Journal of the American Society of Nephrology
2007 JASN IMPACT FACTOR 7.111 HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP 
    advanced
CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Klein, J. D.
Right arrow Articles by Yang, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Klein, J. D.
Right arrow Articles by Yang, B.
J Am Soc Nephrol 15:1161-1167, 2004
© 2004 American Society of Nephrology

BASIC SCIENCE

Upregulation of Urea Transporter UT-A2 and Water Channels AQP2 and AQP3 in Mice Lacking Urea Transporter UT-B

Janet D. Klein*, Jeff M. Sands*,{dagger}, Liman Qian{ddagger}, Xiaodan Wang* and Baoxue Yang{ddagger}

*Renal Division, Department of Medicine, and {dagger}Department of Physiology, Emory University School of Medicine, Atlanta, Georgia; and {ddagger}Department of Medicine, University of California, San Francisco, San Francisco, California

Correspondence to Dr. Baoxue Yang, 1246 Health Sciences East Tower, University of California, San Francisco, San Francisco, CA 94143-0521. Phone: 415-476-8530; Fax: 415-665-3847; E-mail: byang{at}itsa.ucsf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
ABSTRACT. The UT-B urea transporter is the major urea transporter in red blood cells and kidney descending vasa recta. Humans and mice that lack UT-B have a mild urine-concentrating defect. Whether deletion of UT-B altered the expression of other transporter proteins involved in urinary concentration was tested. Fluorescence-based real-time reverse transcription–PCR and Northern blot analysis showed upregulation of the UT-A2 urea transporter and the aquaporin 2 (AQP2) and AQP3 water channel transcripts but no change in other urea transporters or AQP. Western blot analysis showed that UT-A2 protein abundance in the outer medulla of UT-B null mice increased to 122 ± 6% of wild-type control. AQP2 protein abundance increased to 177 ± 32% and 127 ± 7% in the outer and inner medulla, respectively, of UT-B null versus wild-type mice. The abundance of UT-A1, AQP1, renal outer medullary potassium channel, and NKCC2/BSC1 proteins were not significantly different between UT-B null and wild-type mice. The increases in AQP2 and AQP3 would reduce water loss and improve concentrating ability. The lack of UT-B does not result in a change in expression of urea transporters involved in urea reabsorption from the inner medullary collecting duct (UT-A1 and UT-A3). However, UT-B null mice have a selective increase in UT-A2 protein abundance. This may be an adaptive response to the loss of UT-B, because UT-B and UT-A2 are involved in different intrarenal urea recycling pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
In mammals, urea transporters play a central role in the urine-concentrating mechanism. The urea transporters that have been cloned to date belong to two related subfamilies, the renal tubular-type, UT-A, and the erythrocyte-vascular type, UT-B (reviewed in references 1,2). Both genes give rise to proteins that share a high degree of homology and are functionally similar. Five UT-A isoforms have been identified so far, which are produced by alternative splicing of Slc14a2 gene (3,4). UT-A1 and UT-A3 are expressed in the apical membrane of the rat terminal inner medullary collecting duct (IMCD) (5–7). UT-A1 and UT-A3 are also expressed in the mouse terminal IMCD (8,9). In contrast to the rat, mouse UT-A3 is expressed in the basolateral membrane (10). Because the terminal IMCD is the site of vasopressin-regulated urea transport (11), UT-A1 and UT-A3 are thought to be involved in vasopressin-regulated urea transport (12,13). UT-A2 is located in the late part of descending thin limbs of short-looped and long-looped nephrons in rat and mouse (5,7–9,14). UT-A2 expression is increased by chronic dDAVP treatment (14,15) and is thought to facilitate intrarenal urea recycling (16). UT-A4 has been cloned from rat and is expressed in the kidney medulla (12), but it has not been cloned from the mouse (3,8). UT-A5 is not expressed in the kidney (17).

UT-B is expressed in descending vasa recta in kidney outer and inner medulla, in erythrocytes, and in several other tissues (reviewed in reference 1). UT-B has a similar membrane topology to UT-A2 (based on hydropathy analysis) and has similar functional characteristics (reviewed in references 1,18). Both UT-B and UT-A2 are thought to be involved in intrarenal urea recycling, although in different recycling pathways. Humans who lack UT-B are unable to concentrate their urine above 800 mOsm/kg H2O (19). We recently generated UT-B null mice whose ability to concentrate urine is decreased by approximately one third (20), similar to the percentage reduction in concentrating ability in humans (19). The UT-B null mice have a more severely impaired ability to concentrate urea than to concentrate other solutes, resulting in an increase in blood urea concentration and a decrease in maximal urine-concentrating ability (20). 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 urine-concentrating mechanism and is not able to prevent a dose-dependent rise in plasma urea in UT-B null mice, as it does in normal mice (21). This suggests that UT-B deletion in the kidney results in a selective urea-concentrating defect, most likely as a result of impaired countercurrent exchange of urea between ascending and descending vasa recta (20).

