Copyright ©The Histochemical Society, Inc.

Altered Expression of Aquaporins in Bullous Keratopathy and Fuchs' Dystrophy Corneas

M. Cristina Kenney, Shari R. Atilano, Nadia Zorapapel, Bret Holguin, Ronald N. Gaster and Alexander V. Ljubimov

Department of Ophthalmology, University of California Irvine Medical Center, Orange, California (MCK,SRA,BH,RNG); Ophthalmology Research Laboratories, Burns and Allen Research Institute, Cedars-Sinai Medical Center, UCLA Medical School Affiliate, Los Angeles, California (AVL); and Dental School, Columbia University, New York, New York (NZ)

Correspondence to: M. Cristina Kenney, M.D., Ph.D., Dept. of Ophthalmology, University of California Irvine, Medical Center, 101 The City Drive, Orange, CA 92868. E-mail: mkenney{at}uci.edu


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 Literature Cited
 
Corneas with edema-related diseases lose transparency, which causes significant vision loss. This study analyzed seven aquaporins (AQPs) in normal corneas, pseudophakic/aphakic bullous keratopathy (PBK/ABK) corneas, Fuchs' dystrophy corneas, keratoconus corneas, post-cataract surgery (PCS) corneas, and normal organ-cultured corneas. RNA levels for AQP1, AQP4, and ß2-microglobulin were measured by RT-PCR. AQP1 antibody localized to stromal cells of all corneas. PBK/ABK and Fuchs' dystrophy corneas had decreased endothelial cell staining compared with normal. AQP1 mRNA was found in whole corneas and cultured stromal fibroblasts but not in isolated epithelial cells. AQP3 staining was found in basal epithelial cells of the normal, Fuchs' dystrophy, and keratoconus corneas but throughout the entire epithelium of PBK/ABK corneas. AQP4 antibody localized to endothelial cells of all corneas and in stromal cells of PBK/ABK corneas. AQP4 mRNA was identified in whole human corneas. AQP5 was found in epithelial cells of all corneas. AQP0, AQP2, and AQP9 were not found in any corneas. Normal AQP distributions were found in PCS and organ-cultured corneas, although they showed signs of swelling. Our study demonstrates that AQP abnormalities are found in PBK/ABK corneas (decreased AQP1, increased AQP3 and AQP4) and Fuchs' dystrophy corneas (decreased AQP1). Although both have vision-disrupting corneal edema, the mechanisms of fluid accumulation may be different in each disease. (J Histochem Cytochem 52:1341–1350, 2004)

Key Words: aquaporins • bullous keratopathy • cornea • Fuchs' dystrophy • Keratoconus • corneal organ culture • immunohistochemistry • RT-PCR


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
PSEUDOPHAKIC BULLOUS KERATOPATHY (PBK) is a corneal disease that occurs after cataract removal and placement of an intraocular lens. When an intraocular lens is not placed in the eye at cataract surgery and there is associated corneal edema, the disease process is called aphakic bullous keratopathy (ABK). A clinical hallmark of both PBK and ABK is chronic corneal edema, which leads to significant loss of transparency in the central cornea (Figure 1) (Waring et al. 1978Go,1982Go; Kenney and Chwa 1990Go; Ljubimov et al. 1996Go). The fluid accumulation occurs at different locations: (a) within epithelial cells to form microcysts, (b) in subepithelial bullae or blisters, and (c) within the stromal cells and matrix.



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Figure 1

Slit-lamp photograph of pseudophakic bullous keratopathy (PBK) cornea. Note loss of corneal transparency due to fluid accumulation. Posterior aspect of the slitlamp beam is irregular (arrowheads) and thickened because of excess fluid.

 
Fuchs' endothelial dystrophy is a hereditary disease linked to a missense mutation in COL8A2, which codes for the {alpha}2-chain of type VIII collagen (Biswas et al. 2001Go). It is most common in older women and has many clinical features similar to PBK/ABK corneas, except that Fuchs' dystrophy corneas also have many guttae along the Descemet's membrane (Rosenblum et al. 1980Go; Bourne et al. 1982Go). Abnormal fluid accumulation is a major feature of both PBK/ABK and Fuchs' dystrophy corneas, but the mechanism(s) is not understood. During the past 30 years, the number of corneal transplants performed for chronic corneal edema-related diseases has increased significantly, and these diseases are now the most common indications for corneal transplantation in the United States and Canada (Liu and Slomovic 1997Go; Lois et al. 1997Go; Cosar et al. 2002Go).

Aquaporins (AQPs) are recently discovered water channel proteins found in animals, plants and microorganisms. These molecular channels are essential for the regulation of water in all living cells and tissues (Kang et al. 2000Go; King et al. 2000Go; Verkman 2002Go), and excellent review articles on AQPs are available (Kang et al. 2000Go; King et al. 2000Go; Agre et al. 2002Go; Verkman 2002Go,2003Go).

Most of the information regarding AQP molecular structure was obtained on AQP1, a 28-kD monomer with six transmembrane domains and intracellular N- and C-termini (King et al. 2000Go). A common motif defined by Asn-Pro-Ala (NPA) is found in all AQPs. These motif regions fold together to form the aqueous pore in the cell membrane. AQP0 has low water permeability, whereas AQP3, AQP7, AQP9, and AQP10 transport both water and small non-ionic solutes such as urea and glycerol (King et al. 2000Go). The other aquaporins, AQP1, AQP2, AQP4, AQP5, and AQP8, transport only water and exclude small solutes. AQP6 is found in membranes of kidney intracellular vesicles and may function as a nitrate channel rather than a water channel (Ikeda et al. 2002Go). The presence of the cysteine 189 residue in most AQPs confers mercury sensitivity, with the exception being AQP4, which is mercury-insensitive (Han et al. 1998Go).

More than 10 AQPs have been reported (King et al. 2000Go; Agre and Kozono 2003Go;Verkman 2003Go), and at least five are found in the eye. AQP0 (major intrinsic protein, MIP) is a major protein of the vertebrate lens cell membranes (Nemeth-Cahalan and Hall 2000Go; Zampighi et al. 2002Go). Cataracts occur in the AQP0-deficient mouse model, which harbors mutations in AQP0 (Berry et al. 2000Go). AQP1 is reported in the non-pigmented ciliary body, iris, scleral fibroblasts, anterior lens epithelium, corneal endothelium, corneal stromal cells, trabecular meshwork, Schlemm's canal endothelium, and retina (Hamann et al. 1998Go; Kim et al. 1998Go,2002Go; Kang et al. 1999Go; Verkman 2003Go; Macnamara et al. 2004Go). AQP3 is found in the bulbar conjunctival epithelium and the cornea (Patil et al. 1997Go; Hamann et al. 1998Go). AQP4 is in the ciliary body, iris, conjunctival epithelium, retinal Müller cells, and astrocytes (Hasegawa et al. 1994Go; Nagelhus et al. 1999Go). AQP5 is reported in lacrimal gland, cornea, and cultured epithelial cells (Raina et al. 1995Go; Patil et al. 1997Go; Kang et al. 1999Go). Lower AQP1 expression was reported in corneas with endothelial cell dysfunction (Macnamara et al. 2004Go), but a comprehensive evaluation of other AQPs in human diseased corneas has not been performed.

