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Endocrinology, doi:10.1210/en.2004-0073
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Endocrinology Vol. 145, No. 9 4375-4383
Copyright © 2004 by The Endocrine Society

Aquaporin-2 Is Retrieved to the Apical Storage Compartment via Early Endosomes and Phosphatidylinositol 3-Kinase-Dependent Pathway

Yuki Tajika, Toshiyuki Matsuzaki, Takeshi Suzuki, Takeo Aoki, Haruo Hagiwara, Michio Kuwahara, Sei Sasaki and Kuniaki Takata

Department of Anatomy and Cell Biology (Y.T., T.M., T.S., T.A., H.H., K.T.), Gunma University Graduate School of Medicine, Gunma 371-8511, Japan; and Department of Homeostasis Medicine and Nephrology (M.K., S.S.), Tokyo Medical and Dental University, Tokyo, 113-8519, Japan

Address all correspondence and requests for reprints to: Kuniaki Takata, Ph.D., Department of Anatomy and Cell Biology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail: takata{at}med.gunma-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aquaporin-2 (AQP2) is one of the water-channel proteins expressed in principal cells of kidney collecting ducts, where it is stored in the intracellular compartment. Previous studies have demonstrated that AQP2 vesicles constitute a distinct intracellular compartment partially overlapping with early endosomes. In this report, we performed in vitro experiments using the renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, stably expressing AQP2 (MDCK-hAQP2). In nonpolarized cells, AQP2 vesicles were scattered in the cytoplasm and did not colocalize with Golgi 58K or TGN38. Small portions of AQP2 vesicles were positive for the lysosome marker cathepsin D. An early endosome antigen (EEA1) localized around AQP2 vesicles in close proximity, suggesting involvement of the endosomal system in the trafficking of AQP2. AQP2 vesicles are distinct from other recycling molecules, such as glucose transporter 4 (GLUT4) and endocytosed transferrin. In polarized MDCK-hAQP2 cells, AQP2 vesicles were localized in the subapical recycling compartment and distinct from the Golgi apparatus, trans-Golgi network, lysosome, and early endosome in the nonstimulated state. When the cells were treated with forskolin, translocation of AQP2 to the apical membrane was observed. Washout of forskolin induced retrieval of AQP2 into the cytoplasm, and AQP2 was transiently colocalized with EEA1-positive endosomes. Then, AQP2 moved from EEA1-positive endosomes to the subapical AQP2-storage compartment, which is sensitive to wortmannin and LY294002. These results suggest that AQP2 resides in a recycling compartment at the apical side in polarized MDCK-hAQP2 cells, and its retrieval uses the apical endosomal system and the phosphatidylinositol 3-kinase-dependent pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRANSFER OF WATER across the cellular membrane is mediated by water channel proteins named aquaporins (AQPs). More than 10 isoforms of AQPs have been identified in mammalian cells so far (1). AQP2 (2, 3) is expressed in the principal cells of the collecting ducts in the kidney (4) and plays a critical role in the urine concentration. A unique feature of AQP2 is that it is stored in the intracellular compartment, and upon stimulation of an antidiuretic hormone (ADH, vasopressin), AQP2 translocates to the apical plasma membrane, where it serves in the uptake of water from the lumen of the collecting duct (5). The mutation of AQP2 leads to nephrogenic diabetes insipidus, the inability to concentrate urine. In the recessive nephrogenic diabetes insipidus, mutated AQP2 is retained in the endoplasmic reticulum (6, 7), whereas in the dominant types, it is localized in the Golgi complex (8), lysosome (9), or basolateral plasma membrane (10). These findings clearly show the importance of proper localization and trafficking of AQP2 for its function.

