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The objectives were to determine the effects of dietary crude protein (CP) content and corn grain processing on whole-body urea kinetics and the functional roles of urea transporter-B (UT-B) and aquaporins (AQP) in serosal-to-mucosal urea flux (Jsm-urea) in ovine ruminal epithelia. Thirty-two Rideau-Arcott ram lambs were blocked by bodyweight into groups of 4 and then randomly allocated within blocks to 1 of 4 diets (n = 8) in a 2 × 2 factorial design. Dietary factors were CP content (11% [LP] vs. 16% [HP]) and corn grain processing (whole-shelled [WSC] vs. steam-flaked [SFC] corn). Whole-body urea kinetics and N balance were determined using 4-d continuous intrajugular infusions of [15N15N]-urea with concurrent collections of urine and feces with four blocks of lambs (n = 4). After 23 d on diets, lambs were killed to collect ruminal epithelia for mounting in Ussing chambers to determine Jsm-urea and the measurement of mRNA abundance of UT-B and AQP. Serosal and mucosal additions of phloretin and NiCl2 were used to inhibit UT-B- and AQP-mediated urea transport, respectively. Lambs fed HP had a greater (P < 0.01) N intake (29.4 vs. 19.1 g/d) than those fed LP; however, retained N (g/d or % of N intake) was not different. As a % of N intake, lambs fed SFC tended (P = 0.09) to have a lower N excretion (72.2 vs. 83.5%) and a greater N retention (27.8 vs. 16.6%) compared to those fed WSC. Endogenous urea-N production (UER) was greater in lambs fed HP compared to those fed LP (29.9 vs. 20.6 g/d; P = 0.02), whereas urea-N secreted into the gut (GER; g/d) and urea-N used for anabolic purposes (UUA; g/d) were similar. Lambs fed LP tended (P = 0.05) to have greater GER:UER (0.78 vs. 0.66) and UUA:GER (0.23 vs. 0.13) ratios, and a greater Jsm-urea (144.7 vs. 116.1 nmol/[cm2 × h]; P = 0.07) compared to those fed HP. Lambs fed SFC tended to have a lower NiCl2-insensitive Jsm-urea (117.4 vs. 178.4 nmol/[cm2 × h]; P = 0.09) and had a lower phloretin-insensitive Jsm-urea (87.1 vs. 143.1 nmol/[cm2 × h]; P = 0.02) compared to those fed WSC. The mRNA abundance of UT-B (0.89 vs. 1.07; P = 0.08) and AQP-3 (0.90 vs. 1.05; P = 0.07) tended to be lower in lambs fed SFC compared to those fed WSC. Overall, reducing CP content tended to increase the GER:UER ratio with no changes in the expression or function of UT-B and AQP. Although corn grain processing had no effects on GER, feeding SFC increased the portion of urea secretion into the rumen that was mediated via UT-B and AQP., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Hyponatremia is the most common disorder of electrolyte imbalances. It is necessary to develop new type of diuretics to treat hyponatremia without losing electrolytes. Urea transporters (UT) play an important role in the urine concentrating process and have been proved as a novel diuretic target. In this study, rat and mouse syndromes of inappropriate antidiuretic hormone secretion (SIADH) models were constructed and analyzed to determine if UTs are a promising drug target for treating hyponatremia. Experimental results showed that 100 mg/kg UT inhibitor 25a significantly increased serum osmolality (from 249.83 ± 5.95 to 294.33 ± 3.90 mOsm/kg) and serum sodium (from 114 ± 2.07 to 136.67 ± 3.82 mmol/L) respectively in hyponatremia rats by diuresis. Serum chemical examination showed that 25a neither caused another electrolyte imbalance nor influenced the lipid metabolism. Using UT-A1 and UT-B knockout mouse SIADH model, it was found that serum osmolality and serum sodium were lowered much less in UT-A1 knockout mice than in UT-B knockout mice, which suggest UT-A1 is a better therapeutic target than UT-B to treat hyponatremia. This study provides a proof of concept that UT-A1 is a diuretic target for SIADH-induced hyponatremia and UT-A1 inhibitors might be developed into new diuretics to treat hyponatremia., (© 2024 Federation of American Societies for Experimental Biology.)