The goal of the present study was to characterize the AQPs found in PBK/ABK corneas and Fuchs' endothelial dystrophy corneas, both diseases characterized by chronic corneal edema. To verify that any changes of AQPs were specific to PBK/ABK corneas or Fuchs' dystrophy corneas, we also examined corneas with other pathologies. Keratoconus is a corneal disease that is characterized by disruptions in Bowman's layer, progressive steepening of the cornea, excessive stromal thinning, and no known association with abnormal fluid dynamics. Another category of corneas examined were post-cataract surgery (PCS) normal corneas, because previous studies showed that these corneas had specific changes in their epithelial basement membranes similar to those observed in PBK/ABK and Fuchs' dystrophy corneas (Ljubimov et al. 2002Go).

Finally, we wanted to determine if a corneal organ culture model reflected the AQP patterns found in normal in situ corneas. Fortunately, a model was described that allows extended culture of intact whole human corneas (Foreman et al. 1996Go; Harper et al. 1998Go). These corneas can be transplanted successfully even after 1 month in culture (Harper et al. 1998Go). Corneal organ cultures were established and the AQP patterns analyzed.

We found that PBK/ABK corneas had the most alterations in the AQPs, with abnormal staining for AQP1, AQP3, and AQP4. Fuchs' dystrophy corneas, another disease with chronic edema, had changes in AQP1. Otherwise, keratoconus corneas, organ-cultured normal corneas, and PCS corneas had essentially normal AQP staining patterns.


    Materials and Methods
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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Immunohistochemistry
Healthy (normal) and PCS autopsy corneas were collected from National Disease Research Interchange (NDRI; Philadelphia, PA) within 24 hr after enucleation. The PCS corneas were from eyes that had undergone cataract surgery and had an artificial intraocular lens in place. The diseased corneas were received from various surgeons within 24 hr of the corneal transplant surgery. Tissues were sent to our laboratory on ice, were rinsed thoroughly in cold PBS, pH 7.2, and embedded in Tissue Tek OCT compound. The blocks were frozen rapidly in liquid nitrogen and 5-µm sections cut on a Leica cryostat. The sections were treated with Ultra V Block (Neomarkers; Fremont, CA) for 15 min and antibodies were diluted in Diluent (Neomarkers). Total numbers of corneas studied were as follows: normal (n=13), bullous keratopathy (n=15), Fuchs' dystrophy (n=12), keratoconus (n=11), PCS corneas (n=11), and organ-cultured corneas (n=5).

Polyclonal antibodies were used to the following: AQP0 (8 µg/ml, cat #AQP01-A, lot #3232A; Alpha Diagnostic International, Austin, TX); AQP1 (5 µg/ml, cat #AB3272-50UL, lot #21040737, residues 243–261; Chemicon International, Temecula, CA); AQP2 (8 µg/ml, cat. #AB3066, lot #21042068; Chemicon); AQP3 (7 µg/ml, A0303, residues 275–292 with additional N-terminal lysine and tyrosine; Sigma Chemical, St Louis, MO); AQP4 (2 µg/ml, cat. #AB 3594-50UL, lot # 21040738, residues 249–323; Chemicon); AQP5 (4 µg/ml, G-19:sc-9890; Santa Cruz Biotechnology, Santa Cruz, CA); AQP5 (10 µg/ml, polyclonal, AB3069; synthetic peptide (aa 249–265); Chemicon); AQP5 (10 µg/ml, cat. #178615, cytoplasmic 17-amino-acid peptide near the C-terminus with an additional cysteine residue added; Calbiochem-Novabiochem, San Diego, CA); and AQP9 (8 µg/ml, affinity-purified, cat. #AQP91-A, lot #3232A; Alpha Diagnostic International). Rhodamine-conjugated goat anti-rabbit IgG or donkey anti-goat IgG (25 µg/ml; Chemicon) was used as the secondary antibody. As a control for AQP5, we purchased the peptides to which the antibodies were made (Santa Cruz Biotechnology and Chemicon). The primary antibody was neutralized overnight at 4C before staining. Another control was the use of secondary antibodies only. Sections were viewed and photographed using an Olympus BH-2 microscope (Olympus USA; Melville, NY) equipped with epifluorescence.

Corneal Organ Cultures
Some corneas were placed in a long-term organ culture system to determine whether conditions could be established in vitro that allowed the human corneas to maintain their AQP patterns. The technique allows extended culture of intact whole human corneas (Foreman et al. 1996Go; Harper et al. 1998Go; Xu et al. 2000Go; Zieske 2000Go). Postmortem human corneas with the scleral rims were obtained from NDRI within 24 hr after death. Corneas were organ-cultured over agar–collagen gel as described (Kabosova et al. 2003Go). They were first washed in antibiotic–antimycotic mixture (ABAM; Invitrogen) and then placed epithelial side down in sterile Chiron's corneal transportation vials with a small volume of medium to prevent drying. The corneal concavity was filled with serum-free minimal essential medium (MEM; Invitrogen) containing ABAM, 1 mg/ml calf skin collagen (made from stock solution of 10 mg/ml in 0.1 N acetic acid), and 1% agar (both from Sigma Chemical). This mixture was microwaved until boiling to sterilize it and to dissolve the agar and then was cooled to 37–39C. After the addition to corneas, the mixture solidified within 2–3 min. Corneas were then placed agar side down on a sterile 60-mm dish. Serum-free MEM containing ABAM and insulin–transferrin–sodium selenite (ITS supplement; Sigma) was then added dropwise to the central cornea until it reached the limbus. Corneas were kept in a humidified CO2 incubator at 35.5C and 100 µl medium was added one or two times a day to moisten the epithelium. Corneal morphology and cell viability were monitored microscopically with a x4 objective. After 10–14 days in culture, the corneas were harvested, embedded in OCT medium, and rapidly frozen in liquid nitrogen. The tissues were sectioned and stained with AQP antibodies.

