Pflugers Arch - Eur J Physiol (2007) 453:777–785

DOI 10.1007/s00424-006-0157-3

CELL AND MOLECULAR PHYSIOLOGY

Stimulation of aquaporin-5 and transepithelial water

permeability in human airway epithelium
by hyperosmotic stress
Peter Steen Pedersen & Thomas Hartig Braunstein &
Anders Jørgensen & Per Leganger Larsen &
Niels-Henrik Holstein-Rathlou & Ole Frederiksen

Received: 30 April 2006 / Revised: 25 July 2006 / Accepted: 10 August 2006 / Published online: 17 October 2006
# Springer-Verlag 2006

Abstract Osmotic water permeability (Pf) was measured in
spheroid-shaped human nasal airway epithelial explants
pre-exposed to increasing levels of hyperosmotic stress.
The fluid-filled spheroids, derived from nasal polyps, were
lined by a single cell layer with the ciliated apical cell
membrane facing the outside. The Pf was determined from
diameter changes of the spheroids in response to changes in
bathing medium osmolarity forth and back between 300
and 225 mOsm·l−1. Continuous diameter measurements
also allowed determination of spontaneous fluid absorption.
Hyperosmotic pretreatment (increase from 300 up to
600 mOsm·l−1) caused a time- and osmolarity-dependent
increase (up to ∼1.5 times) in epithelial Pf which was of
similar magnitude in cystic fibrosis (CF) and non-CF
spheroids. The effect saturated at ∼450 mOsm·l−1 and at
∼24 h. Expression of aquaporin-5 (AQP5), studied by
immunofluorescence and confocal microscopy, showed an
increase in parallel with the increase in Pf following
hyperosmotic stress. The AQP5 was localized both in
cytoplasmic vesicles and in apical cell membranes. Spon-
P. S. Pedersen (*)
Department of Clinical Genetics, Rigshospitalet,
University of Copenhagen,
Copenhagen DK2100, Denmark
e-mail: psp@mfi.ku.dk
P. S. Pedersen : T. H. Braunstein : A. Jørgensen :
N.-H. Holstein-Rathlou : O. Frederiksen
Department of Medical Physiology, The Panum Institute,
University of Copenhagen,
Copenhagen DK2200, Denmark
P. L. Larsen
Department of Otolaryngology, Head and Neck Surgery,
Rigshospitalet, University of Copenhagen,
Copenhagen DK2100, Denmark
taneous fluid absorption rates were equal in CF and non-CF
spheroids and were not significantly influenced by hyper-
osmotic stress. The results suggest that hyperosmotic stress
is an important activator of AQP-5 in human airway
epithelium, leading to significantly increased transepithelial
water permeability.
Keywords Human airway epithelia . Epithelial spheroids .
Cystic fibrosis . Water permeability . Fluid absorption .
Aquaporin-5 . Hyperosmotic stress
Introduction
Regulation of transepithelial water flow across the airway
epithelium is critical for the maintenance of the airway
surface liquid (ASL) volume, which influences the muco-
ciliary clearance [2]. The ASL receives fluid from distal
airways and from transient local secretion from submucosal
glands. Reduction in the volume of ASL is achieved by
evaporation, by fluid absorption across the surface epithe-
lium and by mucociliary transport of ASL in the oral
direction. Notably, changes in ASL volume and composi-
tion have been implicated in the pathophysiology of cystic
fibrosis (CF) [3], emphasizing the importance of the fluid
transport properties of the airways. Osmotic equilibration
across the epithelium is maintained by movement of water
depending on the degree of active ion transports in the
surface epithelium, glandular secretion and evaporation-
induced changes in ASL tonicity. Under normal physio-
logical conditions, the tonicity of ASL is not above
300 mOsm. However, during periods of increased venti-
lation, especially when breathing dry air, the ASL
osmolarity may exceed this limit. Thus, maximal recorded
osmolarities in the lower airways may reach ∼450 mOsm
778
[10]. Furthermore, hypertonic NaCl inhalation therapy has
been reported to be beneficial for CF patients [11]. It
therefore seems highly relevant to investigate the regulation
of airway epithelial water permeability (Pf) under luminal
hyperosmotic conditions.
Airway transepithelial water flow is favoured by a
relatively high Pf (30–220 μm/s [5, 9, 12, 13, 25, 31, 32,
39, 40]) at a level that indicates the engagement of
molecular water channels aquaporins (AQPs; [39]). Four
AQPs (AQP1, 3, 4 and 5) are expressed in the respiratory
tract [21–23, 27, 29, 37], but only AQP5 is localized to the
apical cell membrane in human airway epithelia [21, 22],
including the tissue used for the present study. In 1998 Jenq
et al. [20] reported that the expression of AQP1 in mouse
IMCD-3 cells was increased at both mRNA and protein
levels in response to a period of hyperosmotic stress. Since
then, similar phenomena have been reported for AQP1
[24, 37, 38], AQP3 [28, 37], AQP4 [1], AQP5 [18, 19] and
AQP9 [1] in established cell lines from kidney, muscle,
fibroblasts and lung [18–20, 24, 28]; in primary cultures
from rat astrocytes and human keratinocytes [1, 36]; and in
in vivo studies performed on rat with subsequent analysis
of AQP in tissues from lung, salivary glands, lacrimal
glands and brain cortex [1, 18]. Although hyperosmotic
stress in general leads to a decrease in total RNA synthesis
in mammalian cells [8], some up-regulation of a limited
number of genes occurs [4, 8], including activation of heat
shock transcription factor 1 and the mitogen-activated
protein (MAP) kinase pathways [14, 16, 25]. Another
mechanism resulting in a net increase in AQP expression in
response to hyperosmotic stress was demonstrated by
Leitch et al. [24] who observed decreased AQP1 ubiqui-
tination leading to an increase in AQP1 protein stability and
subsequent higher expression of AQP1. Finally, an
increased translocation of AQP to the cell membrane was
demonstrated in two of the hyperosmotic stress studies
(AQP1 [20] and AQP3 [28]). Thus, hyperosmotic stress
may act on both transcriptional and post-transcriptional
levels, both leading to an increased expression of AQPs.
