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.

Thienoquinolins exert diuresis by strongly inhibiting UT-A urea transporters

Urea transporters (UT) play an important role in the urine concentration mechanism by mediating intrarenal urea recycling, suggesting that UT inhibitors could have therapeutic use as a novel class of diuretic. Recently, we found a thienoquinolin UT inhibitor, PU-14, that exhibited diuretic activity. The purpose of this study was to identify more potent UT inhibitors that strongly inhibit UT-A isoforms in the inner medullary collecting duct (IMCD). Efficient thienoquinolin UT inhibitors were identified by structure-activity relationship analysis. Urea transport inhibition activity was assayed in perfused rat terminal IMCDs. Diuretic activity of the compound was determined in rats and mice using metabolic cages. The results show that the compound PU-48 exhibited potent UT-A inhibition activity. The inhibition was 69.5% with an IC50 of 0.32 μM. PU-48 significantly inhibited urea transport in perfused rat terminal IMCDs. PU-48 caused significant diuresis in UT-B null mice, which indicates that UT-A is the target of PU-48. The diuresis caused by PU-48 did not change blood Na+, K+, or Cl− levels or nonurea solute excretion in rats and mice. No toxicity was detected in cells or animals treated with PU-48. The results indicate that thienoquinolin UT inhibitors induce a diuresis by inhibiting UT-A in the IMCD. This suggests that they may have the potential to be developed as a novel class of diuretics with fewer side effects than classical diuretics.

Effects of dietary ruminally degradable starch and ruminally degradable protein levels on urea recycling, microbial protein production, nitrogen balance, and duodenal nutrient flow in beef heifers fed low crude protein diets

Davies, K. L., McKinnon, J. J. and Mutsvangwa, T. 2013. Effects of dietary ruminally degradable starch and ruminally degradable protein levels on urea recycling, microbial protein production, nitrogen balance, and duodenal nutrient flow in beef heifers fed low crude protein diets. Can. J. Anim. Sci. 93: 123–136. The objective was to determine the effects of ruminally degradable starch (RDS; 28.6 and 69.2% of total starch) and ruminally degradable protein [RDP; 48.0 and 55.0% of crude protein (CP)] content on urea recycling, nitrogen (N) balance, duodenal nutrient flow, and microbial protein production in beef heifers fed low CP (10%) diets. Four ruminally and duodenally cannulated beef heifers (723±57 kg body weight) were used in a 4×4 Latin square design with a 2×2 factorial arrangement of dietary treatments with 23-d periods. Jugular infusions of [15N15N]-urea (220 mg d−1; 98+ atom percent) were conducted for 4 d (days 18–22) to estimate urea kinetics, with total collection of faeces and urine. Proportions of [15N15N]- and [14N15N]-urea in urinary urea, and 15N enrichment in faeces were used to calculate urea kinetics. Ruminal microbial N production was estimated using 15N as a marker. Ruminal ammonia-N concentration was greater (P=0.01) in heifers fed high RDP as compared with those fed low RDP, and it was also greater (P=0.01) in heifers fed low RDS as compared with those fed high RDS. Microbial N flow to the duodenum increased as RDP level increased on the high RDS diet, but was not affected by RDP level on the low RDS diet (interaction; P=0.04). Urea-N entry rate and urea-N transfer to the gastrointestinal tract were similar (P>0.05) across diets. The amount of recycled urea-N incorporated into microbial N increased as RDP level increased on the high RDS diet, but the opposite was observed on the low RDS diet (interaction; P=0.008). These results indicate that at a low CP level (10%), increasing both RDS and RDP levels can increase microbial N flow to the duodenum and improve the efficiency of use of recycled urea-N for microbial N synthesis.