Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521
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ABSTRACT |
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Fluid transport across epithelial and endothelial barriers occurs in the neonatal and adult lungs. Biophysical measurements in the intact lung and cell isolates have indicated that osmotic water permeability is exceptionally high across alveolar epithelia and endothelia and moderately high across airway epithelia. This review is focused on the role of membrane water-transporting proteins, the aquaporins (AQPs), in high lung water permeability and lung physiology. The lung expresses several AQPs: AQP1 in microvascular endothelia, AQP3 in large airways, AQP4 in large- and small-airway epithelia, and AQP5 in type I alveolar epithelial cells. Lung phenotype analysis of transgenic mice lacking each of these AQPs has been informative. Osmotically driven water permeability between the air space and capillary compartments is reduced ~10-fold by deletion of AQP1 or AQP5 and reduced even more by deletion of AQP1 and AQP4 or AQP1 and AQP5 together. AQP1 deletion greatly reduces osmotically driven water transport across alveolar capillaries but has only a minor effect on hydrostatic lung filtration, which primarily involves paracellular water movement. However, despite the major role of AQPs in lung osmotic water permeabilities, AQP deletion has little or no effect on physiologically important lung functions, such as alveolar fluid clearance in adult and neonatal lung, and edema accumulation after lung injury. Although AQPs play a major role in renal and central nervous system physiology, the data to date on AQP knockout mice do not support an important role of high lung water permeabilities or AQPs in lung physiology. However, there remain unresolved questions about possible non-water-transporting roles of AQPs and about the role of AQPs in airway physiology, pleural fluid dynamics, and edema after lung infection.
water transport; water permeability; alveolus; fluid clearance; transgenic mice
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INTRODUCTION |
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FLUID MOVEMENT BETWEEN THE AIR SPACE and vascular compartments in lung plays an important physiological role in many processes such as regulation of airway hydration, reabsorption of alveolar fluid in the neonatal period in preparation for alveolar respiration, and the resolution of pulmonary edema (5, 46, 56). Fluid movement across epithelial and endothelial barriers occurs in interstitial and alveolar pulmonary edema resulting from numerous etiologies such as congestive heart failure, acute respiratory distress syndrome, infection, acute lung injury after acid aspiration, and subacute hyperoxic lung injury. The ability to control fluid transport in the lung by pharmacological means, gene therapy, or other approaches could have profound consequences in cardiovascular and lung medicine.
Biological fluids consist of salts, macromolecules, and water, with
water as the major component on a molar basis. Water transport can be
driven across membrane barriers by hydrostatic and oncotic or osmotic
driving forces. The general paradigm for the transport of fluid across
tight cell layers such as epithelia in the lung is that active salt
transport drives water transport by creating osmotic gradients.
Hydrostatic fluid movement can occur when a permeability barrier
becomes leaky to both solutes and water. The reader is referred
elsewhere for additional details about the biophysics of fluid
transport across membrane and complex tissue barriers (17, 75). From
the above considerations, it follows that active and secondary active
ion and solute transport processes play a major role in fluid transport
in the lung and extrapulmonary tissues. There is a considerable body of
information about ion pumps (e.g.,
Na+-K+-ATPase), channels (e.g., epithelial Na
channel, cystic fibrosis transmembrane conductance regulator, ClC2),
and transporters (e.g., Cl/HCO
3 and
Na+/H+ exchangers) involved in maintaining
cellular homeostasis and vectorial fluid transport in airways and lung.
This review addresses the issue of how water moves across barriers in
the lung.
The story begins with the definition of permeability barriers in lung and measurement of their water permeability properties. The general observation is that lung water permeabilities are high and in the case of type I alveolar epithelial cells, higher than in other mammalian cell membranes studied to date. The aquaporin (AQP) family of membrane water channels is reviewed, and indirect evidence is described supporting a role for AQPs in lung physiology, such as the specific AQP expression pattern in the lung and developmental expression-function correlates. The generation and phenotype analysis of transgenic knockout mice lacking each of the lung AQPs is then described. The knockout mice have been very informative in defining the role of AQPs in lung physiology. The review then points out important unresolved questions in the field and directions for future research.
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BARRIERS TO WATER TRANSPORT IN LUNG |
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There are several barriers to the transport of water between the air
space, interstitial, and vascular compartments in the lung (Fig.
