Aquaporins in complex tissues: distribution of aquaporins
1-5 in human and rat eye
Steffen
Hamann1,
Thomas
Zeuthen1,
Morten La
Cour2,
Erlend A.
Nagelhus3,
Ole Petter
Ottersen3,
Peter
Agre4, and
Søren
Nielsen5
1 Department of Medical
Physiology, The Panum Institute, University of Copenhagen, DK-2200
Copenhagen; 2 Eye Department and
Eye Pathology Institute, Rigshospitalet, DK-2200 Copenhagen;
5 Department of Cell Biology,
Institute of Anatomy, University of Aarhus, DK-8000 Aarhus,
Denmark; 3 Department of Anatomy,
Institute of Basic Medical Sciences, University of Oslo, 0317 Oslo,
Norway; and 4 Departments of
Biological Chemistry and Medicine, Johns Hopkins School of Medicine,
Baltimore, Maryland 21205
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ABSTRACT |
Multiple physiological fluid movements are
involved in vision. Here we define the cellular and subcellular sites
of aquaporin (AQP) water transport proteins in human and rat eyes by
immunoblotting, high-resolution immunocytochemistry, and immunoelectron
microscopy. AQP3 is abundant in bulbar conjunctival epithelium and
glands but is only weakly present in corneal epithelium. In contrast, AQP5 is prominent in corneal epithelium and apical membranes of lacrimal acini. AQP1 is heavily expressed in scleral fibroblasts, corneal endothelium and keratocytes, and endothelium covering the
trabecular meshwork and Schlemm's canal. Although AQP1 is plentiful in
ciliary nonpigmented epithelium, it is not present in ciliary pigmented
epithelium. Posterior and anterior epithelium of the iris and anterior
lens epithelium also contain significant amounts of AQP1, but AQP0
(major intrinsic protein of the lens) is expressed in lens fiber cells.
Retinal Müller cells and astrocytes exhibit notable
concentrations of AQP4, whereas neurons and retinal pigment epithelium
do not display aquaporin immunolabeling. These studies demonstrate
selective expression of AQP1, AQP3, AQP4, and AQP5 in distinct ocular
epithelia, predicting specific roles for each in the complex network
through which water movements occur in the eye.
water transporters; cornea; uvea; lens; retina
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INTRODUCTION |
THE EYE IS a water-transporting organ. The corneal
endothelium expels water into the anterior chamber, thereby maintaining transparency and counteracting the swelling tendencies of passive water
uptake through corneal epithelium and endothelium (11, 15). Movement of
water from the ciliary stroma across the epithelium, within the
trabecular meshwork, and across the endothelial wall into Schlemm's
canal is essential for the secretion and reabsorption of aqueous humor
(1, 32). In addition to the major drainage route provided by Schlemm's
canal, some aqueous humor leaves the eye by the uveoscleral route (1).
The iris has a high water permeability, which may facilitate rapid
changes in shape during pupillary constriction, and the iris also plays
an important role in changing the composition of the aqueous humor by
diffusion across its surface. The retinal pigment epithelium transports water toward the choroid, thereby enhancing retinal adhesion (18, 36).
Proper balance of ions and water between the cytoplasm and the
extracellular space between the lens fiber cells helps maintain lens
clarity. In addition, the functions of each cell type are dependent on
regulation of cell volume.
The driving forces and the pathways for epithelial water transport are
not completely understood (39). Molecular understandings were advanced
by the identification of aquaporin water channel proteins in the plasma
membranes of multiple epithelia, and at least six different mammalian
aquaporins (AQP) are now recognized. AQP1 is highly expressed in
several epithelia, including kidney proximal tubules and descending
thin limbs and capillary endothelium (26); in the eye, AQP1 is present
in corneal endothelium, iris, and ciliary and lens epithelia (13, 25,
34). AQP2 (8) is the vasopressin-regulated water channel and is
restricted to the kidney collecting duct (23, 31). AQP3 is expressed in the kidney collecting duct and other organs including the conjunctival epithelium (5-7). AQP4 is predominantly expressed in the brain (14, 16), where it is localized in glial cell processes at multiple
sites, including the cerebellum and supraoptic nuclei (24), as well as
ependymal cells lining the ventricles (24). AQP4 is also distributed
outside the central nervous system (7). The cDNA encoding AQP5 was
isolated from salivary glands; in situ hybridization and Northern
blotting revealed expression of the transcript in salivary glands,
corneal epithelium, and lung (29). Major intrinsic protein of the lens
(MIP) is designated AQP0 and is abundantly expressed in lens fiber
cells (37). This protein has recently been shown to be a molecular
water channel (21, 38). Recently, an extensive study using RT-PCR
demonstrated AQP1, AQP3, and AQP4 mRNAs in multiple dissected rat
ocular epithelia (27). AQP1 mRNA was found in almost all ocular
tissues, including the retina, and AQP4 was found in the retina and
ciliary body (27).
Although aquaporin transcripts and immunoreactivities have been
detected in various ocular tissues, a comprehensive analysis of eye
tissues for each member of the aquaporin family of proteins has not
been reported at the protein level. Here we utilize immunoblotting, immunocytochemistry, and immunoelectron microscopy of intact human and
rat tissues to define the cellular and subcellular distribution of
aquaporins (AQP1-AQP5) in the eye.
