Whole-body urea kinetics and functional roles of urea transporters and aquaporins in urea secretion into the rumen in sheep fed diets varying in crude protein content and corn grain processing method.

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.

In ruminants, urea produced in the liver as a nitrogenous waste can be secreted into the rumen where it can be used by rumen microorganisms as a source of nitrogen (N) for their growth. Therefore, urea secretion into the rumen is nutritionally important for ruminants particularly when dietary N intake is deficient. Urea secretion into the rumen occurs via transporter proteins in rumen tissue referred to as urea transporters (UT-B) and aquaporins (AQP). The purpose of this research was to investigate the effects of dietary crude protein (CP) content and corn grain processing on urea secretion into the rumen and the function of UT-B and AQP. Thirty-two Rideau-Arcott lambs were assigned to 1 of 4 diets in a 2 × 2 factorial design. Dietary factors were CP content (11% [LP] vs. 16% [HP]) and corn processing (whole-shelled [WSC] vs. steam-flaked [SFC] corn). When compared to feeding HP, feeding LP tended to increase urea secretion into the rumen, but there were no corresponding changes in UT-B and AQP function. Corn processing did not influence urea secretion into the rumen; however, the portion of urea secretion that was facilitated via UT-B and AQP was greater in lambs fed SFC compared to those fed WSC.

Urea transporter UT-A1 as a novel drug target for hyponatremia.

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.

An update to Kidd blood group system.

Since publication of the original Immunohematology review of the Kidd blood group system in 2015 (Hamilton JR. Kidd blood group system: a review. Immunohematology 2015;31:29-34), knowledge has mushroomed pertaining to gene structure, alleles causing variant and null phenotypes, clinical significance in renal transplant and hemolytic disease of the fetus and newborn, and physiologic functions of urea transporters in non-renal tissues. This review will detail much of this new information.

Functional asymmetry of the urea transporter UT-B in human red blood cells.

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 14C-labeled compounds. Under SE conditions, both urea and thiourea show perfect Michaelis-Menten kinetics with half-saturation constants, K½,SE (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 K½,NI (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.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).

Structural characterization of human urea transporters UT-A and UT-B and their inhibition.

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, UTBinh-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.

Investigating nitrogen movement in North Pacific spiny dogfish (Squalus acanthias suckleyi), with focus on UT, Rhp2, and Rhbg mRNA abundance.

For ureosmotic marine elasmobranchs, the acquisition and retention of nitrogen is critical for the synthesis of urea. To better understand whole-body nitrogen homeostasis, we investigated mechanisms of nitrogen trafficking in North Pacific spiny dogfish (Squalus acanthias suckleyi). We hypothesized that the presence of nitrogen within the spiral valve lumen would affect both the transport of nitrogen and the mRNA abundance of a urea transporter (UT) and two ammonia transport proteins (Rhp2, Rhbg) within the intestinal epithelium. The in vitro preincubation of intestinal tissues in NH4Cl, intended to simulate dietary nitrogen availability, showed that increased ammonia concentrations did not significantly stimulate the net uptake of total urea or total methylamine. We also examined the mRNA abundance of UT, Rhp2, and Rhbg in the gills, kidney, liver, and spiral valve of fasted, fed, excess urea fed, and antibiotic-treated dogfish. After fasting, hepatic UT mRNA abundance was significantly lower, and Rhp2 mRNA in the gills was significantly higher than the other treatments. Feeding significantly increased Rhp2 mRNA levels in the kidney and mid spiral valve region. Both excess urea and antibiotics significantly reduced Rhbg mRNA levels along all three spiral valve regions. The antibiotic treatment also significantly diminished UT mRNA abundance levels in the anterior and mid spiral valve, and Rhbg mRNA levels in the kidney. In our study, no single treatment had significantly greater influence on the overall transcript abundance of the three transport proteins compared to another treatment, demonstrating the dynamic nature of nitrogen balance in these ancient fish.

SLC14A1 is a new biomarker in renal cancer.