The nature of the intracellular compartment of AQP2 is not fully understood. We showed previously that AQP2 vesicles constitute a distinct intracellular compartment mostly in the apical cytoplasm and partially overlapping with early endosomes in collecting duct cells in the rat kidney. The AQP2 compartment is distinct from lysosomes, trans-Golgi network (TGN), Golgi apparatus, and endoplasmic reticulum (11). For further characterization of AQP2 vesicles, we performed immunocytochemical analysis in cultured renal epithelial cells, Madin-Darby canine kidney (MDCK) cells, in this report. AQP2 stably transfected in MDCK cells undergoes translocation and recycling, which provides an ideal system for the detailed analysis of these processes. In addition, a comparison with other recycling molecules, such as transferrin receptor and glucose transporter 4 (GLUT4), was performed to address the presence of unique and common features of intracellular distribution and the trafficking pathway among these molecules.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
Human AQP2 was stably transfected into MDCK cells (MDCK-hAQP2) as previously described (10). Native vectors were also transfected into MDCK cells as controls (MDCK-mock). Cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 500 µM G418.

Cotransfection
GLUT4 was inserted in pcDNA3 (Invitrogen, Carlsbad, CA) (12) and transiently expressed in MDCK-hAQP2 cells. Cells were cultured on coverslips, and 1 d before the staining, the transfection was performed by electroporation (13).

Transferrin uptake
Cells were grown on coverslips or permeable support (Transwell, Costar, Cambridge, MA). They were cultured in serum-free medium for 1 h, incubated with Texas Red-transferrin (20 µg/ml; Molecular Probes, Eugene, OR) for 10 min, and then fixed with 1% acetic acid in ethanol for 5 min on ice.

Translocation and recycling of AQP2
Cells were incubated with 50 µM forskolin (Sigma Chemical Co., St. Louis, MO) for 30 min and then fixed with 3% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 30 min for immunostaining. For observation of AQP2 recycling, cells were treated with forskolin, washed, and then incubated with a medium with or without phosphatidylinositol 3 (PI3)-kinase inhibitors, 100 nM wortmannin (Sigma), or 20 µM LY294002 (Sigma).

Immunoblotting
Cells grown on a 10-cm culture dish were scraped and homogenized in PBS containing 0.05 M EDTA, 2 mg/ml leupeptin, 2 mg/ml pepstatin A, 2 mM phenylmethylsulfonyl fluoride, and 200 KIE/ml aprotinin. Protein concentration was determined with bicinchonic acid protein assay reagent (Pierce, Rockford, IL). Ten micrograms of protein were run on 13% polyacrylamide gel. After being transferred to FluoroTrans polyvinylidene difluoride membrane (Pall Corp., East Hills, NY), blots were blocked with 3% BSA in rinse buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100 for 3 h and incubated with guinea pig antirat AQP2 antibody (diluted at 1:5000 in 0.3% BSA in rinse buffer) (11) or rabbit antirat AQP2 antibody (1:5000). The signal was detected with horseradish peroxidase-conjugated rabbit antiguinea pig Ig antibody (1:1000; Dako, Glostrup, Denmark) or horseradish peroxidase-conjugated goat antirabbit Ig antibody (1:2000; Dako) and visualized using an enhanced chemiluminescence kit (ECL Plus Western blotting detection system; Amersham Biosciences, Little Chalfont, UK). Chemiluminescence was detected with a Typhoon 9210 variable mode imager (Amersham) at a resolution of 200 µm/pixel. Specificity was checked by incubation with anti-AQP2 antibodies in the presence of 2 µg/ml antigen peptide used to raise antibodies.

Immunocytochemistry
Cells grown on coverslips were treated with adequate fixatives for each antibody used for staining (3% paraformaldehyde in 0.1 M phosphate buffer, methanol, ethanol, or 1% acetic acid in ethanol). After washing with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min. Nonspecific bindings were blocked with 5% normal donkey serum (Chemicon International, Temecula, CA). Specimens were sequentially incubated with guinea pig anti-AQP2 antibody (1:500), and Alexa Fluor 594-conjugated goat antiguinea pig IgG antibody (1:500; Molecular Probes) or Alexa Fluor 488-conjugated goat anti-guinea pig IgG antibody (1:500; Molecular Probes). As immunohistochemical controls, incubation with anti-AQP2 antibody was carried out in the presence of 20 µg/ml antigen peptide. Nuclear DNA was stained with 2 µg/ml 4',6-diamidino-2-phenylindole (Roche Diagnostics, Basel, Switzerland).