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MATERIALS AND METHODS |
Tissue preparation.
Human eyes were provided by the Department of Eye Pathology, University
of Copenhagen, and the Eye Department of Rigshospitalet and were fixed
in 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. Male
Wistar rats weighing 200-300 g (Møllegard Breeding Centre,
Eiby, Denmark) were allowed free access to food and water. Rat eyes,
lacrimal glands, sclera, and conjunctiva were fixed by cardiac
perfusion with 4% paraformaldehyde in 0.1 M sodium cacodylate buffer,
pH 7.2. Tissue blocks, including small blocks of human and rat eyes
trimmed from the cornea, ciliary epithelia, iris, and retina, were
further fixed by immersion for 2 h in 4% paraformaldehyde in 0.1 M
sodium cacodylate buffer, pH 7.2, infiltrated for 30 min with 2.3 M
sucrose containing 2% paraformaldehyde, mounted on holders, and
rapidly frozen in liquid nitrogen. Tissues for immunoblotting were
obtained after cardiac perfusion with PBS.
Electrophoresis and immunoblotting.
Tissue from rat eyes or cerebellum were minced finely and homogenized
in dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH
7.2, with 8.5 mM leupeptin and 1 mM phenylmethylsulfonyl fluoride) with
five strokes of a motor-driven Potter-Elvehjem homogenizer at 1,250 rpm. The homogenates were centrifuged in a Beckman L8M centrifuge at
4,000 g for 15 min at 4°C, and the supernatants were centrifuged at 200,000 g for 1 h. The high-speed pellets were
resuspended in dissecting buffer and assayed for protein concentrations
by the method of Lowry.
Membrane samples were solubilized in Laemmli sample buffer containing
2.5% SDS and loaded at 10-50 µg/lane onto 12% SDS-PAGE gels,
run on a Bio-Rad Minigel System, and transferred to nitrocellulose paper by electroelution. Blots were blocked for 1 h with 5% skim milk
in PBS-T (80 mM
Na2HPO4,
20 mM
NaH2PO4,
100 mM NaCl, 0.1% Tween 20, pH 7.5), washed with PBS-T, and then
incubated overnight at 4°C with affinity-purified antibodies in
PBS-T-0.1% BSA as follows: anti-AQP1 [0.1 µg IgG/ml (kindly
provided by Mark Knepper)], anti-rat AQP2 (23), anti-rat AQP3
[0.1-0.2 µg/ml, kindly provided by Mark Knepper
(5)], anti-rat AQP4 [0.5-2 µg IgG/ml (24, 35)], or anti-rat AQP5 (1-5 µg/ml). After they were
washed, the blots were incubated for 1 h with horseradish
peroxidase-conjugated goat anti-rabbit secondary antibody (P-448, Dako;
1:3,000). After a final wash, antibody binding was visualized using the
enhanced chemiluminescence system (Amersham). Controls, which were
prepared with nonimmune IgG or omission of primary or secondary
antibody, revealed no labeling.
An antibody to human AQP4 was prepared using exactly the same
procedures used for the production of anti-rat AQP2, AQP3, AQP4, and
AQP5. A peptide corresponding to the 15 COOH-terminal amino acids
(GenBank g1680707) with an
NH2-terminal cysteine was
conjugated to keyhole limpet hemocyanin and used for immunization in
rabbits. The antibody was affinity purified using methods described
previously (5).
Immunofluorescence and immunoperoxidase cytochemistry.
The procedures have been described previously (23, 26). For light
microscopy, paraffin sections of whole human eyes (2-4 µm thick
prepared using standard techniques) or cryosections of different ocular
epithelia obtained with a Reichert-Jung cryoultramicrotome (0.85 µm
thick) were used. Cryosections or paraffin sections were placed on
gelatin-coated glass slides. After preincubation with PBS containing
1% BSA or 0.1% skim milk and 0.05 M glycine, the sections were
incubated with anti-aquaporin antibodies. The use of the
affinity-purified antibodies against aquaporins for immunocytochemistry has previously been described (referenced above for each antibody): anti-AQP1 (0.1-0.2 µg IgG/ml), anti-AQP2 (0.1-0.8 µg/ml),
anti-AQP3 (0.5 µg/ml), anti-AQP4 (1-5 µg/ml), and anti-AQP5
(5-20 µg/ml). The labeling was visualized by use of
peroxidase-conjugated secondary antibody (P-448, Dako; 1:100). Sections
were counterstained with Meier reagent (an unspecific nuclear stain).
For fluorescence microscopy, the sections were incubated with anti-AQP1
antibodies, washed, incubated with goat anti-rabbit conjugated to FITC
(Z-205, Dako; 1:40), and finally washed and mounted with
Glycergel, as previously described (5, 35). The specimens
were studied in a Leitz Laborlux S fluorescence microscope.
Immunoelectron microscopy.
