Urea transporters UT-A1 and UT-A3 accumulate in the plasma membrane in response to increased hypertonicity

Abstract

The UT-A1 and UT-A3 urea transporters are expressed in the terminal inner medullary collecting duct (IMCD) and play an important role in the production of concentrated urine. We showed that both hyperosmolarity and vasopressin increase urea permeability in perfused rat terminal IMCDs and that UT-A1 and UT-A3 accumulate in the plasma membrane in response to vasopressin. In this study, we investigated whether hyperosmolarity causes UT-A1 and/or UT-A3 to accumulate in the plasma membrane or represents a complimentary stimulatory pathway. Rat IMCD suspensions were incubated in 450 vs. 900 mosM solutions. We biotinylated the IMCD surface proteins, collected, and analyzed them. Membrane accumulation was assessed by Western blotting of the biotinylated protein pool probed with anti-UT-A1 or anti-UT-A3. We studied the effect of NaCl, urea, and sucrose as osmotic agents. Membrane-associated UT-A1 and UT-A3 increased relative to control levels when either NaCl (UT-A1 increased 37 ± 6%; UT-A3 increased 46 ± 13%) or sucrose (UT-A1 increased 81 ± 13%; UT-A3 increased 60 ± 8%) was used to increase osmolarity. There was no increase in membrane UT-A1 or UT-A3 when urea was added. Analogously, UT-A1 phosphorylation was increased in NaCl- and sucrose- but not in urea-based hyperosmolar solutions. Hypertonicity also increased UT-A3 phosphorylation. We conclude that the increase in the urea permeability in response to hyperosmolarity reflects both UT-A1 and UT-A3 movement to the plasma membrane and may be a direct response to tonicity. Furthermore, this movement is accompanied by, and may require, increased phosphorylation in response to hypertonicity.

the inner medulla is often hypertonic, especially during antidiuresis, when plasma vasopressin levels are high (12). Vasopressin stimulates urea transport across perfused rat terminal inner medullary collecting ducts (IMCD) (13). Urea transport is also stimulated by hyperosmolarity (resulting from addition of NaCl) in the absence of vasopressin and is further stimulated in the presence of vasopressin (14). While both vasopressin and hyperosmolarity stimulate urea transport, they do so through different second messenger pathways; vasopressin acts by increasing cAMP while hyperosmolarity acts by increasing intracellular calcium but does not increase cAMP (6, 18).

UT-A1 is the major urea transport protein expressed in the IMCD (15). Vasopressin increases urea flux in Madin-Darby canine kidney (MDCK) cells that are stably transfected with UT-A1 (4). In rat IMCD suspensions, vasopressin increases both UT-A1 phosphorylation and UT-A1 plasma membrane accumulation (8). Vasopressin stimulation of UT-A1 phosphorylation can be blocked by the protein kinase A (PKA) inhibitor, H-89, suggesting that UT-A1 phosphorylation is mediated by PKA, either directly or proximally (22). It is still unclear whether phosphorylation is required for plasma membrane accumulation or is a separate regulatory mechanism.

UT-A3 is also located in the IMCD (1, 20, 21). Like UT-A1, it is a phosphoprotein that accumulates in the plasma membrane in response to vasopressin stimulation (1). It is not, however, physically associated with UT-A1 and therefore offers the possibility of separate, complimentary, or coordinated regulation (1).

The purpose of the present study was to determine the mechanism by which hyperosmolarity stimulates urea transport. To accomplish this goal, we measured the plasma membrane accumulation and the phosphorylation of both UT-A1 and UT-A3 in isolated rat IMCD suspensions that were treated with solutions made hyperosmolar by the addition of NaCl, urea, or sucrose.

METHODS

Animals.

All animal protocols were approved by the Emory University Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), weighing 100–150 g, received free access to water and standard rat chow (Purina) containing 23% protein.

Sample preparation.

