Characterization of mouse urea transporters UT-A1 and UT-A2

R. A. Fenton1, G. S. Stewart1, B. Carpenter1, A. Howorth1, E. A. Potter1, G. J. Cooper2, and C. P. Smith1

1 School of Biological Sciences, University of Manchester, Manchester, M13 9PT; and 2 Department of Biomedical Science, University of Sheffield, Sheffield, S10 2TN, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Specialized transporter proteins that are the products of two closely related genes, UT-A (Slc14a2) and UT-B (Slc14a1), modulate the movement of urea across cell membranes. The purpose of this study was to characterize the mouse variants of two major products of the UT-A gene, UT-A1 and UT-A2. Screening a mouse kidney inner medulla cDNA library yielded 4,047- and 2,876-bp cDNAs, the mouse homologues of UT-A1 and UT-A2. Northern blot analysis showed high levels of UT-A mRNAs in kidney medulla. UT-A transcripts were also present in testes, heart, brain, and liver. Immunoblots with an antiserum raised to the 19 COOH-terminal amino acids of rat UT-A1 (L194) identified immunoreactive proteins in kidney, testes, heart, brain, and liver and showed a complex pattern of differential expression. Relative to other tissues, kidney and brain had the highest levels of UT-A protein expression. In kidney sections, immunostaining with L194 revealed immunoreactive proteins in type 1 (short) and type 3 (long) thin descending limbs of the loop of Henle and in the middle and terminal inner medullary collecting ducts. Expression in Xenopus laevis oocytes showed that, characteristic of UT-A family members, the cDNAs encoded phloretin-inhibitable urea transporters. Acute application of PKA agonists (cAMP/forskolin/IBMX) caused a significant increase in UT-A1- and UT-A3-, but not UT-A2-mediated, urea transport.

kidney; urinary concentration; membrane protein; protein kinase A


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

UREA TRANSPORTERS DERIVED from the UT-A gene belong to a family of specialized proteins that play an integral role in the urinary concentrating mechanism (21, 28). These proteins include aquaporin water channels (15), the Na+-K+-2Cl- cotransporter (NKCC-2) (5), Kir 1.1 K+ channels (4), and CLC chloride channels (19).

In the rat, four UT-A isoforms have been identified in the kidney: UT-A1, UT-A2, UT-A3 and UT-A4 (21, 28). UT-A1 localizes to the apical membrane and cytoplasm of the middle and terminal inner medullary collecting duct (IMCD) (17). It is acutely regulated by cAMP (1, 10, 17) and is proposed to mediate the transepithelial movement of urea across the apical IMCD membrane.

In contrast, UT-A2 is located in type 1 (short) thin descending limbs (tDLs) and type 3 (long) tDLs of the loop of Henle (23, 30). This protein is not acutely regulated by cAMP, although chronic exposure to elevated levels of AVP, as triggered by thirsting, increases UT-A2 mRNA and protein levels (2, 6, 27, 30). By facilitating urea recycling between IMCD and tDL segments, UT-A2 is thought to help maintain the hypertonic medulla that provides the driving force for water reabsorption from the collecting ducts.

Significant amounts of UT-A3 are expressed in the cytoplasm of rat IMCD cells (29), and UT-A3 mRNA levels increase in response to thirsting (2, 6). In addition, a 24-h treatment with forskolin of HEK-293 cells heterologously expressing UT-A3 caused an increase in urea uptake (11). Together, these observations suggest that UT-A3 may play a role in the urinary concentrating mechanism and may also be regulated by PKA agonists. However, in contrast to the stimulatory effects of PKA agonists on UT-A3 regulation reported by Karakashian et al. (11), Shayakul and colleagues (26) characterized the function of UT-A3 by using Xenopus laevis oocyte expression and found that a mixture of cAMP agonists (dibutyryl cAMP, IBMX, and forskolin) did not increase UT-A3 activity. At present, the reason for this discrepancy between the two studies is unknown.

Compared with other members of the UT-A family, relatively little is known about UT-A4. UT-A4 mRNA has been detected in the outer medulla, and a protein proposed to be UT-A4 has been detected in the outer medulla by immunoblotting (11, 30), but the nephron location of UT-A4 has not been determined.

Although the rat remains the species of choice for many renal studies, the mouse is the preferred species for gene deletion or disruption studies. This has led to a demand for basic information regarding murine biology and, in particular, information that adds functional context to molecular data. Compared with rats, relatively little is known about mouse (m)UT-A isoforms. The molecular characteristics of mUT-A3 and a novel isoform, mUT-A5, have been reported (8). mUT-A3 has a high degree of identity with rat UT-A3, and, as in the rat kidney, mUT-A3 mRNA localizes to the kidney inner medulla. mUT-A5 is an NH2 terminally truncated variant of mUT-A3. It lacks the first 139 amino acids of mUT-A3 and is expressed within seminiferous tubules in the testes.

