Immunolocalization of aquaporin-8 in rat kidney, gastrointestinal tract, testis, and airways

Marie-Louise Elkjær1, Lene N. Nejsum1, Veronika Gresz1, Tae-Hwan Kwon1,2, Uffe B. Jensen3, Jørgen Frøkiær1, and Søren Nielsen1

1 The Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C; 2 Department of Physiology, Dongguk University, 780 - 714 Kyungju, Korea; and 3 Institute of Human Genetics, University of Aarhus, DK-8000 Aarhus C, Denmark


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

First published August 8, 2001; 10.1152/ajprenal.00158.2001.---The purpose of this study was to determine the cellular and subcellular localization of aquaporin-8 (AQP8) in rat kidney and other organs by RT-PCR analyses and by immunoblotting and immunohistochemistry using peptide-derived rabbit antibodies to rat AQP8. RT-PCR and Southern blotting revealed the presence of AQP8 mRNA in all kidney zones. LLC-PK1 cells transfected with a rat AQP8 construct exhibited strong labeling with the affinity-purified antibodies, whereas controls using cells transfected with the vector, but without the insert, were negative. The labeling was almost exclusively associated with intracellular vesicles. Immunoblotting of kidney membrane fractions revealed a predominant single band of 26-28 kDa. AQP8 immunoreactivity was mainly present in the cortex and outer stripe of the outer medulla. Sequential ultracentrifugation of rat kidney membrane revealed that AQP8 resides predominantly in intracellular vesicular fractions. Immunocytochemistry revealed modest labeling of proximal tubules and weak labeling of collecting ducts in cortex and medulla of rat kidney. The labeling was confined to cytoplasmic areas with no labeling of the brush border. Immunoblotting and RT-PCR/Southern blotting also revealed the presence of AQP8 protein and mRNA in rat liver, testis, epididymis, duodenum, jejunum, colon, and bronchi/trachea. Consistent with this, immunohistochemistry revealed AQP8 labeling in the hepatocytes and spematogenic cells in testis and in the basal cells in ductus epididymis, trachea, and bronchial epithelia. Moreover, AQP8 labeling was observed in the myoepithelial cells in salivary, bronchial, and tracheal glands with no labeling of acini or ductal epithelial cells. AQP8 is also present in the surface epithelial cells in duodenum, jejunum, and colon. In conclusion, AQP8 is expressed at low levels in rat kidney proximal tubules and collecting ducts, and it is present in distinct cell types in liver, testis, epididymis, duodenum, jejunum, colon, trachea, and principal bronchi as well as in multiple glands, including salivary glands.

intracellular water channel; renal; immunocytochemistry


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TRANSPORT OF WATER AND SOLUTES across cell membranes is vital to all cellular functions as well as to the functioning of many organs, including the kidney, heart, and lung. The molecular basis for water transport across cell membranes was unknown until the pioneering discovery of aquaporin-1 (AQP1) by Agre and colleagues (22). AQP1, which is critically involved in rapid fluid transport (22), is expressed in kidney proximal tubule and descending thin limb cells as well as in red blood cells and a number of epithelia throughout the body. Another nine mammalian aquaporins have been identified since the discovery of AQP1. In general, aquaporins represent a group of small membrane proteins that function as water channels in a variety of different tissues. They have been divided into two subgroups according to their transporting characteristics. The first group, the aquaporins, is to a high degree water selective and consists of AQP0, -1, -2, -4, -5, and -6. The other subgroup, the aquaglyceroporins, is permeable to other small molecules, as well as water, and consists of AQP3, -7, and -9.

AQP8 was first identified by homology cloning in 1997 and has since been sequenced by different groups in rats, mice, and humans (2, 12-14, 17). The amino acid sequence of AQP8 is similar to those of other aquaporins (30-35%), and it shares the highest sequence homology (38-40%) with the plant water channel AQP-TIP (12-14, 17). The transport properties of AQP8 appear to be species dependent. Urea conductance of AQP8 has been identified in mouse (17), but not in rat (14). Ma et al. (17) also reported that mouse AQP8 conducts water and urea but not glycerol. In humans, AQP8 protein has been found to be a water-selective channel without permeability to either urea or glycerol (13). The reason for these differences is still unclear and may represent technical differences, but similar species differences in transport properties have also been encountered in other aquaporins, e.g., AQP9 (see Ref. 5 for further studies).

With regard to tissue expression, a number of studies using RT-PCR, Northern blotting, or RNAse protection assays have revealed that AQP8 mRNA is present in a number of organs. In the rat, AQP8 mRNA has been found in the testis, jejunum, colon, liver, salivary gland, and pancreas (12, 14, 15). Mouse AQP8 mRNA has been localized to the placenta, colon, liver, heart, lung, kidney, submandibular gland, diaphragm, testis, spleen, stomach, and brain (17). Human AQP8 mRNA has been localized in the pancreas and colon, but interestingly it was found to be absent in the testis (13), where AQP8 mRNA has been shown to be abundant in rats and mice. Despite the fact that AQP8 mRNA has been found in a variety of tissues, there have only been a few reports examining the cellular and subcellular expression of AQP8 protein. So far, AQP8 protein has been immunolocalized in rat pancreatic acinar cells (9) and in human colon columnar epithelial cells (6). Recently, it was suggested that AQP8 is present in the basolateral plasma membrane domains of rat submandibular gland acinar cells (27). The present study, therefore, aims to determine the cellular and subcellular localization of AQP8 protein in rat, with the focus on tissues where AQP8 mRNA is known to be expressed. To achieve this, we developed new peptide-derived rabbit antibodies against rat AQP8 and used them for immunoblotting and immunocytochemistry.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental animals. Studies were performed in male Wistar rats, weighing 250-300 g (M & B, Ry, Denmark). The rats were maintained on a standard rodent diet (Altromin, Lage, Germany) with free access to water. The study was approved by the Animal Ethical Committee of the University of Aarhus.