Given that the UT-B null mouse lacks urea recycling in the medullary vasculature, one may have expected these mice (and UT-B null humans) to have a more profound urine-concentrating defect. The purpose of this study was to test the hypothesis that other proteins involved in the urine-concentrating mechanism are upregulated to adapt to the loss of intrarenal urea recycling when urea transport in descending vasa recta and red blood cells is lost as a result of knockout of the UT-B gene in mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Mice
Transgenic knockout mice deficient in UT-B protein were generated by targeted gene disruption as previously reported (20). UT-B null mice did not express detectable UT-B protein in any organ. Adult female wild-type and UT-B null mice used in this study had CD1 genetic background. Measurements were done in litter-matched mice produced by intercrossing heterozygous mice. All animal procedures were approved by the University of California, San Francisco Committee on Animal Research.

Dissection of Renal Medulla Subzones
Mice (aged 6 to 8 wk, body weight 25 to 30 g) were anesthetized with a mixture of ketamine (40 mg/kg) and xylazine (8 mg/kg) and perfused with PBS through a tube placed into the heart. Kidneys were removed, placed into ice-cold PBS, and dissected into inner and outer medulla as described previously (22). Although the kidneys from the UT-B –/– mice tended to be smaller, there was no obvious change in the relative proportions of the different renal zones.

Fluorescence-Based Real-Time Reverse Transcription–PCR
Total RNA from whole mouse kidney was isolated by homogenization in TRIzol reagent (Invitrogen, Carlsbad, CA), and mRNA was extracted using the Oligotex mRNA mini kit (Qiagen, Valencia, CA). cDNA was reverse-transcribed from mRNA with oligo(dT) (SuperScript First Strand Synthesis System for RT-PCR; Invitrogen). Fluorescence-based real-time reverse transcription–PCR (RT-PCR) was carried out by the LightCycler and with LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche Diagnostics, Indianapolis, IN). Primers were as follows: 5'-TGTATGCCTCTGGTCGTACC-3' (sense) and 5'-CAGGTCCAGACGCAGGATG-3' (antisense) for {beta}-actin, 5'-CTCCCTAGTCGACAATTCAC-3' (sense) and 5'-ACAGTACCAGCTGCAGAGTG-3' (antisense) for AQP1, 5'-CTGGCTGTCAATGCTCTCCAC-3' (sense) and 5'-TTGTCACTGCGGCGCTCATC-3' (antisense) for AQP2, 5'-TTGGTGGCTGGCCAAGTGTC-3' (sense) and 5'-GTCTGTGCCAGTGCATAGAT-3' (antisense) for AQP3, 5'-GAGTCACCACGGTTCATGGA-3' (sense) and 5'-CGTTTGGAATCACAGCTGGC-3' (antisense) for AQP4, 5'-GACAGTGAGACGCAGTGAAG-3' (sense) and 5'-ACGGTCTCAGAGCTCTCTTC-3' (antisense) for UT-A1, 5'-TTTCTCCAGTCCTATCTGAG-3' (sense) and 5'-ACGGTCTCAGAGCTCTCTTC-3' (antisense) for UT-A2, 5'-ACGGTCTCAGAGCTCTCTTC-3' (sense) and 5'-AGAGTGGAGGCCACACGGAT-3' (antisense) for UT-A3, 5'-GCCCTTCAACATTGCCTTAACA-3' (sense) and 5'-ATGTTGGGTGGAGTGGTTGT-3' (antisense) for UTA4, and 5'-TCTTCTCAAACAAGGGCGAC-3' (sense) and 5'-TTGCTGAGCACGGAGCTCAA-3' (antisense) for UT-B. The primers were derived from published sequences with GenBank accession numbers NM 007393 ({beta}-actin), NM 007472 (AQP1), NM 009699 (AQP2), NM 016689 (AQP3), NM 009700 (AQP4), AF366052 (UT-A1), AF367359 (UT-A2), AF258602 (UT-A3), AY221737 (UT-A4), and AF448798 (UT-B). Each primer set was checked using a BLAST search to ascertain that the sequences were unique for each mouse AQP or urea transporter. DNA fragments amplified by each primer set are between 100 and 120 bp and cross an exon-intron boundary to prevent genomic DNA contamination. Real-time PCR was carried out according to the manufacturer’s instructions. {beta}-Actin was used as the reference gene, and pooled wild-type cDNA was used as the calibrator. Results are described as a normalized, calibrated ratio. All samples are normalized to the reference gene. Concentration ratios for each sample are then calibrated to calibrator sample such that the quantification results are reported as a normalized ratio with the calibrator sample as the denominator:

Relative mRNA level = ratio of sample (target/reference)/ratio of calibrator (target/reference).

