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Am J Physiol Cell Physiol 290: C298-C304, 2006. First published August 31, 2005; doi:10.1152/ajpcell.00356.2005
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Protein and Vesicle Trafficking, Cytoskeleton

Polarized trafficking of the aquaporin-3 water channel is mediated by an NH2-terminal sorting signal

Tatemitsu Rai, Sei Sasaki, and Shinichi Uchida

Department of Nephrology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan

Submitted 15 July 2005 ; accepted in final form 30 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Epithelial renal collecting duct cells express multiple types of aquaporin (AQP) water channels in a polarized fashion. AQP2 is specifically targeted to the apical cell domain, whereas AQP3 and AQP4 are expressed on the basolateral membrane. It is crucial that these AQP variants are sorted to their proper polarized membrane domains, because correct AQP sorting enables efficient water transport. However, the molecular mechanisms involved in the polarized targeting and membrane trafficking of AQPs remain largely unknown. In the present study, we have examined the polarized trafficking and surface expression of AQP3 in Madin-Darby canine kidney type II (MDCKII) cells in an effort to identify the molecular determinants of polarized targeting specificity. When expressed in MDCKII cells, the majority of the exogenous wild-type AQP3 was found to be targeted to the basolateral membrane, consistent with its localization pattern in vivo. A potential sorting signal consisting of tyrosine- and dileucine-based motifs was subsequently identified in the AQP3 NH2 terminus. When mutations were introduced into this signaling region, the basolateral targeting of the resulting mutant AQP3 was disrupted and the mutant protein remained in the cytoplasm. AQP2-AQP3 chimeras were then generated in which the entire NH2 terminus of AQP2 was replaced with the AQP3 NH2 terminus. This chimeric protein was observed to be mislocalized constitutively in the basolateral membrane, and mutations in the AQP3 NH2-terminal sorting signal abolished this effect. On the basis of these results, we conclude that an NH2-terminal sorting signal mediates the basolateral targeting of AQP3.

epithelial cells; protein sorting; Madin-Darby canine kidney cells; basolateral

AQUAPORINS ARE A FAMILY of integral membrane channel proteins that mediate osmotic water transport across biological membranes. Thirteen members of the aquaporin (AQP) family have been identified to date (25). They are involved in a variety of fundamental physiological processes, such as water handling in the kidney, fluid secretion and absorption in the gastrointestinal and reproductive tracts, fluid balance in the brain and lung, maintenance of fluid and ionic homeostasis in the inner ear, and maintenance of corneal transparency. The functional importance of aquaporins also has been highlighted by studies of patients with mutations in aquaporin genes (3, 6, 14, 29) and by analyses of AQP-knockout mice that have shown various functional abnormalities (17–19, 33).

The structure of the aquaporin protein is well conserved among its members, reflecting the ubiquitous nature of water transport. Aquaporin has six transmembrane domains, with its NH2- and COOH-terminal domains localized in the cytoplasm. The protein forms a homotetrameric complex of four identical subunits. Aquaporins are distributed widely in various tissues, and sometimes multiple variants are expressed in specific cell types. When aquaporins are expressed in the plasma membrane of epithelial cells, they are located in either the apical or basolateral domains. For the aquaporins to exert their function, it is crucial that they be sorted to their correct location. For example, at least three different types of aquaporins are expressed in a polarized manner in renal collecting duct principal cells. AQP2 is targeted to the apical domain, whereas AQP3 and AQP4 are expressed in the basolateral membrane. The fine-tuning of urinary concentration occurs in the collecting duct. AQP2 residing in the intracellular vesicles is inserted into the apical membrane in response to antidiuretic hormone stimulation, and water is subsequently transported according to the osmotic gradient via AQP2 in the apical membrane and via AQP3 and AQP4 in the basolateral membrane.