Kidneys were removed and the inner medulla (IM) was collected. Fresh suspensions of rat IMCDs were prepared as described previously (8). Briefly, rat IMs were minced in 1 ml of suspension buffer (in mM: 118 NaCl, 5 KCl, 25 NaHCO₃, 1.2 MgSO₄, 2 CaCl₂, 5.5 glucose, 5 Na-acetate, and 4 Na₂PO₄) containing 2 mg/ml collagenase and 0.65 mg/ml hyaluronidase and then incubated for 30 min at 37°C. Next, DNase (5 μl of 1 mg/ml) was added and incubated for another 20 min. Finally, the IMCDs were washed free of enzymes by centrifuging three times with exchanges of suspension buffer, and then resuspended in 1 ml of the hypertonic buffers and incubated for 1 h. The original urea permeability studies used isotonic (290 mosM) for controls and doubled the osmolality to study hypertonic conditions. The biotinylation buffer is slightly hypertonic at 450 mosM. To keep that protocol consistent with our previous biotinylation studies (8), but also to have a comparable increase in tonicity to that used for the other hypertonicity studies (14), the hypertonic solutions were made to twice the osmolality (900 mosmol/kgH₂O) of the control (450 mosmol/kgH₂O) buffer rather than matching the absolute tonicity.

Biotinylation.

Rat IMCD suspensions were biotinylated using a modification of the method described in (8). Samples were washed free of excess hypertonic solution 2× with PBS, and 3× 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). Biotinylation buffers prepared at the desired osmolarity (450 and 900 mosM) were used for the incubation with biotin [buffer containing 3 mg/ml Biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester (catalog no. B1022, Sigma)] for 60 min at 4°C. This incubation procedure resulted in the biotinylation of both apical and basolateral plasma membrane proteins in rat IMCD suspensions (8). 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 1 min, and the pool of biotinylated proteins was analyzed by Western blot.

Western blot analysis.

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 our polyclonal antibody to the COOH terminus of UT-A1 (9) or the NH₂ terminus of UT-A1 and UT-A3 (1) overnight at 4°C. 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.

Phosphorylation.

Metabolic labeling with ³²P-orthophosphate was performed as previously published (22). After the 3-h labeling period, IMCDs were incubated for a further 30 min with phosphate-free DMEM: 1) at 290 mosmol/kgH₂O, 2) with sucrose added to a final 600 mosmol/kgH₂O, 3) with urea added to a final 600 mosmol/kgH₂O, and 4) with NaCl added to a final 600 mosmol/kgH₂O. These osmolalities were chosen to mimic those used in the original isolated perfused tubule studies (14). Following incubation in hypertonic solutions, IMCD cell lysates were prepared and UT-A1 (22) and UT-A3 (1) were immunoprecipitated as previously described. Precipitated proteins were separated by SDS-PAGE and radiolabeled UT-A1 and UT-A3 were determined by autoradiography of the dried gel. Parallel Western blots were performed to ensure uniform protein content per lane (data not shown).

Statistics.

All data are presented as means ± SE. To test more than two groups, we used an ANOVA, followed by Fisher's least significant difference (protected t-test) to determine which groups are significantly different. The criterion for statistical significance is P < 0.05.

RESULTS

Effect of increasing osmolality with NaCl as the added osmolyte.

IMCDs were incubated in 450 mosM buffer or a buffer to which NaCl was added to make the buffer 900 mosM. Incubation in the hypertonic buffer significantly increased the amount of UT-A1 that accumulated in the plasma membrane by 37% [450 mosM: 22 ± 1 arbitrary units (au) vs. 900 mosM: 31 ± 2 au]. Figure 1 shows representative Western blots of the biotinylated UT-A1 at 450 and 900 mosM. The bar graph shows the results from three combined experiments with a total of 9 animals per group (P < 0.002). Figure 2 shows that UT-A3 was also increased by 46% over control levels (450 mosM: 7 ± 1 au vs. 900 mosM: 12 ± 1 au) by the NaCl hyperosmolar solution. These data are the combined result of three experiments with a total of six animals per group, P < 0.001.

Fig. 1.

A: representative Western blot of biotinylated UT-A1 in inner medullary collecting ducts (IMCDs) incubated at 450 mosM (left) and 900 mosM (right). Arrows indicate UT-A1 bands at 97 and 117 kDa. Increase in osmolality was achieved by adding NaCl to the 450 mosM buffer. B: bar graph shows densitometry results of 3 separate experiments with a total of 9 animals per group presented as percent of control (450 mosM). *P < 0.002 vs. control. 450 mosM (open bar) = control animals; 900 mosM (filled bar) = hypertonic treatment.