Tissue blocks were subjected to freeze substitution and embedded in
Lowicryl HM-20 by use of procedures previously described (24). The
frozen samples were freeze substituted in a Reichert auto
freeze-substitution unit (Reichert, Vienna, Austria). Samples were
sequentially equilibrated over 3 days in 0.5% uranyl acetate in
methanol at temperatures gradually increasing from
80 to
70°C, rinsed in pure methanol for 24 h at
70 to
45°C, and infiltrated at
45°C with Lowicryl HM-20
and methanol at 1:1, 2:1, and finally pure HM-20 (1 day in each
solution) before ultraviolet polymerization in pure HM-20 for 2 days at
45°C and 2 days at 0°C. Immunolabeling was performed on
ultrathin sections (40-60 nm) incubated with affinity-purified
aquaporin antibodies (described above) and visualized with
goat-anti-rabbit IgG conjugated to 10-nm colloidal gold particles (GAR.EM10, BioCell Research Laboratories, Cardiff, UK). Sections were
stained for 10 min with uranyl acetate and examined in an electron
microscope (model CM100, Philips).
The following controls confirmed specificity of light- and
electron-microscopic immunolabelings:
1) incubation with nonimmune rabbit
IgG and 2) incubation without
primary antibody or secondary antibody.
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RESULTS |
Immunoblotting of AQP1, AQP4, and AQP5 in membranes from the rat
eye.
Specificity of our antibodies for rat ocular tissues was confirmed by
immunoblotting isolated membranes. AQP1 immunolabeling is abundant in
membranes from the cornea and ciliary body. At this exposure no signals
were obtained in membrane fractions from the retina (Fig.
1A),
although higher exposures revealed a weak signal (not shown). Strong
AQP4 immunolabeling is apparent in membranes from the retina, with the
29-kDa band migrating at the same level as in control membranes from
rat cerebellum (Fig. 1B), a site
with abundant AQP4 expression (7, 16, 24). A weak AQP4 signal is also
observed in membranes from the ciliary body. AQP5 immunolabeling is
strong in membranes prepared from the cornea (Fig.
1C), whereas none appeared in
membranes from the ciliary body or retina. Controls prepared with
nonimmune IgG revealed no labeling (Fig.
1D). On the basis of these
immunoblots, immunocytochemical and immunoelectron-microscopic analyses
were performed on the three layers of rat and human eye: fibrous
tunica, uvea, and retina.

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Fig. 1.
Immunoblots of membrane fractions isolated from various rat ocular
tissues (20 µg membrane protein/lane).
A: immunoblot reacted with
affinity-purified anti-aquaporin 1 (anti-AQP1). Antibody selectively
recognizes 28-kDa nonglycosylated and 35- to 50-kDa glycosylated
moieties of AQP1 in membrane fractions of cornea (cor) and ciliary body
(cil). B: immunoblot reacted with
affinity-purified anti-AQP4; 29-kDa band and higher-molecular-weight
band are seen in membranes from cerebellum (cer; control tissue) and in
retina (ret). C: immunoblot reacted
with affinity-purified anti-AQP5; 28-kDa AQP5 band is seen exclusively
in membrane fractions from cornea. D:
immunolabeling of control, where nonimmune IgG used as primary antibody
revealed no labeling.
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Immunolocalization of AQP1, AQP3, and AQP5 in fibrous tunica and
external tissues.
Corneal keratocytes react strongly with anti-AQP1 (Fig.
2,
a and
b), whereas no AQP1 was observed in
the corneal epithelium in the rat eye (Fig.
2a), and controls confirmed the lack
of nonspecific labeling (Fig. 2a,
inset). The apical and basolateral membranes of the
corneal endothelium are also strongly labeled with anti-AQP1 in the rat
(Fig. 2b) and human (Fig.
2b, inset) eye. The corneal epithelium exhibited only modest labeling with anti-AQP3 (Fig. 2c) and none with anti-AQP4 (Fig.
2d), whereas significant AQP5 immunolabeling is present (Fig. 2e).
When probed with anti-AQP3, anti-AQP4, or anti-AQP5, corneal
keratocytes failed to label (Fig. 2,
c-e).

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Fig. 2.
Immunocytochemical localization of aquaporins in fibrous tunica
(a-i) of rat eye and rat
lacrimal gland (j). Original
magnification ×430. a-e are
from cornea, f and
g from conjunctiva,
h from limbal area, and
i from sclera. In
a, arrows indicate AQP1-immunolabeled
keratocytes. Corneal epithelium (EP) did not display AQP1
immunolabeling. Inset: immunolabeling
control. b: AQP1 immunolabeling of
keratocytes (arrows) and corneal endothelium (EN; arrowheads).
Inset: immunofluorescence microscopy
showing AQP1 immunolabeling of keratocytes and endothelium of human
cornea. In c, plasma membranes of
corneal epithelial cells exhibit weak AQP3 immunolabeling (arrows),
with no labeling of corneal stroma (). In
d, corneal epithelium exhibits no AQP4
immunolabeling. , Stroma. In
e, plasma membranes of corneal
epithelium exhibit abundant AQP5 immunolabeling (arrows). ,
Stroma. Inset: AQP5 immunolabeling of
corneal epithelium at low magnification. In
f, sections of conjunctival epithelium
exhibit extensive AQP3 immunolabeling, with no labeling of underlying
stroma (CS). In g, no expression of
AQP4 is detected in conjunctival epithelium or stroma. In
h, limbal area contains abundant AQP1
in scleral cells (S), ciliary epithelia, epithelia of iris (I), and
aqueous outflow apparatus; endothelium of trabecular meshwork (T),
including uveal meshwork (U); and endothelium in Schlemm's canal
(). In contrast, there is no AQP1 immunostaining of stroma of
ciliary body (CB) or conjunctival epithelium. In
i, scleral fibroblasts exhibit
significant AQP1 immunolabeling (arrows). In
j, lacrimal gland acini exhibit AQP5
immunolabeling of apical plasma membrane (arrows), but not basolateral
membranes (arrowheads).