Tissue Culture of Corneal Fibroblasts
Fibroblasts were cultured from normal corneas (n=4) and a PBK cornea (n=1) to determine if AQP1, which is highly expressed in the intact whole cornea stromal cells, was also expressed in vitro. The normal corneas were received within 24 hr after death from the NDRI. The PBK cornea was received within 24 hr after surgery. After removal of the corneal epithelial and endothelial layers, primary stromal cell cultures were established (Kenney et al. 1994Go,2003Go). Duplicate third-passage cultures were grown to confluence in MEM supplemented with 10% fetal bovine serum. Normal and PBK fibroblasts were plated into 60-mm dishes at 5 x 105 cells/plate.

Statistical Analysis
Immunostaining data were analyzed by the two-sided Fisher's exact test using the InStat software program (GraphPad Software; San Diego, CA). The number of cases with abnormal staining pattern in one experimental group (e.g., normal) was compared with the number of cases with abnormal staining pattern in another experimental group (e.g., PBK). P value less than 0.05 was considered significant.

RNA Analysis
Immunohistochemistry with antibodies specific for AQP4 showed positive staining in the human cornea. Because AQP4 had not been previously described in the human cornea, we also analyzed corneas for the presence of AQP4 mRNA. In addition, it has been controversial whether AQP1 is present in epithelial cells (Hamann et al. 1998Go; Bildin et al. 2001Go; Thiagarajah and Verkman 2002Go; Macnamara et al. 2004Go). Therefore, we examined the whole intact cornea, isolated epithelial cells, and cultured stromal fibroblasts for AQP1 mRNA by RT-PCR.

Tissues prepared for RNA analyses were normal whole corneas (n=4), PBK/ABK whole corneas (n=3), isolated epithelial cells (n=4), cultured normal corneal fibroblasts (n=4), and cultured PBK corneal fibroblasts (n=1). RNA analysis was carried out as described previously (Ljubimov et al. 1998Go; Saghizadeh et al. 1998Go; Spirin et al. 1999Go). The whole corneas were frozen in liquid nitrogen and pulverized into powder. The epithelial cell layer was removed from the underlying Bowman's layer by gently scrapping with a sterile spatula and placed in PBS. The corneal fibroblasts were cultured as described above until nearly confluent and rinsed with PBS. Samples were placed in Trizol (Invitrogen), thawed on ice, and homogenized for 5 min. Chloroform was added (1/5 of the volume) and the phases were separated by centrifugation. The aqueous phase was collected, linear acrylamide was added to 10 µg/ml, and the RNA precipitated by the addition of 0.5 vol of isopropanol. RNA was recovered by centrifugation at 4C and the pellet washed with 75% ethanol. The pellet was air-dried, resuspended in buffer, and processed over RNeasy columns (Qiagen; Valencia, CA). RNA was eluted with 50 µl water and 10 µg of linear acrylamide was added. The solution was adjusted with 2 M sodium acetate, pH 5.0, to 10% vol and precipitated with 2.5 vol ethanol. RNA was recovered by centrifugation, washed with 75% ethanol, and resuspended in 11 µl of water. One µl of the RNA was analyzed using an Agilent 2100 Bioanalyzer to verify both quantity and quality of the RNA.

Five hundred ng of RNA was reverse-transcribed in a 50-µl reaction volume containing 0.5 mM dNTPs, 2.5 µM random decamer primers, 20 U RNase inhibitor, and 200 U SuperScript II reverse transcriptase (Invitrogen). Reactions were carried out for 10 min at 25C, 1 hr at 42C, and 5 min at 95C, followed by cooling to 4C. After synthesis, the cDNA was diluted to equivalent input RNA levels with TE (10 ng input RNA/µl) and stored at –20C. The cDNA samples were subjected to PCR using specific primers for AQP4 (Wang et al. 2003Go): forward AQP4, 5'-TGGCTTCTGATGCTGATTTG-3', reverse AQP4, 5'- TTGCAATGCTGAGTCCAAAG-3', product size 237 bp. The reaction mixtures for AQP1 and AQP4 contained 1 µl of the cDNA, 1.5 mM MgCl2,, 0.2 mM dNTP, and 0.2 µM primers. The AQP4 reaction was carried out for 35 cycles, 94C, 30 sec; 56C, 30 sec; 72C; 1 min. The primers for the AQP1 were forward, AQP1 5'-GTCCAGGACAACGTGAAGGT and reverse, AQP1, 5' -GAGGAGGTGATGCCTGAGAG, product size 218 bp. The AQP1 was 35 cycles, using 94C, 30 sec; 60C, 30 sec; 72C, 1 min. Primers for ß2-microglobulin (ß2-MG) were forward 5'-CTCGCGCTACTCTCTCTTTCTG and reverse 5'-GCTTACATGTCTCGATCCCACTT, product size 334 bp. The ß2-MG reaction was 35 cycles, using 94C, 30 sec; 60C, 30 sec; 72C, 45 sec. PCR products were separated by electrophoresis on 1.5% agarose gels, stained, and visualized under UV light with ethidium bromide. The Webcutter program was used to identify restriction sites within the AQP1 and AQP4. PCR products were digested with Pvu II (New England Biolabs; Beverly, MA) and Stu I (Invitrogen) according to the manufacturer's protocols. Predicted fragment sizes for AQP4 after Pvu II were 198 bp and 39 bp and for StuI were 138 bp and 99 bp. Predicted fragment sizes for AQP1 after Pvu II were 122 bp and 96 bp. Routine RT-PCR controls without reverse transcriptase were negative.


    Results
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 Materials and Methods
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 Literature Cited
 
Figure 1 shows a slit-lamp photograph of a PBK cornea. The edges of the slit lamp beam are irregular (arrowheads), consistent with fluid accumulation. Note that the cornea has decreased transparency, which obscures the view of the iris and pupil.

AQPs in Diseased Corneas
AQP1
Immunostaining detected AQP1 in the stromal cells of all corneas examined (Figures 2 and 3 ; Table 1). The endothelial cells of the normal corneas and keratoconus corneas had AQP1. Although the PBK corneas and Fuchs' dystrophy corneas had fewer endothelial cells, when present they also stained with AQP1. The epithelial cells did not stain with the AQP1 antibody (Figure 2). RT-PCR with primers specific for AQP1 showed a single band of 218 bp, the expected size for AQP1 (Figure 4A) . cDNAs from either whole normal corneas (n=4), PBK/ABK corneas (n=3), or isolated epithelial cells (n=4) were analyzed for AQP1 mRNA (Figure 4A). After normalization to the ß2-MG, the AQP1 mRNA levels in normal corneas vs PBK/ABK corneas were found to be similar (Figure 4A, Lanes 1 and 2). The epithelial cells were devoid of AQP1 transcript (Figure 4A, Lane 3), suggesting that this layer did not contribute to the AQP1 mRNA levels found in the whole corneas. Corneal fibroblasts cultured from normal corneas (n=4) or a PBK cornea (n=1) maintained AQP1 mRNA expression in vitro (Figure 4A, Lanes 4 and 5). The 218-bp band was subjected to Pvu II restriction enzyme and yielded two bands of 122 bp and 96 bp, which correspond to the expected sizes for digestion of AQP1 (Figure 4B).



