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J. Biol. Chem., Vol. 277, Issue 12, 10633-10637, March 22, 2002

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Urea-selective Concentrating Defect in Transgenic Mice Lacking Urea Transporter UT-B^*

Baoxue Yang^§, Lise Bankir^¶, Annemarie Gillespie, Charles J. Epstein, and A. S. Verkman

From the Departments of  Medicine and Physiology and  Pediatrics, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521 and ^¶ INSERM Unit 367, Institut du Fer a Moulin, 75005 Paris, France

Received for publication, January 8, 2002, and in revised form, January 12, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Urea transporter UT-B has been proposed to be the major urea transporter in erythrocytes and kidney-descending vasa recta. The mouse UT-B cDNA was isolated and encodes a 384-amino acid urea-transporting glycoprotein expressed in kidney, spleen, brain, ureter, and urinary bladder. The mouse UT-B gene was analyzed, and UT-B knockout mice were generated by targeted gene deletion of exons 3-6. The survival and growth of UT-B knockout mice were not different from wild-type littermates. Urea permeability was 45-fold lower in erythrocytes from knockout mice than from those in wild-type mice. Daily urine output was 1.5-fold greater in UT-B- deficient mice (p < 0.01), and urine osmolality (U_osm) was lower (1532 ± 71 versus 2056 ± 83 mosM/kgH₂O, mean ± S.E., p < 0.001). After 24 h of water deprivation, U_osm (in mosM/kgH₂O) was 2403 ± 38 in UT-B null mice and 3438 ± 98 in wild-type mice (p < 0.001). Plasma urea concentration (P_urea) was 30% higher, and urine urea concentration (U_urea) was 35% lower in knockout mice than in wild-type mice, resulting in a much lower U_urea/P_urea ratio (61 ± 5 versus 124 ± 9, p < 0.001). Thus, the capacity to concentrate urea in the urine is more severely impaired than the capacity to concentrate other solutes. Together with data showing a disproportionate reduction in the concentration of urea compared with salt in homogenized renal inner medullas of UT-B null mice, these data define a novel "urea-selective" urinary concentrating defect in UT-B null mice. The UT-B null mice generated for these studies should also be useful in establishing the role of facilitated urea transport in extrarenal organs expressing UT-B.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Urea is the major end product of nitrogen metabolism in mammals. Urea is synthesized by the liver and excreted by the kidney. In omnivores such as humans and laboratory rodents, urea represents up to 40-50% of all urinary solutes and is markedly concentrated in urine with respect to plasma (up to 100-fold in humans and 250-fold in rodents) (1). Because of a unique intrarenal recycling process enabling urea to accumulate in the medulla, urea contributes to the urinary concentrating mechanism and, thus, to water conservation (1, 2). Abnormally low blood urea concentration in malnutrition results in a urinary concentrating defect because of impaired intrarenal urea accumulation (3).

Physiological studies over 30 years provided evidence for the existence of facilitated urea transporters in erythrocytes and certain segments of the nephron (4). Two major subfamilies of mammalian urea transporters have been identified, the "renal tubular-type" urea transporter (UT)¹-A and the "erythrocyte-vascular type" urea transporter UT-B (4-6). Five UT-A isoforms have been identified that are produced by alternative splicing of a single gene (7-9). UT-A1 (original name UT1) is expressed in the apical membrane of terminal inner medullary collecting duct cells and is thought to be involved in the vasopressin-regulated increase in urea permeability (10, 11). UT-A2 (UT2) is located in the late part of descending thin limbs of short loops of Henle and in the intermediate part of descending thin limbs of long loops, and it may facilitate urea recycling (1, 12, 13). UT-A3 is most abundant in intracellular membranes and in the apical region of inner medullary collecting duct cells (14). The localization of UT-A4 in the kidney is unclear (15). UT-A5 mRNA was localized to the peritubular myoid cells of the testis (16). A UT-B isoform (original names UT11, HUT11, and UT3) was cloned from human bone marrow (17) and rat kidney (18). In rat, UT-B is expressed in the kidney outer and inner medulla, testis, brain, bone marrow, and spleen (18-20). UT-B is a 384-amino acid protein having 62% identity to rat UT-A2. UT-B has similar membrane topology to UT-A2 based on hydropathy analysis and has functional characteristics similar to UT-A1 and UT-A2. We reported that rat UT-B heterologously expressed at a relatively high level in Xenopus oocytes functioned as a urea/water channel utilizing a common aqueous pathway (21), however, the relevance of water transport by UT-B in mammalian physiology is unresolved (22).