Given the need for information pertaining to mouse urea transporters, we have isolated two cDNAs from mouse kidney encoding mUT-A1 and mUT-A2. Here, we report their molecular characteristics and show that PKA agonists acutely stimulate urea transport meditated by UT-A1 or UT-A3 but not UT-A2, and, furthermore, we resolve the discrepancy between previous studies on PKA stimulation of UT-A3 (11, 26).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Clone isolation. cDNA clones were isolated from an unamplified, size-selected (>1.5 kb) MF1 mouse kidney inner medulla lambda  (lambda GT10) µgt10 cDNA library (8). Initially, ~300,000 clones were screened using the following gel-purified, 32P-labeled (Rediprime II; Amersham Pharmacia Biotech) probes: nucleotides 1-219 of mUT-A3 (GenBank accession no. AF258602) probe 1 (8), or a 427-bp PCR product corresponding to bp 281-708 of mUT-A2 (characterized in Ref. 7). Hybridization was at 48°C in a solution containing 10% (wt/vol) dextran sulfate and 50% formamide. Final washes were carried out at 65°C in 0.1× SSC, 0.1% SDS. Resultant clones from these screens were subcloned into the NotI site of pBluescript SK(-) (Stratagene), and both strands were sequenced using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems).

Xenopus oocyte expression experiments. These were performed as previously described (27). Briefly, plasmids containing mUT-A1, mUT-A2, or mUT-A3 were linearized, and cRNA was prepared using the T7 mMessage mMachine (Ambion). Defolliculated oocytes were injected with cRNA or deionized H2O and incubated for 3 days at 18°C. [14C]urea uptakes were measured in the presence or absence of 0.5 mM phloretin as described previously (27). The effect of PKA agonists was tested by preincubation of oocytes for 1 h in 0.5 mM 8-bromoadenosine 3',5'-cyclic monophosphate, 0.5 mM IBMX, and 0.05 mM forskolin.

5'-Rapid amplification of cDNA ends. The 5'-end of the UT-A1 transcript was determined using MF1 mouse inner medullary mRNA and the Generacer Kit (Invitrogen) according to the manufacturer's recommended protocol. First-strand cDNA synthesis was primed using an oligo-(dT) primer, followed by PCR amplification using an antisense primer (5'-GTGACTGGCTATTTCTTACC) corresponding to nucleotides 366-385 of UT-A1 and the 5'-mRNA cap-specific sense primer (Invitrogen). The products of the PCR amplification were gel purified, subcloned into the pCR-TOPO-4 vector (Invitrogen), and sequenced. This procedure was also used to determine the 5'-end of the UT-A2 transcript, except that the antisense PCR primer (5'-GGAAGTTACCTGATTCAGC) corresponded to nucleotides 291-309 of UT-A2 cDNA.

Primer extension analysis. Plasmids containing the UT-Aalpha (MUT9) or UT-Abeta (MUT1) promoter regions (6) and part of the first exon of UT-A1 or UT-A2, respectively, were used in an in vitro transcription assay as described previously (16) to produce an enriched population of UT-A1 and UT-A2 cRNA. Primer extension was carried out using AMV-reverse transcriptase (Roche) and 5 µg of cRNA using gamma -32P-labeled oligonucleotides corresponding to nucleotides 61-82 of mUT-A1 (5'-CTCAAGGAAGACTGCAAGATC) or nucleotides 50-74 of mouse UT-A2 (5'-GGTCCGAAGTTTTCCACAGAAATCC) at 42°C. These primers were also used to sequence MUT9 and MUT1 using 35S dCTP and the Sequenase Quick Denature Plasmid Sequencing Kit (Amersham Pharmacia Biotech). Sequencing reactions and extension products were separated by electrophoresis on a 6% polyacrylamide gel. Autoradiography was performed using a Fuji FLA-3000 PhosphorImager.

Northern blot analysis. RNA was isolated from MF1 mouse tissues by the guanidine isothiocyanate method. Either poly-A+ RNA (3 µg/lane, enriched as described previously) (8) or total RNA (15 µg/lane) was separated in a 1% agarose gel in the presence of 2.2 M formaldehyde and transferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech). Filters were probed using either 32P-labeled probe 1, nucleotides 1-799 of mouse UT-A2 (probe 2), or full-length mUT-A1. Hybridization was for 16 h at 42°C (50% formamide) and washing at 65°C (high) or 60°C (medium) in 0.1× SSC, 0.1% SDS.