For double labeling, sections were incubated with a mixture of guinea pig anti-AQP2 antibody (1:500) or rabbit antirat AQP2 antibody (1:500) followed by the incubation with an appropriate secondary antibody raised in the rat, rabbit, mouse, or goat. Antibodies used were mouse anti-Golgi 58K antibody (1:40; Sigma) (11, 14), rabbit anti-TGN38 antibody (1:200) (11, 15), rabbit anti-GLUT4 antibody (12), goat anti-EEA1 antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) (11), mouse anti-EEA1 antibody (1:200; BD Transduction Laboratories, Franklin Lakes, NJ) (16), rabbit anti-cathepsin D antibody (1:200) (17), rabbit anti-Rab11 antibody (1:20; Zymed, San Francisco, CA) (16, 18, 19, 20), mouse anti-Rab4 antibody (1:100; BD Transduction Laboratories) (21), mouse anti-Rab5 antibody (1:20; BD Transduction Laboratories) (21), rat anti-ZO-1 antibody (1:200; Chemicon) (22), Cy3-donkey antimouse IgG antibody (1:500; Jackson ImmunoResearch, West Grove, PA), Rhodamine Red X-donkey antirabbit IgG antibody (1:500; Jackson ImmunoResearch), Alexa Fluor 594-goat antirabbit IgG antibody (1:500; Molecular Probes), Rhodamine Red X-donkey antiguinea pig IgG antibody (1:500; Jackson ImmunoResearch), lissamine rhodamine sulfonyl chloride-donkey antirat IgG (1:500; Jackson Immunoresearch). In addition, donkey antigoat IgG antibody (1:200; Jackson Immunoresearch) was labeled using an Alexa Fluor 488 protein labeling kit (Molecular Probes) and used as one of the secondary antibodies.

Semithin cryosection
Cells were grown on permeable support, and vertical semithin cryosections (1 µm thick) were cut as described previously (23) and immunostained.

Microscopy
Fluorescently labeled specimens were examined with a conventional fluorescence or a confocal laser scanning microscope. For conventional microscopy, they were examined with an Olympus BX62 fluorescence microscope (Tokyo, Japan). Images were captured with a Micro MAX 1300-Y cooled-CCD camera (Roper Scientific, Trenton, NJ) and analyzed with IP Lab Spectrum software (Signal Analytics, Vienna, VA). Confocal images were obtained with an Axiophot2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with the Bio-Rad MRC1024ES confocal system (Hercules, CA) operated with Laser Sharp software (Bio-Rad). Images were finally processed using Adobe Photoshop 6.0 software (Adobe Systems Inc., San Jose, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of MDCK-hAQP2 cells and anti-AQP2 antibody
We previously raised the polyclonal antibody against the isoform-specific C-terminal peptide sequence of rat AQP2 in the guinea pig and rabbit (11). These antibodies were useful for double-immunofluorescence microscopy in combination with antibodies raised in other species. Because human AQP2 and rat AQP2 share similar C-terminal sequences (Fig. 1AGo), we checked whether these antibodies recognize hAQP2 by immunoblotting and immunohistochemistry. As shown in Fig. 1BGo, antibodies recognized bands at the predicted molecular mass of human AQP2 expressed in MDCK (lanes 1 and 4). A narrow band of 29 kDa and a broad band between 43 and 67 kDa corresponding to the nonglycosylated and the glycosylated forms of AQP2, respectively (3, 10), were detected. The intensity of the glycosylated band is within the range of previous reports (10, 11, 24) Homogenate from MDCK-mock cells gave no band (lanes 2 and 5). The bands disappeared when the incubation was carried out in the presence of the antigen peptide (lanes 3 and 6). For immunofluorescence staining, MDCK cells grown on coverslips were stained with guinea pig antirat AQP2 antibody (Fig. 1CGo, top). The positive labeling was seen mainly in intracellular vesicles. Immunolabeling was abolished when the primary antibody was incubated in the presence of the antigen peptide (Fig. 1CGo, middle). No labeling was observed in MDCK-mock cells (Fig. 1CGo, bottom). The specificity of rabbit antirat AQP2 antibody in immunohistochemistry was also confirmed (data not shown). These results show that these antibodies specifically recognized hAQP2 and that these are useful for immunohistochemical detection of hAQP2.



