1 Division of Respiratory
Diseases, During the perinatal period, a dramatic
reversal of lung transepithelial ion and water transport occurs that
involves the amiloride-inhibitable
Na+ channel (ENaC). Aquaporin
(AQP) water channel proteins facilitate cell membrane water transport.
We now report that AQP-4, localized to basolateral membranes of airway
epithelial cells, increases its mRNA expression in developing lung
eightfold during the 2 days before birth to reach a peak on the first
postnatal day in the lungs but not in brains or kidneys of neonatal
rats. AQP-4 and the
aquaporin water channel; lung development; neonatal pulmonary
function
IN FETAL LIFE, mammalian lungs are filled with a
Cl At present, a total of eight AQPs have been identified from various rat
tissues (6, 10, 14, 16-18, 37). Although their transmembrane
domains share a large degree of structural homology, both
NH2- and COOH-termini of AQPs are
divergent. The specific roles of three AQPs in both adult and perinatal
lung function remain unclear despite considerable effort by multiple
laboratories. In rat lung, AQP-1 is expressed by pulmonary endothelial
cells and may facilitate fluid clearance from lungs during the
perinatal period (22). Despite demonstration that its expression in
lung increases fivefold at parturition and is potentiated by maternal corticosteroid administration (15), evidence that AQP-1 plays a
critical role in lung function is lacking, since human subjects lacking
AQP-1 are phenotypically normal (32). Although AQP-5 is expressed in
type I pneumocytes during the perinatal period, its expression
increases significantly only 2-4 days after birth (18). Thus
timing of maximal AQP-5 expression in perinatal lung does not coincide
with the interval of maximal transepithelial fluid reabsorption in
lung.
AQP-4 [14; also previously referred to as a mercurial-insensitive
water channel (8, 10)] is present in adult rat lung at modest
levels and is significantly increased during the perinatal period (37).
However, a recent demonstration that mice lacking AQP-4 protein display
neither abnormal lung development nor significant lung disease at birth
suggests that AQP-4 is not critical for lung fluid clearance
during the perinatal period (19).
In this report, we quantify the AQP-4 mRNA expression in the lungs,
kidneys, and brain of fetal, neonatal, and adult rats to determine if
lung AQP-4 expression is modulated in a manner similar to that
exhibited by the Isolation of AQP-4 and AQP-5 probes.
RT-PCR was utilized to amplify cDNAs using primers corresponding
to nucleotides (nt) 4-84
(5'-GCTGATCATGGTGGCTTTCAAAGGCGTCTG-3' with an
engineered Bcl I site), nt
934-951
(5'-CCGGCATGCAGTTCTTCCCCTTCTTCTCG-3' with an
engineered Sph I site) for AQP-4 (14),
and nt 104-122 (5'-GGCACCATGAAAAAGGAGGTG-3') and nt 906-924 (5'-CAGTGTGCCGTCAGCTCGATG-3')
for AQP-5 (33). After 10 ng of cDNA from total lung RNA of
Sprague-Dawley rats (Charles River Laboratories, Billerica, MA) were
prepared using the Superscript preamplification system (GIBCO-BRL,
Gaithersburg, MD), PCR amplifications were achieved by 40 cycles
(94°C, 1 min; 55°C, 1 min; 72°C, 2 min plus 72°C, 7 min) using 50 pmol of each primer per reaction. The
resulting 879-bp AQP-4 fragment and the 820-bp AQP-5 fragment were
subcloned into PCR II (Invitrogen, San Diego, CA), and both strands
were sequenced by the Department of Genetics DNA Sequencing Facility
(Children's Hospital, Boston, MA) using the dye terminator technique
(9).
RNA isolation and Northern analyses.