Regular PCR was performed with the same templates and primer sets. PCR products were electrophoresed on a 2% agarose gel.

Northern Blot Analysis
Total RNA from mouse tissues was isolated using TRIzol reagent. mRNA purified by Oligotex mRNA mini kit (Qiagen) were resolved on a 1% formaldehyde-agarose denaturing gel (1.5 µg/lane), transferred to a Nylon+ membrane (Amersham, Piscataway, NJ), and hybridized at high stringency with a 32P-labeled probe corresponding to the mouse UT-A2, UT-A3, UT-B, AQP1, AQP2, AQP3, AQP4, or {beta}-actin cDNA coding sequence. The UT-A3 cDNA coding sequence is used as a UT-A1 probe.

Antibodies
Polyclonal antibodies to UT-A (C-terminus or N-terminus), UT-B, AQP1, AQP2, and NKCC2/BSC1 were generated in rabbit. In mouse, the UT-A C-terminal antibody detects UT-A1 and UT-A2 (22). The UT-A N-terminal antibody detects UT-A1 and UT-A3 (23). Anti–renal outer medullary potassium channel (anti-ROMK) antibody was the gift of Dr. Mark Knepper (National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD) (24). All antibodies used in this study were affinity purified. Secondary antibodies (anti-rabbit IgG) used with the LICOR visualization system were fluorescently labeled with Alexa 680 dye (Molecular Probes, Eugene, OR).

Western Blot Analysis
Tissues were homogenized in isolation buffer (10 mM triethanolamine, 250 mM sucrose, 1 µg/ml leupeptin, 0.1 mg/ml PMSF [pH 7.6], 0.025 to 0.1 g tissue/ml isolation buffer), and concentrated SDS was added to a final concentration of 1%, after which samples were sheared with a 28-G insulin syringe and centrifuged for 15 min at 14,000 x g. Protein was determined in the supernatant fractions using the BioRad DC protein assay kit (BioRad, Richmond, CA). Kidney tissue lysate in Laemmli sample buffer (10 µg/lane) was size-separated by SDS-PAGE using 10 or 15% polyacrylamide gels. Proteins were blotted to polyvinylidene difluoride membranes (Gelman Scientific, Ann Arbor, MI). After transfer, blots were incubated for 30 min at room temperature with blocking buffer: 5% nonfat dry milk suspended in Tris-buffered saline (TBS: 20 mM Tris HCl, 0.5 M NaCl [pH 7.5]). They were then incubated with primary antibody overnight at 4°C. Blots were washed three times in TBS with 0.5% Tween-20 (TBS/Tween), then incubated with goat anti-rabbit IgG linked to Alexa 680 fluorescent dye for 2 h at room temperature. Blots were washed twice with TBS/Tween, then the bound secondary antibody was visualized using infrared laser detection (Alexa-linked 2°Ab; LICOR Odyssey gel scanning system, Lincoln, NE). Laser densitometry was used to quantify the intensity of the resulting bands. Results are expressed as arbitrary units/µg protein loaded. Our laboratory has previously confirmed the specificity of protein recognition by these antibodies by probing with preimmune serum and by performing peptide competition studies with rat tissue samples (25). The specificity of the UT-A C-terminal antibody against mouse proteins was confirmed using the same antibody preadsorption procedure (25). In all cases, parallel gels were stained with Coomassie blue and showed uniformity of loading (data not shown).

Statistical Analyses
All data are presented as mean ± SEM. To test for statistical significance between UT-B null and wild-type mice, we used an unpaired t test. The criterion for statistical significance was P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Body Weight and Kidney Weight
UT-B null mice and wild-type mice had similar body weight up to age 4 wk, but the UT-B null mice had significantly lower body weight than wild-type mice after 6 wk of age (Figure 1A). However, there was no significant difference in the ratio of kidney weight to body weight between UT-B null mice with wild-type mice at any age (Figure 1B).



View larger version (17K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. Body weight and kidney weight of wild-type mice (+/+) and UT-B null mice (–/–). (A) Growth curves of wild-type and UT-B null mice. (B) Ratio of kidney weight to body weight. Data are mean ± SEM; n = 6; *P < 0.05.