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The epithelium of the bulbar conjunctiva shows very strong labeling,
with anti-AQP3 localized over the basolateral plasma membranes;
however, no AQP3 immunolabeling was observed in conjunctival stroma
(Fig. 2f). In contrast, neither AQP4
(Fig. 2g) nor AQP5 immunolabeling of
bulbar conjunctival epithelium or stroma was observed (not shown).
Similar to submandibular glands, lacrimal glands revealed AQP5
immunolabeling restricted to the apical membrane (Fig.
2j), whereas antibodies specific for
AQP1, AQP3, and AQP4 failed to react (not shown). AQP1 immunolabeling
is prominent in the endothelium covering the trabebular meshwork,
including the uveal meshwork and endothelium of Schlemm's canal (Fig.
2h). Anti-AQP1 also labels scleral
fibroblasts (Fig. 2i), whereas no AQP3, AQP4, or AQP5 immunolabeling of scleral fibroblasts was observed
(not shown). Immunoelectron microscopy confirmed that AQP1 is
specifically associated with scleral fibroblast plasma membranes but
not with adjacent collagen fiber bundles (Fig.
3).

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Fig. 3.
Immunoelectron micrograph of AQP1 in a scleral stroma cell. Fibroblast
(F) shows abundant AQP1 immunolabeling over plasma membranes
(arrows). C, closely packed fibrils of collagen.
Original magnification ×80,000.
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Immunolocalization of AQP1 and AQP4 in the uvea.
Immunocytochemistry of the pars plicata of the human ciliary body
reveals strong AQP1 immunolabeling of the internal nonpigmented epithelium, including basal infoldings and lateral plasma membranes (Fig.
4a),
whereas controls showed no specific labeling (Fig. 4b). Although it is clear that there
is no labeling of the basolateral portion of the ciliary pigmented
epithelial cells, melanized pigment granules in the apical portion of
the ciliary pigmented epithelial cells prevent unambiguous
interpretation of immunoperoxidase labeling of the apical membranes. To
resolve this issue, immunofluorescence microscopy was performed
revealing anti-AQP1 labeling of the apical nonpigmented epithelial
plasma membranes (Fig. 4, c and
d). Although autofluorescence
signals were emitted by small granules in the nonpigmented epithelial
cells, no AQP1 immunolabeling was seen in the cytoplasm (Fig. 4,
b and
c). In contrast to the strong immunolabeling of the internal nonpigmented epithelium, anti-AQP1 labeling was not observed in the heavily pigmented external epithelium (CPE, Fig. 4, a, c, and
d). The ciliary stroma was
unlabeled, but the endothelium of capillaries and postcapillary venules
in the stroma exhibit strong AQP1 immunolabeling (Fig.
4d). Immunoelectron microscopy of
ciliary nonpigmented epithelium in the pars plicata confirmed that AQP1
immunolabeling is abundant in basolateral plasma membranes (Fig.
5a) as
well as in apical plasma membranes facing the unlabeled external
ciliary epithelium (Fig. 5b).

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Fig. 4.
Immunocytochemical localization of AQP1 in uveal tract.
a-d are from human ciliary body,
e from human iris, and
f from rat choroid. Original
magnification ×430. In a,
paraffin section of ciliary body shows AQP1 immunolabeling of
basolateral membranes (arrows) of ciliary nonpigmented epithelium
(NPE), but no labeling of external ciliary pigmented epithelium (CPE).
In b, parallel to
a, immunolabeling control shows no
labeling. Brown spots represent melanin granules. In
c, immunofluorescence microscopy of
paraffin section demonstrates AQP1 immunolabeling of basolateral and
apical NPE plasma membranes (arrows) facing unlabeled CPE (arrowhead).
In d, immunofluorescence labeling of a
semithin cryosection shows labeling of plasma membranes of NPE cells
(arrows). Endothelial cells of vessels (V) show significant labeling.
Autofluorescence signals appear over small granules within NPE cells.
In e, a section from iris reveals
significant AQP1 immunolabeling (arrows) of posterior pigmented
epithelium (PE) as well as anterior epithelium (AE). In
f, sections of choroid show AQP1
immunolabeling of choriocapillary endothelium (arrows) with no staining
of retinal pigment epithelium (RPE) or rod-and-cone layer (RCL).
Inset: immunolabeling control, where
nonimmune IgG was used as primary antibody.
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Fig. 5.
Immunoelectron micrograph of AQP1 in pars plicata of rat ciliary NPE.
In a, extensive AQP1 immunolabeling
appears over basolateral plasma membranes (arrows). ILM, inner limiting
membrane; M, mitochondrion; N, nucleus. In
b, extensive AQP1 immunolabeling of
apical plasma membranes extends along cytoplasmic processes (P)
projecting from apical surface into intercellular spaces (ICS).
Original magnification ×85,000.
