Am J Physiol Renal Physiol 307: F1088–F1094, 2014. First published September 10, 2014; doi:10.1152/ajprenal.00284.2014.

Expression and localization of a UT-B urea transporter in the human bladder C. Walpole, A. Farrell, A. McGrane, and G. S. Stewart School of Biology and Environmental Science, Science Centre West, University College Dublin, Belfield, Dublin, Ireland Submitted 21 May 2014; accepted in final form 4 September 2014

Walpole C, Farrell A, McGrane A, Stewart GS. Expression and localization of a UT-B urea transporter in the human bladder. Am J Physiol Renal Physiol 307: F1088 –F1094, 2014. First published September 10, 2014; doi:10.1152/ajprenal.00284.2014.—Facilitative UT-B urea transporters have been shown to play an important role in the urinary concentrating mechanism. Recent studies have now suggested a link between UT-B allelic variation and human bladder cancer risk. UT-B1 protein has been previously identified in the bladder of various mammalian species, but not yet in humans. The aim of the present study was to investigate whether any UT-B protein was present in the human bladder. First, RT-PCR results confirmed that UT-B1 was strongly expressed at the RNA level in the human bladder, whereas UT-B2 was only weakly present. Initial Western blot analysis confirmed that a novel UT-B COOH-terminal antibody detected human UT-B proteins. Importantly, this antibody detected a specific 40- to 45-kDa UT-B signal in human bladder protein. Using a peptide-N-glycosidase F enzyme, this bladder UT-B signal was deglycosylated to a core 30-kDa protein, which is smaller than the predicted size for UT-B1 but similar to many proteins reported to be UT-B1. Finally, immunolocalization experiments confirmed that UT-B protein was strongly expressed throughout all urothelium layers except for the apical membrane of the outermost umbrella cells. In conclusion, these data confirm the presence of UT-B protein within the human bladder. Further studies are now required to determine the precise nature, regulation, and physiological role of this UT-B. urea transporter; bladder; protein

(UT-A and UT-B, respectively) are known to play a key role in the mammalian urinary concentrating mechanism (9, 19, 25). Studies using mouse knockout models have confirmed that renal isoforms UT-A1 (5), UT-A3 (5), UT-A2 (27), and UT-B1 (30) all contribute to the production of concentrated urine. Interestingly, whereas UT-A proteins are mostly expressed in the kidney, UT-B is expressed in numerous tissues, including the brain, testes, gastrointestinal tract, red blood cells, and bladder (25). In humans, it is known that UT-B actually corresponds to the Kidd blood group (14), and a very minor proportion of the population comprise UT-B-null individuals, who have a 20% reduction in maximal urinary concentrating capacity (18). Human (h)UT-A and hUT-B proteins are derived from two distinct genes, namely, SLC14A2 and SLC14A1, respectively, which are both found on chromosome 18 (12). The SLC14A1 gene that encodes hUT-B is ⬃30 kb in length and contains 11 exons (12). A schematic representation of the SLC14A1 gene is shown in Fig. 1. This structure is similar to that reported for Slc14a1 genes in other mammalian species, such as the mouse (31), rat (31), and cow (24). Initially, it was believed that the Slc14a1 gene only encoded one protein, namely, UT-B1 (25).

FACILITATIVE UREA TRANSPORTERS

Address for reprint requests and other correspondence: G. Stewart, School of Biology and Environmental Science, Univ. College Dublin, Science Centre West, Rm. 2.55, Belfield, Dublin 4, Ireland (e-mail: [email protected]). F1088