 
Fluorescence-Based Real-Time RT-PCR
At first, regular RT-PCR was done to determine which AQP and urea transporter transcripts are detectable in kidney and to confirm that the PCR products are specific. Reverse-transcribed cDNA was used as the template for amplification with AQP- or urea transporter–specific primers (which amplify the coding sequences of AQP1 to AQP4, UT-A1 to UT-A4, and UT-B). As expected, each PCR product was a single DNA fragment between 100 and 120 bp, and a UT-B transcript was found in wild-type mice but not in UT-B null mice. Figure 2A shows that DNA fragments for {beta}-actin, AQP1, AQP2, AQP3, AQP4, UT-A1, UT-A2, UT-A3, and UT-B were amplified from kidney cDNA. The identity of each DNA fragment was confirmed by subcloning it and analyzing its sequence. No UT-A4 transcript was found by regular RT-PCR using different combinations of two pairs of primers, consistent with previous studies that did not detect UT-A4 in mouse kidney (3,8). The relative quantification of the changes in the abundance of the mRNA was determined by fluorescence-based real-time RT-PCR. The normalized, calibrated ratios of urea transporter and AQP transcript levels are shown in Figure 2B. In the kidney of UT-B null mice, UT-A2, AQP2, and AQP3 mRNA expression was significantly increased, compared with wild-type mice. There was no change in the mRNA expression of UT-A1, UT-A3, AQP1, and AQP4 in UT-B null mice compared with wild-type mice.



View larger version (35K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. Transcript expression of aquaporins (AQP) and urea transporters in the kidney of wild-type and UT-B null mice. (A) Regular reverse transcription–PCR (RT-PCR) analysis. mRNA was isolated from mouse kidney and reverse-transcribed to cDNA. PCR was performed using kidney cDNA as template and with {beta}-actin–, AQP-, or urea transporter–specific sense and antisense primers. (B) Fluorescence-based real-time RT-PCR. Real-time PCR was carried out by the LightCycler and with LightCycler FastStart DNA MasterPLUS SYBR Green I kit. mRNA expression level for each sample is expressed relative to wild-type kidney, which is arbitrarily considered equal to 1.0 in each individual comparison (see Materials and Methods). Data are mean ± SEM; n = 6 mice of each genotype; *P < 0.01.

 
Northern Blot Analysis
In inner medulla, the mRNA abundance of AQP2 (1.7 kb) was increased in UT-B null mice, compared with wild-type mice (Figure 3). However, there was no difference in the mRNA abundance of UT-A1 (4.1 kb), UT-A3 (2.1 kb), or AQP4 (5.5 kb) between wild-type and UT-B null mice. In outer medulla, UT-A2 (3.1 kb) and AQP3 (1.9 kb) mRNA expression was increased in UT-B null mice. However, there was no difference in the mRNA abundance of AQP1 (3.4 kb). Consistent with our previous study, the UT-B transcripts were 3.8 and 2.0 kb in the wild-type mice, and only very weak, truncated UT-B transcripts were found in the UT-B null mice (20).



View larger version (52K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. Northern blot analysis of kidney mRNA. mRNA was isolated from kidney inner medulla (top) or outer medulla (bottom) of wild-type (+/+) and UT-B null (–/–) mice and probed for the indicated cDNA.

 
Western Blot Analysis
UT-B protein is detected as a 41- to 54-kD smear on Western blot (26,27). Figure 4 confirms the absence of the 41- to 54-kD smeared band in the UT-B null mice. UT-A2 protein abundance was significantly increased in the UT-B null mice to 122 ± 6% of the level in wild-type mice in the outer medulla and to 130 ± 9 in the inner medulla (Figure 5). There was no difference in UT-A1 protein abundance between UT-B null mice and wild-type mice in the inner medulla (Figure 6). The specific identification of the UT-A glycoprotein isoforms in the inner medulla was verified by ablation of the bands upon preadsorption of the antibody with immunizing peptide (Figure 7). In addition, there was no change in UT-A3 protein abundance (data not shown). AQP2 protein abundance was significantly increased (Figure 8) in both the inner medulla (to 127 ± 7% of wild-type) and the outer medulla (to 177 ± 32% of wild-type mice). There was no significant change in the abundance of AQP1 protein in either kidney region (data not shown). There was no change in the protein abundance of either the Na+-K+-2Cl– co-transporter (NKCC2/BSC1) or the ROMK in the outer medulla of UT-B-null mice (Figure 9), both of which are normally expressed in the thick ascending limb.



View larger version (50K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. Western blot of UT-B in wild-type (+/+) and UT-B null (–/–) mice. Each lane was loaded with inner medullary lysate (10 µg) from a single mouse. Blots were probed with our polyclonal antibody to UT-B. Arrows indicate positive antibody identification. The 41- to 54-kD smear of proteins (bracket) represent glycosylated variants of UT-B. These glycosylated UT-B isoforms are absent in the UT-B –/– mice (right). A 98-kD band is detected in both wild-type and UT-B null mice, indicating that it is not UT-B.

 


View larger version (21K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 5. UT-A2 protein in the outer medulla (OM) and inner medulla (IM) of wild-type (+/+) and UT-B null (–/–) mice. Each lane was loaded with protein (10 µg) extracted from a single mouse. The arrows indicate the position of the glycosylated group of bands (45 to 60 kD) that compose UT-A2. The bar graph (right) of the densitometry (arbitrary units/µg protein) provides a quantitative analysis of all samples in this experiment. Data are mean ± SEM; n = 6; *P < 0.05. The figure is representative of two cohorts of mice.