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Immunoblotting revealed distinct AQP4 labeling in membrane fractions
prepared from dissected ciliary body (Fig. 1). Consistent with this
finding, immunocytochemistry revealed low but distinct labeling of AQP4
in the nonpigmented ciliary epithelium (Fig. 6). These data are consistent with the
previous identification of AQP4 mRNA at this site (27).

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Fig. 6.
Immunocytochemical localization of AQP4 in cryostat sections of
immersion-fixed rat ciliary body (from rat strain with pigment in
ciliary pigmented epithelium). Original magnification ×190. In
a, cryostat section of ciliary body
shows AQP4 immunolabeling of basolateral and apical membranes (large
arrows) of ciliary NPE, but no labeling of CPE (small arrows). Large
open arrow, initiation of retina. b:
Parallel view using interference microscopy.
c: Immunolabeling control performed
without primary antibody on a cryostat section from ciliary body.
Background labeling (produced by secondary antibody) is seen in deeper
portions of ciliary body, whereas no staining is found in ciliary
epithelium, confirming selective AQP4 labeling demonstrated in
a. Arrows as described in
a.
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Sections of rat and human iris revealed strong AQP1 immunolabeling in
the posterior pigmented epithelium and the anterior epithelium. Apical
and basolateral plasma membranes are labeled by immunofluorescence
microscopy (Fig. 4c) and
immunoelectron microscopy (not shown). No AQP3 or AQP5 immunolabeling
of ciliary or iris epithelium was observed (not shown). Consistent with
labeling of nonfenestrated capillary endothelium outside the central
nervous system (3, 25), the choriocapillary endothelium exhibited notable AQP1 immunolabeling (Fig.
4f), whereas controls showed a lack
of nonspecific labeling (Fig. 4f,
inset). AQP1 immunolabeling of the endothelium was
further confirmed by immunoelectron microscopy (not shown).
Immunolocalization of AQP4 in the
retina.1
Immunoperoxidase cytochemistry in paraffin sections revealed no
anti-AQP1 labeling of cells in the layers in the neuronal part of rat
retina (layers 2-10) or in the
retinal pigment epithelium (Fig.
7a, layer
1) but distinct labeling of scleral cells. However, use of thin cryosections revealed very low AQP1 labeling in the outer
nuclear layer (Fig. 7a, inset),
presumably of glial elements. The labeling was weak, and it was not
possible with use of standard techniques to obtain significant labeling
at the electron-microscopic level (not shown). In contrast,
immunocytochemistry revealed very abundant AQP4 labeling of
Müller cells and astrocytes (Fig. 7, b-d) extending from the outer
limiting membrane (Fig. 7c, layer 3)
throughout the retina, as demonstrated by immunoperoxidase (Fig. 7,
layers 3-7) and by
immunoelectron microscopy (Figs. 8 and 9). Also, glial
processes in layers 8 and
9 exhibit similar anti-AQP4 labeling
(not shown). Higher magnification reveals strong AQP4 immunolabeling of
Müller cell processes surrounding capillaries (Fig.
7d, arrows) and weaker but significant
labeling of glial processes surrounding neurons (Fig. 7,
b and
d, arrowheads). Very prominent
labeling is seen in the inner plexiform layer (layer 7); significant immunolabeling is also seen in the
inner nuclear layer (layer 6), where
Müller cell bodies reside. All AQP4 immunolabeling controls were
negative (Fig. 7c, inset). In
addition, anti-AQP4 failed to label retinal pigment epithelium or the
rod-and-cone layer (Fig. 7, b and
c, layers 1 and
2), and no anti-AQP3 or anti-AQP5 labeling of the retina was observed (not shown). Immunoelectron microcopy reveals abundant AQP4 immunolabeling by plasma membranes of
Müller cell processes in the inner limiting membrane (lower magnification, Fig.
8a; higher
magnification, Fig. 8, b and
c). Also, perivascular Müller
cell processes exhibit extensive anti-AQP4 labeling, whereas retinal
capillary endothelium and neurons showed no labeling (Fig.
9). Thin cryosections of human retina
labeled with affinity-purified anti-human AQP4 revealed a similar
labeling pattern, with specific labeling of astroglial cells (data not shown).

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Fig. 7.
Immunocytochemical localization of aquaporins in retina of rat eye
(a-d) and human lens
(e). Original magnification
×430 unless otherwise noted. Retinal layers are as follows:
retinal pigment epithelium (1),
rod-and-cone layer (2), outer
limiting membrane (3), outer nuclear
layer (4), outer plexiform layer
(5), inner nuclear layer
(6), inner plexiform layer
(7), ganglion cell layer
(8), nerve fiber layer
(9), and inner limiting membrane
(10).
a: Paraffin section of rat retina.
AQP1 immunolabeling is present in choroid (CH) and in sclera (S) but
weak or absent in layers of retina.
Inset: AQP1 immunolabeling using thin
cryosections. In b, anti-AQP4 shows
extensive labeling of Müller cells (large arrows and arrowheads).
C, capillary. AQP4-labeled astrocytes (arrowhead) are abundant in inner
plexiform layer and ganglion cell layer. AQP4 immunostaining of
Müller cells commences in outer limiting membrane at apexes and
continues internally with staining in outer nuclear layer and outer
plexiform layer. c: Distinct AQP4
immunolabeling of Müller cells (arrows) in outer limiting
membrane, outer nuclear layer, and outer plexiform layer.