We report here the generation of the first transgenic mouse model of facilitated urea transporter deletion. The erythrocyte/vasa recta transporter UT-B was deleted by targeted gene disruption, and the phenotype of the null mice was studied with focus on urinary concentrating ability. Erythrocyte urea permeability in UT-B null mice was remarkably reduced, and the mice manifested a unique type of urea-selective urinary concentrating defect. Our data provide evidence that UT-B-dependent countercurrent exchange of urea in the renal medulla contributes to approximately one-third of the total capacity of the kidney to concentrate urine but contributes even more greatly to the ability of the kidney to concentrate urea itself.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

cDNA Cloning and Genomic Analysis of Mouse UT-B-- Kidney mRNA was isolated (Oligotex direct mRNA kit, Qiagen) from C57/bl6 mice. cDNA was reverse-transcribed from the mRNA using oligo(dT) (SuperScript II preamplification kit, Invitrogen). Polymerase chain reaction amplification was performed with cDNA template and primers designed from the rat UT-B sequence (sense, 5'-AGGTGTGGCCTCAAAGTACTTGGCTA-3'; antisense, 5'-GATAGCAGCGTGCAGGCACATGAGT-3'). The PCR product (~ 0.7 kb) was purified (Qiaex II gel extraction kit, Qiagen) and subcloned into PCRII TA cloning vector (Invitrogen). The full-length insert was sequenced. 5' and 3' end cDNA sequences were obtained by 5'- and 3'-RACE (CLONTECH). For functional measurements of urea and water permeability, the mouse UT-B cDNA (full-length coding sequence) was subcloned into oocyte expression plasmid pSP64T, and in vitro transcribed cRNA (5 ng) was expressed in Xenopus laevis oocytes as described previously (21). The structure of the mouse UT-B gene was analyzed by PCR amplification of exon-intron-exon fragments by using C57/bl6 mouse liver genomic DNA as template. All exons and exon-intron boundaries were sequenced.

Generation of UT-B Null Mice-- A targeting vector for homologous recombination was constructed with a 1.7-kb genomic UT-B DNA fragment containing intron 2 and part of exon 3 (left arm) and a 5.5-kb fragment containing part of exon 6 and intron 7 (right arm) (see Fig. 2A). The left and right arm genomic fragments (surrounding a 1.8-kb PolIIneobpA cassette) were PCR-amplified, and a PGK-tk cassette was inserted upstream for negative selection. The targeting vector was linearized at a unique downstream NotI site and electroporated into CB1-4 embryonic stem cells. Transfected embryonic stem cells were selected with G418 and FIAU for 7 days, yielding two targeted clones from 120 doubly resistant colonies on PCR screening with a neo-specific sense primer and a UT-B gene-specific antisense primer located 50 bp upstream of the targeted region. Homologous recombination was confirmed by Southern hybridization in which 10 µg of genomic DNA was digested with SpeI, electrophoresed, transferred to a Nylon+ membrane (Amersham Biosciences, Inc.), and hybridized with a 1.1-kb genomic fragment. Embryonic stem cells were injected into postcoitus 2.5-day eight-cell morula stage CD1 zygotes, cultured overnight to blastocysts, and transferred to pseudopregnant B6D2 females. Offspring were genotyped by PCR followed by Southern blot analysis as described above. Heterozygous founder mice containing the disrupted UT-B gene in the germ line were bred to produce homozygous UT-B knockout mice.

Northern Blot Analysis-- Total RNA from mouse tissues was isolated using TRIzol reagent (Invitrogen). mRNAs purified by Oligotex direct mRNA kit (Qiagen) were resolved on a 1% formaldehyde-agarose denaturing gel (1.5 µg/lane), transferred to a Nylon+ membrane (Amersham Biosciences, Inc.), and hybridized at high stringency with a ³²P-labeled probe corresponding to the mouse UT-B cDNA coding sequence.

Immunohistochemistry and Immunoblot Analysis-- Immunoperoxidase localization of UT-B protein in fixed frozen kidney sections was done using a 1:500 dilution of rabbit polyclonal serum raised against a C-terminal peptide (DNRIFYLQNKKRMVESPL). Immunoblot analysis of hemoglobin-free ghost membranes from erythrocytes was carried out with the same antibody as described previously (23).

