly phosphorylated by a cAMP-dependent pathway, UT-A3 is found in the plasma membrane, and UT-A3 moves to the apical membrane via a cAMP stimulus.


   METHODS
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RESULTS
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Antibodies. We made the UT-A1/UT-A3 NH2-terminal (NH2- CEEKDLRSSDEDSHIVKIEKPNERC-COOH) and UT-A3 (NH2-CIYFLTVRRSEEEKSPNGD-COOH) polyclonal antibodies used in the studies described blow. Synthetic peptides were designed for specificity, antigenicity, and absence of posttranslational modifications using computer analysis. They were produced by standard solid-phase, peptide-synthesis techniques, purified by high-performance liquid chromatography, and conjugated to maleimide-activated keyhole limpet hemocyanin via covalent linkage to the NH2-terminal cysteine by the Emory University Microchemical facility. For each antibody, two rabbits were immunized by using a combination of Freund's complete and incomplete adjuvants (Spring Valley Laboratories, Woodbine, MD). The antisera obtained were affinity purified by using a column on which 2 mg of the immunizing peptide were immobilized via covalent linkage to agarose beads (SulfoLink Kit; Pierce, Rockford, IL). A UT-A1 COOH-terminal antibody also used in these studies was prepared by this laboratory as described earlier (13).

Protein isolation and Western blot analysis. Kidneys were removed from normal Sprague-Dawley rats and dissected into cortex (CTX), outer medulla (OM), the base of the IM, and the tip of the IM. Tissues were placed into ice-cold isolation buffer (10 mM triethanolamine, 250 mM sucrose, pH 7.6, 1 µg/ml leupeptin, and 40 µg/ml PMSF) and homogenized with glass homogenizers. SDS was added to a final concentration of 1%, and the samples were sheared with a 25-gauge needle. Homogenates were centrifuged at 8,000 g for 15 min, and the protein in the supernatant fractions was measured by a modified Lowry method (DC Protein Assay Kit; Bio-Rad, Hercules, CA). Proteins (20 µg/lane) were size separated by SDS-PAGE by using 10% gels and then electroblotted to polyvinylidene difluoride membranes (Imobilon, Millipore, Bedford, MA). Blots were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS: 20 mM Tris·HCl, 0.5 M NaCl, pH 7.5) at room temperature for 1 h and then incubated with primary antibody overnight at 4°C. The primary antibodies used were a polyclonal antibody to the COOH-terminal of UT-A (13) and the NH2-terminal and MQ2 antibodies. Blots were washed three times in TBS with 0.5% Tween 20 (TBS/Tween) and then incubated with Alexa Fluor 680-linked anti-rabbit IgG (Molecular Probes, Eugene, OR). Blots were washed two times with TBS/Tween, and then the bound secondary antibody was visualized using infrared detection with the Licor Odyssey protein analysis system. For antibody competition studies, primary antibody was preincubated with the immunizing peptide (0.1 µg/ml) for 30 min at room temperature to saturate the primary antibody binding sites and then used to probe the Western blot.

Biotinylation of UT-A3. IMCD suspensions were biotinylated using a method previously described (12). Fresh suspensions of rat IMCDs were prepared by enzyme digest as described (29) and were subsequently treated ex vivo with forskolin (10 µM) for 15 min at 37°C. Then, samples were washed free of excess solution two times with 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, 2.5 mM Na2HPO4). Treatments were added back during the incubation with biotinylation buffer containing 3 mg/ml biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester (Sigma, St. Louis, MO) for 60 min at 4°C. Cells were then washed free of unattached biotin by three washes with biotin quenching buffer (0.1 mM CaCl2, 1 mM MgCl2, 260 mM glycine in PBS) with the last wash incubated for 20 min at 4°C. Next, samples were washed three times with lysis buffer without detergent and the cells were solubilized for 1 h in lysis buffer containing 1% NP-40 (150 mM NaCl, 5 mM EDTA, 50 mM Tris). After centrifugation (14,000 g, 10 min, 4°C) to remove insoluble particulates, streptavidin beads were added to the supernatant fractions and allowed to absorb biotinylated proteins overnight at 4°C. After being washed with high salt and no salt buffers, Laemmli SDS-PAGE sample buffer was added directly to the pellets. Samples were boiled for 2 min, and the pool of biotinylated proteins was analyzed by Western blot.