Epithelial cells are functionally and morphologically polarized, and transepithelial transport is dependent on the asymmetrical distribution of proteins and lipids on the cell surface. The two membrane domains of epithelial cells, the apical and basolateral membranes, are separated by tight junctions, with the former facing the luminal side and the latter facing the interstitium. To maintain polarity, the epithelial cells must sort their newly synthesized proteins to either the apical or the basolateral domain. To date, several determinants of protein sorting have been identified. For the sorting of proteins destined for the apical domain, cytoplasm-transmembrane signals such as rafts, sphingolipids, or oligosaccharides have been shown to be involved (2, 5, 26, 28, 32). On the other hand, basolateral targeting is mediated by specific amino acid motifs in the cytoplasmic tail (11, 12, 23, 24). Recently, several sequence motifs involved in basolateral sorting have been identified, and two examples of these follow. 1) The crucial tyrosine residue in either the NPXY motif or the YXX{phi} motif (with X representing any amino acid and {phi} indicating a bulky hydrophobic residue) has been shown to target several proteins, including the LDL receptor to the basolateral membrane in MDCK cells (23). 2) The dileucine motif guides E-cadherin and the IgG Fc receptor to the basolateral side (11, 24). However, for many proteins, the exact molecular mechanism of polarized sorting has not been elucidated.

Recently, polarized membrane targeting of aquaporins has become a topic of extensive study. In the AQP2 protein, the COOH terminus has been shown to be important for apical targeting (34). In addition, in patients with autosomal dominant nephrogenic diabetes insipidus (NDI) who have mutations in the COOH terminus of AQP2, it has been shown that the mutant AQP2 proteins are not sorted properly to the apical domain, which might be an underlying mechanism of the pathogenesis of defective urinary concentration in these individuals (1, 15). A tyrosine-based motif and a dileucine motif residing in the COOH terminus have been shown to determine basolateral expression of AQP4 (20). However, there have been no other studies published in which the mechanism of polarized targeting of other aquaporin types has been reported.

In our present study, we investigated the cellular localization of AQP3 in cultured epithelial cells. AQP3 was found to be expressed predominantly in the basolateral membrane, consistent with its subcellular localization in vivo. Furthermore, we have identified a combined tyrosine- and dileucine-based motif in the NH2 terminus of AQP3 that mediates the basolateral targeting of AQP3.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
cDNA constructs and mutagenesis. cDNA encoding the mouse AQP3 and AQP2 proteins was amplified by performing PCR from a mouse kidney cDNA library and was cloned into either the pcDNA3.1 or pHM6 expression vector. The mutations listed in Table 1 were generated either by performing PCR-based mutagenesis or by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The chimeric constructs (see Fig. 7) were generated using the Seamless Cloning Kit (Stratagene) according to the manufacturer's instructions. The integrity of each of these constructs was confirmed by performing DNA sequencing.


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  		Table 1. NH2-terminal amino acid sequences of wild-type and mutant mouse AQP3

 


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  		Fig. 7. Schematic of AQP3-AQP2 chimeric constructs. The NH2 terminus of AQP2 was replaced by the NH2 terminus of AQP3. Mutations in the YRLL motif also were introduced into the chimeric constructs. Relevant confocal microscopic images of the expression pattern of these different mutants are shown in the right column.

 
Transfection and stable cell line generation. Both MDCKII and LLC-PK1 cells were grown in DMEM supplemented with 10% FBS. They were transfected with recombinant plasmid expression vectors using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA). Stably transfected MDCKII cell clones were isolated in a selection medium containing 1.2 mg/ml G418 (Sigma-Aldrich, St. Louis, MO) and screened using Western blot analysis.

Antibodies. The antibodies and working dilutions used in this study were as follows: rabbit anti-AQP3 (1:500 dilution; generous gift from Dr. Takata, Gunma University, Japan) (22), 2) rabbit polyclonal anti-hemagglutinin (anti-HA, 1:200 dilution; Zymed Laboratories, South San Francisco, CA), and 3) mouse monoclonal anti-Na+-K+-ATPase (1:200 dilution; Sigma-Aldrich). As secondary antibodies, we used Alexa Fluor 546-conjugated donkey anti-rabbit IgG (1:200 dilution), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:200), and Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:200 dilution; Molecular Probes, Breda, The Netherlands).