Erythrocyte Urea and Water Permeability Measurements-- Fresh erythrocytes obtained by tail bleeding (100-200 µl/bleed) were washed three times in phosphate-buffered saline to remove serum and the cellular buffy coat. Stopped-flow measurements were carried out on a Hi-Tech Sf-51 instrument. For measurement of osmotic water permeability, suspensions of erythrocytes (~0.5% hematocrit) in phosphate-buffered saline were subjected to a 250 mM inwardly directed gradient of sucrose. The kinetics of decreasing cell volume were measured from the time course of 90^o scattered light intensity at 530 nm wavelength. Osmotic water permeability coefficients (P_f) were computed from the light scattering time course (24). For the measurement of urea permeability, the erythrocyte suspension was subjected to a 250 mM inwardly directed gradient of urea. In some experiments, 0.7 mM phloretin was added to the erythrocyte suspension prior to stopped-flow experiments.

Studies of Fluid Turnover and Urinary Concentrating Ability-- Daily water intake and urine output were evaluated in 30-40-day-old mice placed in mouse metabolic cages (Harvard Apparatus). In other experiments, urine samples were obtained by placing mice on a wire mesh platform in a clean glass beaker until spontaneous voiding was observed. Blood samples were obtained by tail bleeding, and plasma was separated from blood cells by centrifugation. In some experiments, urine samples were obtained from the same mice under basal conditions (unrestricted access to food and water) and after a 24-h deprivation of food and water. Inner medulla tissue homogenates were obtained by homogenizing ~2 mg of papillary tissue/kidney in a 15-fold excess of distilled water, and the supernatant after centrifugation was assayed for urea and chloride. Urine osmolality was measured by freezing point osmometry (micro-osmometer, Precision Systems Inc.). Urine and plasma chemistries were measured by the University of California San Francisco Clinical Chemistry Laboratory.

Statistics-- Results obtained in null mice were compared with those of wild-type mice by Student's t test. When three groups were compared, one-way ANOVA was performed followed by Fischer posthoc test. One-way ANOVA with repeated measures was used to compare mice of the two genotypes in the dehydration study. p < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A cDNA clone with homology to rat UT-B was isolated from C57/bl6 mouse kidney. The open reading frame of 1152 bp (GenBank^TM accession number AF448798) encodes a 384- amino acid protein with 10 putative hydrophobic transmembrane domains. The deduced amino acid sequence is 85 and 94% identical to human and rat UT-B, respectively (Fig. 1A). Northern blot analysis revealed transcripts of 3.8 and 2.0 kb in brain, spleen, kidney, ureter, and urinary bladder (Fig. 1B). Functional analysis in Xenopus oocytes expressing mouse UT-B cRNA indicated efficient urea-transport and weak water-transport activity (Fig. 1C) in agreement with results for rat UT-B (21). Also, as found previously, urea and water transport in oocytes expressing mouse UT-B was inhibited by phloretin.

View larger version (49K): [in this window] [in a new window]   Fig. 1.   cDNA cloning, distribution, and function of mouse UT-B. A, comparison of deduced amino acid sequences of mouse, rat, and human UT-B. Conserved residues are shown in capital letters. Arrows indicate intron positions. *, protein kinase C site; #, N-linked glycosylation site. Putative membrane-spanning regions are underlined. B, multi-tissue Northern blot probed with full-length UT-B coding sequence. C, urea (left) and osmotic water (right) permeabilities in Xenopus oocytes microinjected with water or 5 ng of UT-B cRNA (± S.E., n = 10). *, p < 0.05; **, p < 0.01 compared with control.