However, a second isoform, named UT-B2, was identified at both RNA and protein levels in the cow rumen (24). Interestingly, both UT-B1 and UT-B2 mRNA transcripts have now been detected in humans: UT-B1 in various tissues, such as the kidney and gastrointestinal tract (GenBank Accession No. BC050539), and UT-B2 in the caudate nucleus (GenBank Accession No. AK091064). However, while UT-B1 protein has been detected in the human kidney (8), human colon (2), etc., there are no reports of UT-B2 protein in either the caudate nucleus or any other human tissue. UT-B1 protein has been detected in the bladder of various mammalian species, including the mouse (13), rat (20), and dog (21). UT-B1 RNA has also been reported in the bovine urothelium (7). Interestingly, compared with many other tissues, very high UT-B RNA expression has been reported in the mouse bladder (30). Most recently, it has been reported that UT-B knockout mice suffered DNA damage and apoptosis in the bladder, strongly suggesting that UT-B plays a significant physiological role protecting the bladder urothelium (4). Since urea levels were significantly higher in the UT-B-null urothelium compared with wild-type urothelium, it has been suggested that UT-B specifically helps remove intracellular urea from the bladder urothelium (4). Importantly, two studies (6, 15) have previously reported a strong association between human bladder cancer risk and UT-B allelic variation. One explanation suggested by a recent study is that there is a direct association between genetic variance in the SLC14A1 gene and final voided urine concentration, as measured by urinary specific gravity (10). This study (10) also reported the presence of UT-B1 RNA within human bladder tissue. The aim of the present study was therefore to investigate whether UT-B protein was present in the human bladder and, if so, determine the precise localization of this protein. METHODS

RT-PCR. Using purchased human small intestine total RNA and human bladder total RNA samples (AMS Biotechnology), cDNA preparation was performed using a Go-Script reverse transcription kit (Medical Supply). For each RNA sample, reactions were performed with and without reverse transcription enzyme (⫹RT and ⫺RT, respectively) present. All resulting cDNA samples underwent PCR amplification with a Platinum Taq polymerase enzyme (Biosciences) using primers for UT-B, aquaporin (AQP)3, AQP7, AQP9, or AQP10 (Eurofins MWG). Cycling parameters were an initial denaturation at 94°C for 2 min followed by 35 cycles at 94°C for 30 s, 60 or 65°C for 30 s, and 72°C for 30 s. The final extension was at 72°C for 8 min. UT-B primer sequences are all shown in Table 1. Control experiments using actin primers were also performed to confirm cDNA sample integrity. Antibodies. To study hUT-B proteins, a novel “hUTBc19” antibody was raised against a 19-amino acid peptide (NH2-EENRIFYLQAKKRMVESPL-COOH) corresponding to the COOH-terminal sequence of hUT-B1 (using services provided by Thermo Scientific Antibod-

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Fig. 1. Schematic diagram representing the basic structure of the human SLC14A1 gene. It should be noted that exon 3 contains the start codon used for urea transporter (UT)-B2 but is not included in UT-B1. In contrast, exon 4 contains the start codon used for UT-B1 but is also included in UT-B2. Labeled arrows show the location and direction of all UT-B primers used in this study. [Note: diagram based on the gene structure orginally shown by Lucien et al. (12).]

ies). Horseradish peroxidase-conjugated secondary anti-rabbit IgG antibody (65-6120, Invitrogen) was also used. Immunoblot analysis. Purified hUT-B1 protein (TP308509, Origene Technologies), UT-B1 whole cell lysate (LY402465, Origene Technologies), and UT-B2 whole cell lysate (LY426961, Origene Technologies) were purchased. Human protein whole homogenates for the bladder, blood, kidney, ileum, and rectum were also purchased (AMS Biotechnology). Dissected rat kidney protein samples were a kind gift from the laboratory of Prof. Alan Baird (Univ. College Dublin, Dublin, Ireland). For hUT-B1 protein experiments, the purified hUT-B1 protein was heated in the absence or presence of ␤-mercaptoethanol (5% or 10%) at 70°C for 1 h. For deglycosylation experiments, protein samples were incubated with and without peptide-N-glycosidase F enzyme for 3 h at 37°C. For the production of membrane-enriched and cytoplasm-enriched samples, serial centrifugation of whole cell homogenates at 2,500 g for 5 min followed by 16,900 g for 30 min was performed at 4°C. For loading samples onto gels, 2⫻ reducing Laemmli sample buffer [5% SDS, 25% glycerol, 0.32 M Tris (pH 6.8), bromophenol blue, and 5% ␤-mercaptoethanol] was added to all protein samples at a ratio of 1:1 and heated at 70°C for 15 min. SDS-PAGE was performed on minigels of 12% polyacrylamide by loading ⬃10 ␮g protein/lane except for purified hUT-B1 protein, where ⬃0.5 ␮g protein/lane was used. After transfer to nitrocellulose membranes, immunoblots were probed for 16 h at room temperature in 1:1,000 hUTBc19. For peptide inhibition exper-