After RNA was isolated from fetal [embryonic
days 18-21
(E18-E21)], postnatal (postnatal days
1, 3,
7, and
14), and adult rat tissues and
cultured cells using a STAT 60 Kit (Teltest B, Friendswood, TX), it was
size fractionated by electrophoresis in formaldehyde-1.2% agarose
gels, transferred to nylon membranes (Duralon-UV; Stratagene, La Jolla, CA), and ultraviolet cross-linked as described previously (13). After
prehybridization, blots were hybridized with either
32P-labeled AQP-4, AQP-5, or
full-length Preparation of affinity-purified anti-AQP-4
antiserum. A 16-mer peptide corresponding to amino
acids 277-292 of AQP-4 (HVIDIDRGDEKKGKDC) was synthesized and
purified by HPLC (QCB, Hopkington, MA), conjugated to keyhole limpet
hemocyanin, and used to immunize rabbits. Subsequent immune sera were
affinity purified using SulfoLink Coupling gel (Pierce, Rockford,
IL) as described previously (21).
Immunohistochemistry. Tracheas of
anesthetized rats were intubated, and lungs were perfused with optimum
cutting temperature (OCT) compound (Miles, Elkhart, IN). Lung tissue
samples were then embedded in OCT compound and snap-frozen in liquid
nitrogen, and 6-µm-thick frozen sections were mounted on
Superfrost/Plus slides (Fisher Scientific, Pittsburgh,
PA). Slides were incubated at 37°C for 15 min, fixed with acetone
for 5 min, exposed to 0.3% Triton X-100 in phosphate-buffered saline
(PBS) for 5 min, and blocked with PBS containing 2% bovine serum
albumin (BSA) and 10% normal donkey serum (Jackson ImmunoResearch
Laboratories, West Chester, PA) for 1 h at 25°C. After a 1.5-h
exposure to affinity-purified anti-AQP-4 peptide antibody (1:50
dilution in PBS with 2% BSA), slides were incubated (1 h) with
alkaline phosphatase-conjugated affinity-purified donkey anti-rabbit
IgG (1:75; Jackson ImmunoResearch Laboratories), and then washed slides
were developed using fast red chromagen (Sigma Chemicals, St. Louis,
MO) and counterstained with methyl green.
Isolation of FDLE cells and fetal
matrix. FDLE cells were isolated from E20 Wistar rats
as described previously and seeded at confluence (5 × 105
cells/cm2) on either lung
cell-derived matrix [mixed-lung cell (MLC)] derived from
embryonic day 17 (E17) fetal rats or
uncoated filters (31). Previous work (31) has demonstrated that FDLE
cells are derived primarily from prealveolar epithelium. FDLE cell
filters were then cultured in Dulbecco's modified Eagle's medium with
10% FBS in humidified 21% O2-5%
CO2-74%
N2 or 3%
O2-5%
CO2-92%
N2 atmospheres for 24 h (30).
After replacement of the medium in 24 h, adherent cells were harvested
for RNA at 24 h (uncoated vs. MLC experiments) or 72 h (3 vs. 21%
O2 concentration experiments).
Maximal AQP-4 expression occurs in rat lungs during
the perinatal period. RT-PCR amplifications of adult
rat lung cDNA with primers specific for AQP-4 and AQP-5 yielded probes
possessing nucleotide sequences identical to those reported previously
(14, 33). As shown in Fig.
1A,
Northern analyses of brain, lung, and kidney total RNA from adult rats
confirmed (14) high levels of AQP-4 expression in adult rat brain
(100%, n = 4) that were significantly
different (P < 0.001) compared with
either lungs (36 ± 10.8%) or kidneys (17 ± 6%) from the same
animals.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-,
-, and
-subunits of ENaC are both
expressed by cultured rat fetal distal lung epithelial (FDLE) cells.