Protein preparation and immunoblotting. Tissues were homogenized in 5 ml of ice-cold isolation solution in a hand-held Douse homogenizer. Isolation solution contained 300 mM mannitol plus 12 mM HEPES adjusted to pH 7.6. The protease inhibitors, pepstatin (1 µg/ml final concentration, Sigma), leupeptin (2 µg/ml, Sigma), and phenylmethylsulfonyl (1 µg/ml, Sigma) were added before homogenization. Homogenates were centrifuged at 2,000 g for 15 min at 4°C. The supernatants were saved and centrifuged at 200,000 g for 30 min at 4°C. The resulting pellets, containing plasma and vesicular membranes, were resuspended in isolation solution, total protein concentrations were determined using a Bio-Rad protein assay kit, and sample concentrations were adjusted to 10 µg/µl in isolation solution. Laemmli sample buffer (5×) containing 625 mM Tris buffer, pH 6.8, 5% SDS, 25% glycerol, 0.025% bromophenol blue, and 30 mg/ml DTT was added to the samples in a 1:4 ratio. Samples were then heated to 60°C for 15 min before being loaded onto gels. SDS-PAGE was performed on 8% polyacrylamide gels. The Full Range Rainbow Molecular Weight Marker (Amersham Pharmacia Biotech) was used for protein size determination, and proteins were electrophoretically transferred to nitrocellulose membrane (Gelman Sciences). After blocking for 1 h in 5% nonfat dry milk prepared in wash buffer (15 mM Tris, pH 8.0, 150 mM NaCl, 0.01% Tween 20, Sigma), the membranes were probed with an affinity-purified antibody, L194, raised to the 19 COOH-terminal amino acids of rat UT-A1 (17), for 18 h at 4°C. Antiserum L194 was a kind gift from Dr. Mark A. Knepper, National Heart, Lung, and Blood Institute, National Institutes of Health. For peptide preabsorption controls, blots were incubated with primary antibody that had been previously incubated overnight at 4°C with a five times molar excess of immunizing peptide. The membranes were washed three times for 10 min in an excess of wash buffer and incubated at room temperature for 1 h in horseradish peroxidase-conjugated secondary antibody (P448; goat-anti-rabbit, Dako), diluted 1:5,000 in 5% nonfat dry milk/wash buffer. Immunoreactivity was visualized using luminol-based chemiluminescence (Amersham Pharmacia Biotech).

Immunohistochemistry. MF1 mice were anesthetized with Inactin (100 mg/kg ip) and perfused via the descending aorta with PBS, and tissues were perfusion-fixed with 4% paraformaldehyde/PBS. Kidneys were removed, dehydrated in an ascending series of ethanol concentrations (50-100%) and embedded in paraffin wax. Tissue was sectioned at 5 µm, mounted on Superfrost Plus slides (BDH) and allowed to dry overnight at 37°C. After xylene treatment and rehydration in a descending series of ethanol concentrations (100-70%), endogenous peroxidase was blocked by incubating sections for 30 min in 3% hydrogen peroxide in methanol. Antigen retrieval was performed by boiling sections for 10 min in a solution containing 25 mM Tris · HCl (pH 8.0), 10 mM EDTA, and 50 mM glucose before overnight incubation at 4°C with affinity-purified antiserum L194 diluted in 1% BSA, 0.3% Triton X-100 in PBS. Labeling was visualized with a 1:200 dilution of P448 goat-anti-rabbit horseradish peroxidase-conjugated secondary antibody (Dako), followed by incubation with diaminobenzidine.

Statistics. Quantitative data were compared using one-way ANOVA. The Student-Newman-Keuls post hoc test was used to detect significant differences between groups. Values are quoted as means ± SE, and statistical significance was assumed at the 5% level.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Homology screening experiments. High-stringency screening of a mouse kidney inner medulla cDNA library with probe 1, corresponding to nucleotides 1-219 of mUT-A3 (8), yielded a 4,047-bp cDNA (Fig. 1A). Analysis of the nucleotide sequence revealed a putative open reading frame (ORF) from nucleotide 449 to 3,241, a polyadenylation signal (ATTAAA) at position 4,014, and a poly (A) tail. The ORF predicts a 930-residue protein, the mouse homologue of rat UT-A1 (25). mUT-A1 (GenBank accession no. AF366052) has 93 and 85% amino acid identity with rat UT-A1 (25) and human UT-A1 (1), respectively. There are three putative N-glycosylation sites (N-I/N-T-W/G) at N-280, N-424, and N-743; seven potential PKC sites at S24, S88, S206, T448, S495, T546, and S669; and five potential PKA sites at S85, S92, S487, S564 and S919 (Fig. 2).


View larger version (72K):
[in this window]
[in a new window]
 
Fig. 1.   A: mouse (m)UT-A1 urea transporter cDNA and amino acid sequence (GenBank accession no. AF366052). B: mUT-A2 cDNA and amino acid sequence (GenBank accession no. AF367359).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.   Scaled representation of a hypothetical UT-A1 protein. Shaded horizontal bars, hydrophobic domains; , PKC consensus site; , PKA consensus sites; black-lozenge , N-glycosylation sites; nos. (top) not in parentheses, UT-A1 amino acids; nos. in parentheses (top), UT-A2 amino acids; thick solid horizontal lines, amino acid composition of UT-A2, UT-A3, and UT-A5 relative to UT-A1; * , sites not present in human UT-A1.

High-stringency screening of a mouse kidney inner medulla cDNA library with a 427-bp PCR product corresponding to bp 281-708 of mUT-A2 yielded a 2,876-bp cDNA (Fig. 1B). Analysis of the nucleotide sequence revealed a putative ORF from nucleotide 877 to 1,194, a polyadenylation signal (ATTAAA) at position 2,843, and a poly (A) tail. The ORF predicts a 397-residue protein, the mouse homologue of rat UT-A2 (27). mUT-A2 (GenBank accession no. AF367359) has 95 and 91% amino acid identity with rat (27) and human UT-A2 (18), respectively. There is one putative N-glycosylation site (N-I/N-T-W/G) at N-210, two potential PKC sites at T13 and S136, and two potential PKA sites at S31 and S386 (Fig. 2).