Table 1. All specific primers used to investigate UT-B transcripts present in the human bladder

iments, hUTBc19 was preincubated with the specific immunizing peptide (1 ␮g peptide/␮l hUTBc19 antibody) or an equivalent amount of a nonspecific peptide for 24 h using a rotating mixer. After the incubation in primary antibody, immunoblots were washed and then probed with 1:5,000 horseradish peroxidase-conjugated anti-rabbit antibody for 1 h at room temperature. After a further wash, detection of protein was performed using Western Lightning Plus ECL reagents (Perkin-Elmer) and a LAS-4000 Image Reader (Fujifilm). Immunolocalization. Paraffin-embedded human bladder tissue sections (10 ␮m) were purchased (AMS Biotechnology). After neoclear treatment and rehydration of these sections 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 5 min in a solution containing 25 mM Tris·HCl (pH 8.0) and 10 mM EDTA before an overnight incubation at 4°C with 1:500 dilution of hUTBc19 in 0.1% BSA and 0.3% Triton X-100 in PBS. For peptide incubation experiments, hUTBc19 was preincubated with the specific immunizing peptide (1 ␮g peptide/␮l hUTBc19) or an equivalent amount of a nonspecific peptide for 24 h using a rotating mixer. Immunolabeling was visualized with a 1:1,000 dilution of horseradish peroxidase-conjugated anti-rabbit secondary antibody followed by an incubation with diaminobenzidine and counterstaining with hematoxylin. Stained sections were then dehydrated in an ascending series of ethanol concentrations (70 –100%) and treated with neoclear. Finally, coverslips were mounted using Eukitt mounting medium, and slides were stored at room temperature. Detailed images of sections were obtained using a Labophot 2 microscope (Nikon), a MircopublisherRTV-3.3 Digital camera (Q Imaging), and Image-Pro Plus imageanalysis software (MediaCybernetics). RESULTS

A schematic representation of the human SLC14a1 gene that encodes UT-B protein is shown in Fig. 1. Initial RT-PCR experiments investigated for the presence of UT-B and AQP RNA transcripts within human small intestine and bladder samples (see Table 1 for all UT-B primer sequences). For positive controls, PCR products of the expected size were obtained in ⫹RT small intestine cDNA samples with all primer sets tested (see Fig. 2). Further experiments then showed that strong UT-B, AQP3, AQP7, and actin signals were present in UT-B AQP3 AQP7 AQP9 AQP10 Actin

Name

Primer Description

Primer Sequence

F1 F3 F4 R5 F6 R6 R10

Forward, exon 1 Forward, exon 3 Forward, exon 4 Reverse, exon 5 Forward, exon 6 Reverse, exon 6 Reverse, exon 10

5=-GCCAGGAAGCCAGCTAGAGTGGTC-3= 5=-CTAGGGCACACGTCATGCTGATTC-3= 5=-CATGAAAGAACTTGCCAACCAGCTTAAAG-3= 5=-CTGTCCTGGCTGAGCAAGAGG-3= 5=-GGTGGGAGTACTCATGGCTGTCTTTTC-3= 5=-GTAACAGCCACCAGAAATAGTC-3= 5=-GCCATAAAGTTTGCCATGCCG-3=

For each primer, the name, direction of the primer, exon containing the sequence the primer was designed against, and, finally, the primer sequence itself are shown.

