UT-B Protein Research Articles

Regional investigation of UT-B urea transporters in the rat brain

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 < 0.001, N = 3–4, ANOVA), which was also detected by anti-UT-B antibodies. Using the C6 rat astrocyte cell line, Western blot analysis showed that 48-h incubation in either 5 mM or 10 mM significantly increased a 30–45 kDa UT-B protein signal (P < 0.05, N = 3, ANOVA). Furthermore, investigation of compartmentalized C6 protein samples showed the 30–45 kDa signal in the membrane fraction, whilst the 100 kDa non-UT-B signal was predominantly in the cytosolic fraction. Finally, immunolocalization studies gave surprisingly weak detection of rat UT-B, except for strong staining of red blood cells in the cerebellum. In conclusion, this study confirmed that RNA expression and protein abundance of UT-B were equal across all regions of the rat brain, suggesting that urea levels were also similar. However, it also highlighted some of the technical challenges of studying urea transporters at the protein level.

Osmotic regulation of UT-B urea transporters in the RT4 human urothelial cell line.

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.

Urea-modulated UT-B urea transporter internalization is clathrin- and caveolae-dependent in infantile hemangioma-derived vascular endothelial cells.

The aim of this study was to investigate the manner of urea-modulated UT-B urea transporter (UT) internalization in infantile hemangioma-derived vascular endothelial cells (HemECs). The immunohistochemistry assay was performed to identify infancy hemangioma-derived endothelial cell line (XPTS-1) cells. Cell toxicity was detected with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Quantitative real-time polymerase chain reaction and Western blot analysis were measured to analyze the expression of UT-B. UT-B internalization was observed by confocal microscopy. The clathrin inhibitor chlorpromazine (CPZ) and caveolin endocytic disrupter methyl-β-cyclodextrin (MβCD) were used in XPTS-1 cells transfected with UT-B-GFP to repress endocytosis. Urea-promoted UT-B expression in a concentration-dependent manner in an infantile XPTS-1 cell line. CPZ and MβCD significantly inhibited UT-B protein internalization. The pretreatment of UT-B-GFP cells with adaptor protein2 (AP2)-μ2-siRNA and caveolin-siRNA significantly inhibited UT-B protein internalization. Our findings suggested that urea-mediated UT-B UT internalization is clathrin and caveolae dependent in infantile HemECs.

Identification of a Novel UT-B Urea Transporter in Human Urothelial Cancer.

The urea transporter UT-B is widely expressed and has been studied in erythrocyte, kidney, brain and intestines. Interestingly, UT-B gene has been found more abundant in bladder than any other tissue. Recently, gene analyses demonstrate that SLC14A1 (UT-B) gene mutations are associated with bladder cancer, suggesting that urea transporter UT-B may play an important role in bladder carcinogenesis. In this study, we examined UT-B expression in bladder cancer with human primary bladder cancer tissues and cancer derived cell lines. Human UT-B has two isoforms. We found that normal bladder expresses long form of UT-B2 but was lost in 8 of 24 (33%) or significantly downregulated in 16 of 24 (67%) of primary bladder cancer patients. In contrast, the short form of UT-B1 lacking exon 3 was detected in 20 bladder cancer samples. Surprisingly, a 24-nt in-frame deletion in exon 4 in UT-B1 (UT-B1Δ24) was identified in 11 of 20 (55%) bladder tumors. This deletion caused a functional defect of UT-B1. Immunohistochemistry revealed that UT-B protein levels were significantly decreased in bladder cancers. Western blot analysis showed a weak UT-B band of 40 kDa in some tumors, consistent with UT-B1 gene expression detected by RT-PCR. Interestingly, bladder cancer associate UT-B1Δ24 was barely sialylated, reflecting impaired glycosylation of UT-B1 in bladder tumors. In conclusion, SLC14A1 gene and UT-B protein expression are significantly changed in bladder cancers. The aberrant UT-B expression may promote bladder cancer development or facilitate carcinogenesis induced by other carcinogens.

