Expression of the AQP-1 water channel in normal human tissues: a semiquantitative study using tissue microarray technology

A. Mobasheri1 and D. Marples2

1Connective Tissue and Molecular Pathogenesis Research Groups, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ; and 2School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, United Kingdom

Submitted 24 September 2003 ; accepted in final form 23 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aquaporin water channels are a family of membrane proteins that facilitate water movement across biological membranes. Aquaporin-1 (AQP-1) has been found to be important in osmotic water movement across cell membranes of epithelial and endothelial barriers. However, the distribution of AQP-1 in many normal human tissues is still unknown. The aim of this study was to use immunohistochemistry and semiquantitative histomorphometric analysis to determine the tissue distribution and relative expression of AQP-1 in normal human tissues using tissue microarray (TMA) technology. The normal human TMAs employed in this study included cardiovascular, respiratory, gastrointestinal, hepatic and pancreatobiliary, oral, salivary, nasal, mammary, fetal, endocrine, genital tract, central and peripheral nervous systems, urinary tract, skin, cartilage, and other soft connective tissues. Immunohistochemistry and semiquantitative histomorphometric analysis confirmed the presence of AQP-1 in endothelial barriers of almost all tissues and in many epithelial barriers. AQP-1 was highly expressed in the renal cortex, choroid plexus, and pancreatic ducts. AQP-1 expression levels were surprisingly high in the anus, gallbladder, and liver; moderate expression was also detected in the hippocampus and ependymal cells of the central nervous system. This is the first report of AQP-1 protein distribution in normal human TMAs. These findings confirm the presence of AQP-1 in human endothelia and selected water-transporting epithelia and several new locations, including mammary epithelium, articular chondrocytes, synoviocytes, and synovial microvessels where AQP-1 may be involved in milk production, chondrocyte volume regulation, synovial fluid secretion, and homeostasis, respectively.

aquaporin-1; water channel; human tissue microarrays; immunohistochemistry; histomorphometric analysis


LIVING CELLS MUST REGULATE their internal milieu when faced with ionic and osmotic fluctuations in their extracellular environment. The physiological maintenance of water and ion homeostasis is crucial for many basic cellular functions such as proliferation, differentiation, excitability, secretion, and even apoptosis. The molecular basis of membrane water permeability remained elusive until the recent discovery of the aquaporin (AQP) water channel proteins. These proteins are responsible for the water permeability of some biological membranes. More than 200 members of the AQP family have been found in plants (16, 17), microorganisms (11), invertebrates, and vertebrates (1). The importance of aquaporins to the physiology of living organisms has only recently been uncovered (8). Thus far, at least 10 mammalian aquaporins have been identified (8). These proteins are selectively permeated by water (aquaporins) (2) or water plus glycerol (aquaglyceroporins) (13). The aquaporins are located at strategic membrane sites in a variety of epithelia, most of which have well-defined physiological functions in fluid absorption or secretion (10). AQP-1 was the first member of the AQP family to be identified, first in erythrocytes and later cloned from kidney complementary DNA libraries (3). In fluid-transporting epithelia, AQP-1 is permeated by water, driven by osmotic gradients. AQP-1 is abundant in the apical and basolateral membranes of renal proximal tubules and descending thin limbs (24, 26) and plays a key role in setting up and maintaining the counter-current multiplication system (23, 27). AQP-1 is known to be present in a number of extrarenal tissues such as the ciliary body of the eye (3) and the choroid plexus in the brain, where it presumably participates in the formation of cerebrospinal fluid (CSF) (3, 33, 34). AQP-1 is also present in the microvascular structures of the respiratory system and the central nervous system, accounting for the high water permeabilities of endothelial barriers (35).

Evidence suggests that in addition to being an osmotic water channel in water-transporting tissues, AQP-1 is also the structural basis of the Colton blood group antigens (4). Recent electrophysiological studies suggest that AQP-1 may also function as a cation channel gated by cGMP (6). Although AQP-1 is expressed in endothelial barriers of many organs, its expression has never been semiquantitatively compared in normal human tissues using histomorphometric techniques. This lack of knowledge is a potential hindrance to our understanding of the physiological, cell-, and tissue-specific roles of AQP-1 in normal tissues.