Immunofluorescence and confocal microscopy. For immunofluorescence experiments, cells were grown on permeable membranes (0.4-µm pore size) using Transwell chambers (Corning, Corning, NY) until the cells had formed a confluent monolayer. Cells were then washed with PBS and fixed with 2% paraformaldehyde in PBS for 20 min at 37°C. Permeabilization was achieved by treatment with 0.1% Triton X-100 in PBS for 5 min, and blocking was performed using 30-min incubation with 1% BSA in PBS blocking buffer. The cells were then incubated for 1 h in primary antibodies diluted in blocking buffer. Secondary antibodies were diluted in blocking buffer, and the cells were incubated in secondary antibodies for 1 h. After cells were washed with PBS, the filters were mounted in Antifade solution (Molecular Probes). Confocal analysis was performed using an LSM confocal microscope (Zeiss, Oberkochen, Germany).

Side-specific biotinylation and immunoblot analysis. Cells were seeded onto polycarbonate filters (Corning) until they formed a confluent monolayer. Side-specific biotinylation was performed as previously described (9). Briefly, sulfo-N-hydroxysuccinimidobiotin (0.5 mg/ml; Pierce Biotechnology, Rockford, IL) in PBS-CM (PBS with 1 mM MgCl2 and 0.1 mM CaCl2) was applied to the apical or basolateral surface at 37°C for 60 min. The cells were rinsed twice rapidly with cold PBS-CM, and the cells were then incubated for 10 min in 50 mM NH4Cl in PBS. The cells were lysed with 100 mM NaCl and 25 mM Tris·HCl, pH 8.0, supplemented with 1% Triton X-100, and biotinylated proteins were recovered by incubation with avidin beads (Sigma) overnight. The bound proteins were eluted with Laemmli buffer and examined using Western blot analysis with anti-AQP3 antibodies or mouse monoclonal anti-Na+-K+-ATPase antibodies. The blots were quantified using Scion Image software, and statistical analysis was performed using one-way ANOVA, followed by Fisher's post hoc test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Wild-type AQP3 channels are located primarily at the basolateral plasma membrane of cultured epithelial cells. Wild-type mouse AQP3 cDNA was subcloned into the pcDNA3 expression vector and transiently transfected into both MDCKII and LLC-PK1 cells. The subcellular localization of the AQP3 protein was then determined using confocal microscopy with AQP3-specific antibodies. In both MDCKII and LLC-PK1 cells, transiently expressed AQP3 channels were found to be targeted to the basolateral membrane, colocalizing with the staining of Na+-K+-ATPase (Fig. 1). To investigate the steady-state distribution of AQP3 channels, MDCKII cells stably expressing wild-type AQP3 were generated. Four independent cell lines with high levels of transgene expression were isolated. In all four of these cell lines, AQP3 channels were observed almost exclusively in the basolateral membranes of these cells (Fig. 2). Quantification by biotinylation from the basolateral side revealed that 88 ± 5.8% (mean ± SD; n = 5) of the expressed AQP3 was routed to the basolateral membrane (see Fig. 6). Thus exogenously expressed AQP3 was targeted to the basolateral membrane of MDCKII cells, a finding that is consistent with in vivo findings in kidney epithelial cells. This finding indicates that MDCKII cells are an appropriate in vitro cellular model system with which to analyze the molecular determinants of AQP3 basolateral targeting.



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  		Fig. 1. Expression of transiently transfected wild-type aquaporin-3 (AQP3) in both Madin-Darby canine kidney type II (MDCKII) cells and LLC-PK1 cells. Exogenously expressed AQP3 were targeted to the basolateral membrane in both cell types as indicated by their colocalization with the basolateral membrane marker Na+-K+-ATPase.

 


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  		Fig. 2. Expression of stably transfected wild-type AQP3 in MDCKII cells. A: confocal images of wild-type AQP3 protein and Na+-K+-ATPase expression in one representative cell line. The top and bottom images show focal planes that are parallel and perpendicular to the epithelium, respectively. B: steady-state repartition of AQP3 in the apical and basolateral membranes. Western blot analysis of total and plasma membrane fractions of cells biotinylated from either the apical or the basolateral side and probed with anti-AQP3 antibodies or anti-Na+-K+-ATPase antibodies are shown. The 26-kDa nonglycosylated band is shown for AQP3 and was used for quantification. Both proteins were observed almost exclusively in the basolateral membranes.