The study reported here examines the relationship
between hyperosmotic stress, expression of AQP5 and
increased levels of transepithelial Pf in a sphere-shaped
explant preparation of human airway epithelium from the
surface of nasal polyps [30–32]. The morphology and ion
transport properties of this nasal epithelium closely
resemble those of lower airway epithelia [10, 30, 31].
Recently, we demonstrated a mercurial-sensitive high Pf
in this preparation, suggesting that osmotic water flow was
carried by an AQP [32]. The results of this first functional
study of effects of hyperosmotic pretreatment on human
nasal airway epithelium demonstrate an increased ex-
pression of AQP5 in parallel with increased trans-
epithelial Pf with a maximal effect at ∼450 mOsm after
Pflugers Arch - Eur J Physiol (2007) 453:777–785
∼24 h. These effects were equally expressed in non-CF and
CF spheroids.
Methods
Tissue preparation
Nasal polyps from four non-CF subjects (two female and
two male) and five CF patients (three female and two male)
underwent an enzymatic epithelial stripping according to a
procedure described in detail recently [32]. In brief, the
polyps were placed in a Dulbecco’s modified Eagle’s
medium containing protease and antibiotics (penicillin,
streptomycin and gentamycin) and left overnight at 4°C.
The next morning the temperature was raised to 35°C.
After 20–30 min the polyps were shaken gently for
5–10 min (which released large numbers of epithelial
sheets), and the protease effect was quenched by addition
of serum. The solid parts of the polyps were isolated
for later use, and the remaining suspension of small
epithelial sheets was washed twice and suspended in
10 ml HAM-F12 culture medium supplied with a serum
substitute (1% Ultroser-G, IBF Biotechnics, Savage, MD,
USA) and antibiotics (as above) in a 50-ml tissue culture
flask at 37°C with 5% CO2. The solid parts of the polyps
were placed in RPMI medium supplied with collagenase
type II (0.2%) and incubated at 35°C for about 20 min. This
procedure resulted in the release of additional epithelial
sheets which were then treated as described above. During
the first 4–5 h after isolation, the flask was gently tapped
every 30 min to reduce attachment of epithelial sheets to
the bottom of the flask. The medium was changed daily for
the first 2 days and thereafter twice a week by simple
sedimentation for 15 min in a test tube. From the third day
hydrocortisone (5·10−6 M) was added to the medium. During
the development of this airway epithelial preparation [30] we
found that addition of hydrocortisone was advantageous for
the preservation of morphological integrity and function of
the spheroids during experiments. However, hydrocortisone
had no acute effect on spontaneous fluid absorption
(unpublished observation), and data in the present study
demonstrate that lack of hydrocortisone does not affect the
increase in Pf in response to pre-exposure to osmotic stress
(see ‘Results’).
Experimental setup
Experiments were conducted at 36°C in a thermo-con-
trolled chamber (TC-344A, Warner Instrument Corp.,
Hamden, CT, USA) containing 300 μl Ringer solution
(300 mOsm). The spheroids were continuously superfused
with preheated solutions (Inline Heater, Warner Instruments
Pflugers Arch - Eur J Physiol (2007) 453:777–785
Corp.) at a rate of 6.5 ml min−1 using fine-tuned perfusion
pumps (Perfusor, Braun Melsungen AG) equilibrated to
maintain the chamber volume constant. An electronic valve
(Valco Instruments Co. Inc., Houston, TX, USA) was used
for changing solutions during experiments. The chamber
was mounted on the stage of an inverted microscope
(Nikon Diaphot 300 equipped with Hoffman modulation
contrast optics) which was placed on a vibration-free table
(TMC-63-500, TMC, Peabody, MA, USA). Spheroids were
kept in position by a glass pipette with an internal tip
diameter of ∼10 mm by applying gentle suction via a
microinjector (IM-6, Narishige, Japan). The position of the
holding pipette was controlled by a set of hydraulic and
motorized micromanipulators (MM-188 and MO-188,
Narishige, Japan).
Hyperosmotic stress prior to experiments
In order to mimic the physiological conditions in the
airways, where simple evaporation produces significant
hypertonicity, we also used evaporation for production of
varying degrees of hypertonicity. Spheroids in standard
medium were placed in open Petri dishes in a dry incubator,
and water was then allowed to slowly evaporate. In this
way the spheroids were allowed to adapt slowly to the new
osmotic conditions.
