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As part of their osmoregulatory strategy, marine elasmobranchs retain large quantities of urea to balance the osmotic pressure of the marine environment. The main source of nitrogen used to synthesize urea comes from the digestion and absorption of food across the gastrointestinal tract. In this study we investigated possible mechanisms of nitrogen movement across the spiral valve of the cloudy catshark (Scyliorhinus torazame) through the molecular identification of two Rhesus glycoprotein ammonia transporters (Rhp2 and Rhbg) and a urea transporter (UT). We used immunohistochemistry to determine the cellular localizations of Rhp2 and UT. Within the spiral valve, Rhp2 was expressed along the apical brush border membrane, and UT was expressed along the basolateral membrane and the blood vessels. The mRNA abundance of Rhp2 was significantly higher in all regions of the spiral valve of fasted catsharks compared to fed catsharks. The mRNA abundance of UT was significantly higher in the anterior spiral valve of fasted catsharks compared to fed. The mRNA transcript of four ornithine urea cycle (OUC) enzymes were detected along the length of the spiral valve and in the renal tissue, indicating the synthesis of urea via the OUC occurs in these tissues. The presence of Rhp2, Rhbg, and UT along the length of the spiral valve highlights the importance of ammonia and urea movement across the intestinal tissues, and increases our understanding of the mechanisms involved in maintaining whole-body nitrogen homeostasis in the cloudy catshark., Competing Interests: Declaration of Competing Interest The authors declare no conflicts., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Hypertension is a major concern for a wide array of patients. The traditional drugs are commonly referred as 'water pills' and these molecules have been successful in alleviating hypertension. However, this comes at the high expense of precious electrolytes in our body. To dissipate this major adverse effect, the urea transporter inhibitors play especially important roles in maintaining the fluid balance by maintaining the concentration of urea in the inner medullary collecting duct. The purpose of this communication is to provide insights into the structural feature of these target proteins and inhibition of both urea transporter types A (UT-A) and B (UT-B) selectively and non-selectively with a special focus on the UT-A inhibitors as they are the primary target for diuresis. It was observed that a wide class of drugs such as thiourea analogues, 2,7-disubstituted fluorenones can inhibit both the protein non-selectively whereas 8-hydroxyquinoline, aminothiazolone, 1,3,5-triazine, triazolothienopyrimidine, thienoquinoline, arylthiazole, γ-sultambenzosulfonamide and 1,2,4-triazoloquinoxaline classes of compounds inhibit UT-A. The goal of this study is to highlight the important aspects that may be useful to understanding the perspectives of urea transporter inhibitors in rational drug discovery., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The mechanism(s) of ammonia and urea excretion in freshwater fish have received considerable attention; however, parallel investigations of seawater fish, specifically in the early life stages are scarce. The first objective of this study was to evaluate the patterns of ammonia and urea excretion in mahi-mahi (Coryphaena hippurus) up to 102  hours post fertilization (hpf). Similar to other teleosts, mahi embryos are ureotelic before hatch and gradually switch to being ammoniotelic around the time of hatch. The second objective was to characterize mRNA levels of ammonia transporters (Rhag, Rhbg, Rhcg1 and Rhcg2), as well as urea transporter (UT) and sodium hydrogen exchangers (NHE3 and NHE2) during mahi development. As predicted, the mRNA levels of the Rhesus glycoprotein (Rh) genes, especially Rhag, Rhbg and the UT gene were highly consistent with the ontogeny of ammonia and urea excretion rates. Further, the localization of each transporter was examined in larvae collected at 60 and 102 hpf using in situ hybridization. Rhag was expressed in the gills, yolk sac, and operculum. Rhbg was expressed in the gills and upper mouth. Rhcg1 and NHE3 were co-localized in the sub-operculum, and Rhcg2 was expressed in the skin. Together, these results indicate that urea excretion is critical for ammonia detoxification during embryonic development and that Rh proteins are involved in ammonia excretion via gills and yolk sac, possibly facilitated by NHE3., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Gulf toadfish (Opsanus beta) can switch from continuously excreting ammonia as their primary nitrogenous waste to excreting predominantly urea in distinct pulses. Previous studies have shown that the neurotransmitter serotonin (5-HT) is involved in controlling this process, but it is unknown if 5-HT availability is under central nervous control