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Lack of UT-B in vasa recta and red blood cells prevents urea-induced improvement

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发表于 2009-4-22 08:17 |显示全部帖子
作者:Lise Bankir,, Kai Chen, and Baoxue Yang作者单位:1 Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521; and 2 Institut National de la Santé et de la Recherche Médicale Unit 36 Institut du Fer a Moulin, 75005 Paris, France
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          【摘要】1 F  x" @$ @9 F+ a6 B( Y
      Recycling of urea within the renal medulla is known to play an important role in the capacity of the kidney to concentrate urine. This recycling occurs simultaneously through a tubular and a vascular route (i.e., through the loops of Henle and vasa recta, respectively). In the present study, transgenic mice with a selective deficiency in UT-B (the urea transporter protein expressed in descending vasa recta and red blood cells), were used to evaluate the specific contribution of vascular urea recycling to overall urine-concentrating ability (UCA). The renal handling of urea was studied in normal conditions and after acute or chronic alterations in urea excretion (acute urea loading or variations in protein intake, respectively). In normal conditions, UT-B null mice exhibited a 44% elevation in plasma urea (P urea ), a normal creatinine clearance, but a 25% decrease in urea clearance, with no change in that of sodium and potassium. Acute urea loading induced a progressive increase in urinary urea concentration (U urea ) in wild-type mice and a subsequent improvement in their UCA in contrast to UT-B null mice, in which urinary osmolality and U urea did not rise, due to the failure to accumulate urea in the medulla. With increasing protein intake (from 10 to 40% protein in diet, leading to a 5-fold increase in urea excretion), P urea was further increased in null mice while little change was observed in wild-type mice, and null mice were not able to increase U urea as did wild-type mice. In conclusion, this study in UT-B-deficient mice reveals that countercurrent exchange of urea in renal medullary vessels and red blood cells accounts for a major part of the kidney's concentrating ability and for the adaptation of renal urea handling during a high-protein intake. 3 e9 b3 F5 ]% _3 x
          【关键词】 urea transport protein intake urinary concentrating ability creatinine clearance urea clearance
: z( ^. o0 c+ \! @- m# h) J1 h                  UREA, THE MOST ABUNDANT SOLUTE in the urine, is known to participate in the urinary concentrating mechanism by being accumulated in the renal medulla at a high concentration. Urea thus contributes to the osmotic driving force that helps reclaim water from the collecting ducts and concentrate urine ( 4, 11, 16, 17, 31 ). Several urea transporters (UT) are involved in this urea accumulation ( 1, 26, 31 ). Concentrated urea is delivered at the tip of the papilla by the terminal collecting ducts expressing the vasopressin-dependent UT-A1. This urea diffuses in the inner medullary interstitium and is constantly at risk of being washed out from the medulla in the blood flowing in numerous ascending vasa recta and joining the renal vein. A significant fraction of this urea is returned to the inner medulla by countercurrent exchange between venous vasa recta and descending structures. There are two different returning pathways, one through the descending vasa recta involving UT-B, and the other through the descending limbs of Henle's loops involving UT-A2 ( 11, 31 ).
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The existence and intensity of urea recycling in the loops of Henle (the "tubular recycling pathway") have been well documented in a number of micropuncture experiments. The flow of urea in the early accessible part of the distal convoluted tubule (exit of the loop of Henle) was found to exceed that in the late accessible proximal convoluted tubule (upstream of the loop of Henle) in rats, and even more so in some rodents adapted to arid environment ( 3, 12, 14, 18, 25, 34 ). This suggests a net addition of urea in the portion of the nephron located between these two points. In contrast, the magnitude of urea recycling through the descending vasa recta (the "vascular recycling pathway") has never been evaluated directly because vasa recta are accessible to micropuncture only in a very short portion of the inner medulla. However, this vascular recycling is suggested by the parallel arrangement in countercurrent of ascending and descending vasa recta in "vascular bundles" and by the existence of special anatomic adaptations of these bundles in rodents with high urinary concentrating ability (incorporation of thin limbs of short loops in mouse, Mongolian gerbil, and sand rat), likely making countercurrent exchange more efficient ( 3, 12, 34 ). The endothelium of descending vasa recta and the membrane of red blood cells (RBCs) strongly express the facilitated urea transporter UT-B ( 32, 33, 38 ). This transporter confers on them a high permeability to urea, as evaluated directly in isolated perfused vessels ( 21 ) and in RBC suspensions ( 15 ). UT-B has also been found in several cell lines of endothelial origin and in rat aorta, spinotrapezious muscle, and mesenteric artery ( 36 ), but its presence in the endothelium of other renal vessels (outside the descending vasa recta) has not been documented as yet.! ]9 B# F; _: O. w/ ]9 t. ~
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Recently described transgenic mice with selective deletion of UT-B ( 37 ) should be an excellent model to evaluate, for the fist time, the quantitative importance of vascular urea recycling in the urinary concentrating process, independently of any alteration in tubular urea transport. UT-B null mice exhibit no gross phenotypic anomaly. However, urea permeability in their RBCs is markedly reduced (45-fold lower than in wild-type), and it may be assumed that the permeability to urea of their descending vasa recta is also largely reduced. Their overall capacity to concentrate urine is decreased by about one-third, and their capacity to concentrate urea by about one-half ( 37 ).