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  		FIG. 1. A, Amino acid sequence of human and rat AQP2 C-terminals. The underlined sequence was used as an antigen peptide. B, Immunoblotting with antirat AQP2 antibodies. Homogenates from MDCK-hAQP2 or MDCK-mock (10 µg/lane) were resolved with SDS-PAGE (13% polyacrylamide). Two bands (arrow and bracket), corresponding to nonglycosylated and glycosylated forms, respectively, are detected with guinea pig anti-AQP2 or rabbit anti-AQP2 antibody in MDCK-hAQP2 (lanes 1 and 4). Homogenate from the MDCK-mock gives no band (lanes 2 and 5). Bands disappear by adding of antigen peptide (2 µg/ml) (lanes 3 and 6). C, Immunohistochemistry. MDCK-hAQP2 (top and middle) and MDCK-mock (bottom) cells were labeled with guinea pig anti-AQP2 antibody. Addition of antigen peptide (20 µg/ml) completely abolishes the positive labeling (middle). Images were obtained using a cooled-CCD camera. Capturing and processing of images were carried out exactly the same way in these micrographs. Bar, 20 µm.

 
The translocation of hAQP2 in MDCK cells was evaluated by confocal microscopy (Fig. 2Go). Cells were incubated with or without forskolin, an adenylate cyclase activator. AQP2 distributed in the intracellular compartment without forskolin stimulation (Fig. 2Go, A1 and B1). After incubation with forskolin, AQP2 was translocated into the plasma membrane, regardless of the confluency of cells (Fig. 2Go, A2 and B2) as reported previously (10). Still some AQP2 vesicles were scattered in cytoplasm in nonpolarized cells (Fig. 2Go, A2). These results showed that MDCK-hAQP2 cells serve as an appropriate in vitro cellular model system to analyze the intracellular storage compartment and dynamics of AQP2. The foundation of the polarized epithelial cell sheet was examined by monitoring the development of tight junctions. Confluent cells as shown in Fig. 2BGo were double immunostained with AQP2 and tight junction protein ZO-1 (Fig. 2CGo). A continuous meshwork of tight junction (red) was constructed, which divided the plasma membrane into apical and basolateral domains. To further confirm the apical translocation of AQP2, vertical semithin cryosections of cell monolayers were made and double immunostained for AQP2 and ZO-1 (Fig. 2DGo). AQP2 (green) was localized intracellularly (Fig. 2Go, D1) and translocated to the apical plasma membrane after forskolin treatment (Fig. 2Go, D2).



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  		FIG. 2. Effect of forskolin on the hAQP2 localization in MDCK-hAQP2 cells. A, In nonpolarized cells, AQP2 is scattered in the cytoplasm (A1). After incubation with 50 µM forskolin for 30 min, the ridge of the cell is clearly seen, showing the translocation of AQP2 to the plasma membrane (A2). B, In polarized cells, AQP2 is present in the subapical region (B1), and it is translocated to the apical plasma membrane after forskolin treatment (B2). C, Double immunostaining with AQP2 (green) and ZO-1 (red) are shown before and after forskolin treatment (C1 and C2). Multiple confocal images were projected into a single image. D, Confocal images of vertical sections of 1 µm thickness double-stained for AQP2 (green) and ZO-1 (red). The intracellular distribution of AQP2 (D1) and the apical translocation after forskolin treatment (D2) are clearly seen. Bars, 20 µm (A and C) and 10 µm (B and D).