Sequence comparison of mouse UT-B cDNA with a cloned 17-kb genomic DNA indicated 7 introns (lengths 0.4, 1.8, 2.3, 1.5, 0.4, 5.5, and 2.5 kb) (Fig. 2A, top) separating eight exons with boundaries at residues 46, 109, 152, 217, 266, 311, and 332 in the deduced mouse UT-B amino acid sequence, identical to the boundary residues of the human UT-B gene (25). All boundaries correspond to the GT-AG rule. Genomic Southern blot analysis with EcoRI, SpeI, and KpnI-digested mouse genomic DNA indicated a single copy UT-B gene/haploid mouse genome (data not shown).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Genomic cloning of UT-B and generation of UT-B null mice. A, top, organization and restriction map of the mouse UT-B gene based on PCR analysis, restriction digestion, and DNA sequencing. Rectangles indicate exon segments that constitute coding sequences. Middle and bottom, targeting strategy for UT-B gene deletion. Homologous recombination results in the replacement of the indicated segments (thick line) of the UT-B gene by a 1.8-kb polII-neo selectable marker. The probe used for Southern blot analysis is indicated (probe), and the expected sizes (4.3 and 3.7 kb) of hybridized fragments after SpeI digestion are indicated. The 1.7-kb amplified region in PCR analysis is shown. B, Southern blot of mouse liver genomic DNA digested with SpeI and probed as indicated in A. C, Northern blot of mouse kidney probed with the mouse UT-B coding sequence. D, immunoblot of mouse erythrocytes with UT-B polyclonal serum. E, immunoperoxidase localization of UT-B protein in kidney inner medulla of wild-type (left) and UT-B null (right) mouse.

UT-B knockout mice were generated by the gene-targeting strategy shown in Fig. 2A. Heterozygous founder mice containing the disrupted UT-B gene were bred to produce wild-type, heterozygous, and UT-B null mice. Genomic Southern blot analysis of mouse liver genomic DNA digested with SpeI (probed as indicated in Fig. 2A) showed the predicted 4.3-kb fragment in wild-type mice, 3.7-kb fragment in UT-B knockout mice, and both fragments in heterozygous mice (Fig. 2B). Fig. 2C shows only truncated UT-B transcripts in the kidney of knockout mice. Immunoblot analysis revealed UT-B protein in erythrocytes of wild-type but not in the UT-B null mice (Fig. 2D). Immunohistochemistry of kidney showed UT-B protein expression in medullary vasa recta of wild-type mice with no staining in UT-B null mice (Fig. 2E).

Genotype analysis of offspring from breeding of UT-B heterozygous mice indicated a nearly 1:2:1 Mendelian distribution (54 wild-type, 113 heterozygous, and 41 null mice). An analysis of growth by mouse weight (age 1-12 weeks) showed no differences among the genotypes. The UT-B null mice had grossly normal appearance, activity, and behavior. Plasma sodium, potassium, chloride, bicarbonate, and creatinine concentrations were similar in both groups as was hematocrit (48.3 ± 0.4% in wild-type and 49.2 ± 0.5% in UT-B null mice).

Urea and water permeabilities in erythrocytes were measured by stopped-flow light scattering. Urea permeability was measured from the time course of cell swelling in response to a 250 mM inwardly directed urea gradient. There was an initial rapid decrease in cell volume because of osmotically induced water efflux followed by slower cell swelling, which was the result of urea and secondary water influx. At 10 °C, urea equilibrated across erythrocytes from wild-type mice with a half-time of ~27 s, giving a permeability coefficient (P_urea) of 1.1 × 10⁶ cm/s (Fig. 3A, top curve). Urea permeability was inhibited by 96% by 0.7 mM phloretin (Fig. 3A, bottom curve). Fig. 3A, middle curves, shows urea permeability in erythrocytes from heterozygous and UT-B null mice. Urea permeability in the erythrocytes from null mice was 45-fold lower than that from wild-type mice. Fig. 3B shows the time course of osmotic cell shrinking in response to a 250 mM inwardly directed osmotic gradient of sucrose. The data from a series of mice showed that UT-B deletion did not significantly reduce erythrocyte osmotic water permeability (P_f = 0.018 ± 0.0.02 cm/s for wild-type mice; 0.017 ± 0.002 cm/s for UT-B null mice).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Erythrocyte urea and water permeability. Urea permeability was measured from the time course of erythrocyte volume as determined by light scattering in response to a 250 mM inwardly directed urea gradient. A, urea permeability measured in erythrocytes from wild-type mice in the absence (top) and presence (bottom) of 0.7 mM phloretin and from mice of indicated genotypes (middle) at 10 °C. B, water permeability measured from the time course of erythrocyte volume as determined by light scattering in response to a 250 mM inwardly directed sucrose gradient (n = 3).