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% z) v" e- [9 Z% f. x& LUT-B null mice were used in the present study to address the following questions. Does UT-B-mediated urea transport influence renal urea handling? Is UT-B involved in the well-known urea-induced improvement in urinary concentrating ability? After a series of observations performed in basal conditions, selective alterations in urea excretion were induced either by acute administration of urea or by chronic changes in the protein content of the diet. New technical procedures were introduced to improve the reproducibility and accuracy of balance studies and urine collection in mice. These experiments revealed that UT-B deletion does not alter filtration rate nor kidney mass but decreases the clearance of urea. They also showed that urea turnover is much higher in mice than in rats and other mammals, and that the well-known urea-dependent improvement in urinary concentrating ability is totally abolished in the absence of UT-B.
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METHODS
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% H8 m' L3 z$ v) o: J0 T( U! G" ~Experimental animals. Transgenic knockout mice deficient in UT-B protein were generated by targeted gene disruption as previously reported ( 36 ). Knockout mice did not express detectable UT-B protein in any organ. All of the wild-type and null mice used in this study were in the CD1 genetic background. All animal procedures were approved by the University of California San Francisco Committee on Animal Research.
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" e$ |- n4 C$ S; V0 O. ~" tRenal function in basal conditions (experiment A). Adult female wild-type and UT-B null mice (6 mice/group, body wt 25-30 g) were placed in metabolic cages adapted for mice (Harvard Apparatus) and fed a standard synthetic rodent diet (AIN 93G, Bioserv, Frenchtown, NJ) with 20% protein by weight (as casein). To avoid spillage and contamination of urine by powdered food in metabolic cages, food was provided as a paste by mixing the powder with a small amount of water (0.3 ml/g dry food). To ensure similar food intake in the two groups (and thus similar osmolar and urea loads to excrete in the urine), and to reduce interindividual variability in each group, mice were given a limited amount of food per day (5.2 g, corresponding to 4 g dry food). This amount was only slightly lower than their spontaneous intake but was sufficient to maintain stable body weight (note that mice fed solid pellets eat less than 4 g food/day). Drinking water was provided ad libitum during the whole study.
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$ ^  s' B7 S' ]! p7 t4 X8 P" _After 2 days of adaptation to the cages and this type of diet, urine was collected for 24 h under paraffin oil (to prevent evaporation). All parts of the urine collection system were siliconized (Sigmacote, Sigma) to minimize urine losses. After completion of urine collection, mice were anesthetized and blood was collected by puncture of the periorbital venous sinus, in tubes with sodium heparin (Vacutainer, Becton Dickinson Vacutainer Systems).