The recent arrival of tissue microarray (TMA) technology has allowed cell biologists, physiologists, and pathologists to screen large numbers of normal and tumor tissue specimens for protein expression information and to discover novel diagnostic and prognostic correlations. In this study, we have taken advantage of this new technological development to determine, by immunohistochemistry and histomorphometric analysis, the distribution and relative abundance of AQP-1 in normal human TMAs that include almost all of the tissue types present in the human body. The information presented in this paper confirms and reinforces earlier observations by other investigators and provides compelling evidence for the expression of AQP-1 in several tissues in which aquaporins were not previously reported.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and antibodies. Unless otherwise stated, all chemicals were molecular biology grade and were purchased from Sigma/Aldrich (Poole, Dorset, UK). Rabbit polyclonal antibodies against the COOH-terminal 15 amino acids of rat AQP-1 were produced (Sigma-Genosys) and affinity purified in the laboratory of Dr. David Marples (Leeds University, Leeds, UK). The polyclonal antibodies to AQP-1 were tested and found to cross-react with AQP-1 in rat, mouse, sheep, human, and dog kidney. Materials for immunohistochemistry were purchased from Vector Laboratories (Peterborough, UK) and Dako-Cytomation (Ely, Cambridgeshire, UK).

Human tissue microarrays. Human tissue microarrays (TMAs) were obtained from the Cooperative Human Tissue Network (CHTN) of the National Cancer Institute (NCI), the National Institutes of Health, Bethesda, MD (http://faculty.virginia.edu/chtn-tma/home.html). The TMAs contained formalin-fixed paraffin embedded samples of 66 nonneoplastic adult tissues obtained from surgical resection specimens, obtained within 1 h of surgical removal from anonymous donors. All the tissues represented on the TMAs were normal with the exception of parathyroid gland, which was from a benign parathyroid adenoma, and lymphatic tissue, which was from a benign lymphangioma. The central nervous system tissues on the TMAs were obtained from autopsy specimens within 36 h of death. The tissues were grouped into cardiovascular, respiratory, gastrointestinal, hepatic and pancreatobiliary, oral, salivary and nasal, mammary, endocrine, genital tract, central and peripheral nervous systems, urinary tract, skin, cartilage, and synovium. The schematic in Fig. 1 shows the layout of the TMAs used. Local medical ethics committee approval was not required for using TMAs.



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Fig. 1. A: representative tissue microarray (TMA) slide consisting of 0.6-mm diameter spots of 66 normal human tissue types. The slides used in this study were codenamed CHTN2002N1 and obtained from the Cooperative Human Tissue Network of the National Cancer Institute. B: higher magnification views in B illustrate (from left to right) hematoxylin- and eosin-stained spots of water transporting epithelia of the choroid plexus, kidney cortex, and medulla (bars represent 100 µm). C: the tissues cores represented on the microarray are classified by the system from which they originate and are grouped together in distinct square blocks. For a complete list of tissues, refer to Table 1.

 

Immunohistochemistry. The immunohistochemical protocol was initially optimized by using "test" human tissue microarrays, which contained a limited number of selected lymphoid, epithelial, and stromal tissues [i.e., spleen, colonic mucosa and kidney, endometrium, liver, and uterine smooth muscle (TMA code: CHTN2002X1)]. These arrays were used to titrate immunohistochemical assay parameters and antibody dilutions before use of the more comprehensive tissue microarrays. The TMAs were heated at 60°C for 15 min to improve tissue adhesion to the charged glass slides (Fisher Plus). Before immunostaining, TMA slides were deparaffinized in xylene for 20 min to remove embedding medium and washed in absolute ethanol for 3 min. The TMAs were gradually rehydrated in a series of alcohol baths (96, 85, and 50%) and placed in distilled water for 5 min. Endogenous peroxidase activity was blocked for 1 h in a 97% methanol solution containing 3% hydrogen peroxide and 0.01% sodium azide. The TMAs were then incubated for 1 h at room temperature (RT) with 20% normal goat serum (NGS) in PBS containing 1% bovine serum albumin and 0.01% sodium azide to block nonspecific antibody binding. Slides were incubated overnight at 4°C with an affinity-purified rabbit polyclonal antibody to rat AQP-1 diluted 1:200 in PBS containing 1% NGS. After 24 h at 4°C, the slides were washed three times for 5 min each in PBS before incubation with horseradish peroxidase-labeled polymer conjugated to affinity-purified goat anti-rabbit immunoglobulins (DAKO code no. K4010) for 30 min at RT. The sections were washed three times for 5 min in PBS before applying liquid DAB+ Chromogen (DAKO; 3,3'-diaminobenzidine solution) for up to 30 s. The development of the brown-colored reaction was stopped by rinsing in distilled water. The stained slides were immersed for 5 min in a bath of aqueous hematoxylin (Dako-Cytomation, code no. S3309) to counterstain cell nuclei. Finally, the slides were washed for 5 min in running water and dehydrated in a series of graded ethanol baths before being rinsed in three xylene baths and mounted in 1,3-diethyl-8-phenylxanthine (DPX) (BDH laboratories, UK). Control experiments were performed by incubating TMAs with nonimmune serum and by omitting primary antibody.