Urea Transporter B and MicroRNA-200c Differ in Kidney Outer Versus Inner Medulla Following Dehydration

BackgroundUrea transporters (UTs) are important in urine concentration and in urea recycling, and UT-B has been implicated in both. In kidney, UT-B was originally localized to outer medullary descending vasa recta, and more recently detected in inner medullary descending vasa recta. Endogenously produced microRNAs (miRs) bind to the 3′UTR of genes and generally inhibit their translation, thus playing a pivotal role gene regulation. MethodsMice were dehydrated for 24 hours then sacrificed. Inner and outer medullas were analyzed by polymerase chain reaction (PCR) and quantitative PCR for miRNA expression and analyzed by western blotting for protein abundance. ResultsMiRNA sequencing analysis of mouse inner medullas showed a 40% increase in miRNA-200c in dehydrated mice compared with controls. An in silico analysis of the targets for miR-200c revealed that miRNA-200c could directly target the gene for UT-B. PCR confirmed that miR-200c is up-regulated in the inner medullas of dehydrated mice while western blot showed that UT-B protein abundance was down-regulated in the same portion of the kidney. However, in the outer medulla, miR-200c was reduced and UT-B protein was increased in dehydrated mice. ConclusionsThis is the first indication that UT-B protein and miR-200c may each be differentially regulated by dehydration within the kidney outer and inner medulla. The inverse correlation between the direction of change in miR-200c and UT-B protein abundance in both the inner and outer medulla suggests that miR-200c may be associated with the change in UT-B protein in these 2 portions of the kidney medulla.

UT-B Urea Transporter Localization in the Bovine Gastrointestinal Tract.

Facilitative UT-B urea transporters play an important role in the urea nitrogen salvaging process that occurs in the gastrointestinal tract of mammals, particularly ruminants. Gastrointestinal UT-B transporters have previously been reported in various ruminant species-including cow, sheep and goat. In this present study, UT-B transporter localization was investigated in tissues throughout the bovine gastrointestinal tract. RT-PCR analysis showed that UT-B2 was the predominant UT-B mRNA transcript expressed in dorsal, ventral and cranial ruminal sacs, while alternative UT-B transcripts were present in other gastrointestinal tissues. Immunoblotting analysis detected a strong, glycosylated ~50kDa UT-B2 protein in all three ruminal sacs. Immunolocalization studies showed that UT-B2 protein was predominantly localized to the plasma membrane of cells in the stratum basale layer of all ruminal sac papillae. In contrast, other UT-B protein staining was detected in the basolateral membranes of the surface epithelial cells lining the abomasum, colon and rectum. Overall, these findings confirm that UT-B2 cellular localization is similar in all ruminal sacs and that other UT-B proteins are located in epithelial cells lining various tissues in the bovine gastrointestinal tract.

High salt-diet reduces SLC14A1 gene expression in the choroid plexus of Dahl salt sensitive rats

Elevated Na+ concentration ([Na+]) in the cerebrospinal fluid (CSF) contributes to the development of salt-sensitive hypertension. CSF is formed by the choroid plexus (CP) in cerebral ventricles, and [Na+] in CSF is controlled by transporters in CP. Here, we examined the effect of high salt diet on the expression of urea transporters (UTs) in the CP of Dahl S vs Dahl R rats using real time PCR. High salt intake (8%, for 2 weeks) did not alter the mRNA levels of UT-A (encoded by SLC14A2 gene) in the CP of either Dahl S or Dahl R rats. In contrast, the mRNA levels of UT-B (encoded by SLC14A1 gene) were significantly reduced in the CP of Dahl S rats on high salt diet as compared with Dahl R rats or Dahl S rats on normal salt diet. Reduced UT-B expression was associated with increased [Na+] in the CSF and elevated mean arterial pressure (MAP) in Dahl S rats treated with high salt diet, as measured by radiotelemetry. High salt diet-induced reduction in UT-B protein expression in the CP of Dahl S rats was confirmed by Western blot. Immunohistochemistry using UT-B specific antibodies demonstrated that UT-B protein was expressed on the epithelial cells in the CP. These data indicate that high salt diet induces elevations in CSF [Na+] and in MAP, both of which are associated with reduced UT-B expression in the CP of Dahl S rats, as compared with Dahl R rats. The results suggest that altered UT-B expression in the CP may contribute to an imbalance of water and electrolytes in the CSF of Dahl S rats on high salt diet, thereby leading to alterations in MAP.