Inset: immunolabeling control. In
d, a higher magnification of part of
b shows distinct AQP4 immunolabeling
of Müller cells in outer plexiform layer, consistent with known
existence of many branching processes of Müller cells in this
layer. Capillaries (C) are seen in layers
5-7 surrounded by heavily AQP4-stained
perivascular Müller cell processes (small arrows). Large arrows,
Müller cells; arrowheads, astrocytes. Note striking
light-microscopic difference between these 2 types of retinal glia.
Original magnification ×860. e:
Cryosection (0.85 µm thick) of human lens. AQP1 labeling is abundant
in apical and basolateral membranes of lens epithelium (EP). No AQP1
labeling is seen in lens capsule (CA) or lens fiber cells (LC).
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Fig. 8.
Immunoelectron micrographs of AQP4 in Müller cells in inner rat
retina. a: 2 adjacent Müller
cells, their basement membrane (the inner limiting membrane), and
vitreous. Original magnification ×80,000.
b and
c: higher magnifications of
a. Original magnification
×110,000. In a-c, extensive
AQP4 immunogold labeling (arrows) of Müller cell (M) plasma
membranes is shown. Basement membrane (B) is closely related to fibrils
in vitreous (V).
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Fig. 9.
Immunoelectron micrographs showing AQP4 immunolabeling of retinal
perivascular glia (perivascular Müller cells).
a: Thin glial process with dense AQP4
staining of plasma membrane. Original magnification ×80,000.
b: Thicker portion of a glial cell
process with prominent AQP4 staining. Original magnification
×80,000. c: Higher magnification
showing association of immunogold particles with plasma membrane of
glial process. Original magnification ×100,000. G, perivascular
glial cell; P, pericyte; E, endothelial cell; L, capillary lumen.
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Immunolocalization of AQP1 in the lens.
Anterior lens epithelium was strongly labeled by anti-AQP1. Apical and
basolateral plasma membrane domains are immunolabeled, but lens fiber
cells and lens capsule did not label (Fig.
7e). No labeling of the lens was
produced with the antibodies specific for AQP3, AQP4, or AQP5 (not
shown).
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DISCUSSION |
In this study we report the cellular and subcellular localization of
multiple members of the aquaporin family of membrane water channels in
ocular tissues (Fig. 10). We provide
novel information about the distinct expression patterns for each
aquaporin, including the presence of AQP4 in Müller cells and
astrocytes with a polarized distribution, the expression of AQP4 in
ciliary nonpigmented epithelium, the presence of AQP5 and AQP3 in
corneal epithelium, the presence of AQP3 in basolateral membranes of
conjunctival epithelium, and the presence of AQP5 in apical domains of
lacrimal gland epithelium; moreover, AQP1 was shown to be abundant in
scleral cells in addition to the known broad expression in multiple
ocular tissues. Furthermore, we defined the absence or low expression
of aquaporin proteins in certain tissues. The studies were performed in
rat and human tissues at light- and electron-microscopic levels.
Together with previous data, we conclude that each of these aquaporins
is expressed at distinct sites in the eye, predicting specific roles at
each site.
AQP3 was not found in intraocular epithelia but is very abundant in
basolateral plasma membranes of conjunctival epithelium, and AQP5 is
abundant in the apical domain of lacrimal acinus cells and in corneal
epithelial cells. The participation of AQP3 and AQP5 in tear formation
and external moistening is suspected. Naturally occurring mutations in
genes encoding AQP3 and AQP5 are not known. AQP1 is present in corneal
endothelium and corneal keratocytes, consistent with a role in
maintenance of corneal hydration, which is critical for corneal
transparency and refraction.
AQP1 is present in the nonpigmented ciliary and iris epithelium,
suggesting a role in aqueous humor secretion. AQP1 is also present in
the endothelium covering Schlemm's canal, scleral stromal cells, and
capillary endothelium, tissues that are involved in aqueous humor
drainage. AQP1 in the anterior epithelium of the lens suggests
participation in maintaining optical refraction of the lens. Although
humans with knockout mutations in AQP1 do not show a severe clinical
phenotype, their extreme rarity suggests that they may bear a
compensating mutation (28). In contrast, MIP is known to be an
aquaporin (AQP0), and naturally occurring mouse mutations result in
congenital cataracts (33).
Abundant expression of AQP4 is noted in glial elements in the retina,
including Müller cells, with extensive expression in processes
facing the capillary endothelium or the inner limiting membrane facing
the vitreous body. Thus the AQP4 expression pattern in the retina is
very similar to that in the brain (24). Although the role of AQP4 in
glial cells in the retina and brain remains undefined, it may be
speculated that AQP4 participates in volume regulation in response to
potassium siphoning during synaptic transmission. We have no clue from
genetics, since AQP4 mutants are not yet known. Very low levels of AQP1
labeling were also found in nuclear layers of the retina. These
observations are consistent with a previous report by Patil et al.
(27), who found significant levels of AQP4 and AQP1 mRNA using RT-PCR
of dissected retina. The cells expressing low levels of AQP1 are presumably glial cells, since AQP1 has consistently been found to be
absent from neurons elsewhere (13, 24, 25).