Antibodies. Polyclonal AQP8 antibodies were raised in rabbit against a peptide corresponding to COOH-terminal amino acids 249-263 (RA2277/1262; peptide/rabbit nos.) of rat AQP8 (12). The antibodies were affinity purified. Also, commercially available affinity-purified antibodies against a peptide corresponding to COOH-terminal amino acids 249-263 of rat AQP8 were used (AQP81-A; Alpha Diagnostic, San Antonio, TX). In control experiments, polyclonal rat anti-AQP1 antibodies (LL266), which has previously been characterized (26), were used (kindly provided by Dr. M. Knepper).

Transfection of LLC-PK1 cells. LLC-PK1 cells (originating from porcine proximal tubules) were transfected using calcium-phosphate coprecipitation. Cells were transfected with pcDNA3.1 (Invitrogen, Groningen, The Netherlands) containing the coding sequence for rat AQP8 (kindly provided by Dr. G. Calamita, University of Bari, Bari, Italy). As controls, cells were transfected with the vector pcDNA3.1. Fifteen micrograms of DNA were used for each T-25 culture flask, and 6 µg were used for SlideFlasks (NUNC). The cells were incubated with the precipitated DNA for 16 h, followed by a wash in PBS, and returned to culture medium, DMEM (GIBCO Life Technologies, Taastrup, Denmark) containing 10% FBS (GIBCO Life Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were analyzed after 24-48 h. Cells grown in SlideFlasks were prepared for immunostaining by fixation in neutral buffered formalin for 5 min and washed in PBS. The slides were blocked and permealized by incubating in PB-buffer [0.5% skim milk powder, 0.1% Triton X-100 in TBS (0.9% NaCl, 20 mM Tris, pH 7.2)]. Primary antibodies were diluted in PB-buffer, and the slides were incubated for 1-2 h at room temperature. Secondary antibodies were FITC-conjugated goat anti-rabbit IgG (Alexa 488, Molecular Probes Europe, Leiden, The Netherlands). These were diluted in PB-buffer, and slides were incubated for 1 h. Images were captured using a cooled charge-coupled device camera (series 200, Photometrics) controlled by IPLab (Scanalytics) on a Macintosh computer (Apple).

RNA extraction. mRNA was extracted from tissues using poly-T-coated magnetic beads (Dynabeads mRNA DIRECT Micro kit, Dynal).

RT-PCR and Southern blotting. RT was performed on mRNA (directly on the magnetic beads) using Superscript II (GIBCO Life Technologies). PCR (30 cycles) was performed using sequence-specific primers for AQP8 (9): 5'-CAGATATGTCTGGGGAGCAGACGC-3' (sense) and 5'-CTGCCAGCAGTTCTTCACCTCGAC-3' (antisense). A test for beta -actin was performed to confirm the presence of cDNA: 5'-GAGTACAACCTCCTTGCAGCTC-3' (sense) 5'-TTGTAGAAAGTGTGGTGCCAAA-3' (antisense). Both a RT-negative control and a PCR-negative control reaction were performed. PCR products were analyzed by agarose gel electrophoresis and subsequent high-stringency Southern blotting with a fluorescein-labeled, sequence-specific AQP8 cDNA probe.

Membrane fractionation and immunoblotting analyses. Rat tissues were homogenized with an Ultra-Turrax T8 homogenizer (IKA Labortechnik, Staufen, Germany) for 15 s on ice in dissection buffer containing 0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, 0.4 mM pefabloc, and 8.5 µM leupeptin (pH 7.2). The homogenate was centrifuged in an Eppendorf 5403 centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. The supernatant was either used directly (VLSS) or centrifuged at 17,000 g for 1 h to produce a pellet enriched for plasma membrane (LS-P) and a supernatant enriched for intracellular vesicles (LS-S). From the resultant pellets and supernatants, gel samples (in 2% SDS) were made. Gel samples were loaded onto 12% SDS-PAGE gels and run on a Bio-Rad minigel systems (Bio-Rad Mini-Protean II). The proteins were transferred to nitrocellulose paper by electroelution. After being blocked and washed, blots were incubated overnight at 4°C with primary antibodies (see above). Blots were subsequently washed and then incubated at room temperature for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies (diluted 1:3,000 in PBS-T; P0448, DAKO, Glostrup, Denmark). Finally, antibody binding was visualized using the enhanced chemiluminescence system (ECL; Amersham International). Immunolabeling of controls was performed using antibodies preabsorbed with immunizing peptide.