BACKGROUND: Renal cancer is one of the common malignant tumors of the urinary tract, prone to distant metastasis and drug resistance, with a poor clinical prognosis. SLC14A1 belongs to the solute transporter family, which plays a role in urinary concentration and urea nitrogen recycling in the renal, and is closely associated with the development of a variety of tumors. METHODS: Transcription data for renal clear cell carcinoma (KIRC) were obtained from the public databases Gene Expression Omnibus database (GEO) and The Cancer Genome Atlas (TCGA), and we investigated the differences in SLC14A1 expression in cancerous and normal tissues of renal cancer, its correlation with the clinicopathological features of renal cancer patients. Then, we verified the expression levels of SLC14A1 in renal cancer tissues and their Paracancerous tissues using RT-PCR, Western-blotting and immunohistochemistry. Finally, we used renal endothelial cell line HEK-293 and renal cancer cell lines 786-O and ACHN to explore the effects of SLC14A1 on the biological behaviors of renal cancer cell proliferation, invasion and metastasis using EDU, MTT proliferation assay, Transwell invasion assay and scratch healing assay. RESULTS: SLC14A1 was lowly expressed in renal cancer tissues and this was further validated by RT-PCR, Western blotting, and immunohistochemistry in our clinical samples. Analysis of KIRC single-cell data suggested that SLC14A1 was mainly expressed in endothelial cells. Survival analysis showed that low levels of SLC14A1 expression were associated with a better clinical prognosis. In biological behavioral studies, we found that upregulation of SLC14A1 expression levels inhibited the proliferation, invasion, and metastatic ability of renal cancer cells. CONCLUSION: SLC14A1 plays an important role in the progression of renal cancer and has the potential to become a new biomarker for renal cancer.

Nitrogen transporters along the intestinal spiral valve of cloudy catshark (Scyliorhinus torazame): Rhp2, Rhbg, UT.

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.

An HPLC-MS/MS Method for Pharmacokinetic Study of Y-99: A Novel Diuretic Agent Targeting Urea Transporters.

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.

Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter.

Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins.

Study on the role of SLC14A1 gene in biochemical recurrence of prostate cancer.

Prostate cancer (PCa) is a common malignant disease among men and biochemical recurrence (BCR) is considered to be a decisive risk factor for clinical recurrence and PCa metastasis. Clarifying the genes related to BCR and its possible pathways is vital for providing diagnosis and treatment methods to delay the progress of BCR. An analysis of data concerning PCa from previous datasets of The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) was performed. Immunohistochemical (IHC) staining were used to evaluate the expression of SLC14A1 in prostate tissues. Kaplan-Meier analysis, Pearson correlation, and single sample Gene Set Enrichment Analysis (ssGSEA) were used to identify the potential pathway and molecular mechanism of the function of SLC14A1 in BCR of PCa. The expression of SLC14A1 is significantly reduced in prostate cancer cells and tissue comparing to normal prostate epithelial cell and para-cancerous tissue. As indicated by Kaplan-Meier analysis, High expression of SLC14A1 could increase the BCR-free survival time of PCa patients. This effect might be related to the interaction with miRNAs (has-miR-508, has-mir-514a2, and has-mir-449a) and the infiltration of B cells. SLC14A1 is a novel important gene associated with BCR of PCa, and further studies of its molecular mechanism may delay the progress of BCR.

A potential link between AQP3 and SLC14A1 gene expression level and clinical parameters of maintenance hemodialysis patients.

BACKGROUND: The transport of water and urea through the erythrocyte membrane is facilitated by aquaporins such as aquaglyceroporin (AQP3), and type B urea transporters (UT-B). As they may play an important role in osmotic balance of maintenance hemodialysis (HD) patients, the aim of the present study was to determine whether any relationship exists between the expression of their genes and the biochemical / clinical parameters in HD patients. METHODS: AQP3 and UT-B (SLC14A1) gene expression was evaluated using RT-qPCR analysis in 76 HD patients and 35 participants with no kidney failure. RESULTS: The HD group demonstrated significantly higher median expression of AQP3 and UT-B (Z = 2.16; P = 0.03 and Z = 8.82; p < 0.0001, respectively) than controls. AQP3 negatively correlated with pre-dialysis urea serum concentration (R = -0.22; P = 0.049) and sodium gradient (R = -0.31; P = 0.04); however, no significant UT-B correlations were observed. Regarding the cause of end-stage kidney disease, AQP3 expression positively correlated with erythropoietin dosages in the chronic glomerulonephritis (GN) subgroup (R = 0.6; P = 0.003), but negatively in the diabetic nephropathy subgroup (R = -0.59; P = 0.004). UT-B positively correlated with inter-dialytic weight gain% in the GN subgroup (R = 0.47; P = 0.03). CONCLUSION: Maintenance hemodialysis seems significantly modify AQP3 and UT-B expression but their link to clinical and biochemical parameters needs further large-scale evaluation.