Urinary concentrating function was compared in the wild-type, heterozygous, and UT-B null mice. Fig. 4A shows daily fluid consumption and urinary output. The UT-B null mice were moderately polyuric, consuming and excreting approximately 50% more fluid than litter-matched heterozygous and wild-type mice. The difference in fluid intake and urinary output, primarily representing insensible respiratory losses, was similar in all groups. The average urine osmolality in UT-B null mice (1532 ± 71 mosM/kg H₂O) was significantly lower than that in wild-type mice (2056 ± 83 mosM/kg H₂O, Fig. 4B). Urinary concentrating ability was measured in response to a 24 h water deprivation. Urine osmolality in the UT-B null mice increased significantly, although to a significantly lesser extent than in wild-type mice (p < 0.02). Body weight loss during this test was similar in both groups (20.1 ± 1.2% wild-type mice; 21.7 ± 0.8% null mice).

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Urinary concentrating defect in UT-B null mice. A, twenty-four hour fluid consumption (left) and urine output (right) in mice of indicated genotype (± S.E., 6 mice/genotype). B, urine osmolality measured in mice given free access to food and water (basal) and after a 24 h water deprivation. Data for 10 individual mice/group shown together with the means ± S.E. *, p < 0.001 compared with wild-type mice. The rise in urine osmolality in response to water deprivation was significantly smaller in null mice than in wild type (p < 0.02).

Osmolality and urea concentration were measured in plasma and urine of six wild-type and six UT-B null mice under basal conditions (food intake was equal in the two groups). Plasma osmolality was slightly but not significantly higher in UT-B null mice than in wild-type mice (330 ± 4 versus 324 ± 4 mosM/kgH₂O). The urine-to-plasma ratio for osmolality, an index of overall concentrating capacity of the kidney, was lower by one-third in UT-B null mice compared with wild-type mice (4.0 ± 0.2 versus 6.1 ± 0.4, p < 0.001). As shown in Fig. 5A, plasma urea concentration was significantly higher, and urinary urea concentration was significantly lower in the UT-B-deficient mice. These opposing changes suggest that plasma urea increased because the kidney was less able to recycle urea and concentrate urea in the urine. The urine-to-plasma ratio of urea concentration reflects the capacity of the kidney to concentrate urea above its level in body fluids. This ratio was markedly decreased in UT-B null mice from 124 ± 9 to 61 ± 5 (Fig. 5A). Fig. 5B shows the composition of the aqueous component of the inner medulla as measured on the supernatants of centrifuged homogenates of inner medullas. The significantly reduced inner medullary osmolality primarily was because of a reduction in inner medulla urea concentration.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Effect of UT-B deletion on urea recycling. A, plasma (top) and urine (middle) urea concentration, and urea/plasma urea concentration ratio (bottom) (S.E., n = 6) *, p < 0.01; **, p < 0.001. B, Osmolality (top), urea concentration (middle), and chloride concentration (bottom) in homogenized inner medullas (S.E., n = 6). C, diagram of urea recycling in the kidney of UT-B null mice. Urea is delivered normally to the tip of the papilla, but its recycling via the vascular route is compromised in UT-B null mice, leading to a greater return of urea to the general circulation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The cDNA encoding mouse UT-B had 94% amino acid identity to a rat homolog (rUT-B) cloned previously (18). Mouse UT-B and rUT-B transcript expression differed in a few respects. Northern blot analysis revealed mouse UT-B transcripts with two sizes (3.8 and 2.0 kb) in brain, spleen, kidney, ureter, and urinary bladder, whereas a single mRNA band was found for rUT-B. The UT-B transcript was identified in rat testis (18), but it was not seen in mouse testis.

Transgenic null mice deficient in the UT-B were generated and characterized. The UT-B knockout mice expressed truncated UT-B transcript but lacked detectable UT-B protein by C-terminal antibody. Urea permeability in erythrocytes from null mice was 45-fold lower than that from wild-type mice, indicating that UT-B was functionally deleted. We found previously that rat UT-B transported some water along with urea when heterologously expressed in Xenopus oocytes (21). Mouse UT-B also functioned as a weak water channel and an efficient urea transporter in Xenopus oocytes. The osmotic water permeability in UT-B-deficient erythrocytes was not reduced by UT-B deletion, however, the high AQP1-dependent water permeability in mouse erythrocyte precluded the detection of effects of UT-B deletion. The measurement of water permeability in erythrocytes lacking UT-B and AQP1 together, when available, should be informative in this regard.