X. laevis oocyte expression. Oocytes injected with mUT-A1 or mUT-A2 cRNA showed threefold and twofold increases, respectively. Inclusion of 0.5 mM phloretin in the uptake solution did not significantly affect urea uptake in H2O-injected oocytes. However, consistent with previously characterized UT-A transporters (8, 11, 25, 27), phloretin reduced urea influx into oocytes injected with mUT-A1 or mUT-A2 (data not shown). Phloretin completely inhibited mUT-A1- or mUT-A2-mediated urea influx. Titration of mUT-A1 cRNA (data not shown) revealed that urea transport by oocytes injected with <= 1 ng cRNA was increased by cAMP (Fig. 3). Injection of molar equivalent amounts of mUT-A2 or mUT-A3 showed that urea transport via UT-A2 was not affected by cAMP; however, urea transport was significantly increased in oocytes expressing mUT-A3 (Fig. 3). These findings agree, in part, with those previously reported by others for UT-A orthologs. However, our result that mUT-A3 acutely responds to PKA agonists is in contrast to the observations reported by Shayakul et al. (26) using rat UT-A3 but does agree with those reported by Karakashian and colleagues (11). This acute effect of PKA agonists may have implications for AVP-stimulated urea transport in the IMCD.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of PKA agonists on [14C]urea uptake by oocytes expressing mUT-A1, mUT-A2, and mUT-A3. A summary of the urea accumulated in a 90-s period by H2O-injected control oocytes or oocytes expressing mUT-A1, mUT-A2, or mUT-A3 is shown. Open bars, urea uptake under control conditions; filled bars, urea uptake after the preincubation of oocytes for 1 h in 0.5 mM 8-bromoadenosine 3',5'-cyclic monophosphate, 0.5 mM IBMX, and 0.05 mM forskolin. Values are means ± SE; n = 8. +Significant increase in urea uptake compared with the H2O-injected control group (P < 0.05). * Significant increase in uptake caused by PKA agonists compared with the paired control group (P < 0.05).

Identification of transcriptional start site for UT-A1 and UT-A2. The transcriptional start of UT-A1 and UT-A3 was identified using 5' rapid amplification of cDNA ends (RACE) and primer extension. 5'-RACE identified a UT-A transcript that was shorter than mUT-A1 cDNA (corresponding to bp 43 of the UT-A1 cDNA) but 49 bp longer than mUT-A3 cDNA (8). This transcript generated by 5'-RACE had an m7G cap structure at its 5'-end so was likely to represent a full-length transcript (22). With the use of primer extension, three transcriptional start sites were identified 5, 8, and 13 bp 3' to the 5'-terminus of mUT-A1 cDNA and a further start site 12 bp 5' to the 5'-terminus of mUT-A1 cDNA (Fig. 4B). Together, these data indicated that UT-A1 and UT-A3 mRNA transcripts have multiple transcriptional start sites that are driven by the same promoter. In contrast, 5'-RACE identified 39 additional nucleotides at the 5'-end of the mUT-A2 cDNA that are different from the region identified upstream of the mUT-A1 cDNA (Fig. 4B). Independently, primer extension analysis gave the same result. These data show that transcription of the mUT-A2 mRNA transcript is initiated from a single start site, which is distinct from the transcription start site of UT-A1 and UT-A3.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 4.   A: primer extension analysis of UT-A1. The sequence of plasmid MUT1 (6) was used to determine the sequence of the resulting product. B: sequence at the 5'-end of the UT-A1 cDNA (underlined); arrowheads, transcriptional start sites. C: primer extension analysis of UT-A2. The sequence of plasmid MUT9 (6) was used to determine the sequence of the resulting product. D: sequence at the 5'-end of the UT-A2 cDNA (underlined).

Northern blot analysis and tissue distribution. High-stringency Northern blot analysis of kidney inner medulla poly-A+ RNA (3 µg/lane) using probe 1, the unique 5'-untranslated region (UTR) of UT-A1 (see METHODS), resulted in strong signals of 4.1 and 2.1 kb (Fig. 5A). These signals correspond to mUT-A1 and mUT-A3 (8), respectively. No other transcripts were detected in this tissue segment using this probe. High-stringency Northern blot analysis of mRNA using probe 2, the unique 5'-UTR of UT-A2 (see METHODS), resulted in a strong signal of 3.1 kb in kidney inner medulla (Fig. 5B). This signal corresponds to the mUT-A2 transcript and confirmed that the 5'-UTR of UT-A2 is a unique sequence not present in any other major urea transporter isoform. No other signals were apparent, even after prolonged exposure of up to 6 days.


View larger version (75K):
[in this window]
[in a new window]
 
Fig. 5.   A: Northern blot of kidney inner medulla (IM) poly A+ RNA (3 µg/lane) probed with cDNA probe 1 at high stringency. Strong hybridization to mRNA species is evident at 4.1 and 2.1 kb in kidney IM. These signals corresponded to UT-A3 and UT-A1, respectively. B: Northern blot of kidney IM poly A+ RNA (3 µg/lane) probed with cDNA probe 2 at high stringency. A strong 3.1-kb signal is detected in kidney IM that corresponded to UT-A2. C: total RNA Northern blot (15 µg/lane) probed with full-length mUT-A1 at medium stringency. In addition to the signals observed in A and B, hybridization to mRNA species is evident at 2.9 and 3.4 kb in kidney cortex, testes, and heart; 3.1 kb in brain and liver; and 1.5 kb in testes. OM, outer medulla.