 


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  		Fig. 6. Quantification of the basolateral expression of wild-type and mutant AQP3 by specific membrane biotinylation assays. Data are means ± SD of 5 experiments.

 
AQP3 NH2 terminus contains its basolateral sorting signal. Protein sequence alignment of AQP3 from different species (human, rat, and mouse) has identified a conserved, four-amino acid motif, YRLL, in the NH2 terminus that resembles the tyrosine-based and dileucine signals that have been shown to be important for basolateral targeting. To investigate whether this YRLL sequence affects the steady-state localization of the AQP3 protein, three mutant expression constructs, Y19A, LL-AA, and YRLL-AAAA, were generated by performing site-directed mutagenesis (Table 1). Y19 was mutated to alanine in the Y19A construct, the two leucines were mutated to alanines in the LL-AA construct, and all four residues were mutated to alanines in the YRLL-AAAA construct. Stable MDCKII cell lines were established, with each expressing each of these three AQP3 mutants. For each AQP mutant, four independent cell lines were isolated and the subcellular localizations of mutant AQP3 were analyzed using immunocytochemistry and side-specific biotinylation.

Mutations in either the tyrosine residue or the dileucine residue (constructs Y19A and LL-AA) resulted in a partial disruption of the basolateral targeting of AQP3, with a portion of the AQP3 protein remaining in the cytoplasm (Figs. 3 and 4). Quantification by biotinylation from the basolateral side revealed that 49 ± 3.5% (mean ± SD; n = 5) of Y19A and 40 ± 3.8% (mean ± SD; n = 5) of LL-AA mutants were routed to the basolateral membrane (see Fig. 6). Moreover, the substitution of all four YRLL residues (construct YRLL-AAAA) resulted in the complete dysregulation of AQP3 localization. As shown in Fig. 5, basolateral membrane expression in MDCKII-YRLL-AAAA cells was no longer detectable and complete retention of the exogenous mutant AQP3 in the cytoplasm was observed. No YRLL-AAAA AQP3 protein could be detected in the basolateral membrane on the basis of side-specific biotinylation assay (0 ± 0%; n = 5) (Fig. 6). These results were essentially reproducible in each of the cell lines. They indicate that the identified four-amino acid NH2-terminal sequence of AQP3, YRLL, is the basolateral targeting determinant of this aquaporin and that both the tyrosine and dileucine residues in this motif are necessary for the proper sorting of AQP3.



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  		Fig. 3. Expression of stably transfected mutant AQP3 (Y19A) in MDCKII cells. A: confocal images of mutant AQP3 protein and Na+-K+-ATPase expression in one representative cell line. B: steady-state repartition of mutant AQP3 in the apical and basolateral membranes.

 


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  		Fig. 4. Expression of stably transfected mutant AQP3 (LL-AA) in MDCKII cells. A: confocal images of mutant AQP3 protein and Na+-K+-ATPase expression in one representative cell line. B: steady-state repartition of mutant AQP3 in the apical and basolateral membranes.

 


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  		Fig. 5. Expression of stably transfected mutant AQP3 (YRLL-AAAA) in MDCKII cells. A: confocal images of mutant AQP3 protein and Na+-K+-ATPase expression in one representative cell line. B: steady-state repartition of mutant AQP3 in the apical and basolateral membranes.