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3 C( P( y6 x  Y' _: X( hUrinary volume was measured by gravimetry, assuming a density of 1 g/ml. Plasma and urinary osmolality were measured by freezing-point depression (Micro-osmometer, Precision Laboratory). Sodium, chloride, potassium, urea, and creatinine concentrations were measured with automatic analyzers (enzymatic method for creatinine with the Ortho Vitros Analyzer) at the University of California San Francisco Clinical Chemistry Laboratory. Absolute excretion, clearance, and urine-to-plasma concentration ratio (U/P) were calculated for creatinine and the different solutes. Results obtained in the two genotypes were compared by Student's t -test, and differences were considered statistically significant for P - H; M: B8 y/ A

8 `9 t8 o- b  Z3 ~+ eAcute urea load (experiment B). Adult female wild-type and UT-B null mice (6 mice/group, body wt 25-30 g) were adapted for 2 days before the urea load experiment to metabolic cages and to 4 g dry food/day (AIN 93G, 20% casein). To ensure similar basal conditions of urinary concentrating ability in the two genotypes before the acute experiment, urinary osmolality was reduced and urinary flow rate was increased in wild-type mice to the level seen in UT-B null mice. This was achieved by mixing their food with 1.3 ml water/g dry food vs. only 0.3 ml water/g dry food in UT-B null mice (thus yielding 5.2 ml/day in wild-type mice vs. only 1.2 ml/day in UT-B null mice). Food dishes were replaced every day at 6:00 P.M., and most of the food was consumed during the night period. Drinking water was provided ad libitum during the entire study.
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Urine was collected for the 24 h preceding the acute urea load, and urinary volume and osmolality were measured as above. During the acute experiment, urine was collected every 2 h after spontaneous voiding and/or bladder massage as follows. The lower part of the metabolic cage was removed and replaced by a tray on which a large piece of Parafilm was placed. At 8:00 A.M., each mouse had its bladder emptied by gentle abdominal massage, and this urine was discarded. Thereafter, to avoid evaporation, the Parafilm sheets were inspected every 30 min, and any urine found on the sheets was collected and placed in preweighed tubes filled with a little paraffin oil (to prevent evaporation). Every 2 h (at 10:00 A.M., and 12:00, 2:00, 4:00 and 6:00 P.M.), urine was collected by abdominal massage and transferred into preweighed tubes. Urine collected from 8:00 to 10:00 A.M. corresponded to a basal period. The urea load was administered just after the 10:00 A.M. urine collection. Three hundred microliters of a 1 M urea solution was administered by intraperitoneal injection, thus yielding 300 µmol of urea, an amount about one-tenth of the daily urea excretion. Preliminary experiments have shown that intraperitoneal administration of 1 M urea was well tolerated. This high concentration was intended to yield as little water as possible with the urea load.
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Urinary volume and osmolalilty were measured as in experiment A. Urinary urea concentration was measured with a standard kit (Sigma). Osmolar and urea excretions were calculated as well as the concentration and excretion of nonurea solutes (osmolar excretion - urea excretion). Basal conditions in the two groups of mice (previous 24 h and first 2-h period) were compared by Student's t -test. Results observed in the two groups after the urea load were compared by two-way ANOVA followed by Fisher's post hoc test.$ C, Q, ]& V  q- b' s- ~" r
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Chronic alteration in protein intake (experiment C). Adult female wild-type and UT-B null mice (6 mice/group, body wt 21-25 g) were offered the synthetic food AIN 93G (Bioserv) with low (LP)-, normal (NP)-, or high (HP)-protein content (10, 20, or 40% casein, respectively, as the only source of protein). Each diet was given for 1 wk, in order of increasing protein content. These diets produced a fivefold range in daily urea excretion. The starch content of the diet was altered in inverse proportion of casein to ensure an equivalent calorie intake. As in experiment A, each mouse was offered only 4 g dry food/day mixed with 0.3 ml water/g dry food. Drinking water was provided ad libitum during the entire study.9 u* I3 {/ \9 B* z) N

2 k- \: E, |1 W5 S7 QFor the last 3 days of each week, mice were placed in metabolic cages and urine was collected for 24 h on the last day. At the end of each weekly urine collection, a blood sample ( 75 µl) was taken from each mouse in heparinized microtubes by tail bleeding. Urinary volume and osmolality and urinary and plasma urea concentrations were measured as in experiment B. Osmolar and urea excretions as well as the urine-to-plasma concentration ratio of urea (U urea /P urea ) were calculated. Results obtained in the two different genotypes on the three different diets were compared by two-way ANOVA followed by Fisher's post hoc test., m; a6 Q7 y0 \3 g
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RESULTS
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Renal function in basal conditions (experiment A). The consequences of UT-B deletion on basal renal function were studied in mice fed a fixed daily amount of food, with special care taken to limit urine contamination by the food, urine loss in the metabolic cages, and interindividual variability. As already reported ( 37 ), the lack of UT-B results in a significant urinary concentrating defect and in an elevated plasma urea level. In the present experimental setting, daily urinary output was 62% higher and osmolality 33% lower in UT-B null mice than in wild-type mice, and plasma urea was increased by 44% ( Table 1 ). The U/P concentration ratio for creatinine and electrolytes was 30% lower in UT-B null mice than in wild-type mice, whereas the U/P concentration ratio for urea was more severely decreased to 50% of that in wild-type mice ( Table 1 ).