UT-B null mice exhibited normal growth and no overt abnormalities in their main biological functions, behavior, and sensory activity. The sex and genotype ratios were normally distributed, suggesting no influence of UT-B gene deletion on survival or sexual differentiation. As expected, the major phenotypic abnormality was in renal water and urea handling. Fluid intake and urine output were approximately 50% higher, and urine osmolality was approximately one-third lower in null mice than in wild-type mice. Despite this difference in fluid turnover, the concentrations of the major plasma solutes were unaltered. The very low urea permeability in red cells did not influence the hematocrit.

Urea is an important contributor to the urinary concentrating mechanism (1, 2, 26-29). Interestingly, urea contributes mostly to its own concentration, because it is the most abundant solute in the urine, except in herbivores, and because its concentration in blood is much lower than that of the other main urinary constituents. In normal human urine, urea is concentrated approximately 45-fold above plasma, K⁺ (12-fold), and Na⁺ (0.65-fold). The kidney transforms large amounts of urea-poor plasma into a small volume of urea-rich urine. Thus, besides the overall urinary concentrating defect in UT-B null mice, we investigated the possibility of a selective defect in urea concentrating capacity by measurement of the urine-to-plasma urea concentration ratio and inner medulla composition. UT-B deficiency in kidney resulted in decreased urinary urea and increased plasma urea concentrations, producing a 2-fold decrease in the urine-to-plasma urea concentration ratio. UT-B deficiency also produced a >2-fold reduction in inner medullary urea concentration with little effect on inner medullary chloride (salt) concentration.

Mechanistically, as depicted schematically in Fig. 5C, these observations suggest that some of the urea delivered to the tip of the papilla by UT-A1/UT-A3/UT-A4 and carried up by the blood through venous ascending vasa rect is not recycled in UT-B-deficient arterial descending vasa recta and thus returned to the general circulation. In contrast to the remarkably reduced urea concentrating ability in the UT-B null mice, the defect in the concentration of all solutes as assessed by the urine-to-plasma osmolality ratio (lower by only one-third) was more modest, suggesting that UT-B plays a greater role in enabling the kidney to concentrate urea than other solutes in the urine. The selective reduction in inner medullary urea concentration supports this conclusion.

A natural model of UT-B gene deletion occurs in rare human subjects lacking the Kidd blood group J_K antigens, J_K (a,b) (30). Erythrocytes in these subjects have low urea permeability, and it is assumed that they also lack UT-B protein in vasa recta. In two such subjects, Sands et al. (31) found a normal blood urea nitrogen and a modest ~20% decline in maximum urinary concentrating ability after dehydration and vasopressin infusion (790 mosM/kg H₂O versus ~980 mosM/kg H₂O in healthy subjects). The concentrating defect observed in mice in this study is substantially larger than that in humans. Blood urea concentration was elevated, and maximum urinary concentration after water deprivation decreased by 35%. This difference may be accounted for by the difference in overall concentrating ability of the two species partly because of the differences in kidney architecture and development of the renal medulla. The urinary concentrating capacity in different mammalian species is influenced by several factors, including the relative length of the papilla (much longer in mice than in humans) and the anatomy of the vascular bundles in the outer medulla (32, 33). The human kidney has a relatively simple architecture and modest concentrating ability, whereas mice have a more complex vascular architecture and greater concentrating ability.

In conclusion, the UT-B null mice manifest a unique defect in urinary concentrating capacity of the kidney affecting the ability to concentrate urea preferentially over other urinary solutes. The impaired urea recycling in the medullary vasculature results in increased blood urea concentration and decreased urine urea concentration, producing an ~50% reduction in urea concentrating capacity. As reported in rat (18-20), mouse UT-B was also found to be expressed in several other organs in which facilitated urea transport was not suspected to occur previously, including brain, testis, spleen, ureter, and urinary bladder. The UT-B null mice will allow studies to elucidate the role of facilitated urea transport in these extrarenal organs.

	ACKNOWLEDGEMENT

We thank Liman Qian for breeding mice.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants HL58198, DK35124, HL60288, and HL51854 and Grant R613 from the National Cystic Fibrosis Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF448798.

^§ To whom correspondence should be addressed: Cardiovascular Research Institute, 1246 Health Sciences East Tower, University of California, San Francisco, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail: byang@itsa.ucsf.edu or www.ucsf.edu/verklab.

Published, JBC Papers in Press, January 15, 2002, DOI 10.1074/jbc.M200207200

	ABBREVIATIONS

The abbreviations used are: UT, urea transporter; RACE, rapid amplification of cDNA ends; ANOVA, analysis of variance; mRNA, messenger RNA.