In this study, we present the structures of human urea transporters UT-A and UT-B to characterize them at molecular level and to detail the mechanism of UT-B inhibition by its selective inhibitor, UTB inh -14. High-resolution structures of both transporters establish the structural basis for the inhibitor's selectivity to UT-B, and the identification of multiple binding sites for the inhibitor will aid with the development of drug lead molecules targeting both transporters. Our study also discovers phospholipids associating with the urea transporters by combining structural observations, native MS, and lipidomics analysis. These insights improve our understanding of urea transporter function at a molecular level and provide a blueprint for a structure-guided design of therapeutics targeting these transporters.

Theodoxus fluviatilis (Linnaeus, 1758) (Gastropoda: Neritidae) is an oligohaline aquatic gastropod that inhabits most of Europe and adjacent areas of Asia. Two different ecotypes can be distinguished: One in freshwater (FW) and another along the Baltic Sea coast in brackish water habitats (BW). Individuals of either ecotype use free amino acids and urea as organic osmolytes to adjust body fluid osmolality to the external medium; however, the BW ecotype is able to accumulate them in larger quantities. The use of urea as an organic osmolyte in aquatic gastropods such as T. fluviatilis has only recently been initially described and raised the question of how urea transport between body fluids and the environment is balanced. Upon examining transcriptome and preliminary genome sequence data of T. fluviatilis, we identified putative homologues of DUR3 genes, which code for urea transporters (UTs) in other organisms. In this study, we provide evidence for the presence of four different subtypes of DUR3-like UTs that belong to two distinct families. Two of the UT subtypes were subject to qRT-PCR analyses to investigate differences in mRNA expression during the acclimation of individuals of both ecotypes to different salinities. Our results indicate that only BW animals regulate DUR3 gene expression in the context of osmoregulation. [ABSTRACT FROM AUTHOR]

Y-99, a promising first-in-class diuretic, is a novel urea transporter inhibitor with oral diuretic activity. However, little is known about the pharmacokinetic profiles of Y-99 in experimental animals. In this study, a method of quantitative determination of Y-99 in rat plasma based on high-performance liquid chromatography-tandem mass spectrometry was developed and validated in selectivity, linearity, recovery and matrix effect, accuracy and precision, stability, carry-over and dilution integrity. Chromatographic separation was conducted on an ACQUITY BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with gradient elution at a 0.3 mL/min flow rate after protein precipitation. Mass spectrometry was performed by a positive electrospray ionization mass spectrometer in multiple reaction monitoring mode. The method showed standard-compliant linearity (1-1,000 ng/mL, r = 0.9991). The intra-day and inter-day accuracy (relative error < 11.2%) and precision (coefficient of variation <8.4%) were within acceptable criteria. The recovery and matrix effects were 97.3-110.7% and 103.7-107.5%, respectively. The stability, dilution integrity and carry-over of the method were also within the acceptable criteria. Pharmacokinetic profiles of Y-99 in rats were first investigated using this method, which was vital for developing novel diuretics without electrolyte imbalance targeting urea transporters., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Recent studies have reported increased levels of urea in the aging brain and various neurological disorders. Additionally, these diseased tissues also have increased expression of the UT-B transporter that regulates urea transport in the brain. However, little is known regarding the actual UT-B protein distribution across the brain in either normal or diseased states. This current study investigated UT-B protein abundance across three regions of the rat brain – anterior, posterior and cerebellum. Endpoint RT-PCR experiments showed that there were no regional differences in UT-B RNA expression (NS, N = 3, ANOVA), whilst Western blotting confirmed no difference in the abundance of a 35 kDa UT-B protein (NS, N = 3–4, ANOVA). In contrast, there was a significant variation in a non-UT-B 100 kDa protein (P

Theodoxus fluviatilis (Linnaeus, 1758) (Gastropoda: Neritidae) is an oligohaline aquatic gastropod that inhabits most of Europe and adjacent areas of Asia. Two different ecotypes can be distinguished: One in freshwater (FW) and another along the Baltic Sea coast in brackish water habitats (BW). Individuals of either ecotype use free amino acids and urea as organic osmolytes to adjust body fluid osmolality to the external medium; however, the BW ecotype is able to accumulate them in larger quantities. The use of urea as an organic osmolyte in aquatic gastropods such as T. fluviatilis has only recently been initially described and raised the question of how urea transport between body fluids and the environment is balanced. Upon examining transcriptome and preliminary genome sequence data of T. fluviatilis, we identified putative homologues of DUR3 genes, which code for urea transporters (UTs) in other organisms. In this study, we provide evidence for the presence of four different subtypes of DUR3-like UTs that belong to two distinct families. Two of the UT subtypes were subject to qRT-PCR analyses to investigate differences in mRNA expression during the acclimation of individuals of both ecotypes to different salinities. Our results indicate that only BW animals regulate DUR3 gene expression in the context of osmoregulation.