Immunocytochemistry. Male Wistar rats (weighing 250-300 g) were anesthetized with halothane and fixed by retrograde perfusion through the abdominal aorta with 2 or 4% paraformaldehyde in 0.1 M sodium cacodylate buffer or 0.1 M phosphate buffer. Different rat tissues were removed and postfixed for 1 h. For preparation of cryostat sections (10 µm), tissues were cryoprotected in 25% sucrose. For paraffin-embedded preparation (2-3 µm), tissues were dehydrated in ethanol followed by xylene and finally embedded in paraffin. The staining was carried out using indirect immunofluorescence or indirect immunoperoxidase. The sections were dewaxed and rehydrated. For immunoperoxidase labeling, endogenous peroxidase was blocked by 0.5% H2O2 in absolute methanol for 30 min at room temperature. To reveal antigens, sections were incubated in 1 mM Tris solution (pH 9.0) supplemented with 0.5 mM EGTA and heated in a microwave oven for 10 min. Nonspecific binding of Ig was prevented by incubating the sections in 50 mM NH4Cl for 30 min, followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with affinity-purified anti-AQP8 antibodies, and the labeling was visualized with HRP-conjugated secondary antibodies (P0448 goat anti-rabbit, 1:200; DAKO), followed by incubation with diaminobenzidine. For fluorescence microscopy, the labeling was visualized using FITC-conjugated goat anti-rabbit secondary antibodies (Alexa 488, Molecular Probes Europe). Immunolabeling of controls was performed using antibodies preabsorbed with immunizing peptide.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RT-PCR analyses of AQP8 mRNA expression in rat organs. To confirm and further extend knowledge regarding tissue distribution of AQP8 mRNA in rats, RT-PCR analyses were performed. The specificity was confirmed by Southern blotting with a labeled, sequenced AQP8 cDNA probe. Consistent with previous results, abundant AQP8 mRNA was encountered in the liver and testis (Fig. 1). In the kidney, AQP8 mRNA was present in all kidney zones (Fig. 1). AQP8 mRNA was also found in a variety of other tissues, including the salivary glands (not shown), duodenum, jejunum, and bronchi (Fig. 1).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1.   Aquaporin-8 (AQP8) RT-PCR using mRNA isolated from rat kidney (all zones) and liver, colon, duodenum, jejunum, testis, epididymis, and principal bronchi. Top: Southern blot showing AQP8 RT-PCR (811 bp) signal in kidney (all zones), liver, colon, duodenum, jejunum, testis, epididymis, and principal bronchi. Bottom: presence of cDNA in all samples was confirmed with beta -actin RT-PCR (329 bp). OSOM and ISOM: outer and inner stripe of outer medulla, respectively; IM, inner medulla.

LLC-PK1 cells transfected with rat AQP8. To establish the cellular and subcellular localization of AQP8, peptide-derived antibodies to rat AQP8 were designed and produced (see MATERIALS AND METHODS). In addition, a commercially available antibody was used. The specificity of the antibodies was supported by three lines of evidence: 1) immunolabeling of AQP8-transfected cells, 2) immunoblotting of rat tissues where AQP8 mRNA is known to be expressed (revealing the predicted molecular size), and 3) immunolabeling of controls. As demonstrated in Fig. 2, LLC-PK1 cells transfected with an AQP8 construct exhibited significant immunolabeling. All antibodies labeled the cells, whereas nontransfected cells (Fig. 2) or cells transfected only with the vector were not labeled (not shown). The AQP8 immunolabeling was almost exclusively associated with intracellular compartments, resembling small vesicles, whereas plasma membrane domains appeared unlabeled (Fig. 2). This suggests that AQP8 is mainly an intracellular water channel.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2.   Anti-AQP8 (RA2677/1262) recognizes LLC-PK1 cells transfected with an AQP8 construct. A: cells transfected with an AQP8 construct exhibit significant labeling (arrows), whereas nontransfected cells (arrowheads) show no labeling. B: higher magnification of a cell transfected with an AQP8 construct. The labeling is mainly intracellular (arrow), with little or no labeling of the plasma membrane. N, nucleus. Magnification: ×240 (A), ×960 (B).

Immunoblotting analysis of AQP8 in rat kidney. To determine whether AQP8 protein is expressed in the kidney and, if this is the case, to determine in which part of the kidney AQP8 is present, immunoblotting was performed using membrane fractions from different kidney zones: the cortex, outer stripe of the outer medulla, inner stripe of the outer medulla, and inner medulla. Immunoblotting using anti-AQP8 (RA 2677/1262) revealed a single ~26- to 28-kDa band in the cortex and OSOM (Fig. 3). Also, a weak signal was obtained in ISOM and IM (Fig. 3A). Immunolabeling of controls using peptide-preabsorbed antibody revealed an absence of labeling (Fig. 3B). Anti-AQP8 antibody did not recognize purified human AQP1 protein or red blood cells, as did anti-AQP1 antibody (Fig. 3, C-F), eliminating the remote possibility of cross-reactivity between anti-AQP8 antibody and AQP1.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 3.   Immunoblot showing anti-AQP8 (RA2677/1262) immunoreactivity in rat kidney. A: anti-AQP8 immunoblot of membranes from kidney cortex, OSOM, ISOM, and IM. Anti-AQP8 recognizes a 26- to 28-kDa band. B: immunoblot of anti-AQP8 preabsorbed with immunizing peptide. No labeling was detected. C-F: immunoblot demonstrating that anti-AQP8 (RA2677/1262) does not recognize purified human AQP1 protein (C), in contrast to anti-AQP1 antibody that does recognize purified human AQP1 protein (D). Additionally, anti-AQP8 (RA2677/1262) recognizes total kidney but not red blood cells (RBC; E), whereas anti-AQP1 recognizes total kidney and RBC (F). G and H: immunoblot showing anti-AQP8 (RA2677/1262) labeling mainly intracellular vesicles (LS-S; G) and anti-AQP1 (LL266) labeling mainly plasma membranes (LS-P; H) in all zones from kidney. Anti-AQP1 (F and H) recognizes a 28-kDa lower band and a 35- to 45-kDa upper band corresponding to the nonglycosylated and the glycosylated form of AQP1, respectively, as shown previously.