Expression and localization of a UT-B urea transporter in the human bladder.

Facilitative UT-B urea transporters have been shown to play an important role in the urinary concentrating mechanism. Recent studies have now suggested a link between UT-B allelic variation and human bladder cancer risk. UT-B1 protein has been previously identified in the bladder of various mammalian species, but not yet in humans. The aim of the present study was to investigate whether any UT-B protein was present in the human bladder. First, RT-PCR results confirmed that UT-B1 was strongly expressed at the RNA level in the human bladder, whereas UT-B2 was only weakly present. Initial Western blot analysis confirmed that a novel UT-B COOH-terminal antibody detected human UT-B proteins. Importantly, this antibody detected a specific 40- to 45-kDa UT-B signal in human bladder protein. Using a peptide-N-glycosidase F enzyme, this bladder UT-B signal was deglycosylated to a core 30-kDa protein, which is smaller than the predicted size for UT-B1 but similar to many proteins reported to be UT-B1. Finally, immunolocalization experiments confirmed that UT-B protein was strongly expressed throughout all urothelium layers except for the apical membrane of the outermost umbrella cells. In conclusion, these data confirm the presence of UT-B protein within the human bladder. Further studies are now required to determine the precise nature, regulation, and physiological role of this UT-B.

Short communication: Urea transporter protein UT-B in the bovine parotid gland

Ruminant nutrition relies upon the symbiotic relationship that exists with microbial populations in the rumen. Urea transported across the ruminal epithelia and secreted by the salivary glands is a key source of nitrogen for microbial growth in the rumen. As ruminal urea transport can be mediated by specific UT-B urea transporters, this study investigated whether UT-B urea transporters were also present in the bovine salivary gland. Western blotting experiments detected only small amounts of UT-B protein in whole-cell lysate from the bovine parotid gland. In contrast, strong 32 to 34 and 40kDa UT-B proteins were detected in parotid plasma membrane-enriched protein, showing the importance of using enriched samples. These signals were also detected in rumen and correspond to bovine UT-B1 and UT-B2 urea transporters, respectively. Further immunolocalization studies identified that these proteins were located in the ductal system of the parotid gland. This study, therefore, confirmed the presence of UT-B urea transporter protein in the bovine parotid salivary gland.

Urea and Water Permeation across the Human Red Blood Cell Membrane. New Insights into Transport Mechanisms

The human red blood cell (hRBC) has an exceptionally high permeability for water (Pf) and urea (Purea). However, the contribution of several blood group proteins to permeability of both molecules is still a matter of debate. Here, we have taken advantage of 3 extremely rare blood group phenotypes Colton-null, Kidd-null and Gil-null characterized by total deficiency of aquaporin-1 (AQP1), urea transporter (UT-B) and aquaporin-3 (AQP3), respectively. To evaluate the possible contribution of each protein to water and urea permeation, these hRBC were analyzed using a stopped-flow spectrometer. Functional studies showed that AQP1 and UT-B proteins substantially contribute (96%) to the high water permeability of the hRBC membrane. Furthermore, molecular dynamics simulations (MDS) of water diffusion through urea transporter reveal a permeation mechanism different from that observed in AQP1. Interestingly, uptake studies using 14C-urea indicated that the rate of urea transport through UT-B pore was reduced in the absence of AQP1 (Colton-null) and can be modulated by changing the osmolarity conditions. Overall, these data provide evidence that the urea transport through UT-B was controlled by the movement of water molecules into this channel.Taken together, this result allowed to propose molecular mechanisms of permeation and selectivity of UT-B for urea permeability.

