ARTICLE |
Correspondence to: Antonio Frigeri, Dipartimento di Fisiologia Generale ed Ambientale, Università degli Studi di Bari, via Amendola 165/A, I-70126 Bari, Italy. E-mail: a.frigeri@biologia.uniba.it
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Summary |
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Aquaporin-9 (AQP9) is a water channel membrane protein also permeable to small solutes such as urea, glycerol, and 5-fluorouracil, a chemotherapeutic agent. With the aim of understanding the pathophysiological role of AQP9, we performed an extensive analysis by Western blotting, RT-PCR, and immunolocalization in rat tissues. Western blotting analysis revealed a major band of approximately 32 kD in testis, liver, and brain. Immunofluorescence showed strong expression of AQP9 in the plasma membrane of testis Leydig cells. In liver, AQP9 expression was found to be sex-linked. Male rats had higher levels of AQP9 than female in terms of both protein and mRNA. Moreover, in female livers the expression of AQP9 was mostly confined to perivascular hepatocytes, whereas males showed a more homogeneous hepatocyte staining. No differences in AQP9 expression level related to the age or to protein content of the diet were found, indicating that differences in the liver may be gender-dependent. In the brain, AQP9 expression was found in tanycytes mainly localized in the areas lacking a bloodbrain barrier (BBB), such as the circumventricular organs (CVOs) of the third ventricles, the subfornical organ, the hypothalamic regions, and the glial processes of the pineal gland. AQP9 expression in the osmosensitive region of the brain suggests a role in the mechanism of central osmoreception. All these findings show a unique tissue distribution of AQP9 compared to the other known aquaporins. (J Histochem Cytochem 49:15471556, 2001)
Key Words: water channels, aquaporins, AQP9, testis, liver, brain
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Introduction |
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The discovery of water channel proteins (aquaporins) has provided a molecular explanation for the way by which water can cross the cell plasma membrane of highly water permeable epithelia. Ten mammalian aquaporins that belong to the MIP family have so far been cloned (
The most recently cloned water channel, AQP9, has recently been identified in rat liver (
Compared to rat distribution, the human AQP9 mRNA is differently expressed. High AQP9 expression was found in leukocytes and much less in liver. Furthermore, no AQP9 mRNA was found in human testis (
In this study, as a first step toward understanding the functional role of AQP9, the tissue distribution and the membrane localization of AQP9 in rat tissues were determined by immunohistochemistry and the molecular identity of the AQP9 protein was established. Affinity-purified polyclonal antibodies revealed a unique cellular distribution of AQP9, providing insight into the role of this aquaporin in testis and liver. Moreover, the expression of AQP9 in the osmosensitive region of the rat brain suggests a role for this aquaporin in the mechanism of central osmoreception. While this article was in preparation, an immunolocalization study of AQP9 was reported by another group (
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Materials and Methods |
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Antibodies and Animals
Two different commercial affinity-purified rabbit polyclonal antibodies were used for this study. Antibody A was from Alpha Diagnostic International (San Antonio, TX) and antibody C was from Chemicon International (Temecula, CA). Both were produced against an 18-amino-acid synthetic peptide within the C-terminal domain of rat AQP9 predicted to be cytoplasmic.
Male and female rats 1 month and 6 months old fed with a standard diet containing 19% protein (w/w) were used for this study. To analyze the effect of the protein diet on liver expression of AQP9, 6-month-old rats were fed with a 22% (w/w) protein diet for 2 weeks before the experiment. Three to five animals for each experimental condition were used.
Immunocytochemistry
Immunofluorescence.
Rat liver, testis, uterus, and ovaries were removed, washed in PBS, sliced, and fixed with PBS containing 4% paraformaldehyde (
Immunoperoxidase.
Immunoperoxidase staining was performed as previously described (
Preparation of Liver, Brain, and Testis Membrane Vesicles and Western Blotting
Rat brain, liver, and testis were removed and homogenized in 0.25 M sucrose, 10 mM Tris-HCl, pH 7.5, containing 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 mM PMSF. After homogenization in a Potter apparatus and centrifugation at 1000 x g for 10 min, a low-speed pellet was prepared by centrifugation at 17,000 x g for 45 min at 4C. The pellet was resuspended at a final concentration of 1012 µg/µl and overloaded on a discontinuous sucrose gradient consisting of layers of 50% sucrose (7 ml), 23% sucrose (10 ml), 17% sucrose (10 ml), and the low-speed pellet sample (10 ml). The gradient was centrifuged at 55,000 x g overnight using a SW28 rotor and three separated fractions were collected from the bottom and analyzed for Western blotting. F1 (pellet) contained cell debris; F2 collected at the 2350% sucrose interface contained microsomal and ER vesicles and residual plasma membranes; F3 collected at the 1723% sucrose interface contained an enriched plasma membrane fraction (
SDS-PAGE was performed as previously described (
Endoglycosidase Treatment
The fraction 3 of liver and the low-speed pellet of kidney were used for AQP9 and AQP1 deglycosylation experiments, respectively. One hundred µg of each sample was incubated for 10 min at 60C in 50 mM sodium phosphate buffer, pH 7, containing 0.2% SDS and 50 mM ß-mercaptoethanol. The denatured samples were then incubated with 4 U of N-glycosidase F (Boehringer Mannheim; Mannheim, Germany) for 4 hr at 37C and finally analyzed by Western blotting.