To determine whether AQP8 protein is present in intracellular vesicles and/or plasma membranes, membrane fractionation was carried out. Immunoblotting revealed predominant AQP8 labeling of membrane fractions enriched for intracellular vesicles (Fig. 3G, LS-S) although weaker labeling was also observed in membrane fractions enriched for plasma membrane (Fig. 3G, LS-P). In contrast, control experiments revealed that AQP1 was mainly present in membrane fractions enriched with plasma membrane (Fig. 3H, LS-P), as previously shown (21). Thus AQP8 protein is present in the kidney and appears to be present mainly in intracellular compartments.

Immunocytochemical localization of AQP8 in rat kidney. Immunohistochemistry revealed that AQP8 is present in proximal tubules (Fig. 4A), including in S3 proximal tubules (shown in Fig. 4B in the transition from proximal tubule to descending thin limb). In addition, very weak labeling was observed in the collecting ducts (Fig. 4C), whereas other nephron segments and glomeruli did not exhibit labeling in excess of background. In the proximal tubule, the labeling was confined to cytoplasmic domains (in both apical, central, and basal parts of the cells), with no labeling of the brush border (arrowheads in Fig. 4, A and B) and an absence of, or very weak, labeling of basolateral plasma membrane domains. Similarly, in collecting duct cells the weak labeling was associated with intracellular structures. The predominant intracellular localization is consistent with the observations in membrane fractionations (Fig. 3) and with the results obtained by immunolocalization in AQP8-transfected LLC-PK1 cells shown in Fig. 2.


View larger version (175K):
[in this window]
[in a new window]
 
Fig. 4.   Anti-AQP8 (RA2677/1262) labels proximal tubules and collecting ducts in rat kidney. A: immunoperoxidase labeling of paraffin section showing anti-AQP8 labeling (arrows) in the proximal tubule (P). The labeling is mainly intracellular, with no labeling of the brush border (arrowhead). B: transition of proximal tubule to thin descending limb. The labeling is confined to the intracellular part of the proximal tubule (arrows), with no labeling of the brush border (arrowhead) and no labeling of the thin descending limb. C: immunoperoxidase labeling of paraffin section showing weak anti-AQP8 labeling (arrows) in the collecting duct (CD). D and E: immunolabeling control, using anti-AQP8 preabsorbed with immunizing peptide, shows absence of labeling in the proximal tubule (D) and in the collecting duct (E). Magnification: ×870.

Immunoblotting analyses of AQP8 in extrarenal tissues. Immunoblotting using membrane fractions from the liver, testis, epididymis, duodenum, jejunum, colon, and trachea revealed a predominant ~26- to 28-kDa band (Fig. 5A). In addition, we observed a higher molecular weight band in liver and testis and a lower molecular weight band in testis (Fig. 5A). Immunolabeling of controls, using antibodies that were preincubated with the immunizing peptide, was negative (Fig. 5B). The presence of AQP8 protein in the colon is consistent with previous evidence (14, 15, 17). Additionally, evidence for the presence of AQP8 in these tissues was obtained by RT-PCR and Southern blotting, which revealed a signal for AQP8 mRNA in the liver, testis, epididymis, duodenum, jejunum, colon, and principal bronchi (Fig. 1).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   Anti-AQP8 (RA2677/1262) immunoblot of membranes from rat liver, testis, epididymis, duodenum, jejunum, colon, and trachea. A: anti-AQP8 specifically recognizes a 26- to 28-kDa band in liver, testis, epididymis, duodenum, jejunum, colon, and trachea. In addition an ~24-kDa band in testis and a higher molecular weight band in testis and liver was observed. B: immunoblot of anti-AQP8 preabsorbed with immunizing peptide shows an absence of labeling.

Immunocytochemical localization of AQP8 in testis and epididymis. Immunocytochemistry demonstrated the presence of AQP8 protein in the spermatogenic cells of rat testis (Fig. 6), consistent with previous demonstration of AQP8 mRNA by in situ hybridization by Ishibashi et al. (12). In sections from epididymis, AQP8 immunolabeling was seen in the basal cells of the duct epithelium (Fig. 7). The labeling seen was predominantly of cytoplasmic areas, and the labeling pattern was identical in caput and cauda epididymis. There was no labeling of other epithelial duct cells (Fig. 7, A-C). Immunolabeled controls were negative (Fig. 7D).


View larger version (99K):
[in this window]
[in a new window]
 
Fig. 6.   Anti-AQP8 (RA2677/1262) labels spermatogenic cells in rat testis. A: paraffin section showing anti-AQP8 labeling in testis. The labeling is in the spermatogenic cells (arrows). S, seminiferous tubule. B: immunolabeling control, using anti-AQP8 preabsorbed with immunizing peptide shows absence of labeling. Magnification: ×180.