AQP-4 and ENaC expression increase in FDLE cells cultured on uncoated
permeant filters compared with matched control cells cultured on
filters containing extracellular matrix derived from fetal lung
epithelial cells. Similarly, AQP-4 expression increases in FDLE cells
exposed to 21% O2 compared with
cells exposed to 3% O2. These
data demonstrate that AQP-4 expression is highest on the first day
after birth in neonatal rat lungs. Exposure to ambient 21%
O2 may contribute to increases in
AQP-4 and ENaC expression to facilitate water transport across neonatal
airway epithelia in the immediate postnatal period.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-rich fluid secreted by
the epithelium that is necessary for proper lung development (reviewed
in Refs. 3, 23, 36). The switch from placental to pulmonary gas
exchange occurring after parturition requires that fluid be absorbed
from both the alveoli and airways to create an air-liquid interface for
adequate respiratory function. To accomplish this transition,
transepithelial Na+ absorption
occurs across the pulmonary epithelium, accompanied by modulation in
both the expression and activity of basolateral Na+-K+-ATPase
(4, 27, 29) as well as of the apical amiloride-inhibitable epithelial
Na+ channel (ENaC; see Refs. 5,
26, 25, 28). To determine whether membrane aquaporin (AQP) water
channel proteins (6, 38) are modulated concomitant with
Na+ reabsorption during this
interval, we characterized the temporal relationship between the
expression of two lung AQPs, AQP-4 and AQP-5, compared with ENaC
subunits in both intact lungs and fetal distal lung epithelial (FDLE)
cells from neonatal rats.
-,
-, or
-subunits of ENaC. Furthermore, we
quantified alterations in AQP-4 expression in rat FDLE cells cultured
under conditions in which either extracellular matrix (ECM) or ambient
fraction of inspired O2 was
varied. Together, these data demonstrate that AQP-4 expression is
modulated in a manner similar to that displayed by ENaC. The eightfold
increase in AQP-4 within a 24- to 72-h interval from embryonic (E)
day 20 (E20) to postnatal
day 1 suggests that AQP-4 may play
some noncritical role in lung airway transepithelial water
reabsorption, perhaps in conjunction with ENaC during the early
postnatal period.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(3.7 kb)-,
(2.2 kb)-, or
(3.2 kb)-ENaC probes
(5 × 105
counts · min
1 · ml
1;
see Ref. 5), washed with 0.1× SSC + 0.01% SDS at 55°C for 30 min (AQP-4 and AQP-5) or 0.2× SSC + 0.1% SDS at 42°C (
-,
- and
-ENaCs), and then subjected to autoradiography at
70°C (Lightening Plus intensifier screens and XAR-5 film;
Eastman Kodak, Rochester, NY). Individual lanes of autoradiograms were
quantified using either photoanalytic image processing with National
Institutes of Health (Bethesda, MD) Image or laser densitometry as
described previously (34), and the signal for each band was normalized by probing the same blots with a similarly labeled cDNA probe for
glyceraldehyde-3-phosphate dehydrogenase and comparing signal intensity. Results are expressed as means ± SE. Significant
differences between samples were determined using ANOVA. Statistical
significance was considered at P
values of <0.05.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
Aquaporin (AQP)-4 expression in the lung peaks in the perinatal period.
A: expression of AQP-4 in the adult
rat. Twenty micrograms of total RNA prepared from whole brain
(lane 1), lung
(lane 2), and kidney
(lane 3) were subjected to Northern
blotting analyses using a
32P-labeled AQP-4 cDNA probe.
Resulting single autoradiogram (exposed for 18 h) displayed here shows
a single 5.5-kb transcript present in all lanes and is representative
of 4 separate experiments. Bottom of
A shows data from the same blot after
reprobing with a
[32P]glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA probe to correct for differences in RNA
loading. Filled circles denote positions of 28S and 18S ribosomal RNA
bands. B: expression of AQP-4 in
developing rat lung. Total RNA was prepared from embryonic
day 20 (E20; lane
1), embryonic day 21 (E21; lane 2), neonatal
day 1 (D1; lane
3), neonatal day 3 (D3; lane 4), neonatal
day 7 (D7; lane
5), neonatal day 14 (D14; lane 6), and adult lungs
(lane 7) using both AQP-4 and GAPDH
probes. Relative expression of AQP-4 represented by the
autoradiographic densities of AQP-4/GAPDH normalized for the mean AQP-4
expression in adult lung RNA is shown.