Coimmunoprecipitation of UT-A1 and UT-A3. Inner medullary tissue was dissected from normal Sprague-Dawley rat kidneys and cut into small pieces. The pieces were placed into a cold homogenizer containing 600 µl cold gentle lysis buffer (10 mM Tris·HCl, pH 7.5, 10 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin) and homogenized. The resulting homogenate was placed in a 1.5-ml microfuge tube. Insoluble fragments were removed by centrifugation at 12,000 g for 15 min at 4°C. One milliliter of supernatant was removed and placed in a 1.5-ml microfuge tube that contained either 8 µl COOH-terminal antibody (UT-A1 specific) or 10 µl MQ2 (UT-A3 specific). After gentle agitation at 4°C overnight, 20 µl of protein A agarose beads (Pierce) were added to each microfuge tube and agitation was continued for 2 h at 4°C. Beads were collected by centrifugation (1 min, 12,000 g, 4°C) and the supernatant was removed and the beads were washed three times with 1 ml gentle lysis buffer at 4°C. Laemmli SDS-PAGE sample buffer (100 µl) was added directly to the beads, samples were boiled for 2 min, and the resulting pool of proteins was analyzed by Western blot.

[32P]labeling of UT-A3. IMCD pieces were incubated in phosphate-free DMEM containing 0.1 mCi/ml [32P]orthophosphate for 3 h at 37°C and gassed with 5% CO2-95% air as described previously (29). At the end of the 3-h loading period, forskolin (10 µM) was added and the incubation continued for an additional 30 min at 37°C. Unincorporated 32P was removed by three washes with phosphate-free DMEM. Then, the IM tissue pieces were homogenized in 1 ml RIPA buffer (10 mM Tris·HCl, pH 7.4, 2.5 mM EDTA, 50 mM NaF, 1 mM Na4P2O7-10·H2O, 1 mM phenylmethylsulfonyl fluoride; 1% Triton X-100, 10% glycerol, 1% deoxycholate, 1 µg/ml aprotinin, 0.18 mg/ml sodium orthovanadate) and sheared with a 26-gauge needle. After centrifugation at 14,000 g for 15 min to remove insoluble particulates, samples were incubated overnight with the NH2-terminal antibody at 4°C with gentle mixing. Immunocomplexes were precipitated with protein A agarose for 2 h at 4°C; then, the pelleted beads were washed six times with RIPA and once with potassium-free phosphate-buffered saline. Washes were counted to ensure complete removal of unbound radiolabeled material. Laemmli-SDS-PAGE sample buffer was added directly to the pelleted beads, samples were boiled, and proteins were size-separated on two identical SDS-polyacrylamide gels. One gel was dried and [32P]incorporation into UT-A1 and UT-A3 was analyzed by autoradiography. The proteins on the other gel were transferred to polyvinylidene difluoride membrane, and the amount of immunoprecipitated UT-A1 and UT-A3 protein was assayed by Western blot.

Immunohistochemistry. Kidney vibratome sections (50-µm thick) were collected from perfused control Sprague-Dawley rats or rats injected with AVP (5 nmol, American Regent, Shirley, NY) 45 min before perfusion. Sections were washed for 20 min 3 times in 50 mM NH4Cl/PBS at 4°C before being washed for 10 min 3 times in PBS. Tissue sections were incubated at 4°C for 30 min with buffer B (1% BSA, 0.05% saponin, and 0.2% gelatin in PBS) for 6 consecutive washes. The tissue sections were then incubated overnight at 4°C in the polyclonal antibodies against COOH terminal (1:1,000), NH2 terminal (1:300), and MQ2 (1:150) in buffer A (1% BSA in PBS). After six 10-min washes with buffer C (0.1% BSA, 0.05% saponin, and 0.2% gelatin in PBS), the tissue sections were incubated for 2 h at room temperature in peroxidase-conjugated donkey anti-rabbit IgG Fab fragment (Dako, Via Real Carpinteria, CA). Tissues were then washed three times for 10 min with buffer C followed by three 10-min washes with PBS. Tissues were incubated in 0.1% DAB for 5 min before 3% H2O2 was added to detect labeling. Sections were dehydrated with graded alcohol and embedded in Epon mixture (EMS, Fort Washington, PA). Tissues were sectioned at 1.5-µm thickness with a Reichert Ultracut S ultra microtome equipped with a diamond knife (DiATOME, Hatfield, PA). After hematoxylin staining, light microscopy was carried out with Leica DMRE (Leica Microsystems, Herlev, Denmark).