Medium-stringency (60°C) Northern blot analysis of total RNA (15 µg/lane) isolated from mouse kidney inner medulla, kidney outer medulla, kidney cortex, testes, heart, brain, and liver, using a full-length mUT-A1 probe, showed UT-A transcripts were present in all tissues tested (Fig. 5C). Strong signals of 4.1 and 3.1 kb were present in the kidney inner medulla and a weaker band at 2.1 kb. In the kidney outer medulla, a strong 3.1-kb signal was distinguishable, and weak 3.4- and 2.9-kb signals were detected in the kidney cortex. UT-A transcripts were also detected in extrarenal tissues. In testes, signals of 3.4, 2.9, and 1.5 kb were observed. After prolonged exposure (3 days; not shown), signals of 3.4 and 2.9 kb were also detected in heart, and a 3.1-kb transcript was detected in the brain and liver. These results were regarded as semiquantitative because total RNA was analyzed and lanes were equally loaded. From this viewpoint, it was evident that the kidney medulla contains the highest concentration of UT-A mRNA compared with the other tissues analyzed. Weak bands at 1.8 and 4.4 kb evident in total RNA samples, but not in poly-A+ RNA samples, indicated that the mUT-A1 bound weakly to rRNA despite the inclusion of yeast tRNA in the hybridization solution.

Western blot analysis. Antiserum L194 has been used extensively in rats to detect UT-A1 and UT-A2 (17, 30). In mouse kidney outer medulla, it has been reported to detect UT-A2 and, putatively, UT-A4 (30). Immunoblots of the 200,000-g fraction of mouse tissue, containing plasma membranes and subcellular vesicular membranes, showed that antiserum L194 labeled several proteins of various molecular weights (Fig. 6A). Compared with the other tissues we tested, excluding the brain, the kidney inner medulla showed the strongest protein signals. In the kidney inner medulla, L194 detected strong protein bands of 44, 47, 53, 72, 77, 98, 105, and 120 kDa. The predominant protein band in the kidney outer medulla was 53 kDa. Several weaker protein bands were also observed in the kidney outer medulla at 44, 47, 94, and 105 kDa. Surprisingly, the 53-kDa signal was equally strong in kidney cortex, and the 94- and 105-kDa protein bands, although weaker, were also detected. In testes, protein bands of 51, 72, 100, 110, and 120 kDa were detected, along with a protein band at 143 kDa. In the heart, weak protein bands of 53, 70, and 105 kDa were detected, whereas in the liver the predominant signals were at 98, 115, and 154 kDa. Very strong protein bands were observed in the brain at 45, 53, 60, and 154 kDa, and weaker protein bands were detected at 45, 72, 105, and 120 kDa. All signals were absent from immunoblots when L194 was preincubated with immunizing peptide before analysis (Fig. 6B). No signals were observed when L194 was excluded and only the secondary antiserum was applied (not shown).


View larger version (91K):
[in this window]
[in a new window]
 
Fig. 6.   Western blot analysis of 200,000-g mouse tissue fraction containing plasma and vesicular membranes. A: membrane probed with the L194 antibody, raised to the 19 COOH-terminal amino acids of rat UT-A1, showing a complex pattern of differential expression. Within the kidney, the greatest abundance of UT-A protein occurs in the IM. Multiple strong signals are also apparent in testes and brain. B: an identical blot to that shown in A probed with antibody preincubated with the immunizing peptide. All bands are ablated.

Immunocytochemistry. Immunostaining with antiserum L194 strongly labeled structures in the renal medulla (Fig. 7, A-C). Preincubation of L194 with the immunizing peptide abolished immunostaining by L194 (Fig. 7D). In the inner stripe of the outer medulla, at the junction with the inner medulla, the radial pattern of labeling was very similar to that previously reported in rats and mice (30). This labeling corresponds to type 1 tDLs contained in vascular bundles. In the initial part of the inner medulla, L194 labeled tubules of a similar dimension to those labeled in the outer medulla, but the pattern of labeling was very different. Labeled tubules were not clustered in bundles but were sparsely distributed among unlabeled tubules. Previous RT-PCR studies in rat showed that UT-A2 mRNA was present in type 3 long tDLs, proximal to the bend in the loop of Henle and thin ascending limbs (23). On the basis of these data and the pattern of distribution of the L194-labeled tubules in the mouse, we conclude that the structures labeled by L194 in the initial inner medulla are type 3 tDLs. As previously observed in the rat, L194 strongly labeled mouse IMCD in the middle inner medulla and papillary tip. Interestingly, both apical and basolateral membranes were strongly labeled, suggesting that UT-A proteins were present in both membranes of the epithelium.