 


View larger version (14K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 6. UT-A1 protein in the IM of wild-type (+/+) and UT-B null (–/–) mice. Each lane was loaded with protein (10 µg) extracted from a single mouse. The arrows indicate positions of the 117- and 97-kD UT-A1 bands. The bar graph (right) of the densitometry (arbitrary units/µg protein) provides a quantitative analysis of all samples in this experiment. Data are mean ± SEM; n = 6. The figure is representative of two cohorts of mice.

 


View larger version (53K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 7. Specificity of the UT-A antibody. The IM proteins from three wild-type (+/+) mice were duplicated (10 µg/lane) on one 10% polyacrylamide gel. After transfer to polyvinylidene difluoride membranes, the blot was cut into two identical halves, which were probed with normal anti–UT-A (control; left) or UT-A antibody that had been preadsorbed with immunizing peptide (right). The brackets indicate positions of the UT-A1 (117, 97 kD) and UT-A2 (45 to 60 kD) present in the IM of these mice.

 


View larger version (23K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 8. AQP2 protein in the IM and the OM of wild-type (+/+) and UT-B null (–/–) mice. Each lane was loaded with protein (10 µg) extracted from a single mouse. The arrows indicate positions of the glycosylated (35 to 50 kD) and unglycosylated (29 kD) forms of AQP2. The bar graph (right) of the densitometry (arbitrary units/µg protein) provides a quantitative analysis of all samples in this experiment. Each bar represents the combined densities of glycosylated and unglycosylated AQP2 protein bands. Data are mean ± SEM; n = 6; *P < 0.05. The figure is representative of two cohorts of mice.

 


View larger version (19K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 9. NKCC2/BSC1 and renal outer medullary potassium channel proteins in the OM of wild-type (+/+) and UT-B null (–/–) mice. Each lane was loaded with protein (10 µg) extracted from a single mouse. The arrows provide molecular weights of the identified proteins. The bar graph (right) of the densitometry (arbitrary units/µg protein) provides a quantitative analysis of all samples in this experiment. Data are mean ± SEM; n = 6. The figure is representative of two cohorts of mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
The major finding in this study is that UT-A2 is upregulated at both the mRNA and protein levels in the medulla of mice that lack UT-B. The increase in UT-A2 abundance may be an adaptive response to the loss of UT-B, because UT-B and UT-A2 are involved in different intrarenal urea recycling pathways. Urea transporters contribute to the urine-concentrating mechanism in several ways, and the loss of UT-B would be expected to interfere in two ways. First, UT-B speeds up urea transport across the red blood cell membrane as the red blood cells go through the descending and ascending vasa recta, thereby improving the efficiency of countercurrent exchange (28). Second, urea is retained within the medulla by intrarenal recycling pathways (reviewed in references 16,29). Intrarenal urea recycling pathways include reentry of urea along the descending vasa recta, where UT-B is expressed, and along a specialized section of the thin descending limb, where UT-A2 is expressed (5,16,26). The urea-selective urine concentrating defect in UT-B null mice is most likely due to the loss of these two mechanisms as a result of the absence of UT-B (20). The results of the present study suggest that the loss of one urea recycling pathway induces an adaptation that involves upregulation of the other pathway. Upregulation of UT-A2 may reduce the loss of urea from the medulla and thereby limit the decrease in urine-concentrating ability.

The phenotype of the UT-B null mouse, i.e., the urine-concentrating defect, is mild but is similar to that observed in people who lack UT-B (Kidd antigen) (19,20). Because the decrease in concentrating ability is modest, perhaps it is not surprising that the increase in UT-A2 is also modest. Presumably, an animal that lacked both UT-B and UT-A2 would be totally unable to recycle urea and would have a more severe urine-concentrating defect. This hypothesis will need to be tested in a double knockout mouse, assuming that a UT-A2 knockout mouse and then a UT-A2/UT-B double knockout mouse can be produced in the future.

Another important pathway by which urea transporters contribute to the urine-concentrating mechanism is the reabsorption of large quantities of urea across the terminal IMCD, where both UT-A1 and UT-A3 are expressed (5,6). We found no change in either UT-A1 or UT-A3 mRNA or protein abundance in the UT-B null mice. This result is somewhat surprising because an increase in urea reabsorption from the collecting duct would decrease urea’s osmotic effect. This finding suggests the hypothesis that the UT-B null mouse is attempting to adapt to the loss of urea recycling in descending vasa recta by upregulating urea recycling in descending thin limbs, rather than by increasing urea reabsorption across the IMCD.

Another finding in the present study is the demonstration that the 98-kD band that is detected in kidney by some UT-B antibodies (26,30) but not by another UT-B antibody (31) is not UT-B. Although this band can be competed away by immunizing peptide, indicating that antibody recognition is specific, the molecular explanation for this band was unknown (26). The present study shows the presence of this band in kidneys from UT-B null mice, thus demonstrating that the 98-kD band is not UT-B. This finding is consistent with our previous study in which a nonspecific band at a high molecular weight (~90 kD) was found in erythrocytes from UT-B null mice (27). It is not known whether the 98- and 90-kD bands represent the same protein.