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Figure 2

Aquaporins (AQPs) in epithelium and anterior stroma of normal and diseased corneas. Immunofluorescent staining with specific antibodies. (AQP1) Note staining of stromal cells in normal and diseased corneas. (AQP3) Note expression in some of the basal epithelial cells in normal, keratoconus and Fuchs' dystrophy corneas. In PBK/ABK corneas, the AQP3 epithelial staining is generally more uniform and brighter than in any other corneal group. The stromal cells in region of extracellular matrix disruption and fibrosis are AQP3-positive. (AQP4) Note increased staining in the stromal cells of PBK/ABK corneas. (AQP5) Epithelial cells stain in a punctate-like pattern in normal and diseased corneas. Some stromal cells are AQP5-positive. Corneal sections treated with AQP5 blocking peptide were negative. In normal organ-cultured corneas, staining patterns of all studied AQPs are similar to intact normal corneas. NL, normal corneas; PBK, pseudophakic bullous keratopathy; Fuchs', Fuchs' dystrophy; KC, keratoconus corneas; Organ culture, normal corneas cultured for up to 14 days; Negative control, secondary antibodies only. Bar = 60 µm.

 


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Figure 3

AQPs in the endothelium and posterior stroma of normal and diseased corneas. Immunofluorescent staining with specific antibodies to AQPs. (AQP1) Stromal and endothelial cells are positive. PBK/ABK corneas and Fuchs' dystrophy corneas have fewer endothelial cells and decreased AQP1 staining. (AQP3) Endothelial cells show nonspecific staining. (AQP4) Endothelial cells are positive. Note that PBK corneas have many AQP4-positive stromal cells compared with normal or other diseased corneas. (AQP5) Scattered cells of the posterior stroma are positive and staining is eliminated by treatment with AQP5 blocking peptide. Endothelial cells have nonspecific staining for AQP5. In normal organ-cultured corneas, staining patterns of all studied AQPs are again similar to those of intact normal corneas. NL, normal corneas; PBK, pseudophakic bullous keratopathy; Fuchs', Fuchs' dystrophy; KC, keratoconus corneas; Organ culture, normal corneas cultured for up to 14 days; Negative control, secondary antibodies only. Bar = 60 µm.

 

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Table 1

Presence of aquaporins in normal and diseased corneasa

 


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Figure 4

AQP1 mRNA is present in human corneas and corneal fibroblast cultures but not in isolated epithelial cells. (A) Representative RT-PCR analysis of AQP1 (upper panel) and ß2-microglobulin (ß2-MG, lower panel) gene expression in normal corneas, PBK corneas and cultured fibroblasts and normal isolated epithelial cells. Lane 1, normal human cornea; Lane 2, human PBK cornea; Lane 3, human isolated corneal epithelial cells; Lane 4, human cultured corneal fibroblasts; Lane 5, human cultured PBK corneal fibroblasts; Lane 6, marker ladder. AQP1 product appears as a 218-bp band and is absent in the epithelial cell samples. (B) Representative restriction enzyme pattern of AQP1. Lane 1, uncut AQP1 fragment with expected size of 218 bp; Lane 2, AQP1 cut with Pvu II shows expected sizes of 122 bp and 96 bp. (C) Representative restriction enzyme pattern of AQP4; Lane 1, uncut AQP4 fragment with expected size of 237 bp; Lane 2, AQP4 cut with Pvu II shows expected sizes of 198 bp and 39 bp; Lane 3, AQP4 cut with StuI shows expected sizes of 138 bp and 99 bp.

 
AQP3
AQP3 was found mainly in the basal epithelial cells and some stromal cells of the normal corneas, Fuchs' dystrophy corneas, and keratoconus corneas (Figure 2). The PBK/ABK corneas had increased intensity of AQP3 staining with a distribution in both the basal and superficial epithelial cells. The stromal cells associated with subepithelial fibrosis in keratoconus corneas (8/10; p<0.003), Fuchs' dystrophy corneas (9/10; p<0.006), and PBK corneas (8/12; p<0.01), showed increased AQP3 staining compared with normal corneas (2/13) (Figure 2; Table 1). There was nonspecific staining of the endothelial cells with the AQP3 antibody (Figure 3).

AQP4
In the normal corneas AQP4 was absent in epithelial cells and stromal cells (Figure 2) but present in the endothelial cells (Figure 3). There was a significant increase of AQP4 that appeared in the stromal cells of the PBK/ABK corneas (7/8) compared with normal corneas (0/13; p<0.0001) (Figures 2 and 3; Table 1). It has been controversial as to whether AQP4 was expressed in human corneas. Therefore, in addition to the IHC staining with the specific antibody, we also examined the AQP4 mRNA levels (Figure 4C). The mRNA for AQP4 was found in corneas with an expected size of 237 bp (Figure 4C, Lane 1). After Pvu II and Stu I treatments, the fragments showed the expected sizes of 198 bp and 39 bp (Figure 4C, Lane 2) or 138 bp and 99 bp (Figure 4C, Lane 3), respectively. This supports the presence of AQP4 within the human cornea.

AQP5
AQP5 was found in epithelial cells of normal and diseased corneas (Figure 2) and was prominent in the conjunctival cells (not shown). In addition, AQP5 was found in some cells of the anterior stroma of PBK/ABK corneas (4/10) and keratoconus corneas (6/10) and in some posterior stromal cells adjacent to Descemet's membrane in Fuchs' dystrophy corneas (5/10) (Figure 3). This stromal staining was not significant because occasional staining of stromal cells was also seen in normal corneas (5/10).

In this study we tested three different AQP5 antibodies but saw staining only with the goat polyclonal antibody (G-19:sc-9890) from Santa Cruz Biotechnology. However, the AQP5 staining pattern of the epithelial cells was unusual in that it was punctate rather than having a membrane association. To verify the AQP5 specificity, the antibody was combined with the AQP5 blocking peptide before exposure to the tissues. In these cases, the staining was eliminated from the epithelial cells and stromal cells, suggesting specificity. The endothelial cells had nonspecific staining (Figures 2 and 3).

For comparison to diseased corneas, we also examined corneas from patients that had undergone cataract surgery and had an intraocular lens in place. These PCS corneas had AQP patterns similar to those of normal corneas (data not shown).