Data acquisition and analysis. The stained TMA slides were visually scored in a double-blind fashion by two independent investigators. Staining results were then compared and recorded directly into a Microsoft Excel worksheet using a color-coded key as follows: 0 (blue), no expression; 1 (green), low expression; 2 (yellow), moderate expression; 3 (orange), high expression; 4 (red), abundant expression. Variation in histomorphometric scoring between TMAs and between observers was infrequent and never more than one unit. The data obtained were linked to a database of digital images captured using a Nikon Microphot-FX microscope fitted with a Nikon DXM1200 digital camera.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using TMA technology, we examined the expression patterns of the AQP-1 water channel in 66 types of normal human tissue. The results of the semiquantitative immunohistochemical and histomorphometric data presented in this paper have been summarized in Figs. 2 and 5. Selected immunohistochemical micrographs from the TMAs immunostained with polyclonal antibodies raised against AQP-1 are shown in Figs. 3 and 4. The major findings of this study are summarized as follows: 1) AQP-1 is ubiquitously expressed in endothelial barriers of almost all human tissues examined; 2) the most abundant AQP-1 expression was noted in the choroid plexus, kidney, hepatobiliary ducts, and the gallbladder; 3) moderate AQP-1 expression was seen in the hippocampus and ependymal cell layer of the central nervous system, lung, bronchial epithelium, urinary bladder, synovium, articular cartilage, breast epithelium, and anal mucosa; 4) low expression was detected in the lymphatic endothelium of the heart, epididymis, adrenal medulla, fetal membranes, and other central nervous system regions; and 5) test TMA slides were used as controls to demonstrate that nonspecific immunostaining did not occur when the primary antibody was omitted from the immunohistochemical protocol or when slides were incubated with nonimmune serum (data not shown).



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Fig. 2. The complete list of organs and tissue types represented on the CHTN2002N1 tissue microarray, list of abbreviations originally used by the Cooperative Human Tissue Network of the National Cancer Institute adopted for use in this study (see http://faculty.virginia.edu/chtn-tma/index.html), and a summary of the semiquantitative histomorphometric analysis of the immunohistochemical data obtained with antibodies to aquaporin-1 (AQP-1).

 


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Fig. 5. AQP-1 immunostaining results are summarized using a color coded key: 0 (blue), no expression; 1 (green), low expression; 2 (yellow), moderate expression; 3 (orange), high expression; 4 (red), abundant expression.

 


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Fig. 3. Immunohistochemical localization of aquaporin-1 (AQP-1) in selected tissues represented on the CHTN2002N1 TMA. Cardiovascular (A-C: aortic smooth muscle, myocardium, and small muscular artery of the lung), gastrointestinal (D-F: gastric mucosa, small intestine, and anal mucosa), reproductive (G: epididymis), oral (H: parotid salivary gland), breast (I), hepatic and pancreatobiliary (J-L: gallbladder, liver, and pancreas), endocrine (adrenal cortex and medulla, M and N), and uterine (secretory epithelium of the uterus, O).

 


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Fig. 4. Immunohistochemical staining of AQP-1 in skeletal muscle (A), deep zone of articular cartilage (B), synovium (C), choroid plexus, ependymal cells, and hippocampus (D-F, respectively), selected lymphoid tissues (G-I), lung and bronchus (J-L), and the urinary system (kidney cortex, medulla, and urinary bladder, M-O, respectively). Bars represent 100 µm. Abundant AQP-1 expression was detected in the epithelia and endothelia of the choroid plexus and kidney, whereas expression in lymphoid tissues was restricted to endothelial barriers.