Statistics. All data are presented as means ± SE. To test for statistical significance between two groups, we used a Mann-Whitney U-test. The criterion for statistical significance was P < 0.05.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Localization of UT-A3 in the IMCD. Western blot and immunohistochemistry were used to examine the general expression of UT-A3 in the kidney. Blots probed with UT-A1/UT-A3 NH2-terminal antibody (N-Term) showed that the two glycosylated forms of UT-A1 (97 and 117 kDa) were exclusive to the IM as reported earlier (15) (Fig. 1A). The NH2-terminal antibody also detected the two glycosylated forms of UT-A3 (45 and 65 kDa) (24) (Fig. 1A). A 50-µm-thick Vibratome section stained with NH2-terminal antibody strongly detects both UT-A1 and UT-A3 in the renal papilla (Fig. 1B). The UT-A3-specific antibody also identified the two glycoprotein forms of UT-A3 at 45 and 65 kDa in the IM (Fig. 2A) and did not detect higher molecular weight bands consistent with UT-A1. Another band was detected at 50 kDa. All three bands were ablated by preabsorption with immunizing peptide (Fig. 2B). These forms were also slightly detected in the OM (Fig. 2A) which is similar to the finding reported in Terris et al. (24). Light micrograph of a 50-µm-thick Vibratome section stained with the UT-A3 antibody localizes UT-A3 to the IM (Fig. 2C). Labeling was strongest in renal papilla.


Figure 1
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  		Fig. 1. Western blot of whole cell lysates from rat kidney probed with the NH2-terminal (UT-A1/UT-A3) antibody. A: arrows indicate characteristic bands of UT-A1 (solid, 97 and 117 kDa) and UT-A3 (open, 44 and 67 kDa). B: 50-µm-thick Vibratome section stained with NH2-terminal antibody shows staining at inner medulla (IM) tip and base. IM tip, terminal inner medulla (papillary tip); IM base, middle and base of inner medulla; OM, outer medulla; CTX, cortex. Scale bar represents 300 µm. Blot and light micrograph represent n = 1 of n = 3 using different animals for each n.

 

Figure 2
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  		Fig. 2. Western blot of whole cell lysates from rat kidney probed with the UT-A3-specific antibody. A: arrows detected UT-A3 at 44 and 64 kDa (open arrows). B: primary antibody was preincubated with the immunizing peptide (0.1 µg/ml) for 30 min at room temperature to saturate the primary UT-A3 antibody binding sites before probing rat kidney lysates; no protein bands are detected. C: 50-µm-thick Vibratome section stained with UT-A3 antibody showed staining in the IM. Scale bar represents 300 µm. Blots and light micrograph represent n = 1 of n = 3 using different animals for each n.

 
The UT-A3 antibody used in this study was created based on the same peptide sequence described in Stewart et al. (21); both the current antibody and Stewart et al.'s antibody differ somewhat from the original anti-UT-A3 antibody described by Terris et al. (24). Both of these antibodies were shown to specifically detect UT-A3 and not UT-A1 based on ablation by preabsorption with immunizing peptide and the lack of detection of higher molecular weight bands consistent with UT-A1 [Stewart et al. (21) and Terris et al. (24)], as does our antibody. Stewart et al. The antibody of Stewart et al., while specific for UT-A3, did not observe three distinct bands, but rather a smear from 45 to 65 kDa. Considering that the antibody smear encompasses the 50-kDa band demonstrated by our antibody and that all three bands were ablated by preabsorption with immunizing peptide (Fig. 2B), this 50-kDa band appears to be specific to UT-A3 as well.