View larger version (117K):
[in this window]
[in a new window]
 
Fig. 7.   Immunolocalization of UT-A proteins in longitudinal sections of mouse kidney using antiserum L194. A: ×400 magnification of inner stripe of OM showing staining in groups of terminal type 1 thin descending limbs of Henle's loop (tDLs). B: ×400 magnification of initial part of IM showing isolated staining of long tDLs. C: ×250 magnification of papillary tip showing strong immunoreactivity in IMCD. D: ×250 magnification of papillary region showing no immunoreactivity when antiserum was preabsorbed with immunizing peptide.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The proteins UT-A1, UT-A2, and UT-A3 (reviewed in Ref. 28) represent the major urea transporter proteins expressed in the rat kidney. Knowledge of their molecular structure has greatly added to our understanding of renal urea handling and its regulation. A prerequisite to studies incorporating UT-A knockout mice is knowledge of the characteristics and distribution of the target proteins in wild-type animals. Toward this end, we have previously isolated a cDNA encoding mUT-A3 (8). The purpose of this study was to characterize the mouse homologues of two other major UT-A gene products, UT-A1 and UT-A2, and to resolve the controversy between the effects of PKA agonists on UT-A3-mediated urea transport reported by two other groups (11, 26).

Screening a mouse kidney inner medulla cDNA library yielded cDNAs encoding mUT-A1 and mUT-A2. Both proteins share a high degree of identity with their rodent and human homologues. However, mUT-A1 is one amino acid longer than rat UT-A1 due to an extra histidine residue at position 4. The consensus glycosylation sites present in rat and human UT-A1 are also conserved in the mouse homologue. Comparison of mouse, rat, and human PKA and PKC sites showed that PKA and PKC sites are conserved between murine and rodent UT-A1 proteins. However, the two PKA sites at S85 and S92 and the PKC site at T546 in humans are not conserved.

In rat kidney, differential expression of the mRNA transcripts encoding UT-A1 and UT-A2 are regulated in response to several stimuli, such as hydration state and dietary protein content (2, 9, 20, 24, 27). In the mouse, we have recently determined that the expression of both UT-A2 and UT-A3 transcripts was upregulated in response to thirsting (6). The identification of two different transcriptional start sites for the UT-A1/3 and UT-A2 transcripts, in combination with two promoters (6), provides the means to differentially drive transcription. Interestingly, five potential transcriptional start sites were identified for the UT-A1/3 transcripts, whereas only one start site was identified for the UT-A2 transcript. Similarly to the mouse, the initiation of transcription for the UT-A1/3/4 transcripts in the rat was shown to be at a distinct site that is different from the start site of UT-A2 transcription (2). However, seven possible start sites in the rat were identified for the UT-A1/3/4 transcripts, and three potential start sites were proposed for the UT-A2 transcript (2).

Multitissue Northern blot analysis using a full-length mUT-A1 probe detected six transcripts and, based on size and our analysis utilizing UT-A subtype-selective probes, we can account for the molecular identity of four of these transcripts: 1.5 kb (UT-A5), 2.1 kb (UT-A3), 3.1 kb (UT-A2), and 4.1 kb (UT-A1). At present we do not know the molecular identity of the proteins encoded by the 2.9- or 3.4-kb transcripts. In comparison, the rat UT-A gene gives rise to at least seven transcripts of 1.7, 2.1, 2.8, 3.0, 3.1, 3.7, and 4.0 kb (2, 11, 25, 27). In the mouse kidney, like in the rat, the predominant transcripts are UT-A1 and UT-A2, and these transcripts are differentially expressed with respect to kidney region. In the mouse, UT-A1 mRNA is predominant in the kidney inner medulla, whereas levels of UT-A2 mRNA are approximately equal between the kidney inner medulla and outer medulla. UT-A3 mRNA is expressed in low abundance in the kidney inner medulla, similar to the distribution of UT-A3 mRNA in the rat (2).

As previously reported in the rat, UT-A mRNA transcripts were also evident in low abundance in the liver, where urea transporters probably mediate the movement of urea out of hepatocytes (12). In heart, brain, and testes, tissues not associated with urea metabolism, the physiological role of UT-A urea transporters is far from clear and further studies are needed.

In previous studies, antiserum L194/L403 has provided an effective tool for immunolocalizing UT-A1 and UT-A2 in rat kidney (17, 30). Western blot analysis of mouse tissues using L194 showed that L194 recognized multiple protein bands in all tissues tested. Preincubation of L194 with the immunizing peptide abolished these signals and indicted that L194 was recognizing mouse UT-A proteins containing all or part of the COOH terminus of UT-A1. Of the known UT-A proteins, this would include UT-A1, UT-A2, and UT-A4, although we have so far been unable to isolate a cDNA encoding mouse UT-A4. Further analysis is clearly needed with antisera targeted to different UT-A regions before definitive conclusions can be drawn. It should also be considered that some bands may represent isoforms in different states of glycosylation, as has been previously shown in the rat (3), or potentially be the result of other posttranslational modification.

Immunocytochemistry of mouse kidney using L194 revealed strong staining of type 1 and type 3 tDLs in the kidney medulla region. Stained tubules in the kidney outer medulla occurred in groups, characteristic of the special arrangement of type 1 tDLs in mouse kidney outer medulla (13, 14) and very similar to the pattern of labeling using antiserum L403 reported by Wade et al. (30). Interspersed within the initial part of the inner medulla were signals from type 3 tDLs. In the rat, the pattern of distribution is very similar to that seen in the mouse, and RT-PCR of rat nephron segments has shown the presence of UT-A2 mRNA in type 1 and type 3 tDLs (23). On the basis of these data and data from Western blot analysis, we suggest that the pattern of staining in mice, like in rats, represents UT-A2 expression.