The organ-cultured corneas were also examined (Figures 2 and 3). AQP1 was found in the stromal cells and in the endothelial cells. AQP3 antibody stained the basal epithelial cells and some stromal cells. AQP4 antibody stained only the endothelial cells. AQP5 antibody stained the epithelial cells and the conjunctiva. After prolonged cultures, these corneas exhibited swelling. In general, however, the AQP staining patterns of the corneas that were maintained in organ culture were similar to those found in the normal corneas.


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
PBK/ABK and Fuchs' dystrophy are two major corneal diseases that have endothelial cell dysfunction and chronic edema as major components of the pathological process. In these disorders fluid accumulates within epithelial cells (microcysts) and corneal thickness can increase 100–300 µm, leading to loss of transparency and decreased vision. We present a comprehensive study of seven different AQPs comparing normal corneas to three different corneal diseases (PBK/ABK, Fuchs' dystrophy, and keratoconus), post-cataract surgery corneas, and organ-cultured corneas. We find that PBK/ABK corneas have altered distribution in AQP1, AQP3, and AQP4. Fuchs' dystrophy corneas have AQP1 changes. The non-edematous corneas (keratoconus and PCS corneas) are essentially normal in their AQP patterns.

AQPs in Diseased Corneas
AQP1
Our findings for AQP1 antibody staining in normal stromal cells and endothelial cells are in agreement with other studies (Hamann et al. 1998Go; Wen et al. 2001Go; Thiagarajah and Verkman 2002Go; Macnamara et al. 2004Go). The corneal epithelial cells did not have AQP1 immunostaining, and we provide evidence that isolated human epithelial cells lack the AQP1 mRNA. Our results differ from those of a study that reports AQP1 mRNA in bovine epithelial cells (Bildin et al. 2001Go). This discrepancy may be accounted for by species differences (human vs bovine) or isolation procedures. In their study, the bovine epithelial layer was separated by exposure to a lysate buffer containing 5 M guanidine thiocyanate and 0.1 M EDTA. In contrast, we mechanically separated the epithelial cells from underlying stroma with a gentle scraping procedure. To the best of our knowledge, there are no reports of positive immunostaining with AQP1 antibodies in the corneal epithelial cells (Hamann et al. 1998Go; Wen et al. 2001Go; Thiagarajah and Verkman 2002Go; Macnamara et al. 2004Go).

Our results also agree with a recent report showing that AQP1 was decreased in corneas with endothelial cell dysfunction (Macnamara et al. 2004Go). In our study, AQP1 mRNA levels from the whole normal corneas were not different from PBK/ABK corneas. However, we recognize that if PBK/ABK or Fuchs' dystrophy corneas had a decline in the endothelial cell mRNA, it is unlikely that we would have picked it up because we were analyzing all layers of the cornea together rather than isolated endothelium.

It is recognized that corneal endothelial cells have a significant role in pumping fluid out of the cornea via the Na+,K+-ATPase pump (Sasaki et al. 1986Go McCartney et al. 1987aGo,bGo; Guggenheim and Hodson 1994Go). Our studies showed that endothelial cells of PBK/ABK corneas and Fuchs' dystrophy corneas had normal staining for the Na+,K+-ATPase {alpha}-subunits (Ljubimov et al. 2002Go) and lower levels of AQP1. Our findings along with those of others (Thiagarajah and Verkman 2002Go; Verkman 2003Go; Macnamara et al. 2004Go) suggest that AQPs may be more important in corneal fluid dynamics than previously recognized.

A function of AQP1 in the cornea may be fluid elimination from the stroma across the endothelial cells because AQP1 null mice have delayed recovery of corneal transparency and increased thickness after treatment with hypotonic solutions (Thiagarajah and Verkman 2002Go; Verkman 2003Go). This proposed function is supported by our findings and those of others (Macnamara et al. 2004Go) showing that AQP1 is decreased in the endothelial layer of Fuchs' dystrophy and PBK/ABK corneas. We found an overall decrease in AQP1 antibody staining due to the fewer numbers of endothelial cells, but a number of sparse cells present still were positive. AQP1 is also decreased in a mouse model of corneal endothelial cell injury with subsequent corneal thickening (Macnamara et al. 2004Go). One can speculate that, in the PBK/ABK corneas and Fuchs' dystrophy corneas, as AQP1 declines fluid accumulates within the stroma and subepithelial bullae. In PBK/ABK corneas and Fuchs' dystrophy patients, it is not unusual to find corneal thickness increased to 600–850 µm (normal thickness is ~550 µm). Taken together, these studies suggest that upregulation of the endothelial cell AQP1 might lead to fluid elimination, decreased stromal swelling, and increased corneal transparency for these patients.

AQP3
AQP3 belongs to the aquaglyceroporin subset of the AQP family. Others have reported modest levels of AQP3 in corneal epithelium (Hamann et al. 1998Go). We found AQP3 associated with the membranes of the basal epithelial cells in normal corneas, Fuchs' dystrophy corneas, keratoconus corneas, and organ-cultured corneas. This basal cell AQP3 localization is in agreement with that described for epidermal keratinocytes of mouse skin (Ma et al. 2002Go) and pulmonary epithelial cells (Funaki et al. 1998Go). We are the first to report that PBK/ABK corneas have additional AQP3 immunostaining in the superficial epithelial cells. Functionally, AQP3 null mice have decreased "water holding capacity" compared with wild-type mice (Ma et al. 2002Go). If this function holds for the cornea, then the appearance of AQP3 in the superficial epithelium may be associated with increased fluid accumulation. This might be related to the classic epithelial microcysts representing intracellular fluid found in PBK/ABK corneas (Grayson 1983Go). On the basis of both animal and human tissue studies, we hypothesize that inhibition or blocking of AQP3 might decrease the "water holding capacity" of cells and aid in decreasing the numbers of microcysts that form within PBK/ABK corneal epithelial cells.

Surprisingly, we found increased AQP3 associated with stromal cells in areas of fibrosis in PBK/ABK corneas (8/12; p<0.01), Fuchs' dystrophy corneas (9/10; p<0.01), and keratoconus corneas (8/10; p<0.01) compared with normal corneas (2/13). We suspect that this increased focal staining of the AQP3 is associated with the process of scarring or tissue remodeling because it was found also in fibrotic regions of keratoconus corneas, a disease of thinning and focal scarring but not associated with chronic edema. Perhaps specific "wound healing" cells have a higher expression of AQP3 than normal. Further studies are needed to understand this finding.

