173 Characterization of urea transport mechanisms in the intestinal tract of growing pigs

Previous studies have indicated that pigs are capable of nitrogen salvage via urea recycling, which involves the movement of urea into the gastrointestinal tract and incorporation of nitrogen into endogenous or microbially produced amino acids. Aquaporins (AQP) and urea transporter B (UT-B) have been shown to contribute to urea transport in ruminants; however, it is unclear whether the same processes contribute to urea movement in the intestinal tract of the pig. The objective of this study was to characterize the presence and relative contribution of known urea transporters to urea flux in the growing pig. A total of 9 barrows of 50.8±0.9 kg BW were euthanized and samples of intestinal tissue were obtained from the duodenum, jejunum, ileum, cecum, and colon. All tissue samples were analyzed for mRNA abundance of UT-B and AQP-3, 7, and 10 via qPCR. Immediately after tissue collection, samples from jejunum and cecum were placed in Ussing chambers for analysis of serosal-to-mucosal urea flux using 14C-urea (49.95 kBq). Serosal-to-mucosal urea flux was measured across intestinal tissue samples with no inhibition or with addition of phloretin (1 mM) to inhibit UT-B-mediated transport, NiCl2 (1 mM) to inhibit AQP-mediated transport, or both inhibitors. UT-B was most highly expressed in the cecum (P < 0.05), while AQP-3, 7, and 10 were most highly expressed in the jejunum (P < 0.05). Serosal-to-mucosal urea flux occurred in both the jejunum and the cecum and was higher in the cecum (42.7 vs. 67.8±5.01 µmol/cm2/h; P < 0.05), confirming the capacity for urea recycling into the gut in pigs; however, neither flux rate was influenced by urea transporter inhibitors (P > 0.05). The results of this study indicate that while known urea transporters are present in the gastrointestinal tract of pigs, they do not play a significant role in urea transport.

Characterization of urea transport mechanisms in the intestinal tract of growing pigs

Pigs are capable of nitrogen salvage via urea recycling, which involves the movement of urea in the gastrointestinal tract. Aquaporins (AQP) and urea transporter B (UT-B) are involved in urea recycling in ruminants; however, their contribution to urea flux in the intestinal tract of the pig is not known. The objective of this study was to characterize the presence and relative contribution of known urea transporters to urea flux in the growing pig. Intestinal tissue samples (duodenum, jejunum, ileum, cecum, and colon) were obtained from nine barrows (50.8 ± 0.9 kg) and analyzed for mRNA abundance of UT-B and AQP-3, -7, and -10. Immediately after tissue collection, samples from the jejunum and cecum were placed in Ussing chambers for analysis of the serosal-to-mucosal urea flux ( Jsm-urea) with no inhibition or when incubated in the presence of phloretin to inhibit UT-B-mediated transport, NiCl2 to inhibit AQP-mediated transport, or both inhibitors. UT-B expression was greatest ( P < 0.05) in the cecum, whereas AQP-3, -7, and -10 expression was greatest ( P < 0.05) in the jejunum. The Jsm-urea was greater in the cecum than the jejunum (67.8 . 42.7 ± 5.01 µmol·cm−2·h−1; P < 0.05), confirming the capacity for urea recycling in the gut in pigs; however, flux rate was not influenced ( P > 0.05) by urea transporter inhibitors. The results of this study suggest that, although known urea transporters are expressed in the gastrointestinal tract of pigs, they may not play a significant functional role in transepithelial urea transport. NEW & NOTEWORTHY We characterized the location and contribution of known urea transporters to urea flux in the pig. Aquaporins are located throughout the intestinal tract, and urea transporter B is expressed only in the cecum. Urea flux occurred in both the jejunum and cecum. Transporter inhibitors had no affect on urea flux, suggesting that their contribution to urea transport in the intestinal tract is limited. Further work is required to determine which factors contribute to urea flux in swine.

Purinergic signaling is enhanced in the absence of UT-A1 and UT-A3

ATP is an important paracrine regulator of renal tubular water and urea transport. The activity of P2Y2, the predominant P2Y receptor of the medullary collecting duct, is mediated by ATP, and modulates urinary concentration. To investigate the role of purinergic signaling in the absence of urea transport in the collecting duct, we housed wild-type (WT) and UT-A1/A3 null (UT-A1/A3 KO) mice in metabolic cages to monitor urine output, and collected tissue samples for analysis. We confirmed that UT-A1/A3 KO mice are polyuric, and concurrently observed lower levels of urinary cAMP as compared to WT, despite elevated serum vasopressin (AVP) levels. Because P2Y2 inhibits AVP-stimulated transport by dampening cAMP synthesis, we suspected that, similar to other models of AVP-resistant polyuria, purinergic signaling is increased in UT-A1/A3 KO mice. In fact, we observed that both urinary ATP and purinergic-mediated prostanoid (PGE2) levels were elevated. Tissue analysis shows that P2Y2 mRNA levels remain unchanged, while P2Y2 protein levels are elevated in KO mice. Collectively, our data suggest that the reduction of medullary osmolality due to the lack of UT-A1 and UT-A3 induces an AVP-resistant polyuria that is possibly exacerbated by, or at least correlated with, enhanced purinergic signaling.Summary statementPhysiological analyses suggest that mice lacking urea transporters have increased renal purinergic signaling, perhaps contributing to previously observed vasopressin-resistant polyurias.