Fig. 2.

A: representative Western blot of biotinylated UT-A3 in IMCDs incubated at 450 mosM (left) and 900 mosM (right). Arrows indicate UT-A3 bands at 64–48 kDa and UT-A1 at 97 and 117 kDa. Increase in osmolality was achieved by adding NaCl to the 450 mosM buffer. B: bar graph shows UT-A3 densitometry results of 3 separate experiments with a total of 6 animals per group presented as percent of control (450 mosM). *P < 0.001 vs. control. 450 mosM (open bar) = control animals; 900 mosM (filled bar) = hypertonic treatment.

Effect of increasing osmolality with urea as the added osmolyte.

When urea was used to increase the osmolality, there was no difference in the amount of either UT-A1 (Fig. 3; result of 4 experiments, 12 animals/group) or UT-A3 (Fig. 4; result of 4 experiments, 12 animals/group) in the membrane between the two osmolar concentrations, suggesting that tonicity may be involved in the mechanism of UT-A1 and UT-A3 movement.

Fig. 3.

A: representative Western blot of biotinylated UT-A1 in IMCDs incubated at 450 mosM (left) and 900 mosM (right). Arrows indicate UT-A1 bands at 97 and 117 kDa. Increase in osmolality was achieved by adding urea to the 450 mosM buffer. B: bar graph shows densitometry results of 4 separate experiments with a total of 12 animals per group presented as percent of control (450 mosM). 450 mosM (open bar) = control animals; 900 mosM (filled bar) = hypertonic treatment. There were no significant changes.

Fig. 4.

A: representative Western blot of biotinylated UT-A3 in IMCDs incubated at 450 mosM (left) and 900 mosM (right). Arrows indicate UT-A3 bands at 48–64 kDa and UT-A1 at 97 and 117 kDa. Increase in osmolality was achieved by adding urea to the 450 mosM buffer. B: bar graph shows UT-A3 densitometry results of 4 separate experiments with a total of 6 animals per group presented as percent of control (450 mosM). 450 mosM (open bar) = control animals; 900 mosM (filled bar) = hypertonic treatment. There were no significant changes.

Effect of increasing osmolality by adding sucrose.

To determine whether tonicity might be a factor in the movement of UT-A1 or UT-A3 to the plasma membrane, we increased the osmolality by adding sucrose. This resulted in significantly increased movement of both UT-A1 (81% over control levels; 450 mosM: 21 ± 1 au vs. 900 mosM: 38 ± 5 au) and UT-A3 (60% over control levels; 450 mosM: 7 ± 1 au vs. 900 mosM: 11 ± 1 au) to the membrane (Figs. 5 and 6, respectively). The bar graphs show the results from three combined experiments with a total of nine animals per group for UT-A1 (P < 0.004) and six animals per group for UT-A3 (P < 0.02).

Fig. 5.

A: representative Western blot of biotinylated UT-A1 in IMCDs incubated at 450 mosM (left) and 900 mosM (right). Arrows indicate UT-A1 bands at 97 and 117 kDa. Increase in osmolality was achieved by adding sucrose to the 450 mosM buffer. B: bar graph shows densitometry results of 3 separate experiments with a total of 9 animals per group presented as percent of control (450 mosM). *P < 0.004 vs. control. 450 mosM (open bar) = control animals; 900 mosM (filled bar) = hypertonic treatment.

Fig. 6.

A: representative Western blot of biotinylated UT-A3 in IMCDs incubated at 450 mosM (left) and 900 mosM (right). Arrows indicate UT-A3 bands at 48–64 kDa and UT-A1 at 97 and 117 kDa. Increase in osmolality was achieved by adding sucrose to the 450 mosM buffer. B: bar graph shows UT-A3 densitometry results of 3 separate experiments with a total of 6 animals per group presented as percent of control (450 mosM). *P < 0.02 vs. control. 450 mosM (open bar) = control animals; 900 mosM (filled bar) = hypertonic treatment.