In a separate series of experiments, acute exposure to
hypertonic medium was used to obtain information about
the time-dependence of Pf changes (a sudden change from
the normal 300 to 500–600 mOsm sometimes induced a
collapse of the spheroids). Hypertonic medium for this part
of the study was produced by simple evaporation of water
from the medium (as above, but without spheroids) or by
addition of mannitol to the standard medium in order to
study whether changes in medium components alone were
involved in the effect on Pf. Single spheroids were
repeatedly used the following days for Pf measurements,
and the corresponding tonicity of the medium was
measured with an osmometer (Osmomat 030-D, Gonotec,
Berlin, Germany). In some experiments spheroids were
permanently returned to standard tonicity conditions, and
the Pf was measured for some days to get information on
the reversibility of changes in Pf.
Image analysis
Experiments were video-taped (AG-7355-E, Panasonic,
Osaka, Japan) and then replayed through a Matrox IP-8
image board (Matrox Electronic Systems Ltd., Canada)
inserted in an IBM-compatible PC, where video frames were
captured at a rate of approximately 1 s−1. Each image was
contrast enhanced to maximize the difference in pixel
intensity between the spheroid and the surrounding fluid.
779
Initially the maximal diameter was identified by eye on the
monitor using the computer mouse as a pointing device. An
approximately 40-pixel-wide horizontal area, centred around
the indicated border between the spheroid and fluid, was used
as a template to automatically locate the border in a vertical
region spanning 50 lines upwards and downwards in the
following frames. The border was detected in each horizontal
scan line using a cross-correlation technique [26]. Once the
borders were determined on both sides of the spheroid, the
maximal diameter was found as the distance between the
borders in the horizontal scan line. The software was written
in C by the authors. The resulting time-series of diameters
were processed by the use of a commercial graphics software
(Grapher 2.02) by which the maximum slope [reflecting
transepithelial water flow (JV)] for at least 20 s was calculated
using the linear regression module.
Experiments
In order to measure the transepithelial osmotic water
permeability (Pf) under standardized conditions [30], all
spheroids were first transferred from their actual osmotic
condition to Ringer solution at 300 mOsm. Within a few
minutes the spheroid volume adapted to this osmolarity by
swelling to a new steady-state level. Only a small fraction
of the spheroids exhibited leakage and deflated during this
transfer, and most of these resealed again within a few
minutes. The Pf was then determined from the rate of
volume change in response to changes in bathing medium
osmolarity forth and back between 300 and 225 mOsm,
equal to an inwardly or outwardly directed osmotic gradient
of 75 mOsm. The Pf (micrometer per second) was
calculated as:
Pf ¼ JV A Vw1 Δπ 1 ; ð1Þ
where Jv is the transepithelial water flow (cubic micrometer
per second), A is the surface area (square micrometer), Vw
is the partial molar volume of water (18 μm3·mol−1), and
Δπ is the transepithelial osmotic gradient (osmole·per liter).
Assuming a spherical shape, this reduces to
1 dθ
Pf ¼ Vw1 Δπ1 ; ð2Þ
2 dt max
where dθ
dt max is the maximal rate of diameter change during
the osmotic challenge.
Immunostaining and confocal microscopy: quantification
of AQP5
Spheroids were fixed in 2% paraformaldehyde for 15 min,
permeabilized in 0.2% Triton X-100 in PBS with 4% BSA
for 15 min and blocked in PBS/BSA. The spheroids were
incubated overnight with the primary anti-AQP5 antibody
780
(Cat. 178615, Calbiochem; 1:100) at 4°C, washed and
incubated with the secondary antibody (Alexa-488, Molec-
ular Probes) and rhodamine-phalloidin (Molecular Probes)
at room temperature for 45 min. After several washing steps
the spheroids were mounted in Prolong Gold mounting
medium (Molecular Probes) and imaged in a Leica TCS
SP2 confocal microscope. For all comparisons spheroids
were stained simultaneously with the same solutions and
imaged using the same microscope and laser settings. To
verify that the signal was specific spheroids were stained
without primary antibody, resulting in no detectable signal
(data not shown). A single cell layer was reconstructed in
3-D using AutoDeblur ver. X 1.3.3 (AutoQuant Imaging
Inc.) from a stack of 155 images in three channels.
In order to quantify expression of AQP5, single spheroids
were imaged by confocal microscopy with a 10× objective,
encompassing the entire spheroid in question. A region
covering the spheroid was defined, and the image was
grey level thresholded in order to measure the area of the
specific AQP5 staining as compared with the total area of
the region of interest (MetaMorph, Molecular Devices
Corp., Sunnyvale, CA, USA).
Solutions and chemicals
The standard 300-mOsm HEPES-buffered Ringer solution
contained (in millimolars): 140 Na+, 5 K+, 1.2 Ca2+, 1.2
Mg2+, 131.2 Cl−, 1.6 HPO42−, 0.4 H2PO4−, 10 glucose and
10 HEPES. Two different osmolarities of the standard
Ringer was produced simply by diluting the 300-mOsm
Ringer with water to 225 mOsm. Cell culture media were
obtained from ICN Biochemicals Inc. All other chemicals
were purchased from Sigma Chemical Co.
Statistics
All quantitative data are means±SE. Individual values were
compared with Student’s t test or the Mann–Whitney rank
sum test. Since data from the visual rating of AQP5
expression (Fig. 6) did not comply with normal distribution,
the Wilcoxon matched-pairs test was used to calculate
significance (Statistica, ver. 7.0, Statsoft, Tulsa, OK, USA).
A P value of <0.05 was considered significant.