+RT -RT +RT -RT Human Human Small Bladder Intestine Fig. 2. RT-PCR confirming UT-B and aquaporin (AQP) RNA expression in the human bladder. RT-PCR experiments showed that UT-B, AQP3, AQP7, and actin were all strongly expressed in the human bladder, whereas AQP9 was only weakly expressed and AQP10 was absent. In contrast, strong signals with all sets of primers were detected with human small intestine samples. ⫹RT, reverse transcriptase present; ⫺RT, reverse transcriptase absent.

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Fig. 3. RT-PCR showing UT-B1 and UT-B2 RNA expression in the human bladder. RT-PCR experiments with F1/R5 UT-B primers produced a strong signal at the expected size for human (h)UT-B1 but not at the predicted size for hUT-B2. The same basic results were obtained with F1/F6 UT-B primers, although some other faint signals were present. In contrast, a significant UT-B2 signal was detected with the UT-B2-specific primer set F3/R5.

UT-B general F1/R5

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⫹RT bladder cDNA samples but absent from ⫺RT control samples. In contrast, a AQP9 signal was only weakly present and AQP10 was completely absent in ⫹RT bladder cDNA. Currently, there are only two characterized UT-B proteins reported in the literature, namely, UT-B1 and UT-B2 (25). Further PCR experiments were now performed to specifically investigate for the presence of UT-B1 and UT-B2 transcripts in the human bladder. Using the UT-B primer sets F1/R5 and F1/R6, which distinguish between the two UT-B isoforms, strong signals were only obtained at the predicted size for hUT-B1 and not for hUT-B2 (see Fig. 3). In contrast, a primer combination that only detects UT-B2 (i.e., F3/R5) did detect a significant UT-B2 signal (see Fig. 3). The identities of these UT-B1 and UT-B2 products from the human bladder were confirmed through direct sequencing (Eurofins MWG). Finally, additional experiments with a range of further UT-B primers designed to sequences in exons 1, 4, 6, and 10 of the hUT-B gene did not detect any novel transcripts and only detected strong signals at the predicted size for hUT-B1 (see Fig. 4). UT-B1: ~1100 bp F1/R10

UT-B1: ~320 bp F4/R6

UT-B1: ~850 bp F4/R10

UT-B1: ~610 bp F6/R10

+RT -RT

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bp 1000 900 800 700 600 500 400 300 200 100

Human Bladder Fig. 4. RT-PCR showing that UT-B1 is the main UT-B transcript present in the human bladder. A variety of RT-PCR experiments using different UT-B primer sets detected a strong signal at the expected size for hUT-B1 at 1,100, 320, 850, and 610 bp. No other strong signals were obtained for any of the primer sets.

To investigate for the presence of hUT-B protein, a novel antibody raised against the COOH-terminal of hUT-B1 was produced and characterized, namely, hUTBc19. First, hUTBc19 was used to probe an immunoblot containing lanes of purified hUT-B1 protein treated under various conditions (see Fig. 5A). In the control lane, hUTBc19 detected a strong 100- to 150-kDa smear, which remained after the protein had been heated at 70°C for 1 h. In contrast, in lanes of protein heated at 70°C for 1 h in the presence of 5% or 10% ␤-mercaptoethanol, this strong smear almost totally shifted to a 40-kDa signal. Next, hUTBc19 was shown to detect a distinctive 40- to 60-kDa smear in human red blood cell samples, with a much stronger signal detected in membrane-enriched compared with cytoplasm-enriched protein (see Fig. 5B). Immunoblots using human bladder samples showed a strong 40- to 50-kDa signal in membrane-enriched protein and a 40- to 45-kDa signal whole homogenate protein (see Fig. 5C). Finally, hUTBc19 was used to probe an immunoblot containing membrane-enriched and cytoplasm-enriched protein samples from various dissected regions of the rat kidney (see Fig. 5D). As expected, strong signals (namely, at 30, 40 –50, and 100 – 200 kDa) were detected in membrane-enriched inner medulla protein but not in other samples. In contrast, a strong 100-kDa band was detected to the same extent in every rat kidney sample tested. In the next set of experiments, whole homogenates of cells overexpressing hUT-B1 or hUT-B2 protein were probed with hUTBc19 (see Fig. 6). In a control immunoblot, hUTBc19 detected strong signals at 40 and 50 kDa for hUT-B1, whereas signals at 50 and 55 kDa were primarily detected for hUT-B2. Importantly, all these signals were completely blocked by preincubation of the antibody with 1 ␮g/␮l of the specific original hUTBc19 immunizing peptide but not by preincubation with an equivalent amount of a nonspecific peptide. To further investigate hUT-B protein expression in the bladder, a range of whole cell homogenates was purchased and tested with hUTBc19 (see Fig. 7). Interestingly, the strong 40- to 45-kDa smear was detected in the bladder sample but not in whole kidney, ileum, or rectum samples. As predicted, this strong bladder signal was absent when hUTBc19 was preincubated in its immunizing peptide but still present if a nonspecific peptide preincubation was