 

Gastrointestinal and pancreatobiliary systems. In gastrointestinal tissues AQP-1 was localized to endothelial barriers (Fig. 3, D-F). There was no AQP-1 expression in the epithelia and mucosa of the small intestine, colon, and stomach. An unexpected observation, however, was the moderate level of expression in the stromal tissue of the anus (Fig. 3F). It was difficult to identify the source of the AQP-1 labeling in this region.

High AQP-1 expression was noted in the gallbladder, liver, and pancreas (Fig. 3, J, K, and L, respectively). In the gallbladder epithelium AQP-1 was localized to the basolateral membranes. In the pancreas AQP-1 expression was observed in pancreatic ducts and centroacinar cells consistent with recent reports of AQP-1 expression in the ductal system of the rat exocrine pancreas (14, 19). In the liver, moderate expression was seen in intrahepatic cholangiocytes, hepatic ducts, and endothelial barriers as previously reported (21).

Musculoskeletal systems. AQP-1 expression in skeletal muscle, cardiac muscle, uterine, aortic, and intestinal smooth muscle was strictly limited to endothelial barriers. Apart from bronchial capillaries, no AQP-1 expression was detected in hyaline chondrocytes of bronchial cartilage. Expression of AQP-1 in human cartilage and synovium is shown in Fig. 4, B and C. Immunohistochemical staining demonstrated AQP-1 localization in articular chondrocytes, synoviocytes, and synovial capillary vascular endothelial cells. The strongest AQP-1 expression was observed in chondrocytes that resided in the deep zone of articular cartilage adjacent to subchondral bone. The presence of AQP-1 in chondrocytes and synoviocytes supports a role for AQP-1-mediated water transport across the synovial microvessels and the plasma membrane of chondrocytes and synoviocytes in load-bearing joints. In chondrocytes, AQP-1 may be important for cell volume regulation and the flow of matrix and metabolic water across the membrane. In the synovium, AQP-1 water channels may be involved in the transport of water across the fenestrated synovial endothelium, which may be regarded as a specialized bidirectional water-transporting barrier. These observations support early developmental studies in the rat that have demonstrated abundant AQP-1 mRNA in the mesenchyme surrounding developing calcified bone (7), and more recent evidence from studies in developing orofacial tissues confirm that AQP-1 is expressed in human Meckel's cartilage along with several other members of the AQP gene family (36).

Skin and adipose tissue. In the skin, AQP-1 was identified in capillary endothelial cells but not in sweat glands or epidermal, or subepidermal tissue, consistent with recent observations by Nejsum and coworkers (25). AQP-1 was not seen in adipose tissue.

Genital systems. In the male and female genital systems, AQP-1 expression was exclusively limited to endothelial barriers and not seen in any epithelial cells in the uterus, cervix, and prostate. The only exception was the epididymis where low AQP-1 expression levels were detected (Fig. 3G).

Placenta and fetal membranes. Aquaporins-1 and -3 have recently been reported in human fetal membranes and the placenta (22). Our observations confirm the presence of AQP-1 in these fetal barriers, but the levels detected were much lower compared with kidney, choroid plexus, pancreas, and lung. AQP-1 may play a role in water transport from the amniotic cavity across the placenta into the fetal circulation.

Salivary glands and breast tissue. In the parotid salivary gland, AQP-1 expression levels were low and limited to endothelial barriers and myoepithelial cells (Fig. 3H). A novel observation was made in the breast, where AQP-1 was found to be moderately expressed in basolateral membranes of mammary ducts and glands, as well as endothelial barriers (Fig. 3I). The presence of AQP-1 has not previously been studied in the mammary gland despite the potential importance of aquaporins for milk production.

Urinary tract. In the urinary system, the most abundant AQP-1 expression was noted in the renal cortex: AQP-1 was present in the glomerular capillary endothelium and in the apical (brush border) and basolateral membrane domain of proximal tubule segments (Fig. 4M). In the medulla, AQP-1 immunolabeling was observed in the squamous epithelium of the descending thin limbs of the loop of Henle, which accounts for the high water permeability of this nephron segment, and in the descending vasa recta (Fig. 4N). AQP-1 expression was not detected in the thick ascending limb or medullary collecting ducts. These results are in agreement with previous studies in normal human kidney (3, 12, 24).

Respiratory system. AQP-1 expression was only seen in endothelial barriers in the lung and bronchus (Fig. 4, J-L).