C: individual autoradiogram exposed
for 18 h displaying prominent expression of AQP-4 in the lungs of
neonatal rats. Lane designations correspond with bars in
B. D:
expression of AQP-5 in developing lung. This single autoradiogram is
representative of a total of 4 Northern analyses performed as described
in B. Total lung RNA from E20
(lane 1), E21 (lane
2), D1 (lane 3),
D3 (lane 4), D7
(lane 5), and adult lung
(lane 6) was probed with both AQP-4
and GAPDH. Note that AQP-5 expression is not maximal in the immediate
postnatal period.
AQP-4 is detectable on E20 (Fig. 1C, lane 1), and its maximal expression occurs within the immediate perinatal period corresponding from birth (D1; lane 3) to postnatal day 3 (D3; lane 4) as shown in Fig. 1, B and C. Lung AQP-4 expression within 24 h of birth is 830 ± 20% (P < 0.005, n = 3) higher compared with that in E20 rats or 200 ± 10% (P < 0.001, n = 3) higher than levels present in adult lung (Fig. 1C, lane 7). In contrast, AQP-5 expression in developing lung is distinct from that exhibited by AQP-4 (Fig. 1D). AQP-5 is detectable in total lung RNA at E21 (lane 2) and only increases gradually over the first weeks of life such that adult lung AQP-5 expression (Fig. 1D, lane 6) is 299 ± 54% (P < 0.005, n = 4) greater compared with that present in the lungs of D1 rats.
AQP-4 expression in the developing lung is tissue specific, since identical analyses of both brain (Fig. 2, A and B) and kidney (Fig. 2C) reveal different age-specific patterns. For example, expression of AQP-4 in brain reaches adult levels that are 210 ± 32% (n = 3, P < 0.001) of those of D1 rats only after day 14 (Fig. 2A).
|
AQP-4 is localized to the basolateral surfaces of airway epithelial cells in both the adult and neonatal lung. Affinity-purified antibody raised to a 16-mer peptide containing a sequence present in the COOH-terminal domain of AQP-4 revealed distinct staining of the basolateral membranes of epithelial cells lining both cartilaginous large airways and bronchi as well as of medium-sized bronchi and bronchioles in lungs of newborn and adult rats (Fig. , A and C). Staining is not present in other lung structures, including pulmonary vessels and alveolar spaces. In all cases, AQP-4 staining is specific as demonstrated by its ablation after addition of excess (50 µg/ml) corresponding peptide (Fig. 3B). No specific staining is observed in the lungs of E20 or E21 fetal rats (data not shown). As reported previously by others (8, 19, 37), anti-AQP-4 antiserum also identifies AQP-4 protein in the basolateral membranes of epithelial cells lining the tubules in the kidney medulla (Fig. 3D).
|
AQP-4 is expressed in cultured FDLE cells where its
expression is modulated by alterations in fetal ECM and ambient
PO2 in a manner similar to that
exhibited by the -,
-, and
-subunits of
ENaC. Previous work in one of our laboratories has
utilized cultured FDLE cells to demonstrate that amiloride-inhibitable transepithelial Na+ transport is
modulated by multiple factors, including ECM components synthesized by
MLC isolated from fetal rats (30, 31, 36). To determine if the
expression of ENaC subunits and AQP-4 is modulated in FDLE cells in a
similar manner, we incubated FDLE cells on uncoated filters or filters
coated with fetal ECM. As shown in Fig. 4,
A-C,
the expression of the
-,
-, and
-ENaC subunits as well as of
AQP-4 were reduced on coated filters compared with paired FDLE cells
cultured on uncoated filters. In contrast to FDLE cells from uncoated
filters (Fig. 4, lanes 1 and
3), all ENaC subunits of FDLE cells
cultured on fetal ECM filters are reduced significantly
(P < 0.05, n = 4), including the
-subunit (96 ± 3.5% reduction; Fig. 4C,
lanes 2 and
4) as well as the
- and
-subunits (67 ± 9 and 90 ± 3% reductions, respectively,
Fig. 4B, lanes
2 and 4). In a
similar manner, AQP-4 mRNA expression in FDLE cells cultured on fetal
ECM is also significantly reduced by 48 ± 8%
(P < 0.005 n = 4). No AQP-5 expression was
detectable in these FDLE cells despite prolonged autoradiography (data
not shown). In a similar manner, FDLE cells cultured in 21%
O2 express significantly (50.2 ± 5.3%, P < 0.01, n = 4) more AQP-4 compared with FDLE
cells cultured in 3% O2 (Fig.