AQP4
This is the first study to report AQP4 mRNA within normal human corneas. Our IHC studies localized the AQP4 to the endothelial cells in normal and diseased corneas. Our findings are different from those that report no AQP4 in corneal endothelial cells or cultured bovine endothelial cells (Hamann et al. 1998Go; Wen et al. 2001Go). The discrepancies may be related to species, frozen vs paraffin sections, or the specific antibody used. Because human corneal AQP4 had not been described previously, we also analyzed the corneas for AQP4 mRNA. Using RT-PCR with specific primers for AQP4, we found the expected size product for AQP4 that cut properly with two different restriction enzymes (Pvu II and Stu I). Although significant amounts of AQP4 mRNA are found in brain, lower levels have also been found in the eye by Northern blotting and RNase protection assay (Jung et al. 1994Go). The highest levels of AQP4 are found in the retina (Patil et al. 1997Go; Hamann et al. 1998Go), but it is present in lower amounts in ciliary body, iris, and lens (Patil et al. 1997Go). In addition, AQP4 mRNAs are also described in non-ocular tissues such as muscle, Meckel's cartilage, fetal tooth, and submandibular gland (Wang et al. 2003Go; Trujillo et al. 2004Go). In the normal human cornea, AQP1 and AQP5 are in great abundance (Patil et al. 1997Go), with AQP4 being a minor component.

Surprisingly, the IHC staining for AQP4 antibody was different in the PBK/ABK corneas compared with normal corneas or those affected by other diseases. There was a significant increase in the AQP4-positive stromal cells in PBK/ABK corneas (7/8) compared with normal corneas (0/13; p<0.0001). Usually AQP4 is associated with regulation of fluid movement between the vessels and brain parenchyma, and it is not clear what role AQP4 might play in PBK/ABK corneas, especially since the cornea is avascular. Based on AQP4 null mice studies, it has been suggested that AQP4 was associated with tissue swelling and that mice that lack AQP4 are protected from brain edema (Papadopoulos et al. 2002Go). Absence of AQP4 also reduces intraocular pressure by lowering aqueous humor production in these mice (Zhang et al. 2002Go). If this holds for the human cornea, then the increase of AQP4 in stromal cells might contribute in some way to the swelling found in PBK/ABK corneas, although the mechanism is still not clear. Alternatively, AQP4 might be associated with a specific cell type, such as macrophages (CD-14- positive cells), which are found in PBK/ABK corneas and not in keratoconus or normal corneas (Kenney et al. 2001Go). It should also be considered that the altered AQP pattern may be a response to upregulation of growth factors (i.e., IGF-I or BMP-4) present in PBK/ABK corneas (Saghizadeh et al. 2001Go), because studies showed that AQP gene expression can be altered in response to cytokines/growth factors (Borok et al. 1998Go; Smith et al. 1999Go; Fasshauer et al. 2003Go). Using gene silencing techniques, alternative functions have been proposed for AQP4, such as a role in maintaining cell morphology, cell plasticity, and regulation of ischemia-related genes (Nicchia et al. 2003Go). Further investigations of PBK/ABK corneas are necessary to clarify the role of AQP4 in cell behavior and tissue fluid dynamics.

AQP5
There was no significant difference in the staining pattern for AQP5 in epithelial cells of normal corneas and diseased corneas. Within the stroma, a small number of AQP5-positive cells were adjacent to either Bowman's layer or Descemet's membrane, and nonspecific staining of the endothelial cells was found. AQP5 has been described in rat, mouse, and human corneal epithelium (Raina et al. 1995Go; Hamann et al. 1998Go; Thiagarajah and Verkman 2002Go).

AQP5 plays an interesting role in the cornea. It is found in epithelial cells, and although AQP5 null mice have increased corneal thickness, the baseline corneas remain clear (Thiagarajah and Verkman 2002Go). However, after exposure of the epithelial surface to hypotonic saline (100 mOsm), the recovery rate of corneal swelling was significantly decreased, suggesting that AQP5 is important in fluid elimination via the epithelium (Thiagarajah and Verkman 2002Go). It should be considered that epithelial cell dysfunction may play a role in corneal edema-related diseases because in PBK/ABK corneas these cells have abnormal Na+,K+-ATPase {alpha}-subunits (Ljubimov et al. 2002Go) and altered distribution of AQP3. Further studies should be conducted to clarify the role of epithelial cells in corneal fluid dynamics.

It was surprising to us that Fuchs' dystrophy corneas and PBK/ABK corneas had different AQP patterns because both diseases have many of the same clinical features. One explanation for this difference may be that Fuchs' dystrophy has a genetic component, a mutation in the {alpha}2-chain of type VIII collagen, which is present in Descemet's membrane, the substrate for the endothelial cells (Biswas et al. 2001Go). In contrast, all PBK/ABK corneas have had previous surgery (cataract removal) with the loss of endothelial cells, usually suspected to be traumatic (excessive irrigation during phacoemulsification, corneal contact during intraocular lens placement, postoperative contact with an anterior chamber intraocular lens) and may have an inflammatory component that Fuchs' dystrophy corneas lack. Although the common final pathway in both PBK/ABK corneas and Fuchs' dystrophy corneas is an abnormal accumulation of fluid, the underlying mechanisms leading to the disease process might be different.

Long-term corneal organ cultures have similar AQP patterns to those of the normal in situ corneas and may provide a viable model for future studies into AQP function and regulation. Even after 14 days in culture and various degrees of swelling, the organ-cultured corneas had AQP patterns similar to normal corneas. This organ culture model allows up to 1-month culture of intact whole human corneas (Foreman et al. 1996Go; Harper et al. 1998Go). Our recent data showed that normal expression patterns of basement membrane components and integrins were maintained during this extended incubation (Kabosova et al. 2003Go). It can be suggested that the edema observed in PBK/ABK and Fuchs' dystrophy corneas has a different pathological mechanism of swelling than observed in organ-cultured corneas, where it was apparently due to osmosis-related lateral influx through the severed sclera. Corneal swelling in diseased corneas may involve alterations in Na+,K+-ATPase and/or AQPs, some of which have been described here in detail. Understanding the functions of the corneal AQPs may allow the development of therapeutic interventions that would prevent pathological corneal swelling and preserve visual acuity in the PBK/ABK and Fuchs' dystrophy patients.