View larger version (146K):
[in this window]
[in a new window]
 
Fig. 7.   Anti-AQP8 (AQP81-A) labels basal cells in the duct epithelia in epididymis. A-C: paraffin sections showing anti-AQP8 labeling in rat epididymis. Labeling is seen in the basal cells (arrows). D, ductus epididymidis. D: immunolabeling control, using anti-AQP8 preabsorbed with immunizing peptide, shows absence of labeling. Magnification: ×480 (A), ×810 (B-D).

Immunocytochemical localization of AQP8 in airways. To determine the localization of AQP8 in airways, sections were obtained from the trachea, bronchi, and lung. In the trachea (not shown) and main bronchi, distinct AQP8 immunolabeling of basal cells in the mucosa epithelium was observed (Fig. 8A; indicated by arrows in Fig. 8B). Immunolabeled controls were negative (Fig. 8D). In addition, distinct AQP8 immunolabeling was observed in the myoepithelial cells of the tracheal and bronchial glands (arrowheads in Fig. 8, B and C). Immunolabeled controls are negative (Fig. 8D, inset). In addition to the labeling observed in basal cells and in myoepithelial cells, there was weak AQP8 labeling of the apical parts of the ciliated cells just beneath the cilia (Fig. 8B, *). No significant labeling was observed in alveolar and vascular structures of lung (not shown).


View larger version (167K):
[in this window]
[in a new window]
 
Fig. 8.   Anti-AQP8 (AQP81-A) labels basal cells and myoepithelial cells in rat principal bronchi. A: low magnification showing anti-AQP8 labeling in the basal cells of the bronchial epithelia and of the myoepithelial cells in the bronchial glands. B: labeling is seen in the basal cells (arrows) of the epithelia and in the myoepithelial cells of the bronchial glands (arrowheads). Weak labeling of the very apical part of the cell bodies of the ciliated cells (*) was also observed. C: higher magnification of the bronchial glands showing labeling of the myoepithelial cells (arrowheads). D: immunolabeling control, using anti-AQP8 preabsorbed with immunizing peptide, shows an absence of labeling in the basal cells (arrows) and of the myoepithelial cells of the bronchial glands (inset). Magnification: ×190 (A), ×830 (B-D).

Immunocytochemical localization of AQP8 in the digestive tract. In the digestive tract, AQP8 immunolabeling was examined in the salivary glands and in the different segments of the small and large intestine as well as the liver. In the parotid, submandibular, and sublingual salivary glands, immunolabeling was observed in myoepithelial cells surrounding acinar and ductal cells (Fig. 9, A-D), outlining the classic organization of the myoepithelial cells as basket cells (Fig. 9C). No labeling of ductal cells or acinar cells was observed (Fig. 9). Immunolabeled controls were negative (Fig. 9, E-G).


View larger version (181K):
[in this window]
[in a new window]
 
Fig. 9.   Immunoperoxidase (A-G) and immunofluorescence (inset in D) micrographs showing anti-AQP8 (AQP81-A) labeling in myoepithelial cells in rat parotid (A and B), submandibular (C), and sublingual (D) salivary glands. A-D: AQP8 is present in the myoepithelial cells (arrowheads) around the acini (indicated by A) and the intercalated duct cells (indicated by ID). No labeling was observed in the secretory cells or in the excretory duct system, including the granular duct (indicated by GD). E-G: immunolabeling controls of parotid (E), submandibular (F), and sublingual (G) glands, using anti-AQP8 preabsorbed with immunizing peptide, show absence of labeling. Magnification: ×200 (A), ×530 (B-D), ×180 (E-G).

In rat duodenum and jejunum, AQP8 localization was seen to be in the absorptive epithelia cells (Fig. 10). Labeling was localized in the apical part of the cell (Fig. 10, A and C, arrows) but without labeling of the microvilli (Fig. 10, A and C, arrowheads). Immunolabeled controls were negative (Fig. 10, B and D). In rat colon, labeling of the surface epithelial cells including crypt cells was observed (Fig. 11C). Immunolabeled controls were negative (Fig. 11D). In rat liver, AQP8 immunolabeling was confined to the hepatocytes (Fig. 11A), and the localization was distinctly intracellular (Fig. 11A).


View larger version (145K):
[in this window]
[in a new window]
 
Fig. 10.   Anti-AQP8 labeling in surface epithelia cells in jejunum (RA2677/1262) and in duodenum (AQP81-A). A and C: AQP8 is present in the apical part of the columnar epithelia cells (arrows) in jejunum (A) and in duodenum (C), just beneath the microvilli (arrowheads). B and D: no labeling was observed in the controls using anti-AQP8 preabsorbed with immunizing peptide. Magnification: ×490 (A-D).



View larger version (165K):
[in this window]
[in a new window]
 
Fig. 11.   Anti-AQP8 (RA2677/1262) labels hepatocytes in rat liver, and absorptive epithelial cells in rat colon. A: cryostat section showing anti-AQP8 labeling in liver. The labeling is confined to the hepatocytes. V, central vein. B: immunolabeling control, using anti-AQP8 preabsorbed with immunizing peptide, shows an absence of labeling in the hepatocytes. C: paraffin section showing anti-AQP8 labeling in colon. Labeling is seen in the absorptive epithelia cells (arrows). D: immunolabeling control, using anti-AQP8 preabsorbed with immunizing peptide, shows an absence of labeling in the surface epithelia of colon. Magnification: ×200.