Discovery and pharmacological study of a novel diuretic

Diuretics are used widely to raise renal salt and water clearance in a variety of conditions, such as oedema, as well in non-edematous states such as hypertension, which can reduce morbidity and mortality of cardiovascular and cerebrovascular diseases, especially the frequency of stroke and congestive heart failure. However, long-term use of conventional diuretics has several adverse effects including electrolyte disorders, hyperuricemia, hyperlipidemia, and glucose tolerance decrease. Electrolyte abnormalities can induce cardiac arrythmias and sudden death. Therefore, discovering a new diuretic that does not cause electrolyte disturbance becomes a hot issue. Phenotype analysis of knockout mice lacking urea transporter UT-B or various UT-A isoforms has provided evidence for the involvement of UTs in the urinary concentrating mechanism. Functional deletion of UT-B or UT-A isoforms markedly caused polyuria and urea selective low urine concentrating ability. However, deletion of UT-B or UT-As did not affect GFR and clearance rate of the principal solutes (Na+, K+, Cl-) in urine except for urea. Therefore, we suggested a hypothesis that UT inhibitors might be novel diuretics to excrete water without disturbing electrolyte metabolism. Present study discovered a potent small-molecular urea transporter inhibitor, UT-A4, using an erythrocyte osmotic lysis assay. Stopped flow light scattering experiment, a classical assay for measuring water and urea permeability, confirmed that UT-A4 reversibly inhibited UT-B activity. The experiments also showed that UT-A4 targeted the intracellular part of UT-B protein and had the same inhibition activity on influx and efflux of urea across membrane. UTinh-14 has inhibition activity on human, rabbit, rat and mouse UT-B. We used rats as an in vivo test model for determining the diuretic activity of UT-A4. Interestingly, UT-A4 caused dose-dependent polyuria, low urinary osmolality and urea concentration in rats. 18-h water deprivation raised urine concentrating ability in rats with or without UT-A4 treatment. However, urine osmolality and urea concentration remained significantly less in UT-A4 treated rats than that in control rats, except of unchanged non-urea solutes. Osmolality and urea concentration was significantly decreased in inner medullary tissue of UTinh-14 treated rats, but not in HCTZ treated rats. All these results suggest that UTinh-14 caused urea selective diuresis. The excretion of osmoles, urea and non-urea solutes had no significant difference between control and UTinh-14 treated rats. However, HCTZ treated rats had significant higher excretion of osmole and non-urea solutes than control and UTinh-14 treated rats, which made lower blood Na+, K+ and Cl-. These results indicate that UT-A4 is a selective UT inhibitor and has urea selective diuretic activity without disturbing excretion of electrolytes. It might have potential value on drug discovery as a new diuretic without electrolyte imbalance and metabolic disorder. It might also be used as a tool drug to study the physiological roles of UTs in big animal models.

Expression of urea transporters is affected by dietary nitrogen restriction in goat kidney1

Ruminants are known to be able to very effectively recycle urinary urea and reuse it as a source of N for ruminal microbes. It is presumed that urea recycling is accomplished by specialized urea transporters (UT) which are localized in the kidney. This could be especially important in times of increased N requirement, such as during growth or during reduced dietary N intake. The aim of our study was to characterize and to localize UT in the goat (capra hircus) kidney and to investigate its response to reduced dietary N intake in growing goats. Therefore, 12 growing, male goats were fed either a diet containing high (17% CP in complete diet) or low (9% CP in complete diet) N content for 6 wk. After harvesting, blood and kidney samples were taken and analyzed. The mRNA of the different UT isoforms, UT-A1, UT-A2 and UT-B, were detected semiquantitatively in renal tissue by Northern blot analysis. For UT-A2 and UT-B, no statistically significant effect of dietary N restriction on renal mRNA expression could be detected (UT-A2: P = 0.26, UT-B: P = 0.07). However, renal mRNA abundance of UT-A1 significantly increased in the kidney of low-N-fed goats (P = 0.01). Furthermore, protein amounts of UT-B were verified by western blotting; and the localization of UT-A2 and UT-B protein was demonstrated by immunohistochemistry. No significant differences in protein amounts of UT-B could be observed comparing the 2 feeding groups (P = 0.78). The UT-B was localized in renal medulla and papilla, whereas UT-A2 was only found in renal medulla. In addition, comparison of UT-A and UT-BAA sequences of monogastric animals and ruminants showed a high degree of homology, indicating a similar function of the transporters among these species. In summary, we conclude that in ruminants, urea reabsorption in the kidney is most likely increased in response to a low-N diet via an upregulation of UT-A1 mRNA expression. Hypothetically, the reabsorbed urea can then be returned to the rumen via the bloodstream and thus be reused as a source of N for protein synthesis of ruminal microbial community.