We studied urea, thiourea, and methylurea transport and interaction in human red blood cells (RBCs) under conditions of self-exchange (SE), net efflux (NE), and net influx (NI) at pH 7.2. We combined four methods, a four-centrifuge technique, the Millipore-Swinnex filtering technique, the continuous flow tube method, and a continuous pump method to measure the transport of the 14 C-labeled compounds. Under SE conditions, both urea and thiourea show perfect Michaelis-Menten kinetics with half-saturation constants, K ½, (mM), of ≈300 (urea) and ≈20 (thiourea). The solutes show no concentration-dependent saturation under NE conditions. Under NI conditions, transport displays saturation or self-inhibition kinetics with a SE (mM) of ≈210 (urea) and ≈20 (thiourea). Urea, thiourea, and methylurea are competitive inhibitors of the transport of analog solutes. This study supports the hypothesis that the three compounds share the same urea transport system (UT-B). UT-B functions asymmetrically as it saturates from the outside only under SE and NI conditions, whereas it functions as a high-capacity channel-like transporter under NE conditions. When the red blood cell enters the urea-rich kidney tissue, self-inhibition reduces the urea uptake in the cell. When the cell leaves the kidney, the channel-like function of UT-B implies that intracellular urea rapidly equilibrates with external urea. The net result is that the cell during the passage in the kidney capillaries carries urea to the kidney to be excreted while the urea transfer from the kidney via the bloodstream is minimized. K ½,NI The kinetics of urea transport in red blood cells was determined by means of a combination of four methods that ensures a high time resolution. In the present study, we disclose that the urea transporter UT-B functions highly asymmetric being channel-like with no saturation under conditions of net efflux and saturable under conditions of net influx and self-exchange in the concentration range 1-1,000 mM (pH 7.2 and 25-38 °C).NEW & NOTEWORTHY The kinetics of urea transport in red blood cells was determined by means of a combination of four methods that ensures a high time resolution. In the present study, we disclose that the urea transporter UT-B functions highly asymmetric being channel-like with no saturation under conditions of net efflux and saturable under conditions of net influx and self-exchange in the concentration range 1-1,000 mM (pH 7.2 and 25-38 °C).

Urea transporter (UT) inhibitors are a class of promising novel diuretics that do not cause the imbalance of Na + , K + , Cl - , and other electrolytes. In our previous studies, 25a , a promising diuretic candidate inhibiting UT, was discovered and showed potent diuretic activities in rodents. Here, a sensitive liquid chromatography-tandem mass spectrometry method for the quantitation of 25a in rat plasma, urine, feces, bile, and tissue homogenates was developed and validated to support the preclinical pharmacokinetic studies. The tissue distribution, excretion, and plasma protein binding were investigated in rats. After a single oral dose of 25a at 25, 50, and 100 mg/kg, the drug exposure increased linearly with the dose. The drug accumulation was observed after multiple oral doses compared to a single dose. In the distribution study, 25a exhibited a wide distribution to tissues with high blood perfusion, such as kidney, heart, lung, and spleen, and the lowest distribution in the brain and testis. The accumulative excretion rate of 25a was 0.14%, 3.16%, and 0.018% in urine, feces, and bile, respectively. The plasma protein binding of 25a was approximately 60% in rats and 40% in humans. This is the first study on the preclinical pharmacokinetic profiles of 25a .

Urea transporters (UT) play a vital role in the mechanism of urine concentration and are recognized as novel targets for the development of salt-sparing diuretics. Thus, UT inhibitors are promising for development as novel diuretics. In the present study, a novel UT inhibitor with a diarylamide scaffold was discovered by high-throughput screening. Optimization of the inhibitor led to the identification of a promising preclinical candidate, N-[4-(acetylamino)phenyl]-5-nitrofuran-2-carboxamide (1H), with excellent in vitro UT inhibitory activity at the submicromolar level. The half maximal inhibitory concentrations of 1H against UT-B in mouse, rat, and human erythrocyte were 1.60, 0.64, and 0.13 μmol/L, respectively. Further investigation suggested that 8 μmol/L 1H more powerfully inhibited UT-A1 at a rate of 86.8% than UT-B at a rate of 73.9% in MDCK cell models. Most interestingly, we found for the first time that oral administration of 1H at a dose of 100 mg/kg showed superior diuretic effect in vivo without causing electrolyte imbalance in rats. Additionally, 1H did not exhibit apparent toxicity in vivo and in vitro, and possessed favorable pharmacokinetic characteristics. 1H shows promise as a novel diuretic to treat hyponatremia accompanied with volume expansion and may cause few side effects.