	REFERENCES

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				M. H. Levin, R. de la Fuente, and A. S. Verkman Urearetics: a small molecule screen yields nanomolar potency inhibitors of urea transporter UT-B FASEB J, February 1, 2007; 21(2): 551 - 563. [Abstract] [Full Text] [PDF]

				J. P. Kokko and J. M. Sands Significance of urea transport: the pioneering studies of Bodil Schmidt-Nielsen. Am J Physiol Renal Physiol, December 1, 2006; 291(6): F1109 - F1112. [Abstract] [Full Text] [PDF]

				D. Zhao, L. Bankir, L. Qian, D. Yang, and B. Yang Urea and urine concentrating ability in mice lacking AQP1 and AQP3 Am J Physiol Renal Physiol, August 1, 2006; 291(2): F429 - F438. [Abstract] [Full Text] [PDF]

				E. A. Potter, G. Stewart, and C. P. Smith Urea flux across MDCK-mUT-A2 monolayers is acutely sensitive to AVP, cAMP, and [Ca2+]i Am J Physiol Renal Physiol, July 1, 2006; 291(1): F122 - F128. [Abstract] [Full Text] [PDF]

				G.G Pinter and J.L Shohet Two fluid compartments in the renal inner medulla: a view through the keyhole of the concentrating process Phil Trans R Soc A, June 15, 2006; 364(1843): 1551 - 1561. [Abstract] [Full Text] [PDF]

				J. J. Doran, J. D. Klein, Y. H. Kim, T. D. Smith, S. D. Kozlowski, R. B. Gunn, and J. M. Sands Tissue distribution of UT-A and UT-B mRNA and protein in rat Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2006; 290(5): R1446 - R1459. [Abstract] [Full Text] [PDF]

				S.-W. Lim, K.-H. Han, J.-Y. Jung, W.-Y. Kim, C.-W. Yang, J. M. Sands, M. A. Knepper, K. M. Madsen, and J. Kim Ultrastructural localization of UT-A and UT-B in rat kidneys with different hydration status Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2006; 290(2): R479 - R492. [Abstract] [Full Text] [PDF]

				R. A. Fenton, A. Shodeinde, and M. A. Knepper UT-A urea transporter promoter, UT-A{alpha}, targets principal cells of the renal inner medullary collecting duct Am J Physiol Renal Physiol, January 1, 2006; 290(1): F188 - F195. [Abstract] [Full Text] [PDF]

				S. Uchida, E. Sohara, T. Rai, M. Ikawa, M. Okabe, and S. Sasaki Impaired Urea Accumulation in the Inner Medulla of Mice Lacking the Urea Transporter UT-A2 Mol. Cell. Biol., August 15, 2005; 25(16): 7357 - 7363. [Abstract] [Full Text] [PDF]

				H. Inoue, S. D. Kozlowski, J. D. Klein, J. L. Bailey, J. M. Sands, and S. M. Bagnasco Regulated expression of renal and intestinal UT-B urea transporter in response to varying urea load Am J Physiol Renal Physiol, August 1, 2005; 289(2): F451 - F458. [Abstract] [Full Text] [PDF]

				R. A. Fenton, A. Flynn, A. Shodeinde, C. P. Smith, J. Schnermann, and M. A. Knepper Renal Phenotype of UT-A Urea Transporter Knockout Mice J. Am. Soc. Nephrol., June 1, 2005; 16(6): 1583 - 1592. [Abstract] [Full Text] [PDF]

				B. Yang and L. Bankir Urea and urine concentrating ability: new insights from studies in mice Am J Physiol Renal Physiol, May 1, 2005; 288(5): F881 - F896. [Abstract] [Full Text] [PDF]

				N. Lucien, P. Bruneval, F. Lasbennes, M.-F. Belair, C. Mandet, J.-P. Cartron, P. Bailly, and M.-M. Trinh-Trang-Tan UT-B1 urea transporter is expressed along the urinary and gastrointestinal tracts of the mouse Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2005; 288(4): R1046 - R1056. [Abstract] [Full Text] [PDF]

				H. Inoue, S. D. Jackson, T. Vikulina, J. D. Klein, K. Tomita, and S. M. Bagnasco Identification and characterization of a Kidd antigen/UT-B urea transporter expressed in human colon Am J Physiol Cell Physiol, July 1, 2004; 287(1): C30 - C35. [Abstract] [Full Text] [PDF]