The two antibodies used, RA 2277/1262 and AQP81-A (commercially available antibody), labeled identically in all of the different tissues examined.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AQP8 was identified several years ago; however, virtually nothing is known about its cellular and subcellular localization in multiple tissues. The purpose of the present study was to establish this. For this purpose, several peptide-derived antibodies were used for immunoblotting and immunocytochemical approaches. In the kidney, we demonstrated that AQP8 is present in proximal tubule cells and collecting duct cells. AQP8 is almost exclusively present in intracellular vesicles, with no or little labeling of plasma membrane domains. This is consistent with membrane fractionation and immunoblotting experiments and with the intracellular localization of AQP8 in a transfected proximal tubule cell line (LLC-PK1 cells). Thus AQP8 is likely to be involved in ensuring rapid osmoequilibration between the cytoplasmic and vesicular compartments. This study also provides a demonstration of the cellular and subcellular localization of AQP8 in various organs. In the epididymis, distinct labeling of basal cells was observed, and a similar labeling pattern was observed in basal cells of the conductive airways. Moreover AQP8 was found in myoepithelial cells of glands in the airways and in salivary glands, whereas no labeling was seen in acinar and ductal epithelia. Surface epithelial cells of the airways, intestine, and colon exhibited distinct subapical or intracellular labeling of AQP8 but an absence of labeling of plasma membrane domains. In the liver, AQP8 was present in hepatocytes in a predominantly intracellular localization. Thus in multiple cell types AQP8 appears to be predominantly localized in intracellular vesicles, suggesting a role for osmotic equilibration between cytoplasmic and vesicular content.

AQP8 in the kidney. AQP1 is abundant in the proximal tubule and descending limb, where it is localized in the apical and basolateral plasma membranes (21). Here, it appears to provide the chief route for proximal nephron water reabsorption, which is confirmed by studies of transgenic mice lacking AQP1 (16). In addition, AQP6 and -7 have been localized in the proximal tubule, where the former is localized intracellularly, e.g., mainly in the subapical parts of S2 and S3 proximal tubules (28), whereas AQP7 is localized mainly in the brush border of S3 proximal tubules (10, 18). In this study, we found the kidney localization of AQP8 to be intracellular in the proximal tubule, and in addition we observed very weak labeling in the collecting ducts. The absence or low abundance of AQP8 in plasma membranes in the kidney (determined by immunocytochemistry and supported by AQP8-transfected LLC-PK1 cells and membrane fractionation and immunoblotting) is consistent with the occurrence of AQP8 in a nonglycosylated form, or at least in a different glycosylation pattern compared with renal plasma membrane aquaporins AQP1, -2, -3, and -4. In immunoblotting experiments, these four exhibit both a nonglycosylated and a typical 35- to 55-kDa glycosylated form (3, 4, 19-21, 25), unlike AQP8. This would be consistent with the present view that many plasma membrane proteins exist in a glycosylated form (8). However, it should be emphasized that not all plasma membrane proteins are heavily glycosylated, and this is also the case for the plasma membrane protein AQP5. The presence in intracellular vesicles suggests a role in osmotic equilibration between the cytoplasm and the vesicle. However, it cannot be ruled out that there may be regulation of AQP8, which potentially may involve trafficking of AQP8 to plasma membrane domains, as recently shown for hepatocytes (7). To elucidate this, additional studies are warranted.

AQP8 in surface epithelial cells in the gastrointestinal tract and airways. The gastrointestinal tract is a main organ for water transport. Of the 9,000 ml of fluid presented to the human small intestine per day, only 1,000-2,000 ml pass into the colon; thus large amounts of fluid are absorbed in the intestine. In the present study, we found the AQP8 localization to be in the absorptive columnar epithelia in duodenum, jejunum, and colon. It appears that AQP8 is predominantly localized in intracellular compartments, especially in the small intestine, suggesting that AQP8 does not play a major role for transcellular water transport in rat small intestine. In the colon, AQP8 is also mainly present in intracellular vesicles, although some labeling also appears to be at or near the plasma membrane. Thus it cannot be completely ruled out that AQP8 may play some role in intestinal water reabsorption. The presence in surface epithelial cells in the intestine extends the results reported by Koyama et al. (14, 15), wherein the localization of AQP8 mRNA was found by RNase protection assay and in situ hybridization to be in the columnar epithelial cells in rat jejunum and colon. A similar labeling pattern was also observed in the ciliated surface epithelial cells of trachea and bronchi. The absence or low abundance of AQP8 in plasma membrane domains suggests that AQP8 is not involved in the transcellular water transport across epithelial cells.