Results
We used a spheroid-shaped explant preparation of human
nasal airway epithelium as an experimental model to
investigate the effect of pretreatment with hyperosmotic
solutions on transepithelial Pf. This 3-D epithelial prepara-
tion exhibited morphological and functional features re-
sembling those of natural airway epithelia, including the
Pflugers Arch - Eur J Physiol (2007) 453:777–785
specific ion transport properties characteristic for CF [6,
30–32]. The spheroid-shaped explants consisted of a fully
differentiated epithelium enclosing a central lumen filled
with fluid produced by solute-driven water absorption [30–
32]. The epithelium was monolayered and polarized with a
ciliated apical surface always facing the outside. Their
perfect spherical geometry (Fig. 1) allowed simple calcu-
lation of area and volume used for the calculation of fluid
transport rate [32]. Transepithelial Pf was determined from
the change in spheroid volume evoked by shift of bath
osmolarity creating an inwardly or outwardly directed
osmotic gradient of 75 mOsm. A typical trace is presented
in Fig. 2 showing the regular changes of the spheroid
diameter in response to changes in apical tonicity. The
maximum rate of change in diameter (the slope) was used
for the calculation of Pf, and lines aligned to the top and
bottom of the excursions were used for calculation of
spontaneous fluid absorption and, most importantly, for the
control of ideal full osmotic equilibration (Fig. 2). Although
changes in response to both hypo- and hyperosmotic acute
apical challenges theoretically could have been used for Pf
calculations, recent results [32] indicate that the hypertonic
apical challenge is hampered by unstirred layer phenomena.
Consequently, we only used the hypoosmotic challenge for
the present calculations of the Pf.
Fig. 1 Contrast-enhanced screen picture of a spheroid (from a female
CF patient) kept in place by a holding pipette seen at the top. The
cursor lines inserted at both sides of the spheroid defined the
maximum spheroid diameter. The inserted picture shows the same
spheroid with unmodified contrast and the focus plane set at the
bottom of the spheroid illustrating the amount of cells in this particular
preparation. The diameter of the spheroid is 440 μm, corresponding to
∼45 cells, which gives a cell diameter of∼10 μm
Pflugers Arch - Eur J Physiol (2007) 453:777–785
420
Y1
Spheroid Diameter (µm)
400
Y3
380
360
Y2
340
Z1 Z2 Z3
Fig. 2 Typical trace of a non-CF spheroid exposed to regular changes
in osmolarity of the apical solution between 300 and 225 mOsm. The
spheroid had been pre-exposed to high osmolarity (550 mOsm) of the
incubation medium for more than 24 h. Lines X1 and X2 illustrate
spontaneous fluid absorption (3.4 μl·cm−2 h−1). Lines Z1–Z4 are slope
lines for calculation of Pf (207, 193, 200 and 225 μm·s−1, respective-
ly). Lines Y1–Y3 are help lines for the demonstration of perfect
osmotic equilibration on top of the spontaneous volume increase
The results from Pf measurements performed after
incubation of non-CF and CF spheroids in media with
increasing osmolarity (∼300 to ∼600 mOsm) are shown in
Fig. 3. The increased osmolarities were obtained by simple
evaporation of the bathing medium. The pre-experimental
incubation periods in these experiments were 3–6 days,
depending on the degree of osmolarity obtained. The Pf was
increased by a factor of ∼1.5 when the osmolarity during
hyperosmotic pre-incubation was increased from
∼300 mOsm to osmolarities higher than ∼450 mOsm at
which the effect of hyperosmotic stress on Pf seems to
saturate (Fig. 3). The Pf values and the Pf responses were
equal in CF and non-CF spheroids (Fig. 3). Thus, at
∼300 mOsm Pf was 116.3±3.9 μm·s−1 (n=17) in non-CF
and 124.4±5.4 μm·s−1 (n=12) in CF spheroids, and at
osmolarities ≥450 mOsm, Pf was 179.1±5.4 μm·s−1 (n=29)
in non-CF and 180.6±4.1 μm·s−1 (n=44) in CF spheroids.
200
Water Permeability (µm s )
-1
180
160
140
120
100
300 350 400 450
Medium Tonicity (mOsm) prior to Experiment
Fig. 3 Plot of Pf values (micrometer per second) measured in non-CF
and CF spheroids as a function of osmolarity in a pretreatment period
of at least 24 h. Each point represents the average of at least six
experiments. The plot reveals a maximum Pf value obtained at
∼450 mOsm
781
The presence of hydrocortisone did not seem to influence Pf.
X2
Thus, after 5 days without hydrocortisone and adaptation
to an osmolarity of ∼490 mOsm, the Pf in a batch of CF
X1
spheroids was 186.1±8.2 μm·s−1 (n=16), not significantly
different from hydrocortisone-treated spheroids adapted to
10 minutes similar osmolarities.
Z4 As long as the spheroids remained exposed to high
osmolarity (>14 days), the Pf remained high. When they
were returned to normal osmolarity (300 mOsm), the Pf
declined to control values within 24 h (Fig. 4).
During evaporation of the standard 300-mOsm incuba-
tion medium to an osmolarity of, for example, 500 mOsm,
the pH changed from 7.15 to 7.35. We therefore tested the
effect of the change in pH alone on Pf under isosmotic
conditions. The result showed that this smaller pH change
had no effect on Pf. Thus, at pH=7.15 Pf was 127.2±
14.6 μm·s−1 (n=15) and at pH=7.35 Pf was 131.8±
14.0 μm·s−1 (n=14).