Central and peripheral nervous systems. In the peripheral nervous system, AQP-1 expression was relatively low and localized to capillaries running along peripheral nerve autonomic ganglia and nerve bundles in the small intestine. Expression densities in tissues of the central nervous system were varied (see Fig. 2). Heavy AQP-1 immunolabeling was noted in the apical membrane of choroid plexus epithelial cells (Fig.4D). This was the most abundant expression seen in any human tissue. High AQP-1 expression was also seen in ependymal cells and in the hippocampus (Fig. 4, E and F, respectively). Our data are consistent with earlier (3) and more recent observations in the rat choroid plexus (33) and are highly suggestive of a central role for AQP-1 in water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The data relating to aquaporins in the nervous system are scarce, particularly information relating to the regulation of aquaporins in the brain and spinal cord. Several reviews have proposed important functions for AQP-1 and AQP-4 in nervous tissues, including roles in potassium buffering, body fluid homeostasis, central osmoreception, and development and recovery from brain edema (5, 28, 34). Furthermore, recent reports suggest that AQP-1 expression is increased in brain tumors and, together with AQP-4, may be involved in the formation of brain tumor edema (29, 30). This is the only report that semiquantitatively assesses and confirms the presence of AQP-1 in various regions of the human central and peripheral nervous systems.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The data we have presented confirm many of the recently published papers on the distribution of AQP-1 in simple and complex tissues. Our data also confirm the presence of AQP-1 in previously unknown locations such as mammary epithelium, articular cartilage, and synovium. The present understanding of aquaporin physiology is still incomplete, especially since so many new members of the aquaporin family have been identified. Localization of other members of the aquaporin family in human tissues will have a significant impact on our understanding of cell and organ physiology throughout the body.

Impact of TMA technology on physiological research. The recent proliferation of TMA technology has allowed pathologists to screen large numbers of normal and tumor tissue specimens for gene and protein expression information and for discovering novel diagnostic and prognostic correlations. To the best of our knowledge, this is the first time human TMAs have been used from a physiological perspective for a semiquantitative histomorphometric analysis of AQP-1 water channel expression in multiple normal human tissues. Our experience with TMAs suggests that they are highly suitable for such analyses and offer significant technical benefits for answering simple biological questions. The most significant benefit of having samples of human tissue types represented on a single microarray slide is that the expression profile of a specific protein target can be semiquantitatively analyzed across the various tissues by high through-put immunohistochemistry. Laboratory investigators have used multiple tissue Northern and Western blots for many years in the study of gene and protein expression. These methods are expensive, laborious, and time consuming to establish but ultimately, they do not allow high through-put analysis. Unlike Northern blots and quantitative PCR, which can only show differences in the quantity of transcript, TMAs reveal the relative quantities and anatomical site of the functional protein. TMAs also use less human tissue per individual analysis and offer superior internal consistency for comparative and semiquantitative immunohistochemical studies. We therefore consider this technique to provide a significant advance in the study of protein expression in human tissues.

Applicability of TMA technology to pathophysiological conditions. TMA technology may be applied in future studies to the profiling of specific pathophysiological conditions in which the expression of members of the aquaporin gene family might be altered. These conditions include acute renal failure (20), congestive heart disease (32), liver cirrhosis, diabetes insipidus (15), cystic fibrosis, and inflammatory edema of the lungs, brain, and other organs (9, 18, 28, 31). The advent of TMA technology provides a unique opportunity for physiologists and pathologists to collaborate by creating custom "disease progression" TMAs to monitor alterations in the expression of plasma membrane pumps, channels, or transporters that have been implicated in disease states.


    ACKNOWLEDGMENTS
 
We thank Dr. Christopher A. Moskaluk (Departments of Pathology, Biochemistry, and Molecular Genetics, University of Virginia Health System, Charlottesville, VA) and the staff of the Cooperative Human Tissue Network of the National Cancer Institute (National Institutes of Health, Bethesda, MD) for assistance with the CHTN program.

We gratefully acknowledge the contribution of Dr. Peter Agre (Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD), the 2003 Nobel Laureate in Chemistry for the discovery of water channels.

GRANTS

This study was supported by grants from the University of Liverpool Research Development Fund, the Pet Plan Charitable Trust (to A. Mobasheri), and the Medical Research Council (to D. Marples).


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Mobasheri, Connective Tissue and Molecular Pathogenesis Research Groups, Faculty of Veterinary Science, Univ. of Liverpool, Liverpool L69 7ZJ, United Kingdom (E-mail: a.mobasheri{at}liverpool.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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