AQP8 in the liver, submucosal glands, and salivary glands. The liver, the largest gland in the body, has many complex functions, including formation of bile, carbohydrate storage and release, formation of urea, etc. AQP1 has been reported to be present in rat liver (20, 23), where it has been suggested to be involved in the formation of bile. Recently, AQP9 was localized in the plasma membrane of rat hepatocytes (5), and a role of AQP9 in the formation of urea was proposed. In this study, we found that AQP8 is present in hepatocytes of rat liver. This confirmed previous evidence regarding the presence of AQP8 in the liver. Koyama et al. reported the localization of AQP8 mRNA in rat liver using in situ hybridization (14), and AQP8 mRNA has also been found in mouse liver by the use of Northern blotting and RT-PCR followed by Southern blotting (17). Recently, Garcia et al. (7) showed that AQP8 in cultured hepatocytes can be redistributed from an intracellular domain to the plasma membrane via a cAMP-stimulated mechanism, and thus a possible involvement of AQP8 in the formation of regulated bile secretion has been proposed. As shown in Fig. 11, AQP8 appears to be located mainly intracellularly in hepatocytes, suggesting that AQP8 may not directly be involved in transcellular water transport but rather in intracellular homeostasis. Recently, AQP8 was reported to be present in the basolateral plasma membrane of rat submandibular glands (27). We were not able to confirm these findings but found instead clear labeling of the myoepithelial cells in the parotid, the submandibular and the sublingual glands, and in the bronchial and tracheal glands. Thus it appears that AQP8 is present in the myoepithelial cells of many glands. The physiological role of AQP8 here remains elusive.

AQP8 in spermatocytes. The molecular basis for the high water permeability in rat and human sperm is unknown. Recently, AQP7 was reported to be present at sperm in the late state of spermatogenesis (11, 24), and AQP8 mRNA was reported by Ishibashi et al. (12) to be present in rat testis in all stages of spermatogenesis. We confirmed these findings by immunohistochemistry, where we found the localization of AQP8 protein to be in the spermatogenic cells, although presence of AQP8 in the Sertoli cells could not be ruled out.

AQP8 in basal cells in epididymis and airway epithelia. A localization of AQP8 in the basal cells in the ductus epididymis, trachea, and in the principal bronchi is very interesting. A possibility is that AQP8 is involved in the differentiation from basal cells to more differentiated cell types in these epithelia, e.g., during maturation or after recovery by injury in airways. Moreover, AQP1 is present in the efferent duct cells in the apical and basolateral plasma membranes (1), and AQP9 is present in the stereocilia of the principal cells in epididymis (5). Possibly, the function of these two aquaporins in the efferent duct and in the epididymis is to participate in fluid reabsorption leading to sperm concentration. Whether the role of AQP8 in the basal cells in the epididymis and in the airway epithelia is related to maturation, differentiation, or cell volume regulation still needs to be established.


    ACKNOWLEDGEMENTS

The authors thank Inger Merete Paulsen, Zhila Nikrozi, Mette Vistisen, Gitte Christensen, Helle Høyer, and Margit Palmer Lind for expert technical assistance.


    FOOTNOTES

The Water and Salt Research Center at the University of Aarhus was established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond). Support for this study was provided by the Karen Elise Jensen Foundation; the Danish Medical Research Council; the European Commission (TMR and Biotech programs of the 4th Framework program and key action 3.1.3 of the 5th Framework program); the Human Frontier Science Program; the Helen and Ejnar Bjørnows Foundation; the A.P. Møller and Spouse Chastine Mc-Kinney Møllers Foundation; the Mrs. Ruth T. E. Konig-Petersens Research Foundation for Kidney Diseases; and the Research Foundation of the Danish Kidney Association.

Address for reprint requests and other correspondence: S. Nielsen, The Water and Salt Research Center, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus, Denmark (E-mail: sn{at}ana.au.dk).

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.

First published August 8, 2001;10.1152/ajprenal.00158.2001

Received 18 May 2001; accepted in final form 6 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Brown, D, Verbavatz JM, Valenti G, Lui B, and Sabolic I. Localization of the CHIP28 water channel in reabsorptive segments of the rat male reproductive tract. Eur J Cell Biol 61: 264-273, 1993[ISI][Medline].

2.   Calamita, G, Spalluto C, Mazzone A, Rocchi M, and Svelto M. Cloning, structural organization and chromosomal localization of the mouse aquaporin-8 water channel gene (Aqp8). Cytogenet Cell Genet 85: 237-241, 1999[ISI][Medline].

3.   Denker, BM, Smith BL, Kuhajda FP, and Agre P. Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules. J Biol Chem 263: 15634-15642, 1988[Abstract/Free Full Text].

4.   Ecelbarger, CA, Terris J, Frindt G, Echevarria M, Marples D, Nielsen S, and Knepper MA. Aquaporin-3 water channel localization and regulation in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 269: F663-F672, 1995[Abstract/Free Full Text].

5.   Elkjær, M, Vajda Z, Nejsum LN, Kwon T, Jensen UB, Amiry-Moghaddam M, Frøkiær J, and Nielsen S. Immunolocalization of AQP9 in liver, epididymis, testis, spleen, and brain. Biochem Biophys Res Commun 276: 1118-1128, 2000[ISI][Medline].

6.   Fischer, H, Stenling R, Rubio C, and Lindblom A. Differential expression of aquaporin 8 in human colonic epithelial cells and colorectal tumors. BMC Physiol 1: 1, 2001[Medline].

7.   Garcia, F, Kierbel A, Larocca MC, Gradilone SA, Splinter P, LaRusso NF, and Marinelli RA. The water channel aquaporin-8 is mainly intracellular in rat hepatocytes, and its plasma membrane insertion is stimulated by cyclic AMP. J Biol Chem 276: 12147-12152, 2001[Abstract/Free Full Text].