In order to study the time-course of the hypertonicity-
induced increase in Pf, smaller groups of spheroids were
acutely exposed to a 450-mOsm medium for 0, 8, 16, 24
and 48 h. This osmolarity was obtained either by pre-
evaporation of standard medium or by addition of mannitol
to the standard medium. Figure 5 demonstrates that Pf
increased with a time delay of more than 16 h, but at 24 h
the response was fully developed. The time delay indicates
a requirement for synthesis of new protein and seems to
rule out translocation of AQP5 to the cell membrane as the
sole explanation for the increase in Pf.
(516)
200 (530)
(465)
150
100 (300)
Non-CF (300)
CF
0 1 2
500 550 600
Fig. 4 Reversibility of the increase in Pf induced by hyperosmotic
stress in a batch of CF spheroids. The spheroids were pretreated with
•
increased osmolarity of the incubation medium for 3 days. From
day 0, half of the spheroids continued in hypertonic medium ( ),
whereas the other half was returned to standard 300-mOsm medium
○
( ). The osmolarity of the medium is shown in brackets. Each point
represents at least four Pf measurements
782 Pflugers Arch - Eur J Physiol (2007) 453:777–785

As illustrated in Fig. 2 it was possible to measure an

200 increase in spheroid diameter while the spheroids were
repeatedly exposed to changes in bathing medium
Water Permeability (µm s-1)

osmolarity forth and back between 300 and 225 mOsm.

180
This spontaneous fluid absorption was significantly higher
in non-CF than in CF spheroids that had only been
160 exposed to standard (300 mOsm) medium (non-CF, 3.7±
0.4 μl·cm−2 h−1(n=17) vs CF, 2.4±0.2 μl·cm−2 h−1(n=26);
P<0.002). In spheroids that had previously been exposed to
140 hypertonicity (>450 mOsm for 3–6 days), the spontaneous
absorption (measured at 300 mOsm) was slightly but
120 not significantly increased in both non-CF and CF
spheroids (non-CF, 4.5±0.3 μl·cm−2 h−1(n=15) vs CF,
3.4±0.5 μl·cm−2 h−1(n=13); not significantly different,
100 P=0.062).
0 8 16 24 32 40 48 It has recently been suggested that inhibition of apical cell
Time (Hrs. after start of Hypertonic Exposure) membrane Na+ channels (ENaC) by amiloride (100 μM)
Fig. 5 Time study of changes in Pf performed in a batch of non-CF
may at the same time inhibit apical membrane Pf in pri-

•
spheroids acutely exposed to 450 mOsm incubation medium [made
hypertonic by addition of mannitol ( ) or by simple evaporation ( )] □ mary cultures of human airway epithelium [11]. However,
an effect of amiloride (100 μM in the apical bathing

Fig. 6 Typical apical mem-

branes from unstimulated (a)
and hyperosmolarity-stimulated
(b) spheroids (scale bar=8 μm).
Red colour shows the marginal
bundles of actin cytoskeleton
and green colour shows AQP5.
c A transversely sectioned polyp
from which the spheroids de-
rive. The single layer of cells
shows upward-facing apical
AQP5 staining (in green) (scale
bar=4 μm). d Quantification of
AQP5 in unstimulated and
stimulated spheroids (+SE) at
300 and 450 mOsm (*P<0.001)
Pflugers Arch - Eur J Physiol (2007) 453:777–785
medium) on Pf was not observed in the present explant
epithelium (during osmotic stimulation). Without amilo-
ride the Pf in CF spheroids was 190.7±8.5 μm·s−1 (n=14)
vs 186.4±8.9 μm·s−1 (n=9) with amiloride (P=0.74), and
Pf in non-CF spheroids was 182.1±6.5 μm·s−1 (n=32)
without amiloride vs 177.2 ± 8.0 μm·s−1 (n = 19) with
amiloride (P=0.64). On the other hand, spontaneous
fluid absorption was abolished by amiloride as previously
shown [32].
In order to investigate if the increase in Pf in response to
a rise in medium osmolarity could be explained by an
increased expression of AQP5, we examined the spheroids
by immunostaining. Spheroids subjected to either normal
osmolarity (300 mOsm) or high osmolarity (∼450 mOsm)
for 24 h were fixed and immunostained with antibodies
specific for AQP5 (green colour) and actin (red colour) as
demonstrated in Fig. 6. The figure illustrates an increased
expression of AQP5 (panel b) in hyperosmotic stimulated
spheroids as compared with unstimulated spheroids (panel
a). This applies to both density and intensity of the green
AQP5 staining. In an attempt to study the distribution of
AQP5 in the epithelial cells, we did a 3-D reconstruction of
the single cell layer from a stimulated spheroid from a large
series of z-plane scans. This reconstruction showed that
AQP5 was present in the apical membrane as well as in
transport vesicles distributed evenly throughout the cyto-
plasm (data not shown). The reconstruction was in
concordance with the notion that epithelial cells of the
polyp from which the spheroids derive showed a strong
apical AQP5 signal (Fig. 6c). The punctuate nature of
AQP5 labelling probably represents clustering of AQP5 in
apical membranes and transport vesicles [15]. In order to
quantify AQP5 expression in isosmotic and hyperosmotic
medium, a series of images covering full spheroids were
analysed (as described in ‘Methods’). The image analysis
clearly demonstrated an increased AQP5 expression when
spheroids had been subjected to increased osmolarity
(Fig. 6d; P<0.001). The recorded increase is somewhat
dependent on the bit value used for thresholding the
images, and therefore, the ∼7 times increase in expression
of AQP5 in stimulated spheroids is not a precise measure.