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Fig. 5. Immunoblots showing the initial characterization of the novel hUTBc19 antibody. A: hUTBc19 detected a strong signal at 100 –150 kDa for hUT-B1, the predicted size of a functional UT-B1 multimer. As expected, in the presence of 5% or 10% ␤-mercaptoethanol and heating at 70°C for 1 h, this signal shifted to the 40-kDa size representing the hUT-B1 monomer. B: using human red blood cell samples, hUTBc19 detected a 40- to 60-kDa smear strongly in membrane-enriched (Mem) protein but only weakly in cytoplasm-enriched (Cyt) protein. C: in human bladder samples, hUTBc19 detected a strong 40- to 50-kDa signal in membrane-enriched protein and a 40- to 45-kDa signal in whole cell homogenate (Wh). D: using a range of samples from dissected rat kidneys, hUTBc19 detected strong 30-, 40- to 50-, and 100- to 200-kDa signals, but only in membrane-enriched inner medulla protein. In contrast, a 100-kDa protein was detected in all samples tested. IM, inner medulla; IS, inner stripe outer medulla; OS, outer stripe outer medulla; Cx, cortex.

performed. This was not observed for all proteins detected by hUTBc19 (e.g., the distinct 100-kDa band), suggesting that some nonspecific signals are present. Using peptide-Nglycosidase F enzyme, a series of deglycosylation experiments was also performed (see Fig. 8). The 40- to 45-kDa protein in the bladder was deglycosylated to a 30-kDa core protein signal, whereas no shift was observed for other nonspecific bands (e.g., 50- and 100-kDa signals). However, a similar 40- to 50-kDa smear that was present in the rat kidney medulla was indeed also deglycosylated to a strong 30-kDa signal. Intriguingly, in contrast to the human bladder and rat kidney UT-B protein, the hUT-B1 cell lysate signal was deglycosylated to 40 kDa and the hUT-B2 cell lysate signal was deglycosylated primarily to 50 kDa. Importantly, these two deglycosylated signals were very close to the predicted sizes for hUT-B1 (i.e., 42 kDa) and hUT-B2 (i.e., 48 kDa).

Finally, immunolocalization experiments were performed using hUTBc19 and 10-␮m sections of human bladder tissue (see Fig. 9). Strong staining was consistently observed throughout the bladder urothelium (see Fig. 9A). Importantly, given the previous immunoblot results, this staining was mostly prevented by preincubation with the specific immunizing peptide (see Fig. 9B) but unaffected by a nonspecific peptide preincubation (see Fig. 9C). Higher magnification showed that strong UT-B staining was present in all layers of the urothelium (i.e., umbrella, intermediate, and basal cells) except for the apical membrane of umbrella cells (see Fig. 9D). As expected, the plasma membrane of red blood cells within blood vessels was also strongly stained for UT-B protein,

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Fig. 6. Immunoblot showing that hUTBc19 signals are blocked by preincubation with the specific immunizing peptide. As expected, hUTBc19 strongly detected 40- and 50-kDa proteins representing hUT-B1 (B1) and hUT-B2 (B2), respectively. Importantly, these signals were completely blocked by preincubation of hUTBc19 with its immunizing peptide (1 ␮g peptide/1 ␮l antibody) but not by preincubation in an equivalent concentration of nonspecific peptide.