Aquaporins in the cornea.
The abundant expression of AQP1 in corneal endothelium (Fig.
2b) indicates a role of AQP1 in the
water transport necessary to prevent swelling and thereby maintain
transparency. The corneal endothelium (but not the corneal epithelium)
is well established to be essential for maintaining corneal
transparency (4, 15, 20). Active mechanisms are thought to involve
Na+-K+-ATPase
and a bicarbonate-dependent
Mg2+-ATPase (reviewed in Ref. 15).
Inhibition of
Na+-K+-ATPase
activity with ouabain is known to produce marked swelling of the cornea
(9), suggesting that the enzyme is responsible for an outward transport
of salt from the cornea into the aqueous humor. Nevertheless, the
Na+-K+-ATPase
has been localized to the basolateral plasma membranes facing the
stroma, not the anterior chamber (19). If this is the predominant
ouabain-inhibitable ATPase, then the pumping direction appears to be
the reverse of the orientation found in the kidney proximal tubule.
Thus it remains to be established whether the functional inhibition
with ouabain is caused by this basolateral Na+-K+-ATPase
or by another not yet defined ouabain-inhibitable transporter. The
water transport secondary to the gradient established by active ouabain-sensitive salt transport is likely to take place via AQP1, which is abundant in apical and basolateral plasma membranes.
AQP5 is expressed in the plasma membranes of the stratified squamous
corneal epithelium (Fig. 2e). This
epithelium is unique, in that it provides a moist apical surface, which
is the major refractive surface of eye. Its location at the surface of
the translucent cornea further requires that the epithelium must be transparent (11). The current knowledge of the transport capabilities of the corneal epithelium is very limited, and this epithelium has been
considered to represent a barrier for water transport. It may be
speculated that a potential role of AQP5, and possibly AQP3, in the
epithelium, acting in a concerted fashion with AQP1 in the corneal
keratocytes and AQP1 in corneal endothelium, may ensure that water can
be transported rapidly, preventing the formation of gradients within
subcompartments of the cornea that would cause swelling and reduce
transparency. Other potential roles of AQP5 in the corneal epithelium
may include 1) a mechanism for
prompt cell volume regulation to prevent cell swelling,
2) an external port that may
increase hydration of the corneal surface by allowing water to replace
evaporation from the eye surface, or
3) an importer allowing water to
enter from the exterior. Similar roles of AQP1 in keratocytes can be
hypothesized. The abundant expression of aquaporins in the cornea and
in lens epithelium indicates a critical role of aquaporins in
maintaining constant hydration without swelling of the refractive optic
elements of the eye.
Aquaporins in the ciliary epithelium.
As demonstrated in Figs. 4 and 5, AQP1 is abundantly expressed in the
internal nonpigmented epithelium (Fig. 4) of ciliary tissue. In
contrast, no labeling of the heavily pigmented external epithelium was
observed (Fig. 4, a, c, and
d). Immunoelectron microscopy
confirmed the abundant labeling of apical and basolateral plasma
membranes of the nonpigmented epithelium (Fig. 5). A weak expression of
AQP4 was also noted (Fig. 6), indicating that AQP4 may also participate
in conjunction with AQP1. The ciliary body has a major function in
production of aqueous humor (32). The two layers of epithelia are
apposed apex to apex, and the pigmented epithelium represents the
forward continuation of the retinal pigment epithelium, whereas the
nonpigmented epithelium represents a forward continuation of the neural
retina. Although evidence suggests that the nonpigmented epithelium
(which also expresses AQP1) is largely responsible for aqueous humor
production, the double-layered epithelium may very well act as a
functional unit (for a comprehensive review see Ref. 32). The ciliary
epithelium is also the site for enzymes that are known to participate
intimately in the rate of aqueous humor production, including
Na+-K+-ATPase,
adenylyl cyclase (in regulating aqueous humor production), and carbonic
anhydrase (32). Notably the nonpigmented epithelium is the site for the
major fraction of
Na+-K+-ATPase
activity, as demonstrated biochemically after separation of bovine
pigmented and nonpigmented epithelia in density gradients (30).
Immunocytochemistry has also shown abundant labeling of
1-,
2-, and
3-subunits of the
Na+-K+-ATPase
in the basolateral plasma membranes of ciliary nonpigmented epithelial
cells, whereas only the
1-subunit was found in the pigmented ciliary epithelial cells (10). Importantly, there appears to
be a close inverse correlation between aqueous humor production rates
and ouabain inhibition of
Na+-K+-ATPase
activity (2). Thus a major driving force for
Na+ transport, and hence water
transport, is likely to be established mainly in the nonpigmented
epithelium. The presence of AQP1 in the nonpigmented epithelium is
therefore consistent with a distinct role of AQP1 in aqueous humor
production. The expression pattern shares similarities with that in the
kidney proximal tubule, with abundant AQP1 in apical and basolateral
plasma membranes and abundant Na+-K+-ATPase
in basolateral plasma membranes (31). Both epithelia are responsible
for near-isosmotic water reabsorption, with release of water at the
basolateral aspect of the cells. Whether AQP1 is essential for aqueous
humor production or just provides an energetically favorable pathway
for water transport is unknown and awaits experiments in mice with
disruption of the Aqp1 gene. Very
recently, Aqp1 knockout mice were
produced, which in response to thirsting demonstrated a critical role
of AQP1 in the kidney for urinary concentration (A. S. Verkman et al.,
unpublished observations). Potential defects in other organs including
the eye await further studies.