Immunohistochemical detection of urea transporter-A in the tear-producing part of the lacrimal system.

OBJECTIVE: Urea constitutes a physiological and presumably well-regulated constituent of tear fluid. Its lacrimal concentration is significantly decreased in dry eye disease. Urea homeostasis within the tear fluid may also depend on the expression of urea transporters. The present study reports on the expression patterns of urea transporter A (UT-A) in the cells and tissues of the ocular surface and the lacrimal glands. METHODS: UT-A immunohistochemistry was performed on 5 µm paraffin sections of paraformaldehyde-fixed human, porcine, and murine corneas, eyelids, and lacrimal glands (n = 5 each). RESULTS: UT-A immunostaining was largely comparable in all three species. UT-A signals were detected in the corneal epithelium and endothelium, in the conjunctival epithelium, in the acinar cells and excretory ducts of the lacrimal gland, Meibomian gland, and in the glands of Moll and Zeis. The Meibomian glands and the glands of Zeis exhibited a marked UT-A-positive staining in the basal cells of the alveolar epithelia and in the ductal epithelia. CONCLUSION: UT-A shows comparable expression patterns to UT-B (previous study) at the ocular surface and in the lacrimal glands, as determined by immunohistochemistry. The presence of both urea transporters in the lacrimal functional unit suggests that they are essential for the normal function of the lacrimal system and the integrity of the tear film. Potential alterations in urea transporter expression might be associated with the significant reduction of urea found in the tear fluid of dry eye patients. They may thus play an important role in the pathogenesis of dry eye disease.

Urea transporter-B expression on the ocular surface and in the lacrimal glands.

OBJECTIVE: Urea is a component of tear fluid showing a significantly decreased concentration in dry eye disease. The urea content of tear fluid may depend on urea transporters. The purpose of this study was to examine the expression of urea transporter B (UT-B) at the ocular surface and in the lacrimal glands. METHODS: UT-B protein and mRNA expression was investigated in human, porcine, and murine samples. Immunohistochemical staining for UT-B was performed on paraffin sections of human, porcine, and murine corneas, eyelids, and lacrimal glands (n = 5 each). Reverse transcriptase polymerase chain reaction was conducted to detect UT-B mRNA in human and murine cornea, conjunctiva, Meibomian gland, and lacrimal gland (n = 5 each). RESULTS: UT-B protein expression was comparable in all three species. It was found in the corneal epithelium and endothelium, in the conjunctival epithelium, in the end pieces and excretory ducts of the lacrimal gland, Meibomian gland, and in the glands of Moll and Zeis. The glands of Zeis and the Meibomian glands showed intense UT-B signals in the basal layers of the alveolar epithelia and in the cells of the ductal epithelia. UT-B mRNA was detected in all samples analyzed. CONCLUSION: UT-B is expressed by the cells and tissues of the ocular surface and in the lacrimal glands. Potential changes in urea transporter expression might have implications for the pathogenesis of dry eye disease. Since comparable results were obtained for all species investigated, the presented findings may open the door for DED-relevant experimentation on porcine and murine model systems.

Preclinical Pharmacokinetic Studies of a Novel Diuretic Inhibiting Urea Transporters.

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 transporter B downregulates polyamines levels in melanoma B16 cells via p53 activation.

Urea transporter B (UT-B, encoded by the SLC14A1 gene) is a membrane channel protein involved in urea transmembrane transport. Compared with normal tissues, UT-B expression is significantly decreased in most tumours, especially melanoma. However, the UT-B role in tumorigenesis and development is still unclear. Herein, we investigated the effects of UT-B overexpression on polyamine metabolism and the urea cycle in murine melanoma B16 cells, to explore the roles of mitochondrial dysfunction and p53 activation in cell growth and polyamines metabolism. UT-B overexpression in B16 cells decreased cell growth, increased apoptosis, and significantly altered metabolic pathways related to the urea cycle, which were characterized by reduced production of urea and polyamines and increased production of nitric oxide. Subsequently, we observed that activation of the p53 pathway may be the main cause of the above phenomena. The p53 inhibitor pifithrin-α partially restored the production of polyamines, but the mitochondrial morphology and function were still impaired. Further treatment of UT-B-overexpressing B16 cells with reactive oxygen species scavenging agent N-acetyl-l-cysteine and coenzyme Q10 restored cell viability and mitochondrial function and increased polyamine production. In conclusion, UT-B overexpression caused mitochondrial dysfunction and increased oxidative stress in B16 cells, and then activated p53 expression, which may be one of the mechanisms leading to the decrease in intracellular polyamines.