 
AQP3 NH2 terminus can function as a dominant basolateral targeting signal. After establishing that the YRLL motif plays an important role in the basolateral targeting of AQP3, we analyzed whether this motif would be sufficient to redirect the otherwise apically sorted AQP2 protein. The apical sorting of AQP2 was previously shown to be mediated by its COOH-terminal tail (34). To preserve the YRLL motif in its natural context, the entire cytoplasmic NH2 terminus of AQP3, consisting of 36 amino acid residues, was introduced into the AQP2 protein in place of the AQP2 NH2 terminus (AQP3-AQP2 chimera). A construct with mutations in the YRLL motif also was generated (AQP3-AQP2 chimera YRLL-AAAA). Wild-type AQP3 (AQP3-WT), AQP3-AQP2 chimera, and AQP3-AQP2 chimera YRLL-AAAA cDNA were subcloned into an NH2-terminal, HA-tagged pHM6 expression vector (Fig. 7). Stably transfected MDCKII cell lines from each of these constructs were generated. For each construct, four independent cell lines were isolated and the subcellular localization of the expressed HA-tagged proteins was analyzed (Fig. 8). The substitution of the NH2 terminus of AQP2 with the NH2 terminus of AQP3 was found to alter completely the localization of the resulting chimeric protein, which was missorted exclusively to the basolateral membrane. In addition, when the YRLL motif of the AQP3-AQP2 chimera was mutated to AAAA, the missorting phenomenon was abolished and the preferential intracellular localization of AQP3-AQP2 chimera YRLL-AAAA was observed. These results were essentially reproducible in each of the cell lines. They show that the NH2 terminus of AQP3 contains a basolateral targeting signal that is sufficient to redirect the otherwise apically located AQP2 protein. Furthermore, the silencing of this basolateral localization by mutation of the YRLL motif confirmed that this motif plays a pivotal role in the basolateral targeting of AQP3.



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  		Fig. 8. Expression of stably transfected AQP3-AQP2 chimeric proteins in MDCKII cells. AQP3-AQP2 chimeric proteins were detected using hemagglutinin (HA) antibody. Focal planes parallel and perpendicular to the epithelium are shown. Representative images of cell lines are shown. A: wild-type AQP3 with an HA tag is sorted properly to the basolateral membrane. B: AQP2 with a substituted AQP3 NH2 terminus is missorted to the basolateral membrane, an effect that is abolished by mutations in the YRLL motif (C).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
AQP3 water channels are expressed in several epithelial tissue types in which they participate in transepithelial water transport. AQP3 is selectively located at the basolateral membrane in the renal collecting duct principal cells and is considered to play a pivotal role in urinary concentration in concord with AQP2 and AQP4 located in the apical and basolateral domains, respectively. However, no studies published to date have elucidated precisely the mechanisms underlying how these different types of aquaporins are sorted effectively to their destinations in a single type of cell. The aim of the present study was to attempt to characterize the trafficking mechanism of the AQP3 protein in renal epithelial cell lines and to identify the molecular determinants of its basolateral expression pattern.

When AQP3 water channels are expressed in polarized MDCK or LLC-PK1 cells, strict basolateral localization is observed, suggesting that these cells contain the correct machinery for AQP3 plasma membrane targeting. We focused our analysis on internal AQP3 motifs, and subsequent sequencing revealed the presence of a potential sorting motif, YRLL. This motif could be interpreted as a tyrosine-based sorting motif, a dileucine motif, or a combination of both. When either the tyrosine alone or the two leucines contained in this YRLL motif were replaced by alanines, the basolateral targeting of the resulting mutant AQP3 was partially disrupted. Upon complete mutation of YRLL to AAAA, the mutant AQP3 protein was found primarily in intracellular locations. These results indicate that both the tyrosine residue and the dileucine residues of the YRLL motif have a synergistic effect on the basolateral targeting of AQP3.

To substantiate these findings, we next replaced the NH2 terminus of AQP2 with the NH2 terminus of AQP3 to determine whether the apically sorted AQP2 could be retargeted by the basolateral signaling motif of AQP3. AQP2 was shown previously to be sorted to the apical membrane in MDCK cells (1, 34). Chimeric AQP2 containing the NH2 terminus of AQP3 was missorted constitutively to the basolateral membrane in MDCK cells. This finding indicated that the NH2 terminus of AQP3 can indeed function as a dominant basolateral localization determinant. Moreover, when the YRLL motif of the AQP2-AQP3 chimera was mutated to four AAAA, the missorting effect was abolished, further confirming the crucial role of this motif in basolateral targeting. Although these results do not exclude the possibility that other signals cooperating with the YRLL motif might exist in the NH2 terminus of AQP3, the NH2-terminal YRLL motif is essential for the steady-state localization of AQP3 channels in the basolateral membrane. The YRLL basolateral sorting signal motif identified in this study is evolutionarily conserved in human, rat, and mouse AQP3.