The objectives were to determine the effects of forage level and grain processing on whole-body urea kinetics, N balance, serosal-to-mucosal urea flux (Jsm-urea), and messenger ribonucleic acid (mRNA) abundance of urea transporter-B (UT-B; SLC14a1) and aquaporins (AQP) in ovine ruminal, duodenal, and cecal epithelia. Thirty-two wether lambs were blocked by body weight into groups of four and assigned to one of four diets (n = 8) in a 2 × 2 factorial design. Dietary factors were forage level (30% [LF] vs. 70% [HF]) and corn grain processing (whole-shelled [WS] vs. steam-flaked [SF]). Four blocks of lambs (n = 4) were used to determine urea kinetics and N balance using 4-d [15N15N]-urea infusions with concurrent fecal and urine collections. Lambs were killed after 23 d of dietary adaptation. Ruminal, duodenal, and cecal epithelia were collected to determine Jsm-urea and mRNA abundance of UT-B and AQP. Lambs fed LF had greater intakes of dry matter (DMI; 1.20 vs. 0.86 kg/d) and N (NI; 20.1 vs. 15.0 g/d) than those fed HF (P < 0.01). Lambs fed SF had greater DMI (1.20 vs. 0.86 kg/d) and NI (20.6 vs. 14.5 g/d) than those fed WS (P < 0.01). As a percentage of NI, total N excretion was greater in lambs fed HF compared with those fed LF (103% vs. 63.0%; P < 0.01) and was also greater in lambs fed WS compared with those fed SF (93.6% vs. 72.1%; P = 0.02). Retained N (% of NI) was greater in lambs fed LF compared with those fed HF (37.0% vs. -2.55%; P < 0.01). Lambs fed SF had a greater (P = 0.02) retained N (% of NI; 28.0% vs. 6.50%) compared with those fed WS. Endogenous urea production (UER) tended (P = 0.09) to be greater in lambs fed HF compared with those fed LF. As a proportion of UER, lambs fed HF had a greater urinary urea-N loss (0.38 vs. 0.22) and lower urea-N transferred to the gastrointestinal tract (GIT; 0.62 vs. 0.78) or urea-N used for anabolism (as a proportion of urea-N transferred to the GIT; 0.12 vs. 0.26) compared with lambs fed LF (P < 0.01). Ruminal Jsm-urea was unaffected by diet. Duodenal Jsm-urea was greater (P < 0.01) in lambs fed HF compared with LF (77.5 vs. 57.2 nmol/[cm2 × h]). Lambs fed LF had greater (P = 0.03) mRNA expression of AQP3 in ruminal epithelia and tended (P = 0.06) to have greater mRNA expression of AQP3 in duodenal epithelia compared with lambs fed HF. Expression of UT-B mRNA was unaffected by diet. Our results showed that feeding more ruminally available energy improved N utilization, partly through a greater proportion of UER being transferred to the GIT and being used for anabolic purposes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

© 2015 International Society of Nephrology Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics (‘urearetics’) with a different mechanism of action than diuretics that target salt transporters. To study UT inhibi

Small-molecule inhibitors of urea transporter (UT) proteins in kidney have potential application as novel salt-sparing diuretics. The urea analog dimethylthiourea (DMTU) was recently found to inhibit the UT isoforms UT-A1 (expressed in kidney tubule epithelium) and UT-B (expressed in kidney vasa recta endothelium) with IC50 of 2-3 mM, and was shown to have diuretic action when administered to rats. Here, we measured UT-A1 and UT-B inhibition activity of 36 thiourea analogs, with the goal of identifying more potent and isoform-selective inhibitors, and establishing structure-activity relationships. The analog set systematically explored modifications of substituents on the thiourea including alkyl, heterocycles and phenyl rings, with different steric and electronic features. The analogs had a wide range of inhibition activities and selectivities. The most potent inhibitor, 3-nitrophenyl-thiourea, had an IC50 of ~0.2 mM for inhibition of both UT-A1 and UT-B. Some analogs such as 4-nitrophenyl-thiourea were relatively UT-A1 selective (IC50 1.3 vs. 10 mM), and others such as thioisonicotinamide were UT-B selective (IC50>15 vs. 2.8 mM).