Fig. 7. Immunoblot showing specific UT-B protein expresssion in whole cell homogenate samples. Using hUTBc19, a strong 40- to 45-kDa smear was detected in human bladder whole cell homogenate but not in any of the other samples. This strong signal in the bladder was absent when hUTBc19 was preincubated with specific immunizing peptide but still present with an equivalent nonspecific peptide preincubation. In contrast, various nonspecific distinct bands were present between 50 and 100 kDa in all experiments. Bl, bladder; Kd, kidney; Il, ileum; Re, rectum.

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Human Rat Human Human Bladder Kidney UT-B1 UT-B2 Medulla Fig. 8. Immunoblot showing that human bladder UT-B is deglycosylated by peptide-N-glycosidase F (PNGaseF) enzyme to a 30-kDa core protein. Deglycosylation by PNGaseF enzyme reduced the 40- to 45-kDa smear detected in the human bladder with hUTBc19 to a 30-kDa signal. A similar 40- to 50-kDa smear in the rat kidney inner medulla was also deglycosylated to a 30-kDa protein. In contrast, hUT-B1 cell lysate signals shifted to 40 kDa, while hUT-B2 cell lysate signals were primarily reduced to a 50-kDa signal.

whereas the lamina propria connective tissue was not stained (see Fig. 9D). DISCUSSION

The aim of the present study was to investigate whether UT-B protein is expressed in the human bladder. Initial PCR data confirmed the presence of UT-B RNA in the human bladder (see Fig. 2), in agreement with a recent study (10). The observed profile of AQP expression also agreed with previous reports, namely, the presence of AQP3, AQP7, and AQP9, but not AQP10 (16). Further PCR experiments showed that hUT-B1 was the main transcript detected, with hUT-B2 only being detected significantly with hUT-B2-specific primers (see Fig. 3). Importantly, no evidence of any other novel UT-B transcripts could be detected (see Fig. 4). Using the novel hUTBc19 antibody, UT-B1 protein was successfully detected using the Western blot technique (see Fig. 5A). Moreover, the original 100- to 150-kDa signal was shifted to a 40-kDa UT-B1 monomer signal by heated incuba-

Fig. 9. Immunolocalization experiments showing that UT-B is present in the human bladder urothelium. A: bladder section (10 ␮m thick) probed with 1:500 hUTBc19 showed strong staining of the urothelium. B: bladder section probed with 1:500 hUTBc19 preincubated with specific immunizing peptide (1␮g peptide/1 ␮l antibody) showed only minimal staining. C: bladder section probed with 1:500 hUTBc19 preincubated with nonspecific peptide (1 ␮g peptide/1 ␮l antibody) showed strong staining of the urothelium. D: human bladder section probed with 1:500 hUTBc19 showing strong UT-B staining throughout urothelium layers except for the apical membrane of outermost umbrella cells (arrow). As expected, strong UT-B staining was also detected on plasma membranes of red blood cells within blood vessels (*).