Triazolothienopyrimidine Inhibitors of Urea Transporter UT-B Reduce Urine Concentration

Urea transport (UT) proteins facilitate the concentration of urine by the kidney, suggesting that inhibition of these proteins could have therapeutic use as a diuretic strategy. We screened 100,000 compounds for UT-B inhibition using an optical assay based on the hypotonic lysis of acetamide-loaded mouse erythrocytes. We identified a class of triazolothienopyrimidine UT-B inhibitors; the most potent compound, UTB(inh)-14, fully and reversibly inhibited urea transport with IC(50) values of 10 nM and 25 nM for human and mouse UT-B, respectively. UTB(inh)-14 competed with urea binding at an intracellular site on the UT-B protein. UTB(inh)-14 exhibited low toxicity and high selectivity for UT-B over UT-A isoforms. After intraperitoneal administration of UTB(inh)-14 in mice to achieve predicted therapeutic concentrations in the kidney, urine osmolality after administration of 1-deamino-8-D-arginine-vasopressin was approximately 700 mosm/kg H(2)O lower in UTB(inh)-14-treated mice than vehicle-treated mice. UTB(inh)-14 also increased urine output and reduced urine osmolality in mice given free access to water. UTB(inh)-14 did not reduce urine osmolality in UT-B knockout mice. In summary, these data provide proof of concept for the potential utility of UT inhibitors to reduce urinary concentration in high-vasopressin, fluid-retaining conditions. The diuretic mechanism of UT inhibitors may complement the action of conventional diuretics, which target sodium transport.

UT-B-deficient mice develop renal dysfunction and structural damage.

BackgroundUrea transporter UT-B is the major urea transporter in erythrocytes and the descending vasa recta in the kidney. In this study, we investigated the effects of long-term UT-B deficiency on functional and structural defect in the kidney of 16-and 52-week-old UT-B-null mice.MethodsUT-B-knockout mice were generated by targeted gene disruption and lacked UT-B protein expression in all organs. The urinary concentrating ability of mice was studied in terms of daily urine output, urine osmolality, and urine and plasma chemistries. Changes in renal morphology were evaluated by hematoxylin and eosin staining.ResultsThe UT-B-null mice showed defective urine concentrating ability. The daily urine output in UT-B-null mice (2.5 ± 0.1 ml) was 60% higher and urine osmolality (985 ± 151 mosm) was significantly lower than that in wild-type mice (1463 ± 227 mosm). The 52-week-old UT-B-null mice exhibited polyuria after water deprivation, although urine osmolality was increased. At 52 weeks of age, over 31% of UT-B-null mice exhibited renal medullary atrophy because of severe polyuria and hydronephrosis.ConclusionsLong-term UT-B deficiency causes severe renal dysfunction and structural damage. These results demonstrate the important role of UT-B in countercurrent exchange and urine concentration.