 
AQP2 in nonpolarized MDCK cells
Double staining was performed using antibodies and tracers to characterize the intracellular pool of AQP2. Antibodies used to visualize various cytoplasmic organelles were as follows: Golgi 58K (Golgi apparatus), TGN38 (TGN), cathepsin D (lysosome), EEA1 (early endosome). Golgi 58K showed tubular and dotty staining in the perinuclear cytoplasm (Fig. 3AGo). TGN38 gave similar perinuclear staining (Fig. 3BGo). AQP2 was not detected in either Golgi apparatus or TGN. Cathepsin D was seen as small dots scattered in the cytoplasm. A few AQP2 vesicles, if any, colocalized with these lysosomal markers (Fig. 3CGo). EEA1 is a Rab5 effector protein associated with the cytoplasmic side of early endosomes (25). EEA1-positive vesicles were seen as dots scattered in the cytoplasm, and AQP2 vesicles were localized in close proximity to EEA1 (Fig. 3DGo).



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  		FIG. 3. Comparison of the localization of AQP2 with Golgi 58K (A), TGN38 (B), cathepsin D (C), EEA1 (D), GLUT4 (E), and transferrin (F) in nonpolarized MDCK-hAQP2 cells. The left column (A1–F1) shows the labeling for Golgi 58K (A1), TGN38 (B1), cathepsin D (C1), EEA1 (D1), GLUT4 (E1), and transferrin (F1). The middle column shows AQP2 staining. Merged images are shown in the right column (A3–F3). In merged images, AQP2 is shown in green, and organelle marker proteins in red. Single confocal images from the intermediate plane of cells are shown. Bar, 10 µm.

 
GLUT4 and AQP2 exhibit similar characteristics in that they undergo hormone-regulated intracellular trafficking. GLUT4 translocates from the intracellular storage compartment to the plasma membrane upon insulin stimulation (26). To address whether GLUT4 and AQP2 share intracellular compartments and trafficking machinery, transfection of GLUT4 into MDCK-hAQP2 cells was performed (Fig. 3EGo). GLUT4 localized in the perinuclear region, and AQP2 did not colocalize with the GLUT4 compartment.

Transferrin has been used to label coated pit-mediated endocytosis and the subsequent recycling process. To visualize these compartments, cells were incubated with fluorescently labeled transferrin for 10 min. Endocytosed transferrin was localized in cytoplasmic vesicles, mostly early and recycling endosomes (19). AQP2 did not colocalize with these transferrin-labeled endosomes (Fig. 3FGo).

AQP2 in polarized MDCK cells
Next, the intracellular pool of AQP2 was characterized in confluent MDCK-hAQP2 cells. Golgi 58K and TGN38 showed tubular and dotty staining, respectively, in the perinuclear region (Fig. 4Go, A and B). Cathepsin D was found in the apical side of the cytoplasm (Fig. 4CGo). None of these organelle markers colocalized with AQP2. EEA1 was reported to be a marker of apical (16) and basal (27) early endosomes in polarized MDCK cells. In our MDCK-hAQP2 cells, EEA1 was found in the apical side of the cytoplasm (Fig. 4DGo) and absent from the transferrin-recycling compartment in the basal side (data not shown). Although the close localization of AQP2 and EEA1 was seen in nonpolarized cells (Fig. 3Go), AQP2 was not found in EEA1-positive early endosomes in polarized cells (Fig. 4DGo). Rab11 was used as a marker of apical recycling endosome (16, 18, 19, 20), and some AQP2 was localized in the Rab11-positive recycling compartment (Fig. 4EGo). The Rab11-positive recycling compartment was distinct from Rab4- or Rab5-positive endosomes (Fig. 4Go, E4 and E5). Transferrin was reported to be a marker of the basal recycling endosome in polarized MDCK cells (19). When MDCK-hAQP2 cells were incubated with Texas Red-labeled transferrin from the basal side on permeable support, the transferrin was endocytosed and found in vesicles in the basal cytoplasm. AQP2 was distinct from the transferrin-recycling compartment (Fig. 4FGo). The colocalization of AQP2 and Rab11 suggests that the compartment was an apical recycling compartment.