5).
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DISCUSSION |
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It is now generally accepted that ENaC plays an important role
in net fluid absorption from airways and alveoli at the time of birth
(11, 24, 25, 28). Targeted deletion of -ENaC gene function results
in mice that exhibit a lack of amiloride-sensitive Na+ transport, fail to clear their
lung liquid, and die shortly after birth (11). However, the
transepithelial pathways for water reabsorption that accompany the
establishment of ionic gradients in newborn lungs are not known.
AQP proteins have been demonstrated to increase the water permeability of both apical and basolateral membranes in various kidney epithelial cells (2). In the kidney proximal tubule, AQP-1 increases the water permeability of both the apical and basolateral membranes where net water reabsorption is driven by the establishment of small osmotic gradients (1, 16). In contrast, a combination of AQP-2 (apical membrane) and AQP-3 as well as AQP-4 (basolateral membrane) facilitates selective water reabsorption in response to large transepithelial osmotic gradients in kidney collecting duct (1, 12, 16, 21, 38). However, the exact physiological roles of various AQPs in transepithelial water transport in mammalian tissues are currently under active investigation. Whereas humans lacking AQP-1 expression do not exhibit major alterations in either kidney or pulmonary physiology (32), mutations in AQP-2 cause non-X-linked nephrogenic diabetes insipidus (7). At present, no natural human mutations of AQP-3, -4, or -5 have been reported. However, recent targeted deletion of AQP-4 gene function in mice has established that a knockout of AQP-4 gene function does not alter normal morphology of developing lung or cause significant changes in the expression of AQP-5 in lung that could possibly compensate for lack of AQP-4 lung function (19).
To investigate if the expression of AQP-4 and AQP-5 is similar to that exhibited by ENaC, we studied selected aspects of both the temporal expression and location of these AQP water channels in the lungs of fetal and postnatal rats as well as in cultured FDLE cells isolated from lung tissue.
Northern analyses (Fig. 1) show that AQP-4 expression begins by E20 in
fetal rats and increases eightfold over a 48- to 72-h interval to
achieve maximal levels during the first postnatal day. Thereafter, lung
AQP-4 mRNA levels decline over the first week of postnatal life to
adult levels that remain approximately one-half of those present during
the brief interval of maximal AQP-4 expression. This pattern of lung
AQP-4 expression is tissue specific (Fig. 2). These data confirm and
extend those published recently by Umenishi et al. (37) who utilized an
RNase protection technique to quantify the expression of AQP-4 and
AQP-5 compared with -actin mRNA levels. The data reported here
provide additional data on days 3 and
14 of postnatal life not examined by
these investigators (37).
Interestingly, the pattern of AQP-4 expression in developing rat lung
is similar to that described previously for ENaC (25, 36), resulting in
coincident maximal coexpression of -,
-, and
-subunits and
AQP-4 on day 1 of life. Although
-ENaC expression peaks immediately before birth and then declines
transiently during the first week of postnatal life,
- and
-ENaC
expression begin 24-48 h before birth and reach maximal levels of
expression during the first week of life on postnatal
day 1 (36). In contrast, similar
Northern analyses of lung AQP-5 expression (Fig.