Role of thin descending limb urea transport in renal urea handling and the urine concentrating mechanism

Urea transporters UT-A2 and UT-B are expressed in epithelia of thin descending limb of Henle's loop and in descending vasa recta, respectively. To study their role and possible interaction in the context of the urine concentration mechanism, a UT-A2 and UT-B double knockout (UT-A2/B knockout) mouse model was generated by targeted deletion of the UT-A2 promoter in embryonic stem cells with UT-B gene knockout. The UT-A2/B knockout mice lacked detectable UT-A2 and UT-B transcripts and proteins and showed normal survival and growth. Daily urine output was significantly higher in UT-A2/B knockout mice than that in wild-type mice and lower than that in UT-B knockout mice. Urine osmolality in UT-A2/B knockout mice was intermediate between that in UT-B knockout and wild-type mice. The changes in urine osmolality and flow rate, plasma and urine urea concentration, as well as non-urea solute concentration after an acute urea load or chronic changes in protein intake suggested that UT-A2 plays a role in the progressive accumulation of urea in the inner medulla. These results suggest that in wild-type mice UT-A2 facilitates urea absorption by urea efflux from the thin descending limb of short loops of Henle. Moreover, UT-A2 deletion in UT-B knockout mice partially remedies the urine concentrating defect caused by UT-B deletion, by reducing urea loss from the descending limbs to the peripheral circulation; instead, urea is returned to the inner medulla through the loops of Henle and the collecting ducts.

Differential Expression and Function of Urea Transporters in Rat and Human Colon

Gastrointestinal urea transporters play a significant role in urea nitrogen salvaging (UNS) processes between mammalian hosts and gut bacteria. In regions of high bacterial populations (proximal colon), urea is secreted into the colonic lumen and used as a nutrient source by commensal bacteria. Although urea transporters are expressed in human colon, their functional relevance remains to be elucidated. The aim of this study was to investigate expression and function of the facilitative urea transporter UT-B in rat and human colon. Immunoblot analysis demonstrated expression of a 35 kDa glycosylated UT-B protein in colonic mucosa with significantly higher expression in right colon compared to left (P<0.01, N=3). This glycosylated UT-B protein was predominantly located in plasma membrane-enriched samples (P<0.001, N=6). Urea flux studies performed in rat and human colon mounted in Ussing chambers demonstrated higher urea flux in right compared to left colon(P<0.05, N=6). In addition, bi-directional urea flux in right colonic specimens was sensitive to the urea transporter inhibitor, 1,3, dimethylurea (DMU) (P<0.05,N=6). This study demonstrates differential expression and function of UT-B transporters in rat and human colon suggesting that urea transport is an important process in the symbiotic relationship between gut commensal bacteria and their mammalian hosts.

Differential protein abundance and function of UT-B urea transporters in human colon

Facilitative UT-B urea transporters enable the passage of urea across cell membranes. Gastrointestinal urea transporters are thought to play a significant role in the urea nitrogen salvaging process that occurs between mammalian hosts and their gut bacteria. This study investigated the expression of UT-B urea transporters in different segments of human colon. Immunoblot analysis showed that human colon expressed a 35-kDa glycosylated UT-B protein in the colonic mucosa. The 35-kDa UT-B transporter was predominantly located in plasma membrane-enriched samples (P < 0.001; n = 6), and its expression was greater in the ascending colon compared with the descending colon (P < 0.01; n = 3). At the cellular level, UT-B transporters were located throughout colonocytes situated in the upper portion of the colonic crypts. Bidirectional trans-epithelial urea transport was significantly greater in the ascending colon than the descending colon (P < 0.05; n = 6). In addition, the facilitative urea transporter inhibitor 1,3,dimethylurea significantly reduced urea transport in the ascending colon (P < 0.05; n = 6) but had no effect in the descending colon (NS; n = 6). These results illustrate differential protein abundance of functional UT-B protein in different sections of the human colon, strongly correlating to regions that contain the largest populations of intestinal bacteria. This study suggests an important role for UT-B urea transporters in maintaining the symbiotic relationship between humans and their gut bacteria.