Phosphorylation of UT-A3. Urea flux mediated by UT-A3 is increased upon activation of a kinase via a cAMP pathway (10, 23). Although it has been suggested that PKA phosphorylates UT-A3, direct phosphorylation of UT-A3 has never been confirmed. Both UT-A1 and UT-A3 are phosphoproteins in control rat IM tissue (Fig. 3A). Forskolin (10 µM, 15 min) significantly increased UT-A1 phosphorylation by 225% (P < 0.05) and increased UT-A3 phosphorylation by 100% (P < 0.05) (Fig. 3, A and C). Western blot of the samples shows that both lanes have equal protein concentration (Fig. 3B).


Figure 3
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  		Fig. 3. Autoradiogram (A) and Western blot (B) of [32P]orthophosphate labeled IM tissue precipitated by NH2-terminal antibody. IM tissue pieces were incubated with either DMSO (–FSK) or forskolin (10 µM; +FSK) for 30 min at 37°C before lysis. Precipitated proteins were separated by SDS-PAGE and then analyzed by autoradiogram. Arrows indicate the glycosylated forms of both UT-A1 (solid) and UT-A3 (open). C: densitometry was performed using Image J software and plotted as density in arbitrary units. Statistics were performed (*P < 0.05). Error bars represent means ± SE. Shown is a representative result from a total of n = 5 pairs.

 
Biotinylation of UT-A3. Results above indicate that UT-A3 is primarily located in the inner medullary portion of the kidney, which is also the location of UT-A1. Our laboratory showed that UT-A1 accumulates in the IMCD plasma membrane in a cAMP-dependent manner (12). Given reports that the function of UT-A3 is cAMP dependent (10, 19, 23), we investigated whether UT-A3, like UT-A1, moves to the IMCD plasma membrane after cAMP stimulation. Probing the biotinylated protein with the UT-A1/UT-A3 NH2-terminal antibody demonstrated that both UT-A1 and UT-A3 are found in the plasma membrane of IM tubules (Fig. 4A). The addition of forskolin (10 µM) to the suspension led to accumulation of UT-A1 and UT-A3 in the plasma membrane (Fig. 4A). Forskolin significantly stimulates the accumulation of UT-A1 in the membrane by 200% (P < 0.05) and UT-A3 by 70% (P < 0.05; Fig. 4B).


Figure 4
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  		Fig. 4. Representative Western blot of biotinylated proteins from IMCD suspensions probed with NH2-terminal antibody (detects UT-A1/UT-A3; A) and resulting densitometry (B). IM tissue was harvested from 6 rats and tubule suspensions were prepared as described in METHODS. Suspensions were pooled in pairs resulting in 3 distinct pools. Pools (1, 2, and 3) were then equally separated and either treated with vehicle control (no treatment) or 10 µM forskolin (1+, 2+, and 3+). Samples were then subjected to the biotinylation protocol outlined in METHODS. Arrows indicate the 2 glycosylated bands of either UT-A1 (solid) or UT-A3 (open). Densitometry was performed using Licor Odyssey protein analysis system and plotted as density in arbitrary units. Statistics were performed (*P < 0.05). Error bars represent means ± SE. Blot shows 3 suspension pools (n = 3) where this experiment was repeated in triplicate for a total n = 9.