1D) reveal that, although it is
detectable in E21 fetal rats, maximal adult AQP-5 mRNA levels are not
achieved until after the first week of postnatal life (15, 37).
Our immunohistochemistry of AQP-4 protein in adult and developing rat lung is similar to previous reports (8, 10, 37) and reveals that AQP-4 is present on the basolateral surfaces of epithelial cells lining large and small airways but not in alveolar epithelial cells (Fig. 3). The lack of detectable AQP-4 protein in airways of fetal rats is consistent with Northern analyses described above. Localization of AQP-4 to airway epithelial cell basolateral membranes suggests that it may augment transepithelial water in a manner similar to that proposed for AQP-4 in the basolateral membrane of kidney inner medullary collecting duct (8, 16, 19). However, at the present time, no apical membrane AQP (corresponding to AQP-2 in kidney) has been identified for these airway epithelial cells.
To explore the relationships between factors that modulate expression
of both AQP-4 and the ENaC subunits, we utilized primary cultures of
FDLE cells exposed to differences in either ECM and O2 concentrations (Figs. 4 and 5).
Compared with control FDLE cells cultured on uncoated filters, FDLE
cells cultured on fetal lung ECM exhibit a combination of significant
reductions in amiloride-inhibitable Na+ transport (31) as well as
significant decreases in -,
-, and
-ENaC subunits as well as
in AQP-4 (Fig. 4).
Recently, Pitkanen et al. (30) reported that switching cultured FDLE cells from fetal 3% to postnatal 21% O2 concentrations significantly increases both the ENaC and ENaC subunit expression. Data shown in Fig. 5 demonstrate that exposure of FDLE cells to 21% O2 vs. 3% O2 increases AQP-4 expression by ~50%. These findings may suggest the existence of an O2-responsive mechanism that may participate in modulation of both AQP-4 and ENaC subunits at the level of transcription. In contrast, FDLE cells do not display detectable levels of AQP-5 mRNA when cultured under any of these conditions described above.
In summary, the data reported here provide evidence that the expression of ENaC subunits and AQP-4 is highest on the first day after birth in neonatal rats. In this regard, exposure to ambient 21% O2 may contribute to the increases in both AQP-4 and ENaC expression. Because the majority of lung liquid is absorbed during labor and the 6-h interval immediately after birth (when AQP-4 protein levels in lung are low; see Ref. 26), AQP-4 protein is not likely a rate-limiting step in transepithelial water flux. These data may provide a partial explanation for the absence of phenotypic abnormalities of AQP-4 in knockout animals (19). We speculate that AQP-4 may become more important in the overall process of transepithelial water flux in airways after the immediate postnatal period when endogenous hormonal levels such as epinephrine have declined (24). Although these data suggest that coordinate regulation of ENaC and AQP-4 expression may occur and imply that AQP-4 may participate in increasing the water permeability of the basolateral membranes of airway epithelial cells to facilitate Na+-mediated reabsorption of fetal lung liquid, both the role of AQP-4 protein in airway water reabsorption and its relationship to transepithelial Na+ flux mediated by ENaC require further study.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. M. Donovan, M. Baum, and K. Haley for advice on immunohistochemistry.
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FOOTNOTES |
---|
This work was supported in part by National Institutes of Health National Research Service Award 1 F32 HL-09131 01 (M. K. Ruddy) and Grant DK-38874 (H. W. Harris) and by the Medical Research Council (MRC) Group in Lung Development (H. M. O'Brodovich). O. M. Pitkanen is a fellow of the MRC of Canada, and H. M. O'Brodovich is a Career Scientist of the Heart and Stroke Foundation of Ontario.
Address for reprint requests: H. W. Harris, Division of Nephrology, Rm. 1260 Enders Bldg., Children's Hospital, 300 Longwood Ave., Boston, MA 02115.
Received 31 December 1996; accepted in final form 6 March 1998.
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