Surface electrocardiogram and action potential in mice lacking urea transporter UT-B

UT-B is a urea transporter protein expressed in the kidney and in many non-renal tissues including erythrocytes, brain, heart, bladder and the testis. The objective of this study was to determine the phenotype of UT-B deletion in the heart. UT-B expression in the heart was studied in wild-type mice vs UT-B null mice by utilizing RT-PCR and Western blot. A surface electrocardiogram (ECG) recording (lead II) was measured in wild-type mice and UT-B null mice at the ages of 6, 16 and 52 weeks. For the action potential recording, the ventricular myocytes of 16 w mice were isolated and recorded by floating microelectrode method. The sodium current was recorded by the patch clamp technique. RT-PCR and Western blot showed the UT-B expression in the heart of wild-type mice. No UT-B transcript and protein was found in UT-B null mice. The ECG recording showed that the P-R interval was significantly prolonged in UT-B null mice ((43.5 +/- 4.2), (45.5 +/- 6.9) and (43.8 +/- 7.6) ms at ages of 6, 16 and 52 weeks) vs wild-type mice ((38.6 +/- 2.9), (38.7 +/- 5.6) and (38.2 +/- 7.3) ms, P<0.05). The atrial ventricular heart block type II and III only appeared in the aging UT-B null mice (52 w old). The amplitude of action potential and V (max) decreased significantly in UT-B null mice ((92.17 +/- 10.56) and (101.89 +/- 9.54) mV/s) vs those in wild-type mice (vs (110.51 +/- 10.38) and (109.53 +/- 10.64) mV/s, P<0.05). The action potential duration at 50% and 90% (APD(50) and APD(90)) was significantly prolonged in UT-B null mice ((123.83 +/- 11.17) and (195.43 +/- 16.41) ms) vs that in wild-type mice ((108.27 +/- 10.85) and (171.00 +/- 15.53) ms, P<0.05). The maximal sodium current decreased significantly in UT-B null mice (-8.80 +/- 0.92) nA vs that in wild-type mice ((-5.98 +/- 1.07) nA, P<0.05). These results provide the first evidence that UT-B deletion causes progressive heart block in mice.

Tissue distribution of UT-A and UT-B mRNA and protein in rat

Mammalian urea transporters are facilitated membrane transport proteins belonging to two families, UT-A and UT-B. They are best known for their role of maintaining the renal inner medullary urinary concentrating gradient. Urea transporters have also been identified in tissues not typically associated with urea metabolism. The purpose of this study was to survey the major organs in rat to determine the distribution of UT-A and UT-B mRNA transcripts and protein forms and determine their cellular localization. Five kidney subregions and 17 extrarenal tissues were screened by Northern blot analysis using two UT-A and three UT-B probes and by Western blot analysis using polyclonal COOH-terminal UT-A and UT-B antibodies. Immunohistochemistry was performed on 16 extrarenal tissues using the same antibodies. In kidney, we detected mRNA transcripts and protein bands consistent with previously-identified UT-A and UT-B isoforms, as well as novel forms. We found that UT-A mRNA and protein are widely expressed in extrarenal tissues in various forms that are different from the known isoforms. We determined the cellular localization of UT-A and UT-B in these tissues. We found that both UT-A and UT-B are ubiquitously expressed as numerous tissue-specific mRNA transcripts and protein forms that are localized to cell membranes, cytoplasm, or nuclei.

UT-B is expressed in bovine rumen: potential role in ruminal urea transport

The UT-A (SLC14a2) and UT-B (SLC14a1) genes encode a family of specialized urea transporter proteins that regulate urea movement across plasma membranes. In this report, we describe the structure of the bovine UT-B (bUT-B) gene and characterize UT-B expression in bovine rumen. Northern analysis using a full-length bUT-B probe detected a 3.7-kb UT-B signal in rumen. RT-PCR of bovine mRNA revealed the presence of two UT-B splice variants, bUT-B1 and bUT-B2, with bUT-B2 the predominant variant in rumen. Immunoblotting studies of bovine rumen tissue, using an antibody targeted to the NH2-terminus of mouse UT-B, confirmed the presence of 43- to 54-kDa UT-B proteins. Immunolocalization studies showed that UT-B was mainly located on cell plasma membranes in epithelial layers of the bovine rumen. Ussing chamber measurements of ruminal transepithelial transport of (14)C-labeled urea indicated that urea flux was characteristically inhibited by phloretin. We conclude that bUT-B is expressed in the bovine rumen and may function to transport urea into the rumen as part of the ruminant urea nitrogen salvaging process.