RT-PCR Experiments
Total RNA was prepared from rat testis, liver, and brain using the TRIzol reagent (Gibco Life Technologies; Beerse, Belgium) and cDNAs were prepared using random primers as previously described (
Plasmid Construction and Transfection
The cDNA encoding for human AQP9 was amplified from human blood by RT-PCR experiments and directly ligated into a green fluorescence protein containing expression vector (pcDNA3.1/NT -GFP TOPO Cloning kits; Invitrogen, Carlsbad, CA) with the cytomegalovirus promoter and the gene for resistance to geneticin. Transfection was performed in CHO-K1 cells by use of lipofectin (
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Results |
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Western Blotting and RT-PCR
The first set of experiments was devoted to analysis of the presence of AQP9 protein in several rat tissues. To this purpose, membranes from rat liver were prepared as described in Materials and Methods by differential centrifugation and sucrose discontinuous gradient. Fig 1A shows a typical Western blot obtained using affinity-purified antibodies (antibody A). AQP9 was detected as a protein of approximately 32 kD. In some experiments, a second band of faint intensity appeared at 35 kD. A weak signal was obtained in a total membrane fraction (17,000 x g), whereas AQP9 was strongly enriched in fraction 2, and even more so in fraction 3, after sucrose gradient centrifugation. The specificity of the result was confirmed by using another anti-AQP9 antibody (C) or by using immunodepleted antibodies obtained using antibodies previously reacted with the immunizing peptide (Fig 1B). Confirmation of the antibody specificity was also obtained using transfected cells. CHO cells were stably transfected with an expression vector containing both GFP and the human AQP9 cDNA coding sequences. As shown in Fig 1C, all the cells uniformly expressed AQP9GFP on the plasma membrane, confirming the correct targeting of the fusion protein. Moreover, when the two antibodies were used to stain the cells, only antibody C crossreacted with the human AQP9 protein (Fig 1D). These data indicate that the two antibodies recognize different epitopes within the rat AQP9 C-terminus sequence.
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Expression of AQP9 in brain and testis was analyzed using the same membrane fractionation method (Fig 2A). The same pattern was detected in fraction 3 of the sucrose gradient, indicating that AQP9 is expressed in these two tissues.
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RT-PCR experiments were then performed to determine the presence of the specific mRNA for AQP9 in those tissues in which AQP9 protein was found. Using specific rat primers, a fragment of the expected size (900 bp) containing the full-length AQP9 cDNA was obtained in liver, brain, and testis (Fig 2B). The specificity of the PCR amplification product was verified by sequencing analysis (not shown).
AQP9 has a potential N-linked glycosylation site at Asn142. Therefore, experiments were performed to analyze whether AQP9 is glycosylated. AQP9-enriched membrane fraction F3 from liver was incubated with endoglycosidase, which specifically digests the glucose residues. AQP1 containing kidney membranes was used as a positive control. Fig 2C shows that the relative content and the electrophoretic profile of the two AQP9 immunopositive bands did not change after endoglycosidase treatment, indicating that the protein is not glycosylated.
When livers from male and female rats were separately analyzed, the expression of AQP9 was found to be quite different. Western blotting experiments revealed that the intensity of the AQP9 protein band was significantly stronger in males than in females (Fig 3B), indicating that males have higher liver AQP9 expression than females. To determine whether or not this different protein expression is related to a different gene activity, we analyzed the expression of AQP9 mRNA by competitive RT-PCR experiments. Semiquantitative analysis revealed reliably different levels of AQP9 transcript in male and female livers (Fig 3C).
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To analyze whether AQP9 expression in liver was age-dependent or linked to the protein content of the diet, AQP9 protein expression was analyzed by Western blotting experiments. Densitometric analysis results (Fig 3D) showed that no differences were found with age or diet, whereas a significant difference (20%) in AQP9 expression was found only between males and females.
Immunolocalization Studies
Indirect immunofluorescence staining was used to localize AQP9 in liver, testis, and brain. The two antibodies (A and C) gave identical results.