				C. Li, J. D. Klein, W. Wang, M. A. Knepper, S. Nielsen, J. M. Sands, and J. Frokiaer Altered expression of urea transporters in response to ureteral obstruction Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1154 - F1162. [Abstract] [Full Text] [PDF]

				R. A. Fenton, C.-L. Chou, G. S. Stewart, C. P. Smith, and M. A. Knepper Urinary concentrating defect in mice with selective deletion of phloretin-sensitive urea transporters in the renal collecting duct PNAS, May 11, 2004; 101(19): 7469 - 7474. [Abstract] [Full Text] [PDF]

				J. D. Klein, J. M. Sands, L. Qian, X. Wang, and B. Yang Upregulation of Urea Transporter UT-A2 and Water Channels AQP2 and AQP3 in Mice Lacking Urea Transporter UT-B J. Am. Soc. Nephrol., May 1, 2004; 15(5): 1161 - 1167. [Abstract] [Full Text] [PDF]

				G. S. Stewart, R. A. Fenton, W. Wang, T.-H. Kwon, S. J. White, V. M. Collins, G. Cooper, S. Nielsen, and C. P. Smith The basolateral expression of mUT-A3 in the mouse kidney Am J Physiol Renal Physiol, May 1, 2004; 286(5): F979 - F987. [Abstract] [Full Text] [PDF]

				L. Bankir, K. Chen, and B. Yang Lack of UT-B in vasa recta and red blood cells prevents urea-induced improvement of urinary concentrating ability Am J Physiol Renal Physiol, January 1, 2004; 286(1): F144 - F151. [Abstract] [Full Text] [PDF]

				J.-Y. Jung, K. M. Madsen, K.-H. Han, C.-W. Yang, M. A. Knepper, J. M. Sands, and J. Kim Expression of urea transporters in potassium-depleted mouse kidney Am J Physiol Renal Physiol, December 1, 2003; 285(6): F1210 - F1224. [Abstract] [Full Text]

				W. Zhang and A. Edwards Theoretical effects of UTB urea transporters in the renal medullary microcirculation Am J Physiol Renal Physiol, October 1, 2003; 285(4): F731 - F747. [Abstract] [Full Text] [PDF]

				J. M. Sands Urine-Concentrating Ability in the Aging Kidney Sci. Aging Knowl. Environ., June 18, 2003; 2003(24): pe15 - 15. [Abstract] [Full Text]

				T. L. Pallone, M. R. Turner, A. Edwards, and R. L. Jamison Countercurrent exchange in the renal medulla Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2003; 284(5): R1153 - R1175. [Abstract] [Full Text] [PDF]

				T. L. Pallone, Z. Zhang, and K. Rhinehart Physiology of the renal medullary microcirculation Am J Physiol Renal Physiol, February 1, 2003; 284(2): F253 - F266. [Abstract] [Full Text] [PDF]

				S. M. Bagnasco Gene structure of urea transporters Am J Physiol Renal Physiol, January 1, 2003; 284(1): F3 - F10. [Abstract] [Full Text] [PDF]

				J. M. Sands Molecular Approaches to Urea Transporters J. Am. Soc. Nephrol., November 1, 2002; 13(11): 2795 - 2806. [Abstract] [Full Text] [PDF]

				M.-M. Trinh-Trang-Tan, F. Lasbennes, P . Gane, N. Roudier, P. Ripoche, J.-P. Cartron, and P. Bailly UT-B1 proteins in rat: tissue distribution and regulation by antidiuretic hormone in kidney Am J Physiol Renal Physiol, November 1, 2002; 283(5): F912 - F922. [Abstract] [Full Text] [PDF]

				B. Yang and A. S. Verkman Analysis of Double Knockout Mice Lacking Aquaporin-1 and Urea Transporter UT-B. EVIDENCE FOR UT-B-FACILITATED WATER TRANSPORT IN ERYTHROCYTES J. Biol. Chem., September 20, 2002; 277(39): 36782 - 36786. [Abstract] [Full Text] [PDF]

				N. Lucien, F. Sidoux-Walter, N. Roudier, P. Ripoche, M. Huet, M.-M. Trinh-Trang-Tan, J.-P. Cartron, and P. Bailly Antigenic and Functional Properties of the Human Red Blood Cell Urea Transporter hUT-B1 J. Biol. Chem., September 6, 2002; 277(37): 34101 - 34108. [Abstract] [Full Text] [PDF]

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