Urea transporters (UTs) facilitate urea diffusion across cell membranes and play an important role in the urinary concentration mechanisms in the kidney. Herein, we injected cRNAs encoding for c-Myc-tagged murine UT-B, UT-A2 or UT-A3 (versus water-injected control) in Lithobates oocytes and evaluated oocyte surface protein expression with biotinylation and immunoblotting, urea uptake using [ 14 C] counts and water permeability (P f ) by video microscopy. Immunoblots of UT-injected oocyte membranes revealed bands with a molecular weight consistent with that of a UT monomer (34 kDa), and UT-injected oocytes displayed significantly increased and phloretin-sensitive urea uptake and P f when compared to day-matched control oocytes. Subtracting the water-injected urea uptake or P f values from those of UT-injected oocytes yielded UT-dependent values*. We demonstrate for the first time that UT-A2 and UT-A3 can transport water, and we confirm that UT-B is permeable to water. Moreover, the [ 14 C] urea*/P f * ratios fell in the sequence mUT-B>mUT-A2>mUT-A3, indicating that UTs can exhibit selectivity to urea and/or water. It is likely that specific kidney regions with high levels of UTs will exhibit increased urea and/or water permeabilities, directly influencing urine concentration. Furthermore, UT-mediated water transport activity must be considered when developing UT-inhibitors as novel diuretics.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Background: As the glomerular filtrate passes through the nephron and into the renal medulla, electrolytes, water, and urea are reabsorbed through the concerted actions of solute carrier channels and aquaporins at various positions along the nephron and in the outer and inner medulla. Proliferating stem cells expressing the nuclear transcription factor Pax2 give rise to renal epithelial cells. Pax2 expression ends once the epithelial cells differentiate into mature proximal and distal tubules, whereas expression of the related Pax8 protein continues. The collecting tubules and renal medulla are derived from Pax2-positive ureteric bud epithelia that continue to express Pax2 and Pax8 in adult kidneys. Despite the crucial role of Pax2 in renal development, functions for Pax2 or Pax8 in adult renal epithelia have not been established., Methods: To examine the roles of Pax2 and Pax8 in the adult mouse kidney, we deleted either Pax2 , Pax8 , or both genes in adult mice and examined the resulting phenotypes and changes in gene expression patterns. We also explored the mechanism of Pax8-mediated activation of potential target genes in inner medullary collecting duct cells., Results: Mice with induced deletions of both Pax2 and Pax8 exhibit severe polyuria that can be attributed to significant changes in the expression of solute carriers, such as the urea transporters encoded by Slc14a2 , as well as aquaporins within the inner and outer medulla. Furthermore, Pax8 expression is induced by high-salt levels in collecting duct cells and activates the Slc14a2 gene by recruiting a histone methyltransferase complex to the promoter., Conclusions: These data reveal novel functions for Pax proteins in adult renal epithelia that are essential for retaining water and concentrating urine., (Copyright © 2020 by the American Society of Nephrology.)