 
Coimmunoprecipitation of UT-A1 and UT-A3. Biotinylation can identify proteins that are associated with the plasma membrane. This method, however, can also detect proteins that are complexed with a membrane-bound protein but are not embedded in the membrane itself. Given that UT-A1 and UT-A3 are both located in the terminal region of the IM, it is possible that UT-A3 is found in the biotinylated protein population because it is complexed with UT-A1 by a protein-protein interaction. IM tissue was subjected to coimmunoprecipitation with either UT-A1-specific antibody or UT-A3-specific antibody. A Western blot of the UT-A1 immunoprecipitate (Fig. 5, left) probed for UT-A1 (Fig. 5A) and UT-A3 (Fig. 5B) shows that UT-A1, but not UT-A3, was detected in the samples precipitated with the UT-A1-specific antibody. A Western blot of the UT-A3 immunoprecipitate (Fig. 5, right) probed for UT-A1 (Fig. 5A) and UT-A3 (Fig. 5B) shows that UT-A3 was detected in the samples precipitated with the UT-A3-specific antibody, and some fainter bands of approximately the correct size for UT-A1 were detected (Fig. 5, right). Despite the specificity of the UT-A3 antibody demonstrated by Western blot of cell lysate (Fig. 2, A and B), there may be some cross-reaction with UT-A1 that becomes detectable after immunoprecipitation. Specificity of the UT-A3 antibody was reexamined using the UT-A1-MDCK stable cell line established in this laboratory (7, 8). Cells lysates were first probed with UT-A1-specific antibody (Fig. 5C, left lanes). The antibody detected the 97-kDa band corresponding to UT-A1. The same cell lysates were probed with UT-A3-specific antibody (Fig. 5C, right lanes). This antibody failed to detect any band that corresponded to UT-A1. This indicates that the UT-A3-specific antibody does not detect UT-A1. These data, plus the lack of any UT-A3 in the UT-A1 immunoprecipitate, suggest that UT-A1 and UT-A3 are not associated.


Figure 5
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  		Fig. 5. Western blot of proteins precipitated from rat inner medullary collecting ducts (IMCDs) and probed with complimentary antibodies to determine coimmunoprecipitation. COOH-terminal anti-UT-A1 (left)- and UT-A3-specific antibodies (right) were used to precipitate. Identical blots were probed for UT-A1 with COOH-terminal antibody (A) or probed for UT-A3 content (B). UT-A1-MDCK lysates were subjected to Western blot analysis and probed for UT-A1 (C, left 2 lanes) or UT-A3 (C, right 2 lanes). Solid arrows indicate UT-A1 bands and open arrows show UT-A3 band. Shown are 2 representative samples of n = 4 experimental results.

 
Immunolocalization of UT-A1 and UT-A3. Biotinylation data indicate that both UT-A1 and UT-A3 move to the membrane in a cAMP-dependent manner. To confirm this effect in vivo, immunolocalization was examined in the IMCD using the UT-A3-specific antibody. In normal rats, staining is largely cytoplasmic (Fig. 6A). Black arrowheads indicate strong basal and lateral plasma membrane staining in the IMCD cells. UT-A3 labeling was still primarily cytosolic in AVP-treated animals (Fig. 6B). Black arrowheads indicate strong basal and lateral plasma membrane staining in the IMCD cells. In contrast to control rats, AVP-treated rats also show apical plasma membrane staining in some cells (open arrow).


Figure 6
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  		Fig. 6. Immunolocalization of UT-A3 in control rat IMCD (A) and vasopressin (AVP)-treated rat (B). Black arrows indicate basal and lateral staining and open arrows show apical identification. Images are representative of n = 3 control and AVP-treated rats and are taken at x1,000 magnification.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The above studies show the following novel findings in rat IMCDs: 1) UT-A3 is a phosphoprotein; 2) UT-A3 can be trafficked to the plasma membrane in a cAMP-dependent manner; 3) UT-A3 does not form a complex with UT-A1; 4) and AVP may induce UT-A3 expression in the apical plasma membrane of IMCD. The two UT-A isoforms, UT-A1 and UT-A3, are strongly expressed in the IMCD. In the rat, UT-A1 has been immunolocalized to the apical membrane and intracellular compartment of IMCD cells (15) where it has been proven to play an essential role in the urine concentrating mechanism. Urea movement across the IMCD basolateral membrane, while unresolved, is assumed to be a urea transporter protein because in vitro perfusion studies on rat tubules show that addition of phloretin to the basolateral side of isolated IMCDs blocks transepithelial urea flux (20). It has been suggested that the urea transporter responsible for basolateral urea flux is UT-A3 (21). Reports concerning the localization of UT-A3 within the IMCD have been conflicting; some groups report basolateral membrane expression (14, 21) while others report apical membrane expression (24). In this report, we used ultra-thin sections of rat IMCD to show that UT-A3 is primarily located in the basal and lateral membranes in control rats but is also expressed in the apical membrane of some IMCD cells in AVP-treated rats.