The membrane accumulation in response to hypertonicity may be due to phosphorylation of the urea transporter.

To assess the possible involvement of phosphorylation in the response of the urea transporter to hyperosmotic conditions, we metabolically labeled suspended IMCDs with ³²P-orthophosphate and then incubated them at different osmolalities. Figure 7 shows the phosphorylation of UT-A1 in response to hypertonic conditions comparable to those in the membrane accumulation experiments above. The autoradiograms in Fig. 7 show that both the 117- and 97-kDa isoforms of UT-A1 are phosphorylated under isotonic control (Ctl) conditions and that the UT-A1 phosphorylation, normalized to total UT-A protein, is increased when incubated in 600 mosM medium containing sucrose [increased by 84 ± 21% (n = 9, P < 0.005)] or NaCl [increased by 181 ± 80% (n = 6, P < 0.05)]. In contrast, when the osmolality was raised to 600 mosM with urea, there was no increase in UT-A1 phosphorylation and in fact, phosphorylation decreased by 37 ± 6% (n = 6, P < 0.001) relative to control levels. These data reflect a minimum of three experiments per hypertonic condition.

Fig. 7.

A: representative autoradiograms of immunoprecipitated UT-A1 from ³²P-metabolically labeled IMCDs that were exposed to hypertonic conditions. Both the 117- and 97-kDa isoforms of UT-A1 (designated by arrows) are phosphorylated under isotonic control (Ctl) conditions and more phosphorylated under hypertonic conditions when the incubation medium is brought to 600 mosM with sucrose (suc; left gel) or NaCl (right gel). When the osmolality was raised to 600 mosmol/kgH₂O with urea (left gel), there is no increase in UT-A1 phosphorylation. B: bar graph from 7 similar experiments. Data reflect ³²P-UT-A1/total UT-A1 presented as % above average control (set to 100%). Bars = means ± SE. Sucrose: 84 ± 21%, n = 9, *P < 0.005; NaCl: 181 ± 84%, n = 7, *P < 0.05; Urea: −37 ± 6%, n = 6, *P < 0.001.

To determine whether hypertonicity would also increase the phosphorylation of UT-A3, we treated ³²P-labeled IMCDs with a 600 mosM solution with sucrose and immunoprecipitated UT-A1 and UT-A3 (Fig. 8). The autoradiogram shows that incubation in hypertonic conditions increased the phosphorylation of UT-A3 by 83 ± 26% (n = 6, P < 0.05). The autoradiogram also confirms that when probed with a different antibody from that used in Fig. 7, UT-A1 phosphorylation is increased in IMCDs under hypertonic conditions.

Fig. 8.

A: autoradiogram of immunoprecipitated UT-A3 from IMCDs, detected using NH₂-terminal UT-A1/UT-A3 antibody. UT-A3 and UT-A1 bands, identified by arrows, are phosphorylated under isotonic Ctl conditions and more phosphorylated under hypertonic conditions when the incubation medium is brought to 600 mosM with sucrose. These data are representative of 6 samples per condition collected in 2 separate experiments. B: bar graph from 2 similar experiments. Data reflect ³²P-UT-A3/total UT-A3 presented as % above average control (set to 100%). Bars = means ± SE. Sucrose: 83 ± 26%, n = 6, *P < 0.05.

DISCUSSION

This study demonstrates that hypertonicity increases the plasma membrane accumulation of both UT-A1 and UT-A3. Our previous work showed that urea permeability in isolated perfused tubules was stimulated by hyperosmolarity in the form of effective osmoles (NaCl, mannitol) but not ineffective osmoles (urea) and that vasopressin upregulates urea permeability in a similar but independent fashion (14). This study provides a possible mechanism whereby the transepithelial transport of urea may be upregulated in response to a hypertonic stimulus.