Aqueous humor drainage takes place via Schlemm's canal and via flow of
aqueous humor into the uvea and sclera. As shown in Fig. 2, significant
AQP1 labeling was found in 1) the
endothelial cells lining Schlemm's canal,
2) the endothelial cells associated with the uveal meshwork, and 3) the
corneoscleral meshwork. AQP1 is also abundant in keratocytes and
scleral stromal cells. The exact role of AQP1 at these sites remains
unknown, but the labeling of endothelial cells (lining Schlemm's canal
and the meshwork) may be comparable to the labeling of capillary and
venule endothelial cells (Fig. 4d)
and corneal endothelium (Fig. 2b).
The presence of AQP1 may provide these cells with a high water
permeability and allow water to be transported across these barriers
efficiently and drained into scleral or uveal capillaries. For example,
the corneoscleral meshwork consists of several interconnecting sheets extending toward the cornea. These sheets contain holes, but the number
of such openings is limited, so aqueous humor would have to be
transported laterally for certain distances to pass from one hole to
another. It remains possible that AQP1 may provide a more efficient
pathway for water. With respect to the role of AQP1 in capillaries,
recent data indicate that AQP1 probably does not play a major role in
transendothelial water transport, such as in the descending vasa recta
in the kidney medulla. However, aquaporins may play a role in certain
conditions where driving forces other than Starling (nonosmotic) forces
are acting. This is exemplified in a recent study showing that ~50%
of water transported during peritoneal dialysis is likely to involve
mercury-sensitive water channels in capillaries known to express AQP1
abundantly (3).
Lack of AQP1, AQP3, AQP4, and AQP5 expression in the retinal pigment
epithelium.
The retinal pigment epithelium is responsible for bulk water transport
out of the eye into the choroidal blood circulation. This epithelium
does not express any of the mammalian aquaporins cloned so far.
Although a noncharacterized aquaporin may be expressed at this site,
other routes for water transport are likely to exist. Recently, Loo et
al. (17) demonstrated that cotransporters, such as the sodium-coupled
glucose transporter, may carry water along with their primary
substrates. Thus, in addition to aquaporin-mediated water transport,
other routes may exist in the retinal pigment epithelium. Indeed,
extensive physiological and biophysical experiments have documented the
presence of several different cotransporters in the retinal pigment
epithelium. These include identification of cotransport of
H+, lactate, and water in a
membrane protein positioned in the apical plasma membrane of the
retinal pigment epithelium of bullfrog eye (40) and human eye (12).
Although direct evidence for the importance of this system is lacking,
it appears likely that these cotransporters may contribute to transport
of water across distinct ocular epithelia, which is under investigation
in these laboratories.
Aquaporin expression in retinal glial cells.
We report abundant expression of AQP4 in the processes of glial cells
including Müller cells. As recently reviewed (22), Müller
cells represent the principal glial cell of the retina and accomplish
multiple functions. Müller cells express voltage-gated channels
and neurotransmitter receptors, and they modulate neuronal activity by
regulating the extracellular content of neuroactive substances
including K+, glutamate, GABA, and
H+. In the human retina the glial
cells are divided into Müller cells, astrocytes, and microglia.
However, in the rat eye, glial elements are comprised only of
Müller cells and astrocytes, with astrocytes present exclusively
in the innermost layers of the retina. Retinal blood vessels are
entirely surrounded by Müller cell end-feet, except in the inner
portion of the retina, where astrocyte end-feet also line the
capillaries. The end-feet in contact with the capillary endothelium can
release K+, acid equivalents, and
water (22). As shown in the present study, AQP4 is extensively
expressed in the glial processes facing retinal capillaries. This would
be consistent with a role of AQP4 in water release to the capillaries,
thereby contributing to maintenance of the extracellular osmolality
during neuronal activity. AQP4 may also be hypothetically involved in
K+ siphoning, a role that has
recently been discussed in relation to the extensive polarized
expression of AQP4 in glial processes in the cerebellum and elsewhere
in the brain (24).
 |
ACKNOWLEDGEMENTS |
The authors thank Hanne Weiling, Trine Møller, and Mette
Vistisen for expert technical assistance.
 |
FOOTNOTES |
This study was supported by the Novo Nordisk Foundation,
Landsforeningen til Bekæmpelse af Øjensygdomme og Blindhed, the
Carlsberg Foundation, the Karen Elise Jensen Foundation, the Danish
Medical Research Council, the University of Aarhus Research Foundation, the University of Aarhus, and National Institutes of Health Grants EY-11239, HL-33991, and HL-48268.
1
The observation of AQP4 expression in glial
cells of the retina including Müller cells was done in parallel
in three laboratories, including our laboratories at the University of
Aarhus, the University of Oslo, and the laboratory of Evelyn Ralston at
the National Institutes of Health (Bethesda, MD).
Address for reprint requests: S. Nielsen, Dept. of Cell Biology,
Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark.
Received 19 May 1997; accepted in final form 13 January 1998.
 |
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