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.

Facilitative UT-B urea transporters play an important role in the urea nitrogen salvaging process that occurs in the gastrointestinal tract of mammals, particularly ruminants. Gastrointestinal UT-B transporters have previously been reported in various ruminant species-including cow, sheep and goat. In this present study, UT-B transporter localization was investigated in tissues throughout the bovine gastrointestinal tract. RT-PCR analysis showed that UT-B2 was the predominant UT-B mRNA transcript expressed in dorsal, ventral and cranial ruminal sacs, while alternative UT-B transcripts were present in other gastrointestinal tissues. Immunoblotting analysis detected a strong, glycosylated ~50 kDa UT-B2 protein in all three ruminal sacs. Immunolocalization studies showed that UT-B2 protein was predominantly localized to the plasma membrane of cells in the stratum basale layer of all ruminal sac papillae. In contrast, other UT-B protein staining was detected in the basolateral membranes of the surface epithelial cells lining the abomasum, colon and rectum. Overall, these findings confirm that UT-B2 cellular localization is similar in all ruminal sacs and that other UT-B proteins are located in epithelial cells lining various tissues in the bovine gastrointestinal tract.

The process of urea nitrogen salvaging plays a vital role in the symbiotic relationship between mammals and their intestinal bacteria. The first step in this process requires the movement of urea from the mammalian bloodstream into the gastrointestinal tract lumen via specialized proteins known as facilitative urea transporters. In this study, we examined both transepithelial urea fluxes and urea transporter protein abundance along the length of the rat gastrointestinal tract. Urea flux experiments that used rat gastrointestinal tissues showed significantly higher transepithelial urea transport was present in caecum and proximal colon (P < 0.01, n = 8, analysis of variance [ANOVA]). This large urea flux was significantly inhibited by 1,3,dimethylurea (P < 0.001, n = 8, ANOVA) and thiourea (P < 0.05, n = 6, unpaired t-test), both known blockers of facilitative urea transporters. Immunoblotting analysis failed to detect any UT-A protein within rat gastrointestinal tissue protein samples. In contrast, a 30-kDa UT-B1 protein was strongly detected in both caecum and proximal colon samples at significantly higher levels compared to the rest of the gastrointestinal tract (P < 0.01, n = 4, ANOVA). We therefore concluded that UT-B1 mediates the transepithelial movement of urea that occurs in specific distal regions of the rat gastrointestinal tract.

Bacterially derived short chain fatty acids (SCFAs), such as butyrate, are vital in maintaining the symbiotic relationship that exists between humans and their gastrointestinal microbial populations. A key step in this process is the transport of SCFAs across colonic epithelial cells via MCT1 transporters. This study investigated MCT1 protein abundance in various human intestinal tissues. Initial RT-PCR analysis confirmed the expected MCT1 RNA expression pattern of colon > small intestine > stomach. Using surgical resection samples, immunoblot analysis detected higher abundance of a 45 kDa MCT1 protein in colonic tissue compared to ileum tissue (P < 0.001, N = 4, unpaired t-test). Importantly, MCT1 abundance was found to be significantly lower in sigmoid colon compared to ascending colon (P < 0.01, N = 8-11, ANOVA). Finally, immunolocalization studies confirmed MCT1 to be abundant in the basolateral membranes of surface epithelial cells of the ascending, transverse, and descending colon, but significantly less prevalent in the sigmoid colon (P < 0.05, N = 5-21, ANOVA). In conclusion, these data confirm that basolateral MCT1 protein abundance is correlated to levels of bacterially derived SCFAs along the human gastrointestinal tract. These findings highlight the importance of precise tissue location in studies comparing colonic MCT1 abundance between normal and diseased states.

The symbiotic relationship between humans and their intestinal microbiome is supported by urea nitrogen salvaging. Previous studies have shown that colonic UT‐B urea transporters play a significant role in this important physiological process. This current study investigated UT‐A and UT‐B urea transporter expression along the human gastrointestinal tract. Initial end‐point PCR experiments determined that UT‐A RNA was predominantly expressed in the small intestine, while UT‐B RNA was expressed in stomach, small intestine, and colon. Using western blotting experiments, a strong 40–60 kDa UT‐B signal was found to be abundant in both ileum and colon. Importantly, this signal was deglycosylated by PNGaseF enzyme treatment to a core protein of 30 kDa in both tissues. Further immunolocalization studies revealed UT‐B transporter proteins were present at the apical membrane of the villi in the ileum, but predominantly at the basolateral membrane of the colonic surface epithelial cells. Finally, a blind scoring immunolocalization study suggested that there was no significant difference in UT‐B abundance throughout the colon (NS, ANOVA, N = 5–21). In conclusion, this current study suggested UT‐B to be the main human intestinal urea transporter. Intriguingly, these data suggested that the same UT‐B isoform was present in all intestinal epithelial cells, but that the precise cellular location varied.

Urea entry into the gastrointestinal tract is facilitated by the urea transporter UT‐B1 (Stewart 2011). This process is crucial to our intestinal wellbeing as it is the first step in the urea nitrogen salvaging mechanism, which is necessary for maintaining healthy gut bacterial populations. In a previous study using a bovine UT‐B antibody, a 35kDa human UT‐B1 protein was found to be highly expressed in the ascending colon (Collins et al., 2010). The aim of this study was to produce a reliable human UT‐B antibody to be used for further investigations of human UT‐B expression. Initial PCR analysis using primers designed along the UT‐B gene confirmed UT‐B1 as the major isoform expressed at the RNA level in all human gastrointestinal tissues. Three polyclonal human UT‐B antibodies ‐ one designed against the N‐terminal (UT‐B#N) and two against the C‐terminal (UT‐B#C and UT‐Bc19) ‐ were made according to the UT‐B1 amino acid sequence and extensive characterization was carried out using Western blot analysis. While both C‐terminal antibodies detected UT‐B1 in the membrane of erythrocytes as a large glycosylated signal at 45‐60kDa as expected, the UT‐B#N antibody failed to do so and was rejected. As UT‐B transporters are glycoproteins, deglycosylation experiments were performed on UT‐B1 protein using PNGaseF enzyme. While UT‐Bc19 successfully detected 36 kDa deglycosylated UT‐B1, UT‐B#C failed to do so and was also rejected. Next, β‐mercaptoethanol was used to reduce UT‐B1 from its native multimer to a monomer and UT‐Bc19 successfully detected a big shift from a smeared 40‐100kDa signal to a neat 40kDa signal. Finally, UT‐Bc19 also detected a strong 40‐70kDa glycosylated UT‐B1 signal in human ileum protein. In conclusion, these data have shown that UT‐Bc19 is the only consistent human UT‐B antibody. Future investigations of UT‐B1 expression will therefore utilise UT‐Bc19 with confidence and help establish the physiological significance of UT‐B1 in the human gastrointestinal tract. Grant Funding Source: Supported by The Wellcome Trust and Science Foundation Ireland.