The tyrosine-based targeting signal (YXX{phi} or NPXY) and the dileucine motif (LL) are the two major sequence motifs that mediate basolateral targeting. However, it has been suggested that these motifs are interpreted differently, depending on the cell type and the specific protein. The tyrosine-based targeting signal has been shown to direct the LDL receptor to the basolateral membrane in MDCK cells (23), but it has no role in the basolateral targeting of E-cadherin in either MDCK or LLC-PK1 cells (4). Targeting of the well-characterized basolateral H+-K+-ATPase {beta}-subunit is mediated by the tyrosine-based signal in MDCK cells. However, in LLC-PK1 cells, the H+-K+-ATPase {beta}-subunit is sorted inversely to the apical membrane (30). This phenomenon is attributed to the absence of the µ1B-subunit of the adaptor protein complex 1 in LLC-PK1 cells. The µ1B-subunit interacts with the tyrosine-based motif and directs proteins to the basolateral membrane (7, 27). In the case of the dileucines, the dileucine motif has been suggested to be the basolateral sorting determinant for a variety of proteins, such as E-cadherin (24), the IgG Fc receptor (12), and the human norepinephrine transporter hNET (8). However, a dileucine motif in the human equilibrative nucleoside transporter hENT2 does not affect its targeting to the basolateral membrane in MDCK cells, but it has been implied to play a role in the surface expression of hENT2 (21). A leucine residue in a dileucine motif in the COOH terminus of the sodium phosphate cotransporter NaPi type IIb was shown to be involved in apical targeting in opossum kidney cells (13). Although the tyrosine-based and dileucine targeting motifs are the most widely documented, other basolateral sorting motifs have been identified for some proteins (10, 16, 31).

Our present study is the first to report a functional tyrosine-based sorting motif and a dileucine motif that reside in a single four-amino acid motif and exert a synergistic effect. AQP3 was targeted correctly to the basolateral membrane in LLC-PK1 cells, suggesting that the µ1B-subunit is not required in the sorting machinery of AQP3. When the µ1B-subunit and AQP3 were coexpressed in MDCK cells, we could not find any interaction between these two proteins on the basis of coimmunoprecipitation (data not shown). These results add important information elucidating the diverse molecular mechanisms underlying epithelial protein sorting.

Two aquaporins, AQP2 and AQP4, are also expressed in the renal collecting duct principal cells. In AQP4, two COOH-terminal signals were shown to determine its basolateral targeting (20). In AQP2, an apical targeting segment was identified in the COOH terminus (34). Altogether, these previous findings and our present results regarding the basolateral targeting of AQP3 indicate that the renal collecting duct epithelia selectively sort different types of aquaporins by interpreting short amino acid signals in the cytoplasmic termini of these proteins. Mutations in the COOH terminus of AQP2 have been found to cause congenital autosomal dominant NDI, a disease characterized by the inability of the kidneys to concentrate urine in response to vasopressin. In patients with this disease, mutant AQP2 is missorted to intracellular organelles (1, 15). Although extremely rare, humans with AQP3 mutations have been identified (29). Naturally occurring mutants, if found in the YRLL basolateral motif, may lead to inappropriate sorting, which also could form the molecular basis of the disease in patients with autosomal dominant NDI who have no mutations in either the V2 vasopressin receptor or AQP2.

In conclusion, our present study shows that AQP3 is expressed in the basolateral membranes of MDCK and LLC-PK1 cells. A potential basolateral sorting signal, YRLL, was identified within the NH2 terminus of AQP3. This finding makes an important contribution to the understanding of the mechanisms underlying the sorting of aquaporins and provides greater knowledge of their involvement in renal water transport.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


   ACKNOWLEDGMENTS
 
We thank Dr. Kuniaki Takata of Gunma University for the generous gift of anti-AQP3 antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Rai, Dept. of Nephrology, Graduate School of Medicine, Tokyo Medical and Dental Univ., 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan (e-mail: trai.kid{at}tmd.ac.jp)

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.


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 DISCUSSION
 GRANTS
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