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  		FIG. 4. Comparison of the localization of AQP2 with Golgi 58K (A), TGN38 (B), cathepsin D (C), EEA1 (D), Rab11 (E), and transferrin (F) in polarized MDCK-hAQP2 cells. Single confocal images are shown. The left column (A1–C1) shows the labeling for Golgi 58K (A1), TGN38 (B1), and cathepsin D (C1). The middle column (A2–C2) shows AQP2 staining. Merged images are shown in the right column (A3–C3). In merged images, AQP2 is shown in green, and organelle marker proteins in red. D, EEA1 (D1 and D4; red in D3 and D5), AQP2 (D2; green in D3 and D5), and merged images (D3 and D5). D4 and D5 are single confocal images of semithin cryosections showing the vertical plane of the cell monolayer. EEA1 is mainly scattered in the apical side of the cell and does not colocalize with AQP2. Nuclear DNAs are stained with TO-PRO-3 and shown in blue (D5). E, Rab11 (E1; red in E3), AQP2 (E2; green in E3), and a merged image (D3). Specificity of anti-Rab11 antibody is checked by double immunostaining with Rab4 and Rab5. Rab11-positive compartment (red in E4 and E5) is negative for Rab4 (green in E4) or Rab5 (green in E5). F, Fluorescently labeled transferrin (F1 and F4; red in F3 and F6) and AQP2 (F2 and F5; green in F3 and F6) and merged images (F3 and F6). Single confocal images from the intermediate plane between the nucleus and the apical surface (F1–F3) and the basal cytoplasm (F4–F6) are shown. Bars, 10 µm.

 
Retrieval of AQP2 to the intracellular compartment
AQP2 was found in close proximity to EEA1 in some nonpolarized cells. To address whether such localization represents a certain state of AQP2 trafficking, we examined the translocation and recycling of AQP2 (Fig. 5AGo). Polarized MDCK-hAQP2 cells were incubated with forskolin for 30 min and then washed. Cells were fixed at various time points after the removal of forskolin, and double immunostaining for AQP2 and EEA1 was performed.



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  		FIG. 5. Translocation and recycling of AQP2. Cells were treated with forskolin for 30 min (A0), washed, and incubated in forskolin-free medium (A1–C4). AQP2 is shown in green, and EEA1 in red. Multiple confocal images are projected onto a single image. AQP2 is translocated to the apical plasma membrane after incubation with forskolin for 30 min (A0). Cells were washed and incubated in forskolin-free medium (A1–A4). Retrieval of AQP2 to the cell is seen within 30 min and colocalized with EEA1 in larger vesicles. Thereafter, AQP2 is localized in the subapical AQP2-storage compartment by 90 min after washout. During this AQP2 retrieval process, distribution of EEA1 remains unchanged, but the size of the EEA1-positive vesicles is enlarged after forskolin washout. Cells were incubated with 100 nM wortmannin (B1–B4) or 20 µM LY294002 (C1–C4) after washout of forskolin. The colocalization of AQP2 with EEA1 lasted. Multiple confocal images were projected onto a single image. Typical results of three independent experiments are shown. Bar, 20 µm.