Background: Urea Transporters are a family of membrane channel proteins that facilitate the passive transport of urea across the plasma membrane. UTs are divided into two subgroups, UT-A and UT-B. UT-As are primarily located in renal tubule epithelia and UT-Bs are highly expressed in renal descending vasa recta and extrarenal multiple tissues. Various urea transporter knockout mice exhibit low urine concentrating ability, which suggests that UTs are novel diuretic targets. With highthroughput screening of small molecule drug-like compound libraries, various potent UT inhibitors with IC50 at nanomolar level were identified. Furthermore, selective UT inhibitors exhibit diuretic activity without disturbing electrolyte and metabolism balance, which confirms the potential of UTs as diuretic targets and UT inhibitors as novel diuretics that do not cause electrolyte imbalance., Objective: This review article summarizes the identification and validation of urea transporter as a potential diuretic target and the discovery of small molecule UT inhibitors as a novel type of diuretics., Conclusion: UTs are a potential diuretic target. UT inhibitors play significant diuresis and can be developed to diuretics without disturbing electrolyte balance., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Facilitative UT-B urea transporters play important physiological roles in numerous tissues, including the urino-genital tract. Previous studies have shown that urothelial UT-B transporters are crucial to bladder function in a variety of mammalian species. Using the RT4 bladder urothelial cell line, this study investigated the potential osmotic regulation of human UT-B transporters. Initial end-point PCR experiments confirmed expression of both UT-B1 and UT-B2 transcripts in RT4 cells. Western blotting analysis revealed glycosylated UT-B protein to be highly abundant and immunolocalization experiments showed it was predominantly located on the plasma membrane. Further PCR experiments suggested that a 48 hr, NaCl-induced raise in external osmolality increased expression of UT-B transcripts. Importantly, these NaCl-induced changes also significantly increased UT-B protein abundance (p < .01, n = 7, ANOVA), whereas mannitol-induced changes in external osmolality had no effect (NS, n = 4, ANOVA). Finally, similar increases in both UT-B RNA expression and protein abundance were observed with urea-induced changes to external osmolality (p < .05, n = 4, ANOVA). In conclusion, these findings strongly suggest that increases in external osmolality, via either NaCl or urea, can regulate human urothelial UT-B transporters., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Urea transporters (UT) play a vital role in the mechanism of urine concentration and are recognized as novel targets for the development of salt-sparing diuretics. Thus, UT inhibitors are promising for development as novel diuretics. In the present study, a novel UT inhibitor with a diarylamide scaffold was discovered by high-throughput screening. Optimization of the inhibitor led to the identification of a promising preclinical candidate, N -[4-(acetylamino)phenyl]-5-nitrofuran-2-carboxamide (1H), with excellent in vitro UT inhibitory activity at the submicromolar level. The half maximal inhibitory concentrations of 1H against UT-B in mouse, rat, and human erythrocyte were 1.60, 0.64, and 0.13 μmol/L, respectively. Further investigation suggested that 8 μmol/L 1H more powerfully inhibited UT-A1 at a rate of 86.8% than UT-B at a rate of 73.9% in MDCK cell models. Most interestingly, we found for the first time that oral administration of 1H at a dose of 100 mg/kg showed superior diuretic effect in vivo without causing electrolyte imbalance in rats. Additionally, 1H did not exhibit apparent toxicity in vivo and in vitro , and possessed favorable pharmacokinetic characteristics. 1H shows promise as a novel diuretic to treat hyponatremia accompanied with volume expansion and may cause few side effects. In the present study, a novel urea transporters (UT) inhibitor with a diarylamide scaffold was discovered by high throughput screening. Optimization of the hit compound E04 led to the identification of 1H , with orally diuretic activity, more powerful inhibition activity on UT-A1, favorable pharmacokinetic characteristics and without electrolyte disturbance. Image 1 [ABSTRACT FROM AUTHOR]

Urea transporter (UT) proteins, which include isoforms of UT-A in kidney tubule epithelia and UT-B in vasa recta endothelia and erythrocytes, facilitate urinary concentrating function. Inhibitors of urea transporter function have potential clinical applications as sodium-sparing diuretics, or 'urearetics,' in edema from different etiologies, such as congestive heart failure and cirrhosis, as well as in syndrome of inappropriate antidiuretic hormone (SIADH). High-throughput screening of drug-like small molecules has identified UT-A and UT-B inhibitors with nanomolar potency. Inhibitors have been identified with different UT-A versus UT-B selectivity profiles and putative binding sites on UT proteins. Studies in rodent models support the utility of UT inhibitors in reducing urinary concentration, though testing in clinically relevant animal models of edema has not yet been done.

Urea transporters (UTs) facilitate urea diffusion across cell membranes and play an important role in the urinary concentration mechanisms in the kidney. Herein, we injected cRNAs encoding for c-Myc-tagged murine UT-B, UT-A2 or UT-A3 (versus water-injected control) in Lithobates oocytes and evaluated oocyte surface protein expression with biotinylation and immunoblotting, urea uptake using [14C] counts and water permeability (Pf) by video microscopy. Immunoblots of UT-injected oocyte membranes revealed bands with a molecular weight consistent with that of a UT monomer (34 kDa), and UT-injected oocytes displayed significantly increased and phloretin-sensitive urea uptake and Pf when compared to day-matched control oocytes. Subtracting the water-injected urea uptake or Pf values from those of UT-injected oocytes yielded UT-dependent values*. We demonstrate for the first time that UT-A2 and UT-A3 can transport water, and we confirm that UT-B is permeable to water. Moreover, the [14C] urea*/Pf* ratios fell in the sequence mUT-B>mUT-A2>mUT-A3, indicating that UTs can exhibit selectivity to urea and/or water. It is likely that specific kidney regions with high levels of UTs will exhibit increased urea and/or water permeabilities, directly influencing urine concentration. Furthermore, UT-mediated water transport activity must be considered when developing UT-inhibitors as novel diuretics. This article has an associated First Person interview with the first author of the paper.