A critical analysis of urea transporter B inhibitors: molecular fingerprints, pharmacophore features for the development of next-generation diuretics.

Urea transporter is a membrane transport protein. It is involved in the transferring of urea across the cell membrane in humans. Along with urea transporter A, urea transporter B (UT-B) is also responsible for the management of urea concentration and blood pressure of human. The inhibitors of urea transporters have already generated a huge attention to be developed as alternate safe class of diuretic. Unlike conventional diuretics, these inhibitors are suitable for long-term therapy without hampering the precious electrolyte imbalance in the human body. In this study, UT-B inhibitors were analysed by using multi-chemometric modelling approaches. The possible pharmacophore features along with favourable and unfavourable sub-structural fingerprints for UT-B inhibition are extracted. This information will guide the medicinal chemist to design potent UT-B inhibitors in future.

N-(4-acetamidophenyl)-5-acetylfuran-2-carboxamide as a novel orally available diuretic that targets urea transporters with improved PD and PK properties.

Urea transporters (UTs) have been identified as new targets for diuretics. Functional deletion of UTs led to urea-selective urinary concentrating defects with relative salt sparing. In our previous study, a UT inhibitor with a diarylamide scaffold, which is denoted as 11a, was demonstrated as the first orally available UT inhibitor. However, the oral bioavailability of 11a was only 4.38%, which obstructed its clinical application. In this work, by replacing the nitro group of 11a with an acetyl group, 25a was obtained. Compared with 11a, 25a showed a 10 times stronger inhibitory effect on UT-B (0.14 µM vs. 1.41 µM in rats, and 0.48 µM vs. 5.82 µM in mice) and a much higher inhibition rate on UT-A1. Moreover, the metabolic stability both in vitro and in vivo and the drug-like properties (permeability and solubility) of 25a were obviously improved compared with those of 11a. Moreover, the bioavailability of 25a was 15.18%, which was 3 times higher than that of 11a, thereby resulting in significant enhancement of the diuretic activities in rats and mice. 25a showed excellent potential for development as a promising clinical diuretic candidate for targeting UTs to treat diseases that require long-term usage of diuretics, such as hyponatremia.

Urea transporter and its specific and nonspecific inhibitors: State of the art and pharmacological perspective.

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.

Role of the cardiovascular system in ammonia excretion in early life stages of zebrafish (Danio rerio).

The purpose of this study was to investigate if the cardiovascular system is important for ammonia excretion in the early life stages of zebrafish. Morpholino knockdowns of cardiac troponin T (TNNT2) or vascular endothelial growth factor A (VEGFA) provided morphants with nonfunctional circulation. At the embryonic stage [30-36 h postfertilization (hpf)], ammonia excretion was not constrained by a lack of cardiovascular function. At 2 days postfertilization (dpf) and 4 dpf, morpholino knockdowns of TNNT2 or VEGFA significantly reduced ammonia excretion in all morphants. Expression of rhag, rhbg, and rhcgb showed no significant changes but the mRNA levels of the urea transporter (ut) were upregulated in the 4 dpf morphants. Taken together, rhag, rhbg, rhcgb, and ut gene expression and an unchanged tissue ammonia concentration but an increased tissue urea concentration, suggest that impaired ammonia excretion led to increased urea synthesis. However, in larvae anesthetized with tricaine or clove oil, ammonia excretion was not reduced in the 4 dpf morphants compared with controls. Furthermore, oxygen consumption was reduced in morphants regardless of anesthesia. These results suggest that cardiovascular function is not directly involved in ammonia excretion, but rather reduced activity and external convection may explain reduced ammonia excretion and compensatory urea accumulation in morphants with reduced cardiovascular function.