    Footnotes
 
Received for publication May 7, 2003; accepted May 15, 2004


    Literature Cited
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 Literature Cited
 

Agre P, King LS, Yasui M, Guggino WB, Ottersen OP, Fujiyoshi Y, Engel A, et al. (2002) Aquaporin water channels–from atomic structure to clinical medicine. J Physiol 542:3–16[Abstract/Free Full Text]

Agre P, Kozono D (2003) Aquaporin water channels: molecular mechanisms for human diseases. FEBS Lett 555:72–78[CrossRef][Medline]

Berry V, Francis P, Kaushal S, Moore A, Bhattacharya S (2000) Missense mutations in MIP underlie autosomal dominant ‘polymorphic’ and lamellar cataracts linked to 12q. Nature Genet 25:15–17[CrossRef][Medline]

Bildin VN, Iserovich P, Fischbarg J, Reinach PS (2001) Differential expression of Na:K:2Cl cotransporter, glucose transporter 1, and aquaporin 1 in freshly isolated and cultured bovine corneal tissues. Exp Biol Med 226:919–926[Abstract/Free Full Text]

Biswas S, Munier FL, Yardley J, Hart-Holden N, Perveen R, Cousin P, Sutphin JE, et al. (2001) Missense mutations in COL8A2, the gene encoding the alpha2 chain of type VIII collagen, cause two forms of corneal endothelial dystrophy. Hum Mol Genet 10:2415–2423[Abstract/Free Full Text]

Borok Z, Lubman RL, Danto SI, Zhang XL, Zabski SM, King LS, Lee DM, et al. (1998) Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression of aquaporin 5. Am J Respir Cell Mol Biol 18:554–561[Abstract/Free Full Text]

Bourne WM, Johnson DH, Campbell RJ (1982) The ultrastructure of Descemet's membrane. III. Fuchs' dystrophy. Arch Ophthalmol 100:1952–1955[Abstract]

Cosar CB, Sridhar MS, Cohen EJ, Held EL, Alvim Pde T, Rapuano CJ, Raber IM, et al. (2002) Indications for penetrating keratoplasty and associated procedures, 1996–2000. Cornea 21:148–151[CrossRef][Medline]

Fasshauer M, Klein J, Lossner U, Klier M, Kralisch S, Paschke R (2003) Suppression of aquaporin adipose gene expression by isoproterenol, TNFalpha, and dexamethasone. Horm Metab Res 35:222–227[CrossRef][Medline]

Foreman DM, Pancholi S, Jarvis-Evans J, McLeod D, Boulton ME (1996) A simple organ culture model for assessing the effects of growth factors on corneal re-epithelialization. Exp Eye Res 62:555–564[CrossRef][Medline]

Funaki H, Yamamoto T, Koyama Y, Kondo D, Yaoita E, Kawasaki K, Kobayashi H, et al. (1998) Localization and expression of AQP5 in cornea, serous salivary glands, and pulmonary epithelial cells. Am J Physiol 275:C1151–1157[Medline]

Grayson M (1983). Diseases of the Cornea. St Louis, CV Mosby.

Guggenheim JA, Hodson SA (1994) Localization of Na+/K(+)-ATPase in the bovine corneal endothelium. Biochim Biophys Acta 1189:127–134[Medline]

Hamann S, Zeuthen T, La Cour M, Nagelhus EA, Ottersen OP, Agre P, Nielsen S (1998) Aquaporins in complex tissues: distribution of aquaporins 1–5 in human and rat eye. Am J Physiol 274:C1332–1345[Medline]

Han Z, Wax MB, Patil RV (1998) Regulation of aquaporin-4 water channels by phorbol ester-dependent protein phosphorylation. J Biol Chem 273:6001–6004[Abstract/Free Full Text]

Harper CL, Boulton ME, Marcyniuk B, Tullo AB, Ridgway AE (1998) Endothelial viability of organ-cultured corneas following penetrating keratoplasty. Eye 12:834–838[Medline]

Hasegawa H, Ma T, Skach W, Matthay MA, Verkman AS (1994) Molecular cloning of a mercurial-insensitive water channel expressed in selected water-transporting tissues. J Biol Chem 269:5497–5500[Abstract/Free Full Text]

Ikeda M, Beitz E, Kozono D, Guggino WB, Agre P, Yasui M (2002) Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63. J Biol Chem 277:39873–39879[Abstract/Free Full Text]

Jung JS, Bhat RV, Preston GM, Guggino WB, Baraban JM, Agre P (1994) Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance. Proc Natl Acad Sci USA 91:13052–13056[Abstract/Free Full Text]

Kabosova A, Kramerov AA, Aoki AM, Murphy G, Zieske JD, Ljubimov AV (2003) Human diabetic corneas preserve wound healing, basement membrane, integrin and MMP-10 differences from normal corneas in organ culture. Exp Eye Res 77:211–217[CrossRef][Medline]

Kang F, Kuang K, Li J, Fischbarg J (1999) Cultured bovine corneal epithelial cells express a functional aquaporin water channel. Invest Ophthalmol Vis Sci 40:253–257[Abstract]

Kang F, Kunyan K, Fischbarg J (2000) Cultured bovine corneal epithelial cells express functional water channel [in Chinese]. Chung Hua Yen Ko Tsa Chih 36:381–383

Kenney MC, Chwa M (1990) Abnormal extracellular matrix in corneas with pseudophakic bullous keratopathy. Cornea 9:115–121[Medline]

Kenney MC, Chwa M, Lin B, Huang GH, Ljubimov AV, Brown DJ (2001) Identification of cell types in human diseased corneas. Cornea 20:309–316[CrossRef][Medline]

Kenney MC, Chwa M, Opbroek AJ, Brown DJ (1994) Increased gelatinolytic activity in keratoconus keratocyte cultures. A correlation to an altered matrix metalloproteinase-2/tissue inhibitor of metalloproteinase ratio. Cornea 13:114–124[Medline]

Kenney MC, Zorapapel N, Atilano S, Chwa M, Ljubimov A, Brown D (2003) Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) modulate tenascin-C and fibrillin-1 in bullous keratopathy stromal cells in vitro. Exp Eye Res 77:537–546[CrossRef][Medline]

Kim IB, Lee EJ, Oh SJ, Park CB, Pow DV, Chun MH (2002) Light and electron microscopic analysis of aquaporin 1-like-immunoreactive amacrine cells in the rat retina. J Comp Neurol 452:178–191[CrossRef][Medline]

Kim IB, Oh SJ, Nielsen S, Chun MH (1998) Immunocytochemical localization of aquaporin 1 in the rat retina. Neurosci Lett 244:52–54[CrossRef][Medline]

King LS, Yasui M, Agre P (2000) Aquaporins in health and disease. Mol Med Today 6:60–65[CrossRef][Medline]

Liu E, Slomovic AR (1997) Indications for penetrating keratoplasty in Canada, 1986–1995. Cornea 16:414–419[Medline]

Ljubimov AV, Atilano SR, Garner MH, Maguen E, Nesburn AB, Kenney MC (2002) Extracellular matrix and Na+,K+-ATPase in human corneas following cataract surgery: comparison with bullous keratopathy and Fuchs' dystrophy corneas. Cornea 21:74–80[CrossRef][Medline]

Ljubimov AV, Burgeson RE, Butkowski RJ, Couchman JR, Wu RR, Ninomiya Y, Sado Y, et al. (1996) Extracellular matrix alterations in human corneas with bullous keratopathy. Invest Ophthalmol Vis Sci 37:997–1007[Abstract]