Urea transporter (UT) inhibitors are a class of promising novel diuretics that do not cause the imbalance of Na+, K+, Cl−, and other electrolytes. In our previous studies, 25a, a promising diuretic candidate inhibiting UT, was discovered and showed potent diuretic activities in rodents. Here, a sensitive liquid chromatography–tandem mass spectrometry method for the quantitation of 25a in rat plasma, urine, feces, bile, and tissue homogenates was developed and validated to support the preclinical pharmacokinetic studies. The tissue distribution, excretion, and plasma protein binding were investigated in rats. After a single oral dose of 25a at 25, 50, and 100 mg/kg, the drug exposure increased linearly with the dose. The drug accumulation was observed after multiple oral doses compared to a single dose. In the distribution study, 25a exhibited a wide distribution to tissues with high blood perfusion, such as kidney, heart, lung, and spleen, and the lowest distribution in the brain and testis. The accumulative excretion rate of 25a was 0.14%, 3.16%, and 0.018% in urine, feces, and bile, respectively. The plasma protein binding of 25a was approximately 60% in rats and 40% in humans. This is the first study on the preclinical pharmacokinetic profiles of 25a.

Abstract Facilitative UT‐B urea transporters play important physiological roles in numerous tissues, including the urino‐genital tract. Previous studies have shown that urothelial UT‐B transporters are crucial to bladder function in a variety of mammalian species. Using the RT4 bladder urothelial cell line, this study investigated the potential osmotic regulation of human UT‐B transporters. Initial end‐point PCR experiments confirmed expression of both UT‐B1 and UT‐B2 transcripts in RT4 cells. Western blotting analysis revealed glycosylated UT‐B protein to be highly abundant and immunolocalization experiments showed it was predominantly located on the plasma membrane. Further PCR experiments suggested that a 48 hr, NaCl‐induced raise in external osmolality increased expression of UT‐B transcripts. Importantly, these NaCl‐induced changes also significantly increased UT‐B protein abundance (p

Although the role of urea in urine concentration is known, the effect of urea handling by the urea transporters (UTs), UT-A1 and UT-A3, on sodium balance remains elusive. Serum and urinary sodium concentration is similar between wild-type mice (WT) and UT-A3 null (UT-A3 KO) mice; however, mice lacking both UT-A1 and UT-A3 (UT-A1/A3 KO) have significantly lower serum sodium and higher urinary sodium. Protein expression of renal sodium transporters is unchanged among all three genotypes. WT, UT-A3 KO, and UT-A1/A3 KO acutely respond to hydrochlorothiazide and furosemide; however, UT-A1/A3 KO fail to show a diuretic or natriuretic response following amiloride administration, indicating that baseline epithelial Na + channel (ENaC) activity is impaired. UT-A1/A3 KO have more ENaC at the apical membrane than WT mice, and single-channel analysis of ENaC in split-open inner medullary collecting duct (IMCD) isolated in saline shows that ENaC channel density and open probability is higher in UT-A1/A3 KO than WT. UT-A1/A3 KO excrete more urinary nitric oxide (NO), a paracrine inhibitor of ENaC, and inner medullary nitric oxide synthase 1 mRNA expression is ~40-fold higher than WT. Because endogenous NO is unstable, ENaC activity was reassessed in split-open IMCD with the NO donor PAPA NONOate [1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine)], and ENaC activity was almost abolished in UT-A1/A3 KO. In summary, loss of both UT-A1 and UT-A3 (but not UT-A3 alone) causes elevated medullary NO production and salt wasting. NO inhibition of ENaC, despite elevated apical accumulation of ENaC in UT-A1/A3 KO IMCD, appears to be the main contributor to natriuresis in UT-A1/A3 KO mice.