A

tion with ␤-mercaptoethanol. This lends support to the suggestion that hUT-B1 protein forms a functional multimer, as has been reported for bovine UT-B1 (11). In red blood cell membrane protein, hUTBc19 detected a strong 40- to 60-kDa signal (see Fig. 5B), very similar to the 45- to 55-kDa (8) and 45- to 65-kDa (26) signals previously reported. Our novel UT-B antibody also detected 40- to 50-kDa and 40- to 45-kDa signals in human bladder membrane protein and whole homogenate protein, respectively (see Fig. 5C). The stronger, more glycosylated signal found in membrane-enriched bladder protein suggests the UT-B protein is predominantly located in cell membranes, and not the cytoplasm, as expected. Once more, these signals are very similar to the reported 40- to 45-kDa UT-B in the mouse bladder (4), 40- to 65-kDa UT-B in the dog bladder (21), and 45- to 55-kDa UT-B in the rat bladder (20). Finally, in dissected rat kidney samples, hUTBc19 detected 30, 40- to 50-, and 100- to 200-kDa signals in inner medulla membrane-enriched protein (see Fig. 5D). We interpret these signals as unglycosylated, glycosylated, and multimer UT-B signals, respectively. These signals are also reminiscent of those previously reported for UT-B in the rat kidney by Timmer et al. (26). Finally, the specificity of hUTBc19 was confirmed by the fact that UT-B1 and UT-B2 whole cell lysates signals were prevented by preincubation with the specific immunizing peptide but not by an equivalent amount of a nonspecific peptide (see Fig. 6). Using whole cell homogenates, hUTBc19 successfully detected a strong 40- to 45-kDa UT-B signal in the human bladder protein sample (see Fig. 7). This signal was not found in other whole cell homogenates of the human kidney, ileum, or rectum, suggesting that more detailed samples may be required to detect UT-B protein in these tissues (e.g., membrane-enriched protein from the human kidney medulla). The 40- to 45-kDa signal in the bladder whole homgenate was shown to be specifically blocked by preincubation with the immunizing peptide, unlike certain other proteins (e.g., 50- and 100-kDa distinct bands) that can therefore be classified as nonspecific signals. As is characteristic for UT-B1 (25), the 40to 45-kDa bladder signal was deglycosylated to a smaller, core UT-B protein (see Fig. 8). The fact that bladder UT-B is glycosylated suggests that it is a functional protein (2). Surprisingly, the size of the unglycosylated bladder UT-B protein was 30 kDa. It was thus smaller than either the 40- or 50-kDa

D

B

C

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proteins observed for deglycosylated hUT-B1 and hUT-B2 cell lysates, respectively, which are very close to the predicted sizes for these isoforms. Importantly, a 30-kDa UT-B signal was also detected in deglycosylated rat kidney inner medulla protein, which is almost identical to the 32-kDa signal previously reported in the rat kidney (26). The 30-kDa size of the “UT-B1” core protein signal detected in the human bladder is an intriguing result. In agreement with the findings of this study, previous studies have shown 29-kDa unglycosylated UT-B protein in the mouse bladder (13) and 32-kDa UT-B protein in the rat bladder (20). Similar, smaller than expected UT-B1 protein signals have also previously been found in other tissues, for example, 30 kDa in the human colon (2), 32 kDa in human red blood cells (26), and 30 kDa in the rat cecum (3). Indeed, it is difficult to find any reports of UT-B1 deglycosylated, core protein signals of the predicted size of ⬃42 kDa. Since the 30-kDa bladder protein in this study was detected by a COOH-terminal hUTBc19 antibody, the simplest explanation is that this protein represents a truncated version of UT-B1 in which the NH2-terminal has been removed. It is tempting to speculate that this NH2-terminal truncation is actually a normal part of the physiological regulation of all UT-B1s, hence explaining the fact that 1) UT-B1 signals generally appear to be smaller than predicted and 2) there are minimal reports of successful UT-B antibodies targetted to the NH2-terminal of UT-B1. However, further investigation of the precise nature of this 30-kDa UT-B bladder protein is now urgently required, for example, by performing experiments on human bladder protein using a successfully characterized NH2-terminal UT-B antibody. Using the hUTBc19 antibody, immunolocalization experiments detailed a strong, specific UT-B protein signal throughout the human bladder urothelium (except at the apical membrane of umbrella cells) and in the plasma membrane of red blood cells (see Fig. 9). This urothelium UT-B staining is a similar location to that previously reported in the mouse (13), rat (20), and dog bladder (21). Importantly, in all these species, strong UT-B basolateral staining has been observed in umbrella cells but with little UT-B staining on the apical membrane. This has led to the suggestion that urea enters umbrella cells across the apical membrane from the urine, via the endosomal pathway (1), and that UT-B then enables the exit of this urea out of the urothelium. The physiological relevance of this hypothesis is supported by the observation that there is a large increase in urea levels within the urothelium of UT-B knockout mice (4). An alternative hypothesis is that lack of UT-B in the kidneys will decrease urine concentrating ability (18, 30) and hence increase urine volume. This volume increase will lead to greater bladder distension and stimulate cellular proliferation, potentially increasing long-term tissue damage (10). Evidence for this second hypothesis comes from a recent study in humans by Koutros et al. (10), which reported a link between UT-B allelic variation, decreased urine concentration, and increased bladder cancer risk. Short-term regulation of UT-B1 has been reported in the mouse bladder, where 2 days of dehydration significantly reduced UT-B1 abundance (13). In contrast, water restriction had no such effect on UT-B1 expression in the rat bladder (20), although rat urothelial urea transport does seem to be regulated by hydration status (22) and dietary protein (23). Future experiments should therefore investigate the effect of dehydration