Ljubimov AV, Saghizadeh M, Spirin KS, Khin HL, Lewin SL, Zardi L, Bourdon MA, et al. (1998) Expression of tenascin-C splice variants in normal and bullous keratopathy human corneas. Invest Ophthalmol Vis Sci 39:1135–1142[Abstract]

Lois N, Kowal VO, Cohen EJ, Rapuano CJ, Gault JA, Raber IM, Laibson PR (1997) Indications for penetrating keratoplasty and associated procedures, 1989–1995. Cornea 16:623–629[Medline]

Ma T, Hara M, Sougrat R, Verbavatz JM, Verkman AS (2002) Impaired stratum corneum hydration in mice lacking epidermal water channel aquaporin-3. J Biol Chem 277:17147–17153[Abstract/Free Full Text]

Macnamara E, Sams GW, Smith K, Ambati J, Singh N, Ambati BK (2004) Aquaporin-1 expression is decreased in human and mouse corneal endothelial dysfunction. Mol Vis 10:51–56[Medline]

McCartney MD, Robertson DP, Wood TO, McLaughlin BJ (1987a) ATPase pump site density in human dysfunctional corneal endothelium. Invest Ophthalmol Vis Sci 28:1955–1962[Abstract]

McCartney MD, Wood TO, McLaughlin BJ (1987b) Immunohistochemical localization of ATPase in human dysfunctional corneal endothelium. Curr Eye Res 6:1479–1486[Medline]

Nagelhus EA, Horio Y, Inanobe A, Fujita A, Haug FM, Nielsen S, Kurachi Y, et al. (1999) Immunogold evidence suggests that coupling of K+ siphoning and water transport in rat retinal Muller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains. Glia 26:47–54[CrossRef][Medline]

Nemeth-Cahalan KL, Hall JE (2000) pH and calcium regulate the water permeability of aquaporin 0. J Biol Chem 275:6777–6782[Abstract/Free Full Text]

Nicchia GP, Frigeri A, Liuzzi GM, Svelto M (2003) Inhibition of aquaporin-4 expression in astrocytes by RNAi determines alteration in cell morphology, growth, and water transport and induces changes in ischemia-related genes. FASEB J 17:1508–1510[Abstract/Free Full Text]

Papadopoulos MC, Krishna S, Verkman AS (2002) Aquaporin water channels and brain edema. Mt Sinai J Med 69:242–248[Medline]

Patil RV, Saito I, Yang X, Wax MB (1997) Expression of aquaporins in the rat ocular tissue. Exp Eye Res 64:203–209[CrossRef][Medline]

Raina S, Preston GM, Guggino WB, Agre P (1995) Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal, and respiratory tissues. J Biol Chem 270:1908–1912[Abstract/Free Full Text]

Rosenblum P, Stark WJ, Maumenee IH, Hirst LW, Maumenee AE (1980) Hereditary Fuchs' dystrophy. Am J Ophthalmol 90:455–462[Medline]

Saghizadeh M, Chwa M, Aoki A, Lin B, Pirouzmanesh A, Brown DJ, Ljubimov AV, et al. (2001) Altered expression of growth factors and cytokines in keratoconus, bullous keratopathy and diabetic human corneas. Exp Eye Res 73:179–189[CrossRef][Medline]

Saghizadeh M, Khin HL, Bourdon MA, Kenney MC, Ljubimov AV (1998) Novel splice variants of human tenascin-C mRNA identified in normal and bullous keratopathy corneas. Cornea 17:326–332[Medline]

Sasaki Y, Tuberville AW, Wood TO, McLaughlin BJ (1986) Freeze fracture study of human corneal endothelial dysfunction. Invest Ophthalmol Vis Sci 27:480–485[Abstract]

Smith JK, Siddiqui AA, Modica LA, Dykes R, Simmons C, Schmidt J, Krishnaswamy GA, et al. (1999) Interferon-alpha upregulates gene expression of aquaporin-5 in human parotid glands. J Interferon Cytokine Res 19:929–935[CrossRef][Medline]

Spirin KS, Ljubimov AV, Castellon R, Wiedoeft O, Marano M, Sheppard D, Kenney MC, et al. (1999) Analysis of gene expression in human bullous keratopathy corneas containing limiting amounts of RNA. Invest Ophthalmol Vis Sci 4:3108–3115

Thiagarajah JR, Verkman AS (2002) Aquaporin deletion in mice reduces corneal water permeability and delays restoration of transparency after swelling. J Biol Chem 277:19139–19144[Abstract/Free Full Text]

Trujillo E, Gonzalez T, Marin R, Martin-Vasallo P, Marples D, Mobasheri A (2004) Human articular chondrocytes, synoviocytes and synovial microvessels express aquaporin water channels: upregulation of AQP1 in rheumatoid arthritis. Histol Histopathol 19:435–444[Medline]

Verkman AS (2002) Physiological importance of aquaporin water channels. Ann Med 34:192–200[Medline]

Verkman AS (2003) Role of aquaporin water channels in eye function. Exp Eye Res 76:137–143[CrossRef][Medline]

Wang W, Hart PS, Piesco NP, Lu X, Gorry MC, Hart TC (2003) Aquaporin expression in developing human teeth and selected orofacial tissues. Calcif Tissue Int 72:222–227[CrossRef][Medline]

Waring GO III, Bourne WM, Edelhauser HF, Kenyon KR (1982) The corneal endothelium. Normal and pathologic structure and function. Ophthalmology 89:531–590[Medline]

Waring GO III, Rodrigues MM, Laibson PR (1978) Corneal dystrophies. I. Dystrophies of the epithelium, Bowman's layer and stroma. Surv Ophthalmol 23:71–122[Medline]

Wen Q, Diecke FP, Iserovich P, Kuang K, Sparrow J, Fischbarg J (2001) Immunocytochemical localization of aquaporin-1 in bovine corneal endothelial cells and keratocytes. Exp Biol Med 226:463–467[Abstract/Free Full Text]

Xu KP, Li XF, Yu FS (2000) Corneal organ culture model for assessing epithelial responses to surfactants. Toxicol Sci 58:306–314[Abstract/Free Full Text]

Zampighi GA, Eskandari S, Hall JE, Zampighi L, Kreman M (2002) Micro-domains of AQP0 in lens equatorial fibers. Exp Eye Res 75:505–519[CrossRef][Medline]

Zhang D, Vetrivel L, Verkman AS (2002) Aquaporin deletion in mice reduces intraocular pressure and aqueous fluid production. J Gen Physiol 119:561–569[Abstract/Free Full Text]

Zieske JD (2000) Expression of cyclin-dependent kinase inhibitors during corneal wound repair. Prog Retin Eye Res 19:257–270[CrossRef][Medline]





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