Previous studies attempting to inhibit urea transport using thiourea suggested that one mechanism by which urea transport might be stimulated in the IMCD is through an increase in the number of urea transporters (2). They also established that vasopressin and hyperosmolarity independently upregulated the same urea transporter because of their similar inhibitor profiles and because they affected the same terminal portion of the IMCD (5). Recently, we showed that it is possible to stimulate the trafficking of both UT-A1 and UT-A3 to the plasma membrane using vasopressin and forskolin (1, 8). In that study, we used biotinylation to show membrane accumulation of UT-A1. In that study, we showed that biotin was able to access the tubule lumen and that it did not enter the cells. Although hypertonic shrinkage in nonperfused IMCDs could alter the access of the biotin to the tubule lumen, there was an increase in UT-A1 biotinylation with both sucrose- and NaCl-induced hypertonicity, and since UT-A1 is known to be apically oriented, we believe that access was not prevented.

Forskolin stimulates adenylyl cyclase which increases cAMP (16). Vasopressin also stimulates the production of cAMP but does this through a series of reactions where vasopressin binds to the V₂ vasopressin receptor on the basolateral surface of the cell. The binding results in stimulation of a G protein signaling pathway that leads to activation of adenylyl cyclase and increased cAMP. By either route, the increased cAMP then stimulates PKA to phosphorylate its substrates (11, 13, 14, 18). UT-A1 and UT-A3 both have consensus PKA phosphorylation sites (7, 15). The phosphorylation of UT-A1 is PKA dependent (22). Attempts to prove that UT-A3 is directly phosphorylated by PKA at a consensus PKA phosphorylation site have failed (17). This suggests that UT-A3 is phosphorylated downstream of the PKA-mediated phosphorylation of another protein or at a nonconsensus PKA phosphorylation site.

The possibility of alternative phosphorylations of these transporters suggests that although both vasopressin and hypertonicity increase urea permeability, plasma membrane accumulation, and phosphorylation, hypertonicity might be acting through an alternate or complimentary pathway. The relationship between membrane accumulation and phosphorylation of UT-A1 or UT-A3 has not been established. It is possible that phosphorylation is a method of activating the transporter once it has already been inserted into the membrane or that phosphorylation helps to target the transporter to be inserted into the membrane. Further studies are needed to elucidate the role of phosphorylation in urea transporter activation.

Physiologically, these results suggest that the hypertonic NaCl present in the IM upregulates the plasma membrane accumulation of UT-A1 and UT-A3 to increase urea transport across the IMCD. Because the tubules responded similarly to sucrose, this effect can be attributed to hypertonicity rather than stimulation by the NaCl molecule specifically. This process would be beneficial to the urine concentrating mechanism by creating a positive feedback system. In times of diuresis, plasma vasopressin concentration is low, as is the concentration of solutes in the IM, which would minimize stimulation of urea transporters through either of these mechanisms. During times of antidiuresis, however, vasopressin and interstitial NaCl could act synergistically to increase the concentration of urea in the IM. According to the passive mechanism hypothesis proposed by Kokko and Rector (10) and by Stephenson (19), this would stimulate NaCl reabsorption from the thin ascending limb of the loop of Henle, creating a positive feedback loop to deliver even more urea to the IM. This would provide the hypertonic IM that is necessary to concentrate urine during antidiuresis.

Although hypertonic NaCl and hypertonic sucrose both stimulated the membrane accumulation of UT-A1 and UT-A3, the membrane accumulation of UT-A1 and UT-A3 was not increased by hyperosmolar urea. Previous findings by Chou et al. (3) showed that high levels of urea actually inhibited urea permeability in isolated perfused tubules. Because hyperosmolar urea does not significantly decrease the membrane accumulation of UT-A1 or UT-A3, it must act through some other pathway to decrease urea transport.

In conclusion, we found that hypertonicity increases the plasma membrane accumulation and the phosphorylation of both of the urea transporters, UT-A1 and UT-A3, which are present in the IMCD. Hyperosmolarity alone is insufficient to cause these changes. The mechanism by which the increased phosphorylation occurs in response to hypertonicity remains to be determined. These results are consistent with hypertonic stimulation of urea transport being complimentary to the stimulation of urea transport by vasopressin.

GRANTS

This work was supported by National Institutes of Health Grants P01-DK-61521, R01-DK-62081, R01-DK-41707, and American Heart Association (AHA) Grant-in-Aid 0655280B and AHA postdoctoral Grant 0725545B.