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0 b8 O! f  M" f$ b' W! Y  _5 qTable 1. Urea handling in UT-B KO and WT mice in normal conditions
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Interestingly, plasma creatinine and creatinine clearance (an index of glomerular filtration rate) were similar in both groups, suggesting that UT-B deletion has no influence on glomerular hemodynamics. Kidney weight in the two genotypes was obtained in a separate series of six UT-B null mice and six wild-type mice placed in conditions similar to those in experiment A. Body weight was 25.0 ± 1.3 and 25.9 ± 1.4 g in wild-type mice and UT-B null mice, respectively, and kidney weight (2 kidneys) was 364 ± 45 and 367 ± 21 mg, respectively (not significant). Thus kidney weight was also not influenced by UT-B deletion. The clearance of sodium and potassium were similar in both groups. In contrast, urea clearance was significantly lower in UT-B null mice than in wild-type mice by 25% ( Table 1 ).( R- Y" W6 c, i* p' g  s

1 Y6 a2 ~, W8 ?Acute urea load (experiment B). To evaluate the contribution of UT-B in the capacity of the kidney to excrete a load of urea and to use urea to improve water conservation mechanisms, mice were subjected to an acute modest urea load ( 1/10 of their daily urea excretion). Before this load, mice of the two genotypes had received the same amount of food, but the fluid content of the food was increased in wild-type mice slightly above that in null mice. As shown in Table 2, this resulted in very similar initial conditions in the two genotypes with respect to their urinary and urea concentrating ability during the previous 24 h and the 2-h period preceding the load.& I0 Q1 l; n, z/ a

7 V1 Z5 h# O5 p$ W# }* R+ S- eTable 2. Urine-concentrating activity and urea excretion before urea load" z* {+ |0 b  i( S7 S3 C7 h9 o
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In the first 2 h after administration of the urea load, urea excretion rose in the two groups in a very similar manner ( Fig. 1 D ). However, this increase resulted from a very different pattern of changes in urinary flow rate and urea concentration in the two genotypes. Wild-type mice increased their urea concentration and urinary osmolality after the first postload period and exhibited only a small rise in urinary flow rate, whereas UT-B null mice almost doubled their urinar flow rate but showed only a modest rise in urinary osmolality and urinary urea concentration ( Figs. 1, A - C ). The cumulated amount of water excreted above the basal level during the first 4 h after the load was 111 ± 23 µl in null mice vs. only 32 ± 9 µl in wild-type mice ( P
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Fig. 1. Effect of acute urea loading on urinary concentrating ability and renal handling of urea in 6 wild-type ( / ; ) and 6 UT-B null mice (UT-B -/-; ). Values are means ± SE. Three hundred micromoles of urea were injected ip just after the first 2-h urine collection ( time 0 ). A : urinary osmolality (U osm ). B : urinary output. C : urinary urea concentration (U urea ). D : urea excretion (excr.). E : non-urea solute concentration (U non-urea solutes ). F : excretion of non-urea solutes. Analysis by 2-way ANOVA (genotype and 2-h periods) indicated significant effects for U osm ( P
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After urea excretion returned to basal level (6 and 8 h after the load), urinary osmolality and urea concentration in wild-type mice remained far higher than during the basal period. Their urinary output declined significantly to less than half of basal level (61 µl/2 h, 8 h after the load vs. 138 µl/2 h in the basal period). In UT-B null mice, urinary osmolality was only modestly elevated and urinary flow rate declined by only one-third (102 µl/2 h, 8 h after the load vs. 152 in basal period) ( Fig. 1, A and B ). Administration of exogenous urea also improved the ability of the kidney to concentrate other urinary solutes in wild-type mice, but this improvement was not observed in UT-B-deficient mice ( Fig. 1 E ). During the last period of the experiment, the concentration of non-urea solute plateaued at 316 ± 47 mmol/l in null mice but reached 611 ± 70 mmol/l in wild-type mice ( P