The present study supports previous findings from several laboratories showing that UT-A3 is expressed in the IMCD (16, 21, 24). Since UT-A3 and UT-A1 are both expressed in the IMCD, we investigated whether these two transporters formed a complex through a protein-protein interaction. We discovered that UT-A1 and UT-A3 are not physically associated with one another suggesting that transporter function is unique to each protein.

Phosphorylation of UT-A1 is an important mechanism by which AVP rapidly increases urea permeability in vivo (29) and has been suggested to be mechanistically involved in UT-A1 trafficking to the plasma membrane (12). The present study confirms that forskolin increases UT-A1 phosphorylation in rat IMCDs. From what we and others observed, AVP regulation of UT-A3 is strikingly similar to UT-A1. MDCK-mUT-A3 cells treated with the PKA-specific inhibitor H89 failed to transport urea even in the presence of AVP. However, when the two PKA consensus sites within UT-A3 were mutated (S to A), the transporter still remained sensitive to cAMP suggesting that phosphorylation occurs at a nonclassical PKA site (19). Another possibility is that while PKA may not directly phosphorylate UT-A3, the kinase can still affect the function of this transporter by affecting trafficking machinery or by activating another signaling cascade such as the MAPK pathway. To examine this, we investigated whether UT-A3 was in fact a phosphoprotein. We observed that not only can UT-A3 be directly phosphorylated but that phosphorylation of the transporter can also be stimulated by forskolin.

Previous reports show that UT-A3 is responsive to cAMP stimulation. Forskolin stimulates urea flux in HEK-293 cells that express rUT-A3 (10) while AVP significantly stimulates urea uptake in mUT-A3-injected oocytes as well as increases urea transport in MDCK-mUT-A3 cells (23). Thus UT-A3 appears to be regulated by cAMP-dependent mechanisms, similar to UT-A1. Since acute AVP or forskolin administration significantly increases UT-A1 accumulation in the plasma membrane of rat IMCD suspensions and MDCK-rUT-A1 cells (12), we investigated whether UT-A3 was trafficked to the plasma membrane of the IMCD in response to forskolin. Biotinylation studies indicate that UT-A3 is expressed in the plasma membrane and that forskolin treatment does significantly increase the abundance of UT-A3, although this increase is not as great as that observed for UT-A1. The limited increase observed by surface biotinylation is consistent with the changes observed by immunohistochemistry. UT-A3 is present in the basal and lateral membranes, but a majority of staining is in the cytosol. AVP-treated rat sections also localized UT-A3 in the cytosol, basal and lateral membranes, but also showed staining of the apical membrane. This newly detected apical staining suggests the appearance of UT-A3 in the apical membrane of AVP-treated rats. The high amount of UT-A3 that remains in the cytosol is consistent with the small increase in membrane insertion we observed using the biotinylation method. Taken together, these data suggest that under normal conditions, UT-A3 functions as the basolateral transporter but in a high cAMP environment, the transporter moves from the cytosol to all plasma membranes to increase urea flux in the IMCD cell.

In summary, we confirm that UT-A3 is located in the inner medullary tip where it is basolateraly expressed. We are the first to show in rat IMCD that UT-A3 is a phosphoprotein, UT-A3 can be trafficked to the plasma membrane in a cAMP-dependent manner, UT-A3 does not form a complex with UT-A1, and AVP may induce UT-A3 expression in the apical plasma membrane.


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This work was supported by National Institutes of Health Grants R01-DK-62081, P01-DK-61521, R01-DK-41707, American Heart Association Grant-in-Aid 0655280B, National Kidney Foundation of Georgia, Edward Noble-David Lowance, M. D. Research Fellowship, and The Emory University School of Medicine Cottrell Foundation Fellowship.


   ACKNOWLEDGMENTS
 
We thank Dr. Y. H. Kim for technical assistance and helpful comments in preparation of this manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. Blount, Renal Division, Emory Univ. School of Medicine, 1639 Pierce Drive, Atlanta, GA 30322 (e-mail: mabloun{at}emory.edu)

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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