or if the 5-HT signal originates from a peripheral source. Following up on a previous study, cranial nerves IX (glossopharyngeal) and X (vagus) were sectioned to further characterize their role in controlling pulsatile urea excretion and 5-HT release within the gill. In contrast to an earlier study, nerve sectioning did not result in a change in urea pulse frequency. Total urea excretion, average pulse size, total nitrogen excretion, and percent ureotely were reduced the first day post-surgery in nerve-sectioned fish but recovered by 72h post-surgery. Nerve sectioning also had no effect on toadfish urea transporter (tUT), 5-HT transporter (SERT), or 5-HT 2A receptor mRNA expression or 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) abundance in the gill, all of which were found consistently across the three gill arches except 5-HIAA, which was undetectable in the first gill arch. Our findings indicate that the central nervous system does not directly control pulsatile urea excretion or local changes in gill 5-HT and 5-HIAA abundance., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Urea transporters (UTs) belonging to the solute carrier 14 (SLC14) family comprise two genes with a total of eight isoforms in mammals, UT-A1 to -A6 encoded by SLC14A2 and UT-B1 to -B2 encoded by SLC14A1. Recent efforts have been directed toward understanding the molecular and cellular mechanisms involved in the regulation of UTs using transgenic mouse models and heterologous expression systems, leading to important new insights. Urea uptake by UT-A1 and UT-A3 in the kidney inner medullary collecting duct and by UT-B1 in the descending vasa recta for the countercurrent exchange system are chiefly responsible for medullary urea accumulation in the urinary concentration process. Vasopressin, an antidiuretic hormone, regulates UT-A isoforms via the phosphorylation and trafficking of the glycosylated transporters to the plasma membrane that occurs to maintain equilibrium with the exocytosis and ubiquitin-proteasome degradation pathways. UT-B isoforms are also important in several cellular functions, including urea nitrogen salvaging in the colon, nitric oxide pathway modulation in the hippocampus, and the normal cardiac conduction system. In addition, genomic linkage studies have revealed potential additional roles for SLC14A1 and SLC14A2 in hypertension and bladder carcinogenesis. The precise role of UT-A2 and presence of the urea recycling pathway in normal kidney are issues to be further explored. This review provides an update of these advances and their implications for our current understanding of the SLC14 UTs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the effects of N-acetylcysteine (NAC) on renal function, as well as on sodium and water transporters, in the kidneys of aged rats. Normal, 8-month-old male Wistar rats were treated (n=6) or not (n=6) with NAC (600 mg/L in drinking water) and followed for 16 months. At the end of the follow-up period, we determined inulin clearance, serum thiobarbituric acid reactive substances (TBARS), serum cholesterol, and urinary phosphate excretion. In addition, we performed immunohistochemical staining for p53 and for ED-1-positive cells (macrophages/monocytes), together with Western blotting of kidney tissue for NKCC2, aquaporin 2 (AQP2), urea transporter A1 (UT-A1) and Klotho protein. At baseline, the two groups were similar in terms of creatinine clearance, proteinuria, cholesterol, and TBARS. At the end of the follow-up period, NAC-treated rats presented greater inulin clearance and reduced proteinuria, as well as lower serum cholesterol, serum TBARS, and urinary phosphate excretion, in comparison with untreated rats. In addition, NAC-treated rats showed upregulated expression of NKCC2, AQP2, and UT-A1; elevated Klotho protein expression, low p53 expression, and few ED-1 positive cells. In conclusion, we attribute these beneficial effects of NAC (the significant improvements in inulin clearance and in the expression of NKCC2, AQP2, and UT-A1) to its ability to decrease oxidative stress, inhibit p53 expression, minimize kidney inflammation, and stimulate Klotho expression., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The present study has examined the role of the colon in regulating ammonia and urea nitrogen balance in two species of chondrichthyans, the ratfish, Hydrolagus colliei (a holocephalan) and the spiny dogfish, Squalus acanthias (an elasmobranch). Stripped colonic tissue from both the dogfish and ratfish was mounted in an Ussing chamber and in both species bi-directional urea flux was found to be negligible. Urea uptake by the mucosa and serosa of the isolated colonic epithelium through accumulation of (14)C-urea was determined to be 2.8 and 6.2 fold greater in the mucosa of the dogfish compared to the serosa of the dogfish and the mucosa of the ratfish respectively. Furthermore, there was no difference between serosal and