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+ T+ l: I8 a2 V9 v8 D+ cChronic alteration in protein intake (experiment C). To evaluate how UT-B null mice chronically adapt their renal function and plasma urea level to different levels of urea excretion, wild-type mice and UT-B null mice were fed for 1 wk a diet containing 10, 20, or 40% protein. Body weight did not vary throughout the study in either group (23.4 ± 1.4, 23.8 ± 1.7, and 24.8 ± 1.0 g in UT-B null mice and 24.5 ± 0.5, 24.4 ± 0.5, and 24.8 ± 1.3 g in wild-type mice on the LP, NP, and HP diet, respectively). This shows that the slight restriction in food intake had no consequence on energy or nitrogen balance. The different levels of protein intake did not induce significant changes in urinary osmolality in either group, this osmolality being, however, consistently lower in UT-B null mice than in wild-type mice ( Fig. 2 A ). In contrast, protein intake had a marked influence on urine output ( Fig. 2 B ), which increased in the two groups with the amount of protein in the diet, thus leading to parallel increases in osmolar excretion ( Fig. 2 C ). Urinary flow rate was consistently higher in UT-B null mice than in wild-type mice on each diet, but osmolar excretion was similar in the two groups for each diet ( Fig. 2 C ), as can be expected because of the similar food intake imposed on both groups.
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9 @5 _! z4 ~0 b; s/ D3 Z5 sFig. 2. Urinary concentrating ability in 6 wild-type and 6 UT-B null mice fed diets containing 10, 20, or 40% casein during 3 successive wk (urine collected on the last day of each week). Values are means ± SE. A : urine osmolality (U osm ). B : daily urine output. C : daily urinary osmolar excretion. Analysis by 2-way ANOVA: a, effect of genotype; b, effect of diet; c, interaction between genotype and diet. NS, not significant.- X6 T2 ?. |) z: s
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The level of protein intake did not significantly alter plasma urea level in wild-type mice. In contrast, plasma urea in UT-B null mice was very sensitive to protein intake and was 48 and 75% higher on the NP and HP diets, respectively, than on the LP diet ( Fig. 3 A ). As a result, the UT-B null mice-to-wild-type mice ratio for plasma urea rose from 1.30 with the LP diet, to 1.55 with the NP diet, and to 1.80 with the HP diet. As expected, urea excretion rose to the same extent in both groups of mice in response to graded increases in protein intake ( Fig. 3 D ). However, in wild-type mice, part of this increase in urea excretion was achieved by a significant rise in urea concentration in the urine ( Fig. 3 B ), whereas in UT-B null mice it was only due to an increase in urine output ( Fig. 2 B ) with no change in urinary urea concentration ( Fig. 3 B ). The U/P ratio of urea concentration was not significantly affected by the level of protein intake but was much lower in UT-B null mice than in wild-type mice on each of the three diets ( Fig. 3 C ).
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7 e- o, O$ p/ Z5 y6 t0 N- U+ OFig. 3. Urea handling in 6 wild-type and 6 UT-B null mice fed diets containing 10, 20, or 40% casein during 3 successive wk (urine collected on the last day of each week). Values are means ± SE. A : plasma urea concentration (P urea ). B : U urea. C : urine-to-plasma ratio (U urea /P urea ) of urea concentration. D : daily urea excretion. Analysis by 2-way ANOVA: a, effect of genotype; b, effect of diet; c, interaction between genotype and diet. NS, not significant./ B$ T1 c4 y9 ~( ?, L
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DISCUSSION% y2 |5 f& _% `. A
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The present study reveals that countercurrent exchange of urea between vasa recta supplying and draining the renal medulla plays a critical role in the renal concentrating process. Mice with UT-B deletion (and thus with a reduced permeability to urea in RBCs and probably also in descending vasa recta) were completely unable to take advantage of urea for improving urinary concentrating ability acutely. When challenged chronically with a high-protein intake, they were unable to prevent an ample rise in plasma urea and to enhance urea concentration in urine relative to plasma, as did wild-type mice. In addition, this study reveals that UT-B deletion does not alter glomerular hemodynamics and kidney mass but selectively reduces urea clearance by 25% without altering the clearance of the other main urinary solutes.