Strong staining was also seen in the middle IMCD and papillary tip. In the rat, this staining corresponds to UT-A1 in the apical membrane and cytosolic compartment of principal cells. Interestingly, we observed staining of both apical and basolateral membranes of some IMCD cells. This suggests that UT-A proteins mediate translocation of urea not only across the apical membrane but also across the basolateral membrane in some IMCD cells.

Acute application of a "cocktail" of PKA agonists significantly increased urea transport in X. laevis oocytes expressing UT-A1 and UT-A3 but not when applied to oocytes that expressed UT-A2. The actions of AVP on IMCD urea permeability occur within 10 min of AVP application and continue to increase over a time frame of ~60 min (21). Over a comparable time frame, application of PKA agonists has been shown to increase urea transport by rat and human UT-A1 in X. laevis oocytes (1, 25). Two previous studies have reported conflicting data regarding the effects of PKA agonists on UT-A3-mediated urea uptake. One study by Shayakul and colleagues (26) reported that rat UT-A3, which has the same complement of PKA sites as mUT-A3, did not respond to treatment with a cocktail of cAMP agonists. However, 24-h treatment of HEK-293 cells expressing rat UT-A3 with forskolin was reported to increase urea uptake (11). This stimulatory effect can be considered to be due to an acute regulation of the UT-A3 protein, and not to possible interference by transcriptional regulation, because the transfecting construct did not include a promoter sequence. Our findings with mUT-A3 agree with those of Karakashian et al. (11) and provide strong evidence that UT-A3 is stimulated by PKA. Furthermore, our result suggests that UT-A3 may be involved in the AVP-stimulated increase in IMCD urea permeability. One possible explanation for the conflicting results observed between our data and that of Shayakul et al. (26) is that both we and other groups (1) have found that a titration of the amount of UT-A cRNA injected into oocytes is necessary before the cAMP effect can be observed. In the light of our findings, knowledge of the cellular location of UT-A3 in mIMCD would represent a major step toward understanding the role this protein plays in renal urea handling and in the urinary concentrating mechanism.

In summary, we have isolated cDNAs encoding the mouse homologues of UT-A1 and UT-A2 and begun to characterize the proteins they encode. We have shown that mUT-A1 and mUT-A3 have a different transcriptional start site to that driving mUT-A2 transcription and that mUT-A proteins are differentially expressed in the kidney, testes, heart, brain, and liver. Finally, we report urea transport mediated via UT-A1 or UT-A3 is increased by PKA agonists.


    ACKNOWLEDGEMENTS

The authors thank Dr. Stefan Roberts, University of Manchester, for expert advice regarding in vitro transcription assays; Dr. Mark Knepper, National Heart, Lung, and Blood Institute, National Institutes of Health, for the antiserum L194; and Martin Brew for helpful comments.


    FOOTNOTES

This work was supported by Biotechnology and Biological Sciences Research Council Grant 34/D10935, National Environment Research Council Grant GR3/10585, Wellcome Trust Grant 043322/Z/94, and the Royal Society (C. P. Smith).

Address for reprint requests and other correspondence: C. P. Smith, School of Biological Sciences, Univ. of Manchester, G.38, Stopford Bldg., Oxford Rd., Manchester, M13 9PT United Kingdom (E-mail: cpsmith{at}man.ac.uk).

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.

June 4, 2002;10.1152/ajprenal.00263.2001

Received 20 August 2001; accepted in final form 30 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bagnasco, SM, Peng T, Janech MG, Karakashian A, and 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].

2.   Bagnasco, SM, Peng T, Nakayama Y, and Sands JM. Differential expression of individual UT-A urea transporter isoforms in rat kidney. J Am Soc Nephrol 11: 1980-1986, 2000[Abstract/Free Full Text].

3.   Bradford, AD, Terris JM, Ecelbarger CA, Klein JD, Sands JM, Chou CL, and Knepper MA. 97- and 117-kDa forms of 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].

4.   Ecelbarger, CA, Kim GH, Knepper MA, Liu J, Tate M, Welling PA, and Wade JB. Regulation of potassium channel Kir 1.1 (ROMK) abundance in the thick ascending limb of Henle's loop. J Am Soc Nephrol 12: 10-18, 2001[Abstract/Free Full Text].

5.   Ecelbarger, CA, Terris J, Hoyer JR, Nielsen S, Wade JB, and Knepper MA. Localization and regulation of the rat renal Na+-K+-2Cl- cotransporter, BSC-1. Am J Physiol Renal Fluid Electrolyte Physiol 271: F619-F628, 1996[Abstract/Free Full Text].

6.   Fenton, RA, Cottingham CA, Stewart GS, Howorth A, Hewitt JA, and Smith CP. Structure and characterization of the mouse UT-A gene (Slc14a2). Am J Physiol Renal Physiol 282: F630-F638, 2002[Abstract/Free Full Text].