Urea is present in all ecosystems, as a result of the metabolism of different organisms and also of human activity, being the world's most common form of nitrogen fertilizer. Fungi and plants can use urea as a nitrogen source, taking it up from the environment through specialized active transport proteins. These proteins belong to a subfamily of urea/H + symporters included in the Solute:Sodium Symporter (SSS) family of transporters. In this review we summarize the current knowledge on this group of transporters, based on our previous studies on Aspergillus nidulans UreA. We delve into its transcriptional and post-translational regulation, structure-function relationships, transport mechanism, and certain aspects of its biogenesis. Recent findings suggest that this urea transporter subfamily is more expanded than originally thought, with representatives found in organisms as diverse as Archaea and mollusks, which raises questions on evolutionary aspects. A. nidulans ureA knockout strains provide a valuable platform for expressing urea transporters from diverse sources, facilitating their characterization and functional analysis. In this context, given the close relationship between plant and fungal active urea transporters, this knowledge could serve to develop strategies to improve the efficiency of applied urea as fertilizer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The original UT-1 transporter gene was initially identified in the spiny dogfish ( Squalus acanthias ), but localization of the UT-1 protein was not determined. Subsequent UT-1 expression was shown to localize to the collecting tubule (CT) of the shark nephron in other shark species, with expression in a closely related chimaera species also located additionally at a lower level in the intermediate-I segment (IS-I) of the nephron. In spiny dogfish, two UT-1 splice variants are known (UT-1 long and short), and there was also a second UT-1 gene described (here termed Brain UT). In this study, a second splice variant of the second Brain UT gene was discovered. Expression profiles (mRNA) of UT-1 long and short and Brain UT were determined in a number of spiny dogfish tissues. Quantitative PCR in kidney samples showed that the level of the short variant of UT-1 was around 100 times higher than the long variant, which was itself expressed around 10 times higher than Brain UT cDNA/mRNA (in kidney). For the long variant, there was a significantly higher level of mRNA abundance in fish acclimatized to 75% seawater. Ultimately, three UT-1 antibodies were made that could bind to both the UT-1 short and long variant proteins. The first two of these showed bands of appropriate sizes on Western blots of around 52.5 and 46 kDa. The second antibody had some additional lower molecular weight bands. The third antibody was mainly bound to the 46 kDa band with faint 52.5 kDa staining. Both the 52.5 and 46 kDa bands were absent when the antibodies were pre-blocked with the peptide antigens used to make them. Across the three antibodies, there were many similarities in localization but differences in subcellular localization. Predominantly, antibody staining was greatest in the intermediate segment 1 (IS-I) and proximal (PIb) segments of the first sinus zone loop of the nephron, with reasonably strong expression also found at the start and middle of the late distal tubule (LDT; second sinus zone loop). While some expression in the collecting tubule (CT) could not be ruled out, the level of staining seemed to be low or non-existent in convoluted bundle zone nephron segments such as the CT. Hence, this suggests that spiny dogfish have a fundamentally different mode of urea absorption in comparison to that found in other shark species, potentially focused more on the nephron sinus zone loops than the CT.

Urea transporters (UTs) are transmembrane urea-selective channel proteins that include two UT subfamilies, UT-A and UT-B. UT-A subfamily includes six members, UT-A1 to UT-A6, which are mainly expressed in kidney. UT-B subfamily has only one member that has a wide distribution in the body. UTs have been confirmed to play important roles in urinary concentration via the phenotypic analysis of 6 UT selective knockout mouse models. Experimental results suggest that UTs might be diuretic targets and that UT inhibitors might be developed as novel diuretics. This article reviews the physiological function and drug discovery of UT.

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 UTB 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. [ABSTRACT FROM AUTHOR]

This paper aims to study the effect of the dietary treatments on mRNA expression of urea transporter B (UT-B) and some aquaporins (AQP) in rumen epithelium of Italian Simmental young bulls. Eighty animals allocated to 16 pens were fed from about 500 to 650 kg body weight with four experimental diets, which resulted from the combination of two crude protein levels (125 and 110 g/kg dry matter, diets M and L, respectively) and two nitrogen sources (soybean meal (SBM) or SBM partly replaced by an isonitrogenous mixture of corn and urea; diets -U and +U, respectively). At slaughtering samples of blood and rumen epithelium were collected from six bulls for each diet. Blood samples were analysed for haematological parameters and quantitative PCR was carried out on the mRNA extracted from the rumen epithelium samples. The bulls fed diets M had lower plasma concentrations of aspartate aminotransferase than those receiving diets L (78.9 vs. 88.3 U/l, p = 0.04). Plasma urea was higher (p = 0.03) for diets M and lower for diets +U (2.0 vs. 2.5 and 1.73 vs. 2.00 mmol/l, respectively, in M and L diets, p = 0.04). The effect of dietary treatments on rumen UT expression were limited to AQP3, which was down regulated (p = 0.01) in diets +U. Finally, a high positive correlation (R 2  = 0.871) between the expressions of AQP7 and AQP10 was found. In conclusion, the AQP3 appears very responsive to dietary treatments and therefore it is a candidate to be further studied in rumen metabolism experiments. The close relationship between mRNA expression of AQP7 and AQP10 indicates a similar function of these two proteins.