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of the abundance and localization of UT-B1 protein in the human bladder. Finally, UT-B1 has also been reported to be localized in cells lining both the mouse ureter and urethra (13), and hence these structures should now be investigated in humans for potential UT-B protein abundance. The findings of this study agree with the hypothesis that UT-B1 plays a significant physiological role in the human bladder. If proven to be correct, this may help explain the observed link between UT-B1 and bladder cancer risk, namely, that alterations in UT-B1 function could severely affect the long-term health of the human urothelium. At this point, it is interesting to note a previous report (29) investigating UT-B1 protein in human red blood cells, in which allelic variation had a significant effect on UT-B membrane abundance levels. Future investigation of the specific relationship between UT-B alleles and potential variations in urea transport across epithelial layers are therefore urgently required. Such studies may not only provide vital information to help understand the development of cancer in the human bladder but also in prostate cancer, where UT-B1 has also been reported to be altered (28). Finally, these studies should not be restricted to UT-B1 but should also include other transporters potentially involved in the movement of urea out of the urothelium, such as the aquagylceroporins known to be present in human bladder (AQP3, AQP7, and AQP9) (16). Interestingly, human AQP3 protein expression has already been reported to be correlated to tumour stage and grade in urothelial cancer (17). In conclusion, this is the first study to report the presence of UT-B protein in the human bladder. We have confirmed that UT-B1 is the main RNA transcript present and identified 40- to 45-kDa glycosylated UT-B. Similar to previous studies in other mammalian species, this UT-B protein was found to be highly abundant in the human bladder urothelium. Further studies are now required to elucidate the precise nature, physiological role, and regulation of human bladder UT-B. GRANTS This work was funded by Science Foundation Ireland Grant 11/RFP.1/ BMT/3088. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: C.W., A.F., and G.S.S. conception and design of research; C.W., A.F., and A.M. performed experiments; C.W., A.F., and A.M. analyzed data; C.W., A.F., and A.M. interpreted results of experiments; C.W. and G.S.S. prepared figures; G.S.S. drafted manuscript; G.S.S. edited and revised manuscript; G.S.S. approved final version of manuscript. REFERENCES 1. Apodaca G. The uroepithelium: not just a passive barrier. Traffic 5: 117–128, 2004. 2. Collins D, Winter DC, Hogan AM, Schirmer L, Baird AW, Stewart GS. Differential protein abundance and function of UT-B urea transporters in human colon. Am J Physiol Gastrointest Liver Physiol 298: G345– G351, 2010. 3. Collins D, Walpole C, Ryan E, Winter D, Baird A, Stewart G. UT-B1 mediates trans-epithelial urea flux in the rat gastrointestinal tract. J Membr Biol 239: 123–130, 2011. 4. Dong Z, Ran J, Zhou H, Chen J, Lei T, Wang W, Sun Y, Lin G, Bankir L, Yang B. Urea transporter UT-B deletion induces DNA damage and apoptosis in mouse bladder urothelium. PLOS ONE 8: e76952, 2013.

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