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7 y/ ]  m) U0 G& B. HUrea accumulation in the renal medulla, which contributes to water conservation, involves complex movements of urea within the kidney in selected nephron segments and vessels expressing several urea transporters, as shown in Fig. 4 A ( 4, 20, 26, 31 ). The contribution of countercurrent exchange in vasa recta (as opposed to that in loops of Henle) has not yet been evaluated experimentally because early descending and late ascending vasa recta are not accessible to micropuncture, as are the entry and exit of the loops of Henle (at least for superficial nephrons). The selective deletion of UT-B transporter protein thus offers an appropriate model for evaluating the importance of the vascular recycling route of urea in the urinary concentrating mechanism and its possible influence on other aspects of renal function.$ _& Q. c2 K; I, d
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Fig. 4. Diagram depicting the vascular and tubular routes of urea recycling within the kidney. A short and a long loop of Henle are depicted within the four kidney zones (C, cortex; OS and IS, outer stripe and inner stripe of the outer medulla, respectively; IM, inner medulla), along with an arterial descending and a venous ascending vasa recta (DVR and AVR, respectively). Urea delivery to the IM and urea transit in AVR are shown in purple. The pathways allowing urea to return to the IM are indicated by red arrows for the vascular route and by blue arrows for the tubular route. A : in normal mice, concentrated urea is delivered to the tip of the papilla by the terminal part of the collecting ducts expressing the vasopressin-regulated urea transporter UT-A1. Urea is taken up by AVR, and a significant fraction of it is returned to the IM by being reintroduced either in the descending vasa recta (DVR; expressing UT-B) or in the descending thin limbs of Henle's loops (expressing UT-A2). B : in UT-B null mice, urea is delivered normally to the IM and can be normally recycled in the loops of Henle, but the possibility to recycle urea through DVR is compromised (thin red arrows). Thus more urea is returned to the general circulation through AVR (thick red arrow exiting AVR at the corticomedullary border).
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Because studies of renal function in mice are hampered by the difficulty of urine collection and by large interindividual variability, several technical improvements were introduced in this study. They were designed to avoid food spillage, to reduce possible differences in food intake between groups, and to perform accurate short-term urine collection in mice. Care was taken to have mice of the two genotypes in the same basal condition before a specific stress was induced and to avoid too severe stresses that could introduce confounding factors.+ Q0 a9 p; a* R; V0 d+ |; L- w

4 ]! j: B6 L" uConsequences of UT-B deletion in basal conditions. Countercurrent exchange of urea is thought to take place between descending and ascending vasa recta in the outer and inner medulla ( 2, 13 ). Venous vasa recta exhibit a fenestrated endothelium that represents no barrier for water and solute movements ( 28 ). Descending vasa recta have a tight endothelium that exhibits a poor permeability to sodium but a much higher permeability to water and urea ( 21, 28 ), due to the expression of aquaporin-1 and UT-B, respectively. The lack of UT-B in vasa recta in UT-B null mice most likely reduces their urea permeability as it does in RBCs. As a consequence, urea transfer from ascending vasa recta (rich in urea coming from the inner medulla) to descending vasa recta (with the same concentration of urea as that in peripheral blood) must be largely reduced. However, the delivery of urea to the inner medulla by the terminal collecting duct (via vasopressin-dependent UT-A1) and the recycling of urea through thin descending limbs (via UT-A2) should remain unaltered ( Fig. 4 B ). As shown previously, urea accumulation in the inner medulla of UT-B null mice is reduced by about one-half compared with wild-type mice ( 37 ). Countercurrent exchange of urea through the vasa recta and RBCs is thus a crucial step in the accumulation of urea in the renal medulla. It allows normal mice to increase their urinary osmolality by as much as 870 mosmol/kgH 2 O (2,650-1,780; see Table 1 ) and to spare 1.2 ml water/day, i.e., 50% of the normal urine output.