 
AQP2 was localized at the subapical region without forskolin treatment. Forskolin treatment resulted in the translocation of AQP2 to the apical membrane (Fig. 5A0Go). EEA1 was scattered in large vesicles in the apical cytoplasm and was not affected by forskolin treatment. Retrieval of AQP2 from the apical membrane began within 30 min after forskolin removal (Fig. 5A1Go). AQP2 was transiently found in large vesicles positive for EEA1. Then, AQP2 segregated from EEA1-positive vesicles and went back to the original subapical compartment of small vesicles (Fig. 5Go A2–A4). After 90 min, AQP2 was restricted to subapical AQP2-storage vesicles, which were small and negative for EEA1.

EEA1 is a functional molecule for vesicle trafficking in cooperation with PI3-phosphate (PI3P). To assess the involvement of PI3P in the trafficking of AQP2, cells were treated with PI3-kinase inhibitors (Fig. 5Go, B and C). After the treatment with forskolin, cells were incubated with wortmannin or LY294002. Three experiments were indepen-dently performed for each PI3-kinase inhibitor, and similar results were obtained. AQP2 was colocalized with EEA1 within 30 min after washout, as was seen in the absence of PI3-kinase inhibitors. The colocalization of AQP2 and EEA1 lasted longer and still remained 120 min after washout. PI3-kinase inhibitors did not affect the forskolin-induced translocation of AQP2 to the plasma membrane (data not shown). These data suggest that the retrieval of AQP2 from the apical plasma membrane to the subapical AQP2-storage compartment is dependent on PI3-kinase.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Model system
Cultured renal cells have been used as a model system to study the intracellular distribution and trafficking of AQP2, although aberrant distribution and trafficking of AQP2 were sometimes encountered. Thus, it is important to develop an in vitro model system that represents the nature of AQP2. In LLC-PK1 cells, the porcine proximal tubule-like cell line, AQP2 was reported to be localized in the TGN (28), and the involvement of coated vesicles in the trafficking of AQP2 was proposed (29, 30). Translocation of AQP2 in LLC-PK1 cells occurs upon ADH stimulation similar to that in the principal cells of kidney collecting ducts, although it distributes in the basolateral membrane. In CD8 cells, the rabbit cortical collecting duct cell line, trafficking of AQP2 is regulated by VAMP-2, a member of SNARE complex (31), and the Rho/actin system (32). However, the perinuclear distribution of AQP2 does not correspond to the distribution in the kidney. The MDCK cell is a kidney epithelial cell line used as an alternative model system. The localization of AQP2 expressed in MDCK cells was similar to that in the principal cells (33, 34). In addition, the translocation of AQP2 into the apical membrane occurred through the protein kinase A signaling system, which is similar to the process in principal cells in the kidney (24). These results indicate that MDCK cells provide an adequate model system to study the intracellular localization and trafficking of AQP2. AQP2 expressed in MDCK cells could translocate from an intracellular compartment to the apical membrane upon forskolin stimulation, and its intracellular localization was distinct from the Golgi apparatus, TGN, and lysosome. The pattern of intracellular distribution of AQP2 and cytoplasmic organelles was compatible with that observed in the principal cells of collecting ducts of the rat kidney (11). Thus, AQP2 in our MDCK-hAQP2 cell system exhibits physiological properties to a large extent. Furthermore, the cells possess AQP2 recycling characteristics. A transient colocalization of AQP2 and the early endosome marker EEA1 was observed in MDCK cells during the retrieval of AQP2. The colocalization of these molecules was also seen in the kidney (11). Because AQP2 is a recycling molecule (35), the colocalization of AQP2 and EEA1 in the kidney may show the recycling process, and MDCK-hAQP2 cells can provide a unique system to analyze this process in vitro.