AQP
The present study found that AQP2 and AQP3 are upregulated in the UT-B null mice. AQP2 and AQP3 both are expressed in the collecting duct, with AQP2 in the apical membrane and AQP3 in the basolateral membrane (reviewed in reference 32). Because the UT-B null mice have a urine-concentrating defect, they may have mild volume depletion and elevated plasma vasopressin levels. Vasopressin, acting through cAMP, increases the transcription and the protein abundance of AQP2 and AQP3 (reviewed in reference 32), as well as UT-A2 (3,4). cAMP also increases the transcription of the UT-A promoter that controls the transcription of UT-A1 and UT-A3 in mice (3), although it does not do so in rats (4). In addition, water restriction increases UT-A3 mRNA abundance but not UT-A1 abundance in both mice (3) and rats (33).

The UT-B null mice weighed significantly less than wild-type mice from 6 wk of age onward, which could be consistent with volume depletion. However, plasma osmolality is only slightly but not significantly higher in UT-B null mice than in wild-type mice (20), which suggests that if the UT-B –/– mice are volume depleted, then the degree of volume depletion is mild. It is tempting to speculate that an increase in vasopressin may be a mechanism that increases AQP2 and AQP3. However, if this were true, then one would have expected UT-A3 mRNA abundance to have increased as well. Unfortunately, measuring vasopressin levels in mice is difficult because of their small size. Regardless of whether an increase in vasopressin is the mechanism that increases AQP2 and AQP3 (and UT-A2), an increase in AQP2 and AQP3 protein abundance will tend to improve water conservation and urine-concentrating ability.

In contrast, AQP1 mRNA and protein abundance did not change. AQP1 is expressed in proximal tubule, thin descending limbs, descending vasa recta, and red blood cells (reviewed in reference 32). Because UT-B and AQP1 are normally expressed in descending vasa recta and red blood cells, one might have expected a change in AQP1. However, we cannot exclude the possibility that AQP1 was up- or downregulated in descending vasa recta and varied in the other direction in thin descending limbs, resulting in no overall change.


   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
UT-B is the major urea transporter in red blood cells and descending vasa recta. Humans and mice that lack UT-B have a mild urine-concentrating defect (19,20). The present study found that the mRNA and protein abundance of UT-A2, AQP2, and AQP3 are upregulated in UT-B null mice. These changes are specific as UT-A1, UT-A3, AQP1, AQP4, NKCC2/BSC1, and ROMK abundance do not change. The increases in AQP2 and AQP3 will tend to reduce water loss, thereby preventing an even greater reduction in urine-concentrating ability than would occur if these proteins were not increased. The increase in UT-A2 protein abundance may be an adaptive response to the loss of UT-B that attempts to decrease the loss of urea from the renal medulla, because UT-B and UT-A2 are involved in different intrarenal urea recycling pathways.


   Acknowledgments
 
This work was supported by American Heart Association Grant 0365027Y and National Institutes of Health Grants DK35124, DK41707, DK66194, and DK63657.

We thank Drs. A.S. Verkman (University of California, San Francisco, San Francisco, CA) and Lise Bankir (INSERM, Paris, France) for critical reading of this manuscript and valuable comments.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