However, the increased expression in stimulated vs unstim-
ulated spheroids was always clearly visible to the naked eye
when visualized by fluorescence microscopy.
Discussion
The present study demonstrates that hyperosmotic stress
induces an increase in osmotic water permeability (Pf) in
spheroid-shaped explant preparation of natural non-CF and
CF human nasal airway epithelium, and that the increase in
Pf is associated with increased cellular levels of AQP5 and
783
localization of AQP5 in the apical cell membrane. Thus, the
results present a functional correlate to a number of recent
studies in other tissues and species demonstrating an
increase in AQPs at the mRNA and protein levels in
response to hyperosmotic stress (see ‘Introduction’).
Control Pf values were in agreement with our recently
reported values in this preparation and were likewise equal
in non-CF and CF spheroids [32]. Hyperosmotic pretreat-
ment induced a maximal increase in Pf by a factor of 1.5
(Figs. 3 and 5). This is in reasonable agreement with the
reported range of maximal increase in expression of AQPs
at the protein (factor 2–4) and mRNA levels (factor 2–6) in
response to hyperosmotic stress [1, 17, 18]. Based on a
reconstruction of a spheroid cell layer, we found that AQP5
was evenly distributed in the cytoplasm as well as in the
apical membrane of the osmotically stimulated spheroids
(data not shown).
The maximal effect of hyperosmolarity on Pf was
obtained at ∼450 mOsm (Fig. 3). Although reported hy-
pertonicities for maximal effects on AQP protein and
mRNA levels varied between different AQPs [1], the
osmolarities for maximal effects on AQPs [1, 17–20, 24,
36, 38] were generally close to the level reported here. In
some studies a further increase in extracellular osmolarity
to more than 500–550 mOsm reduced AQP mRNA and
protein levels [17, 18, 36, 38]. An analogous reduction in Pf
was not observed at these high osmolarities in the present
study (Fig. 3).
Increasing AQP protein levels have been reported to
occur after 6–12 h of hyperosmotic exposure, whereas peak
values occurred after 12–48 h, with corresponding mRNA
levels rising a few hours ahead [1, 17–20, 28, 35, 36, 38].
A similar time delay was seen in the present study where
the Pf of spheroids began to increase after 16 h and was
maximal at 24 h (Fig. 5). This time delay suggests an
involvement of protein synthesis. Conflicting data, howev-
er, have been reported on the effect of protein synthesis
inhibition by cycloheximide on AQP increase by hyper-
osmotic stimulation [1, 38]. In the present study it was not
possible to study this issue since even a very low
concentration of cycloheximide (1 μg·ml−1) destroyed the
3-D structure of the spheroids (data not shown). On the
other hand, the fact that only a fraction of AQP5 was
located in the apical membrane of osmotically stimulated
spheroids indicates that new protein synthesis is required
for an increase in Pf. However, an additional participation
from translocation of water channels to the plasma
membrane or decreased retraction from the plasma mem-
brane can of course not be excluded.
During continuous hyperosmotic treatment, the spher-
oids exhibited a sustained increase in Pf for several days.
This is in contrast to some studies demonstrating a decline
in elevated levels of AQP mRNA to control levels over
784
24–48 h despite continuous exposure to hyperosmolarity
[28, 36]. Our results are, on the other hand, in concordance
with other studies demonstrating that hyperosmolarity-
induced increased AQP expression remained high for
24–166 h [1, 18, 35]. Our observation that the hyper-
osmolarity-induced effect on spheroid Pf disappeared
within hours when isotonic medium was reintroduced
agrees with studies demonstrating a steep decline in AQP
mRNA and protein levels [18, 35, 36, 38].
Hyperosmotic gradients created by ‘impermeable’ osmo-
lytes such as mannitol, sorbitol, glucose, fructose or NaCl
are required in order to increase the expression of AQPs,
whereas cell membrane permeating agents such as DMSO,
urea and glycerol are ineffective [35]. In the present study
no significant difference in the effects on Pf was seen when
two different types of impermeable agents were used to
increase osmolarity. Thus the effects on Pf were similar
when standard medium (300 mOsm) was supplied with
mannitol up to an osmolarity of 450 mOsm and when
hyperosmolarity was obtained simply by evaporation of
standard medium (leaving NaCl as the major osmotic
component). This is in contrast to observations demonstrat-
ing a delayed effect of NaCl compared with mannitol on
induction of AQP4 and AQP9 proteins [1].
Recent observations suggest an interaction between AQPs
and CF transmembrane conductance regulator (CFTR).
Thus, the presence of CFTR has been demonstrated to
amplify expressions of AQP3 in airway epithelial cells [34]
and AQP9 in epididymis [7]. However, AQP3 is constitu-
tively present in basolateral cell membranes of the surface
epithelium in human airways (together with AQP4) [29],
whereas CFTR is localized in apical cell membranes [33].
As pointed out by Matsui et al. [27], the rate limiting
barrier in airway transepithelial water movement is the
apical cell membrane where AQP5 is present. This notion
supports the general opinion that transepithelial fluid
transport predominantly occurs transcellularly. Together
with our previous [32] and present observations that Pf is
equal in non-CF and CF epithelial spheroids and equally
increased by hyperosmolarity in both types of spheroids
(Fig. 3), we therefore hypothesize that the up-regulation of
airway epithelial Pf occurs via an increase in apical cell
membrane AQP5 which obviously does not involve an
interaction with CFTR.