mucosal accumulation of (14)C-urea in the ratfish. Through the addition of 2mM NH(4)Cl to the mucosal side of each preparation the potential for ammonia flux was also examined. This was again found to be negligible in both species suggesting that the colon is an extremely tight epithelium to the movement of both urea and ammonia. Plasma, chyme and bile fluid samples were also taken from the agastric ratfish and were compared with solute concentrations of equivalent body fluids in the dogfish. Finally molecular analysis revealed expression of 3 isoforms of the urea transport protein (UT) and an ammonia transport protein (Rhbg) in the gill, intestine, kidney and colon of the ratfish. Partial nucleotide sequences of the UT-1, 2 and 3 isoforms in the ratfish had 95, 95 and 92% identity to the equivalent UT isoforms recently identified in another holocephalan, the elephantfish, Callorhinchus milii. Finally, the nucleotide sequence of the Rhbg identified in the ratfish had 73% identity to the Rhbg protein recently identified in the little skate, Leucoraja erinacea., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The nature of ammonia and urea excretion was examined in the Pacific hagfish (Eptatretus stoutii), which, under resting conditions, excreted similar quantities of nitrogen as either ammonia or urea. In the presence of high external ammonia (HEA) concentrations, ammonia was taken up at high rates and then excreted at similarly high rates upon return to normal water. However, although elevated by HEA, plasma ammonia levels were maintained at approximately 1-4 μmolNg⁻¹, reflecting time-dependent decreases in the rates of ammonia uptake, the possible conversion of ammonia to urea, and the potential active excretion of ammonia against a gradient. Internal injections of NH₄Cl caused marked increases in the rate of ammonia excretion and a delayed increase in urea excretion that may have resulted from increasing urea levels in the plasma. Conversely, when the rate of urea excretion was reduced in the presence of 0.1 mM phloretin, ammonia excretion was significantly elevated. Rates of urea excretion were initially increased by approximately 1000-fold following internal urea injections while the presence of high external urea levels (5-100 mM final concentration) resulted in associated linear increases in plasma urea levels. Using hagfish skin mounted in Ussing chambers, the rate of diffusion of ammonia across the skin exceeded that of urea by approximately four times when equivalent gradients were imposed. Based on western blotting and immunocytochemistry, hagfish gill appears to possess Rh proteins (Rhag, Rhbg and Rhcg1) and urea transporter proteins. Despite the tolerance of hagfish to high levels of ammonia and urea, it is suggested that the presence of ammonia and urea transporter proteins may be required during the period of time hagfish spend in burrows or while feeding, when conditions of high ammonia and/or urea might be encountered., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The present study reports aspects of GI tract physiology in the white-spotted bamboo shark, Chiloscyllium plagiosum, little skate, Leucoraja erinacea and the clear nose skate, Raja eglanteria. Plasma and stomach fluid osmolality and solute values were comparable between species, and stomach pH was low in all species (2.2 to 3.4) suggesting these elasmobranchs may maintain a consistently low stomach pH. Intestinal osmolality, pH and ion values were comparable between species, however, some differences in ion values were observed. In particular Ca(2+) (19.67+/-3.65mM) and Mg(2+) (43.99+/-5.11mM) were high in L. erinacea and Mg(2+) was high (130.0+/-39.8mM) in C. palgiosum which may be an indication of drinking. Furthermore, intestinal fluid HCO(3)(-) values were low (8.19+/-2.42 and 8.63+/-1.48mM) in both skates but very high in C. plagiosum (73.3+/-16.3mM) suggesting ingested seawater may be processed by species-specific mechanisms. Urea values from the intestine to the colon dropped precipitously in all species, with the greatest decrease seen in C. plagiosum (426.0+/-8.1 to 0mM). This led to the examination of the molecular expression of both a urea transporter and a Rhesus like ammonia transporter in the intestine, rectal gland and kidney in L. erinacea. Both these transporters were expressed in all tissues; however, expression levels of the Rhesus like ammonia transporter were orders of magnitude higher than the urea transporter in the same tissue. Intestinal flux rates of solutes in L. erinacea were, for the most part, in an inward direction with the notable exception of urea. Colon flux rates of solutes in L. erinacea were all in an outward direction, although absolute rates were considerably lower than the intestine, suggestive of a much tighter epithelia. Results are discussed in the context of the potential role of the GI tract in salt and water, and nitrogen, homeostasis in elasmobranchs.