7.   Fenton, RA, Hewitt JE, Howorth A, Cottingham CA, and Smith CP. The murine urea transporter genes Slc14a1 and Slc14a2 occur in tandem on chromosome 18. Cytogenet Cell Genet 87: 95-96, 1999[ISI][Medline].

8.   Fenton, RA, Howorth A, Cooper GJ, Meccariello R, Morris ID, and Smith CP. Molecular characterization of a novel UT-A urea transporter isoform (UT-A5) in testis. Am J Physiol Cell Physiol 279: C1425-C1431, 2000[Abstract/Free Full Text].

9.   Hu, MC, Bankir L, and Trinh-Trang-Tan MM. mRNA expression of renal urea transporters in normal and Brattleboro rats: effect of dietary protein intake. Exp Nephrol 7: 44-51, 1999[ISI][Medline].

10.   Inoue, T, Terris J, Ecelbarger CA, Chou CL, Nielsen S, and 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].

11.   Karakashian, A, Timmer RT, Klein JD, Gunn RB, Sands JM, and Bagnasco SM. Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4. J Am Soc Nephrol 10: 230-237, 1999[Abstract/Free Full Text].

12.   Klein, JD, Timmer RT, Rouillard P, Bailey JL, and 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].

13.   Kriz, W. Structural organization of the renal medulla: comparative and functional aspects. Am J Physiol Regul Integr Comp Physiol 241: R3-R16, 1981[Abstract/Free Full Text].

14.   Kriz, W, and Koepsell J. The structural organization of the mouse kidney. Z Anat Entwicklungsgesch 144: 137-163, 1974[ISI][Medline].

15.   Kwon, TH, Hager H, Nejsum LN, Andersen ML, Frokiaer J, and Nielsen S. Physiology and pathophysiology of renal aquaporins. Semin Nephrol 21: 231-238, 2001[ISI][Medline].

16.   Lin, YS, and Green MR. Mechanism of action of an acidic activator in vitro. Cell 64: 971-981, 1991[ISI][Medline].

17.   Nielsen, S, Terris J, Smith CP, Hediger MA, Ecelbarger CA, and Knepper MA. Cellular and subcellular localization of the vasopressin- regulated urea transporter in rat kidney. Proc Natl Acad Sci USA 93: 5495-500, 1996[Abstract/Free Full Text].

18.   Olives, B, Martial S, Mattei MG, Matassi G, Rousselet G, Ripoche P, Cartron JP, and Bailly P. Molecular characterization of a new urea transporter in the human kidney. FEBS Lett 386: 156-160, 1996[ISI][Medline].

19.   Reeves, WB, Winters CJ, and Andreoli TE. Chloride channels in the loop of Henle. Annu Rev Physiol 63: 631-645, 2001[ISI][Medline].

20.   Rouillard, P, Klein JD, and Sands JM. UT-A protein abundance is altered in response to dietary protein in liver and kidney (Abstract). J Am Soc Nephrol 10: 24A, 1999.

21.   Sands, JM, Timmer RT, and Gunn RB. Urea transporters in kidney and erythrocytes. Am J Physiol Renal Physiol 273: F321-F339, 1997[Abstract/Free Full Text].

22.   Schaefer, BC. Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends. Anal Biochem 22: 255-273, 1995.

23.   Shayakul, C, Knepper MA, Smith CP, DiGiovanni SR, and Hediger MA. Segmental localization of urea transporter mRNAs in rat kidney. Am J Physiol Renal Physiol 272: F654-F660, 1997[Abstract/Free Full Text].

24.   Shayakul, C, Smith CP, Mackenzie HS, Lee WS, Brown D, and Hediger MA. Long-term regulation of urea transporter expression by vasopressin in Brattleboro rats. Am J Physiol Renal Physiol 278: F620-F627, 2000[Abstract/Free Full Text].

25.   Shayakul, C, Steel A, and Hediger MA. Molecular cloning and characterization of the vasopressin-regulated urea transporter of rat kidney collecting ducts. J Clin Invest 98: 2580-2587, 1996[Abstract/Free Full Text].

26.   Shayakul, C, Tsukaguchi H, Berger UV, and Hediger MA. Molecular characterization of a novel urea transporter from kidney inner medullary collecting ducts. Am J Physiol Renal Physiol 280: F487-F494, 2001[Abstract/Free Full Text].

27.   Smith, CP, Lee WS, Martial S, Knepper MA, You G, Sands JM, and Hediger MA. Cloning and regulation of expression of the rat kidney urea transporter (rUT2). J Clin Invest 96: 1556-1563, 1995[ISI][Medline].

28.   Smith, CP, and Rousselet G. Facilitative urea transporters. J Membr Biol 183: 1-14, 2001[ISI][Medline].

29.   Terris, JB, Knepper MA, and Wade JM. 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].

30.   Wade, JB, Lee AJ, Liu J, Ecelbarger CA, Mitchell C, Bradford AD, Terris J, Kim GH, and Knepper MA. UT-A2: a 55-kDa urea transporter in thin descending limb whose abundance is regulated by vasopressin. Am J Physiol Renal Physiol 278: F52-F62, 2000[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 283(4):F817-F825
0363-6127/02 $5.00 Copyright © 2002 the American Physiological Society