In a recent paper a decrease in epithelial Pf induced by
luminal amiloride in both CF and non-CF human airway
epithelial primary cultures was reported [11]. Such an effect
of amiloride on Pf was not confirmed in the present study.
This discrepancy may disclose a change in the functional
expression of water channels in response to culturing
conditions. In the same paper the authors concluded that
dissipation of luminal salt concentration following apical
deposition of hypertonic NaCl (and thus the return of an
Pflugers Arch - Eur J Physiol (2007) 453:777–785
initially increased ASL back to the control volume) is
limited in CF cultures by the absence of CFTR. This,
however, implies that net salt absorption should be smaller
in CF cultures than in non-CF cultures, a conclusion that
disagrees with the authors’ previous conclusion [2, 3], but
are in agreement with the results from our previous study
[32] and with the present study where spontaneous fluid
absorption, reflecting net salt absorption [30, 31], is higher
in non-CF than in CF spheroids whether or not they have
been exposed to hyperosmotic stress. The notion that net
salt and water absorption is decreased in natural CF airway
epithelium may not conflict with the fact that short-circuit
current (primarily representing active Na+ absorption) is
increased in both spheroid CF airway epithelium [31] as
well as in conventional CF airway cell cultures [2, 3].
In conclusion, the present study in spheroid-shaped
explants of human non-CF and CF nasal airway epithelium
represents the first direct measurement of transepithelial
water permeability following exposure to hypertonic stress.
The combined functional and immunochemical results
reveal a correlation between the level of Pf and expression
of AQP5, the rate limiting water channel in airway epithelial
apical cell membranes.
Acknowledgements We thank the staff from the ENT surgery RH,
Dr. N. Rasmussen and Dr. H. Nielsen for their help during this study.
This work was supported by grants from the Danish Research Council
(SSVF), the Novo-Nordisk Foundation, the Lundbeck Foundation,
Zealand Pharmaceutical and the Velux Foundation.
References
1. Arima H, Yamamoto N, Sobue K, Umenishi F, Tada T, Katsuya
H, Asai K (2003) Hyperosmolar mannitol simulates expression
of aquaporins 4 and 9 through a p38 mitogen-activated protein
kinase-dependent pathway in rat astrocytes. J Biol Chem 278:
44525–44534
2. Boucher RC (2003) Regulation of airway surface liquid volume
by human airway epithelia. Pflugers Arch 445:495–498
3. Boucher RC (2004) New concepts of the pathogenesis of cystic
fibrosis lung disease. Eur Respir J 23:146–158
4. Burg MB, Kwon ED, Kultz D (1996) Osmotic regulation of gene
expression. FASEB J 10:1598–1606
5. Carter EP, Umenishi F, Matthay MA, Verkman AS (1997)
Developmental changes in water permeability across the alveolar
barrier in perinatal rabbit lung. J Clin Invest 100:1071–1078
6. Castillon N, Hinnrasky J, Zahm JM, Kaplan H, Bonnet N, Corlieu
P, Klossek JM, Taouil K, Avril-Delplanque A, Peault B, Puchelle
E (2002) Polarized expression of cystic fibrosis transmembrane
conductance regulator and associated epithelial proteins during the
regeneration of human airway surface epithelium in three-
dimensional culture. Lab Invest 82:989–998
7. Cheung KH, Leung CT, Leung GP, Wong PY (2003) Synergistic
effects of cystic fibrosis transmembrane conductance regulator
and aquaporin-9 in the rat epididymis. Biol Reprod 68:1505–1510
8. Cohen DM, Wasserman JC, Gullans SR (1991) Immediate early
gene and HSP70 expression in hyperosmotic stress in MDCK
cells. Am J Physiol 261:C594–C601
Pflugers Arch - Eur J Physiol (2007) 453:777–785
9. Crews A, Taylor AE, Ballard ST (2001) Liquid transport prop-
erties of porcine tracheal epithelium. J Appl Physiol 91:797–802
10. Davies MG, Geddes DM, Alton EFW (2005) The effect of
varying tonicity on nasal epithelial ion transport in cystic fibrosis.
Am J Respir Care Med 171:760–763
11. Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R,
Boucher RC (2006) Mucus clearance and lung function in cystic
fibrosis with hypertonic saline. N Engl J Med 354:241–250
12. Farinas J, Kneen M, Moore M, Verkman AS (1997) Plasma
membrane water permeability of cultured cells and epithelia
measured by light microscopy with spatial filtering. J Gen Physiol
110:283–296
13. Folkesson HG, Matthay MA, Frigeri A, Verkman AS (1996)
Transepithelial water permeability in microperfused distal air-
ways. Evidence for channel-mediated water transport. J Clin
Invest 97:664–671
14. Galcheva-Gargova Z, Derijard B, Wu IH, Davis RJ (1994) An
osmosensing signal transduction pathway in mammalian cells.
Science 265:806–808
15. Gresz V, Kwon T-H, Gong H, Agre P, Steward MC, King L,
Nielsen S (2004) Immunolocalization of AQP-5 in rat parotid and
submandibular salivary glands after stimulation or inhibition of
secretion in vivo. Am J Physiol 287:G151–G161
16. Han J, Lee JD, Bibbs L, Ulevitch RJ (1994) A MAP kinase
targeted by endotoxin and hyperosmolarity in mammalian cells.