The red cell membrane has an exceptionally high permeability for CO2, PCO2 approximately 0.15 cm/s, which is two to three orders of magnitude greater than that of some epithelial membranes and similarly greater than the permeability of the red cell membrane for HCO3-. As shown previously, this high PCO2 can be drastically inhibited by 10 microM 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate (DIDS), indicating that membrane proteins may be involved in this high gas permeability. Here, we have studied the possible contribution of several blood group proteins to CO2 permeation across the red cell membrane by comparing PCO2 of red cells deficient in specific blood group proteins with that of normal red cells. While PCO2 of normal red cells is approximately 0.15 cm/s and that of Fy(null) and Jk(null) red cells is similar, PCO2's of Colton null (deficient in aquaporin-1) and Rh(null) cells (deficient in Rh/RhAG) are both reduced to about 0.07 cm/s, i.e. to about one half. In addition, the inhibitory effect of DIDS is about half as great in Rh(null) and in Colton null red cells as it is in normal red cells. We conclude that aquaporin-1 and Rh/RhAG proteins contribute substantially to the high permeability of the human red cell membrane for CO2. Together these proteins are responsible for 50% or more of the CO2 permeability of red cell membranes. The CO2 pathways of both proteins can be partly inhibited by DIDS, which is why this compound very effectively reduces membrane CO2 permeability.

We used a perfused gill preparation from dogfish to investigate the origin of low branchial permeability to urea. Urea permeability (14C-urea) was measured simultaneously with diffusional water permeability (3H2O). Permeability coefficients for urea and ammonia in the perfused preparation were almost identical to in vivo values. The permeability coefficient of urea was 0.032 x 10(-6) cm/sec and of 3H2O 6.55 x 10(-6) cm/sec. Adrenalin (1 x 10(-6) M) increased water and ammonia effluxes by a factor of 1.5 and urea efflux by a factor of 3.1. Urea efflux was almost independent of the urea concentration in the perfusion medium. The urea analogue thiourea in the perfusate had no effect on urea efflux, whereas the non-competitive inhibitor of urea transport, phloretin, increased efflux markedly. The basolateral membrane is approximately 14 times more permeable to urea than the apical membrane. We conclude that the dogfish apical membrane is extremely tight to urea, but the low apparent branchial permeability may also relate to the presence of an active urea transporter on the basolateral membrane that returns urea to the blood and hence reduces the apical urea gradient.

The gene encoding for the human erythrocyte urea transporter (HUT11 clone) has recently been cloned (Olives et al., J. Biol. Chem., 269, 3/649, 1994). It has been localized at 18q12-q21, like the Kidd blood group gene. In vitro translation experiments indicated that cDNA HUT11 could induce the synthesis of a 36 kDa protein which can be immunoprecipitated by an anti-Jk3 antibody (a human antibody produced by Jk(a-b-) individuals). This antibody also precipitates a 46-60 kDa protein from human red blood cells, except from those with Jk(a-b-) phenotype. After N-glycanase digestion, the length of the glycoprotein was reduced from 46-60 kDa to 36 kDa. Moreover, a rabbit antibody directed against the N-terminal end of HUT11 protein reacts in western-blot with 46-60 kDa proteins present in all human red blood cells, except those with Jk(a-b-) phenotype. Jk(a-b-) cells have neither Kidd protein nor HUT11 urea transporter and they are characterized by a selective defect of urea transport whereas water transport and aquaporin-1 associated Colton antigens are normally expressed. All these findings provide evidence that the Kidd locus encodes for the human erythrocyte urea transporter, offering new prospects for biological study of urea transporters and their tissue-specific regulation.