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- \) ]: [8 {$ G" l. [( L  \Why is urea less well accumulated in the medulla in mice with UT-B deletion? The present study, performed in free living animals, cannot bring an answer to this question. However, a possible mechanism may be proposed, based on well-accepted concepts deduced from micropuncture and microper-fusion experiments. Urea permeability is likely reduced in descending vasa recta as it is in RBCs ( 37 ) as a consequence of UT-B deletion. Thus less of the urea flowing up toward the cortex in ascending vasa recta can diffuse into descending vasa recta, and more is returned to the venous blood in the main renal veins at the corticomedullary border. This interpretation is compatible with the fact that plasma urea is higher and urea clearance lower in UT-B null mice.* m: H5 E3 \4 ~0 f& x
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Deletion of UT-B did not induce any change in either plasma creatinine or creatinine clearance. Even if creatinine clearance is not a reliable marker of glomerular filtration rate (GFR), this observation suggests that UT-B deletion did not alter GFR. In good agreement with this assumption is the fact that kidney weight was also not altered in UT-B null mice.* }  |0 j4 k. Q! H7 I
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Plasma urea was significantly higher and urea clearance significantly lower in UT-B-deficient mice than in wild-type mice ( Table 1 ). The combination of a higher plasma urea, a lower urea clearance, and an unaltered GFR indicates that more urea was filtered in UT knockout mice but that a higher fraction of the filtered urea was returned to the blood.- b( m/ R0 c- \

3 I0 h* B( }! k, z4 j/ e' rThis study reveals that mice have a much higher turnover of urea than larger mammals. As shown in Table 3, they exhibit a much higher rate of urea excretion and a higher daily load of urea per gram of kidney weight than rats. These differences are due to allometric scaling involving a proportionately more intense metabolic rate and higher food consumption in smaller animals. As a result, the mouse kidney needs to excrete a considerably higher relative load of urea than that of larger mammals. Daily urea excretion in mice represents 25 times their body pool of urea vs. only 9 times in rats and about 1 time in humans ( 4 ).' `# \6 L( n4 N; M/ H" `6 [( e
% T- l, s6 v+ c% g/ V9 {
Table 3. Comparison of urea handling in rats and mice9 q' n! U! m+ n8 O! D) a

9 x% ~7 W* t; v; I/ W6 vConsequences of UT-B deletion in the face of acute or chronic increases in urea excretion. Urinary concentrating ability is known to be enhanced by a HP diet ( 6, 8, 10, 23, 27 ) and by addition of urea to the food or administration of exogenous urea ( 7, 9, 22, 30 ). Experiments B and C show that this enhancement is largely due to urea recycling through the vascular route in the medulla. UT-B null mice were unable to elevate their urinary urea concentration when infused acutely with a load of urea or even when given a HP diet for 1 wk. Their plasma urea level increased markedly with the protein content of the diet and thus with increasing needs for urea excretion. The protein-induced increase in plasma urea seen in UT-B null mice in experiment C is most probably due to a greater return of urea to peripheral blood through ascending vasa recta. The U/P ratio for urea, an index of the kidney's capacity to selectively concentrate urea, increased in wild-type mice (from LP to HP) but remained unchanged or even slightly decreased in UT-B null mice. With a LP intake, the difference between the two groups of mice was modest, but it increased dose dependently with protein intake.. B2 a* K( k* X, p2 X
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In experiment B, the acute load of urea, although a relatively small fraction of the daily urea excretion, was very large compared with the body pool of urea ( Table 3 ). Assuming the infused urea was mixed instantaneously with this pool, it should have increased plasma urea concentration about threefold and plasma osmolality by about 18 mosmol/l. Even if urea is a less efficient osmole than sodium for stimulating vasopressin secretion ( 39 ), a significant rise in plasma vasopressin can be expected to occur in response to this load.
3 y  g& W1 U, ]) z3 q* t
4 Y1 E2 C9 P: o( bThe combination of an increased availability of both urea and vasopressin allowed a rise in urinary osmolality and urinary urea concentration in wild-type mice but not in UT-B null mice. The latter required a much larger amount of water to excrete the urea load than the former, thus revealing the crucial role of the vascular recycling route in the dynamic phase of progressive urea accumulation in the medulla. Moreover, in the last two periods of the experiment, osmolality and urea concentration in the urine of wild-type mice remained stable and much higher than in basal conditions, whereas the urinary flow rate was significantly diminished and the concentration of non-urea solutes significantly increased. In contrast, this late effect did not occur in UT-B null mice in which urea accumulation in the inner medulla is compromised ( 37 ). Altogether, these results illustrate the "economy of water referable to urea" described by Gamble et al. ( 9 ) and reveal the importance of UT-B in this process.
. H9 ^0 `1 x. n8 ~3 C: |1 ]
" A, p9 S8 [& x$ o- EThe respective contribution of plasma urea and of RBC urea content to this overall process cannot be deduced from the present study because UT-B deletion affected simultaneously urea permeability in the endothelium of vasa recta and in the membrane of RBCs. However, one should note that hematocrit is lower in the medulla than in the peripheral circulation ( 24 ) and that plasma and vascular endothelium remain inevitable intermediates between RBCs and the surrounding interstitium. Thus the contribution of UT-B-mediated urea transport in RBCs, although certainly not negligible, should probably be less intense than that in the vascular endothelium of descending vasa recta.
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In the aggregate, the present results suggest that the recycling of urea in vasa recta (and RBCs) plays a much greater role in the urinary concentrating process than its recycling in the loops of Henle ( Fig. 4 ). Suppression of the former almost completely prevented the kidney from taking advantage of urea for improving its concentrating ability. This preponderant influence of vascular recycling over tubular recycling was not anticipated. Vascular recycling may be quantitatively more important than tubular recycling for two reasons. First, much more blood than tubular fluid is potentially available for urea transfer because blood velocity in vasa recta is far higher than that of tubular fluid in the loops of Henle and because vasa recta largely outnumber the loops of Henle. Second, UT-B is expressed along the whole length of vasa recta and thus offers a long distance for countercurrent exchange, whereas UT-A2 is expressed only for a short distance along descending limbs ( 19, 29, 31, 35 ). Mice with selective deletion of UT-A2, when available, will allow a more precise evaluation of the relative importance of tubular vs. vascular urea recycling in the urinary concentrating process.* h  T1 O+ H; N) u1 c% P" ^

; H" D9 D' L! k0 M9 _In summary, the present studies revealed that urea turnover and urea clearance are much greater in mice than in other, larger mammals. They also showed that UT-B deletion results in an elevation of plasma urea level and reduced urea clearance without an alteration in GFR and kidney weight. These defects are most likely due to the failure to sequester urea in the renal medulla by countercurrent exchange between ascending and descending vasa recta, and thus to a greater return of renal urea to the peripheral circulation. Defective vascular urea recycling also results in a significant impairment of the capacity to concentrate urea and non-urea solutes in the urine. When challenged with an increase in urea excretion, the kidney is not able to take advantage of this urea to improve the efficiency of the urinary concentrating mechanism and is not able to prevent a dose-dependent rise in plasma urea, as in normal mice. This study in UT-B deficient mice demonstrates the critical role of countercurrent exchange of urea in the renal medullary vessels in the urinary concentrating mechanism and in the adaptation of the kidney to a high-protein intake. Further experiments are required to determine the respective contribution of RBCs and vasa recta to urea-induced water economy.+ I! t' c6 Y8 `; Y

5 t+ a; p1 t" ?8 G- r/ {! F4 {ACKNOWLEDGMENTS2 H( m% \4 \" o/ k
7 J4 ~; f' _) y/ u. b- C
The authors thank Dr. A. S. Verkman (UCSF) and Nadine Bouby (INSERM Unit 367, Paris) for a critical reading of the manuscript and Liman Qian (UCSF) for mouse breeding and genotyping.5 e$ a, y" ]) n2 `) D' J
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GRANTS
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35124 and American Heart Association Grant 0365027Y.
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: l, s) L2 V4 B5 L, w- \( BLassiter WE, Gottschalk CW, and Mylle M. Micropuncture study of net transtubular movement of water and urea in nondiuretic mammalian kidney. Am J Physiol 200: 1139-1146, 1961.
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( c2 g% [7 T# I) U+ T/ r  wZerbe RL and Robertson GL. Osmoregulation of thirst and vasopressin secretion in human subjects: effect of various solutes. Am J Physiol Endocrinol Metab 244: E607-E614, 1983.

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