Science 265:808–811
17. Hasler U, Vinciguerra M, Vandewalle A, Martin PY, Feraille E
(2005) Dual effects of hypertonicity on aquaporin-2 expression in
cultured renal collecting duct principal cells. J Am Soc Nephrol
18. Hoffert JD, Leitch V, Agre P, King LS (2000) Hypertonic
induction of aquaporin-5 expression through an ERK-dependent
pathway. J Biol Chem 275:9070–9077
19. Hwang SM, Lee RH, Song JM, Yoon S, Kim YS, Lee SJ, Kang
SK, Jung JS (2002) Expression of aquaporin-5 and its regulation
in skeletal muscle cells. Exp Mol Med 34:69–74
20. Jenq W, Mathieson IM, Ihara W, Ramirez G (1998) Aquaporin-1:
an osmoinducible water channel in cultured mIMCD-3 cells.
Biochem Biophys Res Commun 245:804–809
21. Krane CM, Fortner CN, Hand AR, McGraw DW, Lorenz JN,
Wert SE, Towne JE, Paul RJ, Whitsett JA, Menon AG (2001)
Aquaporin 5-deficient mouse lungs are hyperresponsive to
cholinergic stimulation. Proc Natl Acad Sci U S A 98:14114–
14119
22. Kreda SM, Gynn MC, Fenstermacher DA, Boucher RC, Gabriel
SE (2001) Expression and localization of epithelial aquaporins in
the adult human lung. Am J Respir Cell Mol Biol 24:224–234
23. Lee MD, King LS, Nielsen S, Agre P (1997) Genomic
organization and developmental expression of aquaporin-5 in
lung. Chest 111:111S–113S
24. Leitch V, Agre P, King LS (2001) Altered ubiquitination and
stability of aquaporin-1 in hypertonic stress. Proc Natl Acad Sci
U S A 98:2894–2898
785
25. Lu J, Park JH, Liu AY, Cnhen KY (2000) Activation of heat shock
factor 1 by hyperosmotic or hypo-osmotic stress is drastically
attenuated in normal human fibroblasts during senescence. J Cell
Physiol 184:183–190
26. Marsh DJ, Jensen PK, Spring KR (1985) Computer-based
determination of size and shape in living cells. J Microsc 137
(Pt 3):281–292
27. Matsui H, Davis CW, Tarran R, Boucher RC (2000) Osmotic
water permeabilities of cultured, well-differentiated normal and
cystic fibrosis airway epithelia. J Clin Invest 105:1419–1427
28. Matsuzaki T, Suzuki T, Takata K (2001) Hypertonicity-induced
expression of aquaporin 3 in MDCK cells. Am J Physiol Cell
Physiol 281:C55–C63
29. Nielsen S, King LS, Christensen BM, Agre P (1997) Aquaporins
in complex tissues. II. Subcellular distribution in respiratory and
glandular tissues of rat. Am J Physiol 273:C1549–C1561
30. Pedersen PS, Frederiksen O, Holstein-Rathlou NH, Larsen PL,
Qvortrup K (1999) Ion transport in epithelial spheroids derived
from human airway cells. Am J Physiol 276:L75–L80
31. Pedersen PS, Holstein-Rathlou NH, Larsen PL, Qvortrup K,
Frederiksen O (1999) Fluid absorption related to ion transport in
human airway epithelial spheroids. Am J Physiol 277:L1096–
L1103
32. Pedersen PS, Procida K, Larsen PL, Holstein-Rathlou NH,
Frederiksen O (2005) Water permeability in human airway
epithelium. Pflugers Arch 451:464–473
33. Pilewski JM, Frizzell RA (1999) Role of CFTR in airway disease.
Physiol Rev 79:S215–S255
34. Schreiber R, Nitschke R, Greger R, Kunzelmann K (1999) The
cystic fibrosis transmembrane conductance regulator activates
aquaporin 3 in airway epithelial cells. J Biol Chem 274:11811–
11816
35. Storm R, Klussmann E, Geelhaar A, Rosenthal W, Maric K
(2003) Osmolality and solute composition are strong regulators of
AQP2 expression in renal principal cells. Am J Physiol Renal
Physiol 284:F189–F198
36. Sugiyama Y, Ota Y, Hara M, Inoue S (2001) Osmotic stress up-
regulates aquaporin-3 gene expression in cultured human kerati-
nocytes. Biochim Biophys Acta 1522:82–88
37. Umenishi F, Schrier RW (2002) Identification and characterization
of a novel hypertonicity-responsive element in the human aqua-
porin-1 gene. Biochem Biophys Res Commun 292:771–775
38. Umenishi F, Schrier RW (2003) Hypertonicity-induced aquaporin-
1 (AQP1) expression is mediated by the activation of MAPK
pathways and hypertonicity-responsive element in the AQP1
gene. J Biol Chem 278:15765–15770
39. Verkman AS, Matthay MA, Song Y (2000) Aquaporin water
channels and lung physiology. Am J Physiol Lung Cell Mol
Physiol 278(5):L867–L879 278:L867–L879 May
40. Widdicombe JH, Bastacky SJ, Wu DX, Lee CY (1997) Regula-
tion of depth and composition of airway surface liquid. Eur Respir
J 10:2892–2897