Liver AQP9 expression was analyzed to determine cell distribution and membrane localization and whether AQP9 expression/distribution was dependent on the age and/or the diet of the rats. Affinity-purified antibodies were used to stain cryostat rat liver sections (Fig 4). AQP9 was found only expressed in the hepatocytes. In 1-month (Fig 4A) and 6-month-old (Fig 4B) female rats the expression was strongest in those cells closest to the central vein. In male rats the expression of AQP9 was found to be more homogeneous and involved all the hepatocytes in both 1-month (Fig 4C) and 6-month (Fig 4D) rats. No differences were found in male and female livers of animals fed with a hyperprotein diet (not shown). At higher magnification, the staining was localized in the basolateral membrane of the cells, whereas the apical bile canalicular membrane was not labeled (Fig 4E). No other structures were specifically recognized. Control experiments performed after preabsorption of antibodies with the immunizing peptide gave no staining, confirming the specificity of the result (Fig 4F).
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Immunofluorescence of rat testis is shown in Fig 5. At lower magnification, AQP9 antibodies recognized interstitial cells of seminiferous tubules (Fig 5A). These cells were histochemically identified as Leydig cells, both isolated or in groups close to blood and lymphatic vessels that surround the seminiferous tubules. No staining was found in the lumen of the seminiferous tubules or in the immature spermatocytes or Sertoli cells. At higher magnification (Fig 5B) the staining was confined to the plasma membrane of the cells. The specificity of the staining was demonstrated by a preabsorbed control (Fig 5B, inset). Immunofluorescence performed in female organs, such as uterus and ovaries, gave no specific staining for AQP9 expression (not shown).
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In brain, the localization of AQP9 was performed using immunoperoxidase staining. The immnocytochemical localization of AQP9 in the brain was performed in the forebrain, including the cerebrum, the diencephalon, and the circumventricular organs (CVOs), and in the pineal gland. Of all brain areas analyzed, a positive signal was found in the CVOs, including the subfornical organ and the hypothalamic regions (Fig 6). Staining was found in a subpopulation of GFAP-positive ependymal cells lacking cilia, and recognized as specialized glial cells (tanycytes) lining the floor of the third ventricle (Fig 6A). Expression of AQP9 was found in the tanycyte bodies and tail process that extended from the ventricular wall into the neuropil and formed end-feet at the vessel surfaces (Fig 6B) in the deep layer. AQP9 immunolabeling was also found in the tanycyte processes ending in the vicinity of the neuronal cells of hypothalmic nuclei (Fig 6C). Strong AQP9 expression was found in the glial limiting membrane of the pineal gland and around the ingrowing vessels (Fig 6D). Fine staining was detectable in the neuroglial processes usually scattered in a random pattern in the pineal stroma or terminating in vessel walls (Fig 6E) or in bulb-like endings on pinealocytes (Fig 6F). No AQP9 labeling was found in the pinealocytes (Fig 5E and Fig 5F) or in the hypothalamic neuronal cells. The specificity of the staining was demonstrated by a preabsorbed control (Fig 6G).
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No differences were found between male and female brains (not shown).
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Discussion |
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In this study we examined the tissue distribution and the cellular and subcellular localization of the AQP9 water channel in rat using affinity-purified polyclonal antibodies raised in rabbit against a C-terminal synthetic peptide (see Table 1). Western blotting results show that a major non-glycosylated band was observed, with electrophoretic mobility of approximately 32 kD. The specificity of our results was confirmed by using two different antibodies that, as demonstrated using transfected cells, recognize different AQP9 epitopes, by immunodepletion, and also by immunoprecipitation experiments (not shown). Contrasting with our finding is the molecular weight reported for AQP9 by the group that recently published a similar immunolocalization study (
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AQP9 was first cloned from rat and in situ hybridization studies indicated strong expression in hepatocytes (
AQP9 is also highly permeable to urea in addition to having water transport properties. The liver is a major site of production and elimination of urea suggesting that AQP9 in liver may function as a urea channel (
In testis, AQP9 was found to be expressed in the plasma membrane of interstitial Leydig cells. These endocrine cells secrete the male sex hormone testosterone under the control of endocrine, paracrine, and autocrine factors (
In the brain, we found an AQP9 expression mainly localized in the areas lacking a bloodbrain barrier, such as the CVOs of the third ventricles, the subfornical organ, and the hypothalamic regions. It is important to note that CVOs share common structural features, such as a weakened BB barrier and specialized ependymal cells, which are important for CNS homeostasis and for regulating osmoreceptor functions. Therefore, the presence of AQP9 staining in the ventricle ependymal layer and in the tanycytic glial cells that form a foot-like expansion close to the blood vessels of the subfornical organs suggests that this water channel may be involved in the production/reabsorption of cerebrospinal fluid and in the control of the specific neuronal activity of the CNS. A close relation between the absence of BB barrier and AQP9 expression is further demonstrated by the presence of this water channel in the perivascular glial processes of the pineal gland, whose topographical relationship to the cavity of the third ventricle has been demonstrated to play an important role in the CSF (
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Acknowledgments |
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Support by Telethon-Italy (grant no. 983).
We thank Raffaello Peragine, Alessandra Colella, and Dr Luca Liuzzi for excellent technical assistance.
Received for publication January 31, 2001; accepted June 27, 2001.
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