  1. Sands JM: Molecular mechanisms of urea transport. J Membr Biol 191: 149–163, 2003[CrossRef][Medline]
  2. Bagnasco SM: Gene structure of urea transporters. Am J Physiol Renal Physiol 284: F3–F10, 2003[Abstract/Free Full Text]
  3. Fenton RA, Cottingham CA, Stewart GS, Howorth A, Hewitt JA, Smith CP: Structure and characterization of the mouse UT-A gene (Slc14a2). Am J Physiol Renal Physiol 282: F630–F638, 2002[Abstract/Free Full Text]
  4. Nakayama Y, Naruse M, Karakashian A, Peng T, Sands JM, Bagnasco SM: Cloning of the rat Slc14a2 gene and genomic organization of the UT-A urea transporter. Biochim Biophys Acta 1518: 19–26, 2001[Medline]
  5. Nielsen S, Terris J, Smith CP, Hediger MA, Ecelbarger CA, Knepper MA: Cellular and subcellular localization of the vasopressin-regulated urea transporter in rat kidney. Proc Natl Acad Sci U S A 93: 5495–5500, 1996[Abstract/Free Full Text]
  6. Terris JM, Knepper MA, Wade JB: UT-A3: Localization and characterization of an additional urea transporter isoform in the IMCD. Am J Physiol Renal Physiol 280: F325–F332, 2001[Abstract/Free Full Text]
  7. Kim Y-H, Kim D-U, Han K-H, Jung J-Y, Sands JM, Knepper MA, Madsen KM, Kim J: Expression of urea transporters in the developing rat kidney. Am J Physiol Renal Physiol 282: F530–F540, 2002[Abstract/Free Full Text]
  8. Fenton RA, Stewart GS, Carpenter B, Howorth A, Potter EA, Cooper GJ, Smith CP: Characterization of the mouse urea transporters UT-A1 and UT-A2. Am J Physiol Renal Physiol 283: F817–F825, 2002[Abstract/Free Full Text]
  9. Jung J-Y, Madsen KM, Han K-H, Yang C-W, Knepper MA, Sands JM, Kim J: Expression of urea transporters in potassium-depleted mouse kidney. Am J Physiol Renal Physiol 285: F1210–F1224, 2003[Abstract/Free Full Text]
  10. Stewart GS, Fenton RA, Wang W, Kwon TH, White SJ, Collins VM, Cooper G, Nielsen S, Smith CP: The basolateral expression of mUT-A3 in the mouse kidney. Am J Physiol Renal Physiol 2004, in press
  11. Sands JM, Nonoguchi H, Knepper MA: Vasopressin effects on urea and H20 transport in inner medullary collecting duct subsegments. Am J Physiol 253: F823–F832, 1987
  12. Karakashian A, Timmer RT, Klein JD, Gunn RB, Sands JM, Bagnasco SM: Cloning and characterization of two new mRNA isoforms of the rat renal urea transporter: UT-A3 and UT-A4. J Am Soc Nephrol 10: 230–237, 1999[Abstract/Free Full Text]
  13. Shayakul C, Steel A, Hediger MA: Molecular cloning and characterization of the vasopressin-regulated urea transporter of rat kidney collecting ducts. J Clin Invest 98: 2580–2587, 1996[Medline]
  14. Wade JB, Lee AJ, Liu J, Ecelbarger CA, Mitchell C, Bradford AD, Terris J, Kim G-H, Knepper MA: UT-A2: A 55 kDa urea transporter protein in thin descending limb of Henle’s loop whose abundance is regulated by vasopressin. Am J Physiol 278: F52–F62, 2000
  15. Promeneur D, Bankir L, Hu MC, Trinh-Trang-Tan M-M: Renal tubular and vascular urea transporters: Influence of antidiuretic hormone on messenger RNA expression in Brattleboro rats. J Am Soc Nephrol 9: 1359–1366, 1998[Abstract]
  16. Knepper MA, Roch-Ramel F: Pathways of urea transport in the mammalian kidney. Kidney Int 31: 629–633, 1987[Medline]
  17. Fenton RA, Howorth A, Cooper GJ, Meccariello R, Morris ID, Smith CP: Molecular characterization of a novel UT-A urea transporter isoform (UT-A5) in testis. Am J Physiol Cell Physiol 279: C1425–C1431, 2000[Abstract/Free Full Text]
  18. Sands JM, Timmer RT, Gunn RB: Urea transporters in kidney and erythrocytes. Am J Physiol 273: F321–F339, 1997
  19. Sands JM, Gargus JJ, Fröhlich O, Gunn RB, Kokko JP: Urinary concentrating ability in patients with Jk(a-b-) blood type who lack carrier-mediated urea transport. J Am Soc Nephrol 2: 1689–1696, 1992[Abstract]
  20. Yang B, Bankir L, Gillespie A, Epstein CJ, Verkman AS: Urea-selective concentrating defect in transgenic mice lacking urea transporter UT-B. J Biol Chem 277: 10633–10637, 2002[Abstract/Free Full Text]
  21. Bankir L, Chen K, Yang B: Lack of UT-B in vasa recta and red blood cells prevents urea-induced improvement of urinary concentrating ability. Am J Physiol Renal Physiol 286: F144–F151, 2004[Abstract/Free Full Text]
  22. Klein JD, Quach DL, Cole JM, Disher K, Mongiu AK, Wang X, Bernstein KE, Sands JM: Impaired urine concentration and the absence of tissue ACE: The involvement of medullary transport proteins. Am J Physiol Renal Physiol 283: F517–F524, 2002[Abstract/Free Full Text]
  23. Bradford AD, Terris J, Ecelbarger CA, Klein JD, Sands JM, Chou C-L, Knepper MA: 97 and 117 kDa forms of the collecting duct urea transporter UT-A1 are due to different states of glycosylation. Am J Physiol Renal Physiol 281: F133–F143, 2001[Abstract/Free Full Text]
  24. Ecelbarger CA, Kim GH, Knepper MA, Liu J, Tate M, Welling PA, Wade JB: Regulation of potassium channel Kir 11 (ROMK) abundance in the thick ascending limb of Henle’s loop. J Am Soc Nephrol 12: 10–18, 2001[Abstract/Free Full Text]
  25. Naruse M, Klein JD, Ashkar ZM, Jacobs JD, Sands JM: Glucocorticoids downregulate the rat vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts. J Am Soc Nephrol 8: 517–523, 1997[Abstract]
  26. Timmer RT, Klein JD, Bagnasco SM, Doran JJ, Verlander JW, Gunn RB, Sands JM: Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues. Am J Physiol Cell Physiol 281: C1318–C1325, 2001[Abstract/Free Full Text]
  27. Yang B, Verkman AS: Analysis of double knockout mice lacking aquaporin-1 and urea transporter UT-B. J Biol Chem 277: 36782–36786, 2002[Abstract/Free Full Text]
  28. Macey RI: Transport of water and urea in red blood cells. Am J Physiol 246: C195–C203, 1984
  29. Bankir L, Trinh-Trang-Tan M-M: Urea and the kidney. In: The Kidney, 6th Ed., edited by Brenner BM, Philadelphia, W.B. Saunders, 2000, pp 637–679
  30. Fenton RA, Cooper GJ, Morris ID, Smith CP: Coordinated expression of UT-A and UT-B urea transporters in rat testis. Am J Physiol Cell Physiol 282: C1492–C1501, 2002[Abstract/Free Full Text]
  31. Trinh-Trang-Tan M-M, Lasbennes F, Gane P, Roudier N, Ripoche P, Cartron J-P, Bailly P: UT-B1 proteins in rat: Tissue distribution and regulation by antidiuretic hormone in kidney. Am J Physiol Renal Physiol 283: F912–F922, 2002[Abstract/Free Full Text]
  32. Nielsen S, Frokiaer J, Marples D, Kwon ED, Agre P, Knepper M: Aquaporins in the kidney: From molecules to medicine. Physiol Rev 82: 205–244, 2002[Abstract/Free Full Text]
  33. Bagnasco SM, Peng T, Nakayama Y, Sands JM: Differential expression of individual UT-A urea transporter isoforms in rat kidney. J Am Soc Nephrol 11: 1980–1986, 2000[Abstract/Free Full Text]
Received for publication November 5, 2003. Accepted for publication February 20, 2004.