8.   Helenius, A, and Aebi M. Intracellular functions of N-linked glycans. Science 291: 2364-2369, 2001[Abstract/Free Full Text].

9.   Hurley, PT, Ferguson CJ, Kwon TH, Andersen ML, Norman AG, Steward MC, Nielsen S, and Case RM. Expression and immunolocalization of aquaporin water channels in rat exocrine pancreas. Am J Physiol Gastrointest Liver Physiol 280: G701-G709, 2001[Abstract/Free Full Text].

10.   Ishibashi, K, Imai M, and Sasaki S. Cellular localization of aquaporin 7 in the rat kidney. Exp Nephrol 8: 252-257, 2000[ISI][Medline].

11.   Ishibashi, K, Kuwahara M, Gu Y, Kageyama Y, Tohsaka A, Suzuki F, Marumo F, and Sasaki S. Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol, and urea. J Biol Chem 272: 20782-20786, 1997[Abstract/Free Full Text].

12.   Ishibashi, K, Kuwahara M, Kageyama Y, Tohsaka A, Marumo F, and Sasaki S. Cloning and functional expression of a second new aquaporin abundantly expressed in testis. Biochem Biophys Res Commun 237: 714-718, 1997[ISI][Medline].

13.   Koyama, N, Ishibashi K, Kuwahara M, Inase N, Ichioka M, Sasaki S, and Marumo F. Cloning and functional expression of human aquaporin 8 cDNA and analysis of its gene. Genomics 54: 169-172, 1998[ISI][Medline].

14.   Koyama, Y, Yamamoto T, Kondo D, Funaki H, Yaoita E, Kawasaki K, Sato N, Hatakeyama K, and Kihara I. Molecular cloning of a new aquaporin from rat pancreas and liver. J Biol Chem 272: 30329-30333, 1997[Abstract/Free Full Text].

15.   Koyama, Y, Yamamoto T, Tani T, Nihei K, Kondo D, Funaki H, Yaoita E, Kawasaki K, Sato N, Hatakeyama K, and Kihara I. Expression and localization of aquaporins in rat gastrointestinal tract. Am J Physiol Cell Physiol 276: C621-C627, 1999[Abstract/Free Full Text].

16.   Ma, T, Yang B, Gillespie A, Carlson EJ, Epstein CJ, and Verkman AS. Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels. J Biol Chem 273: 4296-4299, 1998[Abstract/Free Full Text].

17.   Ma, T, Yang B, and Verkman AS. Cloning of a novel water and urea-permeable aquaporin from mouse expressed strongly in colon, placenta, liver, and heart. Biochem Biophys Res Commun 240: 324-328, 1997[ISI][Medline].

18.   Nejsum, LN, Elkjær M, Hager H, Frøkiær J, Kwon TH, and Nielsen S. Localization of aquaporin-7 in rat and mouse kidney using RT-PCR, immunoblotting, and immunocytochemistry. Biochem Biophys Res Commun 277: 164-170, 2000[ISI][Medline].

19.   Nielsen, S, DiGiovanni SR, Christensen EI, Knepper MA, and Harris HW. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc Natl Acad Sci USA 90: 11663-11667, 1993[Abstract].

20.   Nielsen, S, Smith BL, Christensen EI, and Agre P. Distribution of the aquaporin CHIP in secretory and resorptive epithelia and capillary endothelia. Proc Natl Acad Sci USA 90: 7275-7279, 1993[Abstract].

21.   Nielsen, S, Smith BL, Christensen EI, Knepper MA, and Agre P. CHIP28 water channels are localized in constitutively water-permeable segments of the nephron. J Cell Biol 120: 371-383, 1993[Abstract].

22.   Preston, GM, Carroll TP, Guggino WB, and Agre P. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256: 385-387, 1992[ISI][Medline].

23.   Roberts, SK, Yano M, Ueno Y, Pham L, Alpini G, Agre P, and LaRusso NF. Cholangiocytes express the aquaporin CHIP and transport water via a channel-mediated mechanism. Proc Natl Acad Sci USA 91: 13009-13013, 1994[Abstract/Free Full Text].

24.   Suzuki-Toyota, F, Ishibashi K, and Yuasa S. Immunohistochemical localization of a water channel, aquaporin 7 (AQP7), in the rat testis. Cell Tissue Res 295: 279-285, 1999[ISI][Medline].

25.   Terris, J, Ecelbarger CA, Marples D, Knepper MA, and Nielsen S. Distribution of aquaporin-4 water channel expression within rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 269: F775-F785, 1995[Abstract/Free Full Text].

26.   Terris, J, Ecelbarger CA, Nielsen S, and Knepper MA. Long-term regulation of four renal aquaporins in rats. Am J Physiol Renal Fluid Electrolyte Physiol 271: F414-F422, 1996[Abstract/Free Full Text].

27.   Wellner, RB, Hoque AT, Goldsmith CM, and Baum BJ. Evidence that aquaporin-8 is located in the basolateral membrane of rat submandibular gland acinar cells. Pflügers Arch 441: 49-56, 2000[ISI][Medline].

28.   Yasui, M, Kwon TH, Knepper MA, Nielsen S, and Agre P. Aquaporin-6: an intracellular vesicle water channel protein in renal epithelia. Proc Natl Acad Sci USA 96: 5808-5813, 1999[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 281(6):F1047-F1057
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society