AQP2-storage compartment
Using the MDCK-hAQP2 cell model, a comparison with other recycling molecules was carried out. We compared the distribution of AQP2 and GLUT4 by cotransfection. Localization and intracellular trafficking of GLUT4 were often discussed together with those of AQP2. GLUT4 is stored in the intracellular vesicles and is translocated to the plasma membrane by an exocytotic process upon insulin stimulation (26). Moreover, GLUT4 and AQP2 contain a common targeting sequence, a dileucine motif, which is important in protein trafficking (36). In MDCK cells, transfected GLUT4 distributed in the perinuclear region, and AQP2 in the peripheral region (37). Although GLUT4 expression in renal epithelial cells does not necessarily reflect its physiological nature, its perinuclear distribution resembled that found in adipocytes and muscle cells (38, 39), where GLUT4 is expressed and functions physiologically. Our data indicate that GLUT4 and AQP2 are stored in different compartments in nonstimulated MDCK cells. Additional experiments are needed to rule out the possibility that GLUT4 and AQP2 share any trafficking system.

A comparison of AQP2 and the transferrin recycling pathway was also performed both in nonpolarized and polarized cells, with the result that they did not colocalize. In polarized MDCK cells, endocytosed transferrin is mainly scattered in basal recycling endosomes as reported previously (40). These transferrin compartments are localized in the basal side and hence are clearly distinct from the AQP2 compartment in the apical side.

Recycling pathway
Numerous studies have focused on AQP2 trafficking from the intracellular compartment to the apical plasma membrane in response to ADH, and a signaling pathway via phosphorylation by protein kinase A and protein kinase G has been proposed (41). Nevertheless, the cellular mechanism of retrieval and recycling of AQP2 has not been fully elucidated. We have shown here that AQP2 is stored in the distinct subapical compartment and is translocated to the apical plasma membrane upon forskolin stimulation. After the washout of forskolin, AQP2 is retrieved to the cell by endocytosis and enters EEA1-positive endosomes on its way back to the subapical AQP2-storage compartment. Thus, AQP2 uses the early endosomal system as an early event of the retrieval from plasma membrane.

EEA1 is not only a marker of early endosomes but also a functional molecule for the tethering and docking of endosomal vesicles, interacting with other molecules such as PI3P, a product of PI3-kinase (42). PI3-kinase has been firmly implicated in endosomal membrane traffic in various types of cells through the study of many different trafficking events (43). Inhibition of PI3-kinase activity by wortmannin or LY294002 leads to the delay of AQP2 trafficking from the apical membrane to the subapical compartment, suggesting that PI3-kinase is involved in AQP2 trafficking back to its storage compartment. As well as AQP2 trafficking, endocytosis and recycling of other proteins between plasma membrane and cytoplasmic compartment has been reported to be PI3-kinase dependent. For example, receptors for platelet-derived growth factor (44) and angiotensin II (45) undergo ligand-induced endocytosis. Their postendocytic recycling to the plasma membrane or movement into late endosomes/lysosomes were altered by inhibition of PI3-kinase activity.

AQP2 also seems to travel via a PI3-kinase-independent pathway, because PI3-kinase inhibitors did not completely inhibit AQP2 trafficking back to the storage compartment, and some AQP2 went back to the subapical compartment 120 min after the washout of forskolin in the presence of PI3-kinase inhibitors. Similar dual recycling pathways have been proposed in the case of angiotensin II receptor trafficking (45) and were described as wortmannin-dependent/rapid and wortmannin-independent/slow pathways. Additional studies are needed to elucidate whether PI3-kinase and its relatives are involved in AQP2 trafficking specifically and directly.


   Acknowledgments
 
We thank Ms. Yukiko Tajika-Takahashi for secretarial and technical assistance.


   Footnotes
 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Abbreviations: ADH, Antidiuretic hormone; AQP2, aquaporin-2; GLUT4,glucose transporter 4; MDCK, Madin-Darby canine kidney; PI3, phosphatidylinositol 3; PI3P, PI3-phosphate; TGN, trans-Golgi network.

Received January 22, 2004.

Accepted for publication May 11, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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