This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
L. Guo, D. Zhao, Y. Song, Y. Meng, H. Zhao, X. Zhao, and B. Yang
Reduced urea flux across the blood-testis barrier and early maturation in the male reproductive system in UT-B-null mice
Am J Physiol Cell Physiol, July 1, 2007; 293(1): C305 - C312.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
C. Schmidt, K. Hocherl, and M. Bucher
Cytokine-mediated regulation of urea transporters during experimental endotoxemia
Am J Physiol Renal Physiol, May 1, 2007; 292(5): F1479 - F1489.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
R. A. Fenton and M. A. Knepper
Urea and Renal Function in the 21st Century: Insights from Knockout Mice
J. Am. Soc. Nephrol., March 1, 2007; 18(3): 679 - 688.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
J. M. Sands
Critical Role of Urea in the Urine-Concentrating Mechanism
J. Am. Soc. Nephrol., March 1, 2007; 18(3): 670 - 671.
[Full Text] [PDF]

Home page
 FASEB J.Home page
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]

Home page
 Am. J. Physiol. Renal Physiol.Home page
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]

Home page
 Am. J. Physiol. Renal Physiol.Home page
B. Yang, D. Zhao, L. Qian, and A. S. Verkman
Mouse model of inducible nephrogenic diabetes insipidus produced by floxed aquaporin-2 gene deletion
Am J Physiol Renal Physiol, August 1, 2006; 291(2): F465 - F472.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
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]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
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]

Home page
 Am. J. Physiol. Renal Physiol.Home page
R. M. Seshadri, J. D. Klein, S. Kozlowski, J. M. Sands, Y.-H. Kim, K.-H. Han, M. E. Handlogten, J. W. Verlander, and I. D. Weiner
Renal expression of the ammonia transporters, Rhbg and Rhcg, in response to chronic metabolic acidosis
Am J Physiol Renal Physiol, February 1, 2006; 290(2): F397 - F408.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
J. C. Marini, B. Lee, and P. J. Garlick
In Vivo Urea Kinetic Studies in Conscious Mice
J. Nutr., January 1, 2006; 136(1): 202 - 206.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
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]

Home page
 Am. J. Physiol. Renal Physiol.Home page
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]

Home page
 Am. J. Physiol. Renal Physiol.Home page
D. Kim, J. D. Klein, S. Racine, B. P. Murrell, and J. M. Sands
Urea may regulate urea transporter protein abundance during osmotic diuresis
Am J Physiol Renal Physiol, January 1, 2005; 288(1): F188 - F197.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Klein, J. D.
Right arrow Articles by Yang, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Klein, J. D.
Right arrow Articles by Yang, B.

HOME CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP