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Originally published In Press as doi:10.1074/jbc.M200207200 on January 15, 2002
J. Biol. Chem., Vol. 277, Issue 12, 10633-10637, March 22, 2002
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
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ABSTRACT |
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
(Uosm) was lower (1532 ± 71 versus 2056 ± 83 mosM/kgH2O,
mean ± S.E., p < 0.001). After 24 h of
water deprivation, Uosm (in
mosM/kgH2O) was 2403 ± 38 in UT-B null
mice and 3438 ± 98 in wild-type mice (p < 0.001). Plasma urea concentration (Purea) was
30% higher, and urine urea concentration
(Uurea) was 35% lower in knockout mice than in
wild-type mice, resulting in a much lower
Uurea/Purea 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.
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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 90o
scattered light intensity at 530 nm wavelength. Osmotic water permeability coefficients (Pf) 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.
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RESULTS |
A cDNA clone with homology to rat UT-B was isolated from
C57/bl6 mouse kidney. The open reading frame of 1152 bp
(GenBankTM 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.

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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.
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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).

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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.
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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 (Purea) of 1.1 × 10 6 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
(Pf = 0.018 ± 0.0.02 cm/s for wild-type
mice; 0.017 ± 0.002 cm/s for UT-B null mice).

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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).
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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).

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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).
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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.

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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.
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DISCUSSION |
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 JK
antigens, JK (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.
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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 GenBankTM/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 |
| 1.
|
Bankir, L.,
and Trinh-Trang-Tan, M. M.
(2000)
in
The Kidney
(Brenner, B. M., ed), 6th Ed.
, pp. 637-679, W. B. Saunders Co., Philadelphia, PA
|
| 2.
|
Masilamani, S.,
Knepper, M. A.,
and Burg, M. B.
(2000)
in
The Kidney
(Brenner, B. M., ed), 6th Ed.
, pp. 595-635, W. B. Saunders Co., Philadelphia, PA
|
| 3.
|
Klahr, S.,
Tripathy, K.,
Garcia, F. T.,
Mayoral, L. G.,
Ghitis, J.,
and Bolaños, O.
(1967)
Am. J. Med.
43,
84-96[CrossRef][Medline]
[Order article via Infotrieve]
|
| 4.
|
Sands, J. M.,
Timmer, R. T.,
and Gunn, R. B.
(1997)
Am. J. Physiol.
273,
F321-F339[Abstract/Free Full Text]
|
| 5.
|
Sands, J. M.
(1999)
J. Am. Soc. Nephrol.
10,
635-646[Abstract/Free Full Text]
|
| 6.
|
Bankir, L.,
and Trinh-Trang-Tan, M. M.
(2000)
Exp. Physiol.
85,
243-252[Abstract]
|
| 7.
|
Nakayama, Y.,
Naruse, M.,
Karakashian, A.,
Peng, T.,
Sands, J. M.,
and Bagnasco, S. M.
(2001)
Biochim. Biophys. Acta
1518,
19-26[Medline]
[Order article via Infotrieve]
|
| 8.
|
Bagnasco, S. M.,
Peng, T.,
Janech, M. G.,
Karakashian, A.,
and Sands, J. M.
(2001)
Am. J. Physiol.
281,
F400-F406[Abstract/Free Full Text]
|
| 9.
| Fenton, R. A., Cottingham, C. A., Stewart G. S., Howorth
A., Hewitt J. A., and Smith C. P. (2002) Am. J. Physiol., in press
|
| 10.
|
Nielsen, S.,
Terris, J.,
Smith, C. P.,
Hediger, M. A.,
Ecelbarger, C. A.,
and Knepper, M. A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5495-5500[Abstract/Free Full Text]
|
| 11.
|
Shayakul, C.,
Steel, A.,
and Hediger, M. A.
(1996)
J. Clin. Invest.
98,
2580-2587[Medline]
[Order article via Infotrieve]
|
| 12.
|
Smith, C. P.,
Lee, W. S.,
Martial, S.,
Knepper, M. A.,
You, G.,
Sands, J. M.,
and Hediger, M. A.
(1995)
J. Clin. Invest.
96,
1556-1563[Medline]
[Order article via Infotrieve]
|
| 13.
|
Shayakul, C.,
Knepper, M. A.,
Smith, C. P.,
DiGiovanni, S. R.,
and Hediger, M. A.
(1997)
Am. J. Physiol.
272,
F654-F660[Abstract/Free Full Text]
|
| 14.
|
Terris, J. M.,
Knepper, M. A.,
and Wade, J. B.
(2001)
Am. J. Physiol.
280,
F325-F332[Abstract/Free Full Text]
|
| 15.
|
Karakashian, A.,
Timmer, R. T.,
Klein, J. D.,
Gunn, R. B.,
Sands, J. M.,
and Bagnasco, S. M.
(1999)
J. Am. Soc. Nephrol.
10,
230-237[Abstract/Free Full Text]
|
| 16.
|
Fenton, R. A.,
Howorth, A.,
Cooper, G. J.,
Meccariello, R.,
Morris, I. D.,
and Smith, C. P.
(2000)
Am. J. Physiol.
279,
C1425-C1431
|
| 17.
|
Olives, B.,
Neau, P.,
Bailly, P.,
Hediger, M. A.,
Rousselet, G.,
Cartron, J. P.,
and Ripoche, P.
(1994)
J. Biol. Chem.
269,
31649-31652[Abstract/Free Full Text]
|
| 18.
|
Tsukaguchi, H.,
Shayakul, C.,
Berger, U. V.,
Tokui, T.,
Brown, D.,
and Hediger, M. A.
(1997)
J. Clin. Invest.
99,
1506-1515[Medline]
[Order article via Infotrieve]
|
| 19.
|
Couriaud, C.,
Ripoche, P.,
and Rousselet, G.
(1996)
Biochim. Biophys. Acta
1309,
197-199[Medline]
[Order article via Infotrieve]
|
| 20.
|
Timmer, R. T.,
Klein, J. D.,
Bagnasco, S. M.,
Doran, J. J.,
Verlander, J. W.,
Gunn, R. B.,
and Sands, J. M.
(2001)
Am. J. Physiol.
281,
C1318-C1325[Abstract/Free Full Text]
|
| 21.
|
Yang, B.,
and Verkman, A. S.
(1998)
J. Biol. Chem.
273,
9369-9372[Abstract/Free Full Text]
|
| 22.
|
Sidoux-Walter, F.,
Lucien, N.,
Olives, B.,
Gobin, R.,
Rousselet, G.,
Kamsteeg, E. J.,
Ripoche, P.,
Deen, P. M.,
Cartron, J. P.,
and Bailly, P.
(1999)
J. Biol. Chem.
274,
30228-30235[Abstract/Free Full Text]
|
| 23.
|
Yang, B.,
Fukuda, N.,
van Hoek, A.,
Matthay, M. A., Ma, T.,
and Verkman, A. S.
(2000)
J. Biol. Chem.
275,
2686-2692[Abstract/Free Full Text]
|
| 24.
|
Yang, B., Ma, T.,
and Verkman, A. S.
(2001)
J. Biol. Chem.
276,
624-628[Abstract/Free Full Text]
|
| 25.
|
Lucien, N.,
Sidoux-Walter, F.,
Olives, B.,
Moulds, J., Le,
Pennec, P. Y.,
Cartron, J. P.,
and Bailly, P.
(1998)
J. Biol. Chem.
273,
12973-12980[Abstract/Free Full Text]
|
| 26.
|
Crawford, J. D.,
Doyle, A. P.,
and Probst, J. H.
(1959)
Am. J. Physiol.
196,
545-548
|
| 27.
|
Lassiter, W. E.,
Gottschalk, C. W.,
and Mylle, M.
(1961)
Am. J. Physiol.
200,
1139-1146
|
| 28.
|
Valtin, H.
(1977)
Am. J. Physiol.
233,
F491-F501
|
| 29.
|
Knepper, M. A.,
and Roch-Ramel, F.
(1987)
Kidney Int.
31,
629-633[Medline]
[Order article via Infotrieve]
|
| 30.
|
Frohlich, O.,
Macey, R. I.,
Edwards-Moulds, J.,
Gargus, J. J.,
and Gunn, R. B.
(1991)
Am. J. Physiol.
260,
C778-C783[Abstract/Free Full Text]
|
| 31.
|
Sands, J. M.,
Gargus, J. J.,
Fröhlich, O.,
Gunn, R. B.,
and Kokko, J. P.
(1992)
J. Am. Soc. Nephrol.
2,
1689-1696[Abstract]
|
| 32.
|
Kriz, W.
(1981)
Am. J. Physiol.
241,
R3-R16[Abstract/Free Full Text]
|
| 33.
|
Bankir, L.,
and deRouffignac, C.
(1985)
Am. J. Physiol.
249,
R643-R666[Abstract/Free Full Text]
|
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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|
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|

|
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|
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|
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|
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