Investigation of facilitative urea transporters in the human gastrointestinal tract.

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.

Differential protein abundance of a basolateral MCT1 transporter in the human 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.

Hyperosmolality regulates UT-A6 urea transporter expression in the Caco-2 cell line.

Gastrointestinal facilitative urea transporters play a significant role in the urea nitrogen salvaging process, which supports the symbiotic relationship between mammals and their gut microbial populations. UT-A6 urea transporters have been previously reported in the human gastrointestinal tract, specifically in the colon. As renal UT-A transporters can be regulated by external osmolality, this study investigated whether UT-A6 expression could also be regulated in this manner. Initial end-point RT-PCR experiments confirmed UT-A6 expression along the human gastrointestinal tract (colon > small intestine â‰« stomach) and also in the Caco-2 intestinal cell line. Using Caco-2 cells exposed for 24 hours to changed external osmotic conditions (from 350 to 250, 500, or 600 mOsm), end-point PCR suggested UT-A6 expression increased in hyperosmotic conditions. Using quantitative PCR, it was confirmed that 24 h exposure to 600 mOsm stimulated a significant ~15-fold increase in UT-A6 expression (P < 0.001, N = 5, ANOVA). Finally, inhibitory experiments suggested that protein kinase C and calcium were involved in this hyperosmotic-stimulated regulatory pathway. In conclusion, these data demonstrated UT-A6 expression was indeed regulated by external osmolality. The physiological significance of this regulatory process upon gastrointestinal urea transport has yet to be determined.