1). The trachea and large airways contain
an epithelial cell layer but represent a small fraction of the total
surface area available for movement of fluid into and out of the air
spaces (30, 51, 69, 80). The more numerous smaller airways also contain
an epithelial cell layer but still represent a relatively small surface
area. In human lung, the total airway surface area is ~1.4
m2, only ~1% of the alveolar surface area of 143 m2 (80). The alveolar epithelium thus provides the major
surface lining the air space. The alveolar epithelium contains type I cells, which are flat cells comprising the majority of the alveolar epithelial surface, and type II cells, which transport salt actively and produce surfactant. Movement of water between the air space and
capillary compartments through the alveolar epithelium also encounters
potential permeability barriers in the interstitium and capillary
endothelia. As shown in Fig. 1, the pulmonary artery runs with the
airways and divides as the airways divide (48, 65). The capillary bed
is then formed in the alveolar acinar region beyond the terminal
bronchiolus. The bronchial artery forms a capillary plexus that
supplies the muscle and submucosa of the airways. This plexus
communicates with branches of the pulmonary artery and empties into the
pulmonary veins. Figure 1 also shows the complex cluster organization
of alveolar acini arising from distal airways. Heterogeneity in
vascular geometry, blood flow, and gas exchange throughout the lung
provide yet an additional level of complexity. Thus although the basic
concept of vascular, interstitial, and air space compartments is valid,
the description of lung fluid movement in terms of passage across
well-demarcated uniform barriers is an oversimplification.
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WATER PERMEABILITIES IN LUNG |
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Water permeability across a simple barrier separating two compartments
is characterized by an osmotic water permeability coefficient (Pf), defined as the volume flux
(JV) induced by an osmotic gradient: Pf = JV/(SVwC), where
S is barrier surface area, Vw is the partial molar
volume of water (18 cm3/mol), and
C is the
osmotic gradient. High Pf (generally
>0.005-0.01 cm/s) suggests a facilitated water pathway involving
molecular water channels (17). The Arrhenius activation energy
(Ea), which is deduced from the temperature
dependence of Pf, also provides potentially useful
information. Ea is generally <4-6 kcal/mol for a pore-mediated water pathway (or unstirred layer-limited water
transport) and >8-10 kcal/mol for lipid-mediated water
transport. Many biological membranes contain parallel pore and lipid
pathways that warrant consideration in the interpretation of
Pf and Ea values. Other
biophysical properties describing a water-transporting barrier include
the diffusional water permeability (Pd) and solute reflection coefficient. However, as discussed by Verkman (75), Pd and solute reflection coefficient probably have
little utility in describing complex structures like the lung because
of unstirred layer effects and uncertainties in barrier geometry.
Characterization of a water-transporting pathway also generally
involves the testing of mercury compounds (e.g., HgCl2)
known to inhibit many of the AQP water channels, with the caveat that
mercury compounds are highly toxic to living cells. The reader is
referred to Refs. 16, 17, and 75 for a more detailed description of the
biophysics of water permeability.
An initial estimate of osmotic water permeability (Pf) between the air space and the capillary compartments utilized an air space "instillation and sample" approach in the in situ perfused sheep lung (19). The vascular compartment was perfused continuously with isosmolar saline, and the air space was filled rapidly with hypertonic saline. Serial samples of air space fluid showed that osmolality equilibrated rapidly, with a half-time of ~45 s. Osmotic equilibration was reversibly slowed approximately threefold on addition of HgCl2 to the perfusate. Pf was estimated to be 0.02 cm/s, a very high value that was similar to that of erythrocytes and epithelial cell membranes from kidney tubules. It was concluded that water movement between the air space and the capillary compartments in the intact lung was transcellular and facilitated by mercurial-sensitive water channels. Another approach to study air space-capillary water permeability involves perfusing isolated lungs with an isosmolar and then an anisosmolar solution and measuring serial osmolalities of fluid exiting the pulmonary vein (14); however, this approach does not provide quantitative permeability values and is subject to concerns about the effects of heterogeneity in vascular flow. Newer quantitative methods are described below to measure water permeability across specific barriers in the lung.
Pleural surface fluorescence recordings. For studies in small
animals, where rapid air space fluid instillation and sampling are not
practical, a pleural surface fluorescence method was developed in which
air space osmolality is deduced from the fluorescence of an
aqueous-phase indicator added to the air space fluid (7). The principle
of the method is shown in Fig. 2A.
The air space is filled with fluid containing a membrane-impermeant
fluorophore, and the pulmonary artery is perfused with solutions of
specified osmolalities. Because of the finite penetration depth of the
excitation light, only lung tissue within 100-200 µm of the
pleural surface is illuminated. Under these conditions, the surface
fluorescence signal is directly proportional to the air space
fluorophore concentration. In response to an osmotic gradient, water
flows between the air space and the perfusate compartments, resulting
in a change in fluorophore concentration that is detected continuously
by measurement of pleural surface fluorescence. This approach has
excellent time resolution without the need for invasive fluid sampling.
The representative pleural surface recording shown in Fig.
2B indicates a stable signal with remarkably
little experimental noise. In response to doubling of perfusate
osmolality from 300 to 600 mosM, the pleural surface fluorescence
signal approximately doubles as predicted theoretically.
Pf in the mouse lung was 0.017 cm/s at 23°C,
independent of the solute used to induce osmosis, independent of
osmotic gradient size and direction, weakly temperature dependent, and
inhibited by HgCl2. The pleural surface fluorescence method
was used in a developmental study to show increased air space-capillary
water permeability in the first 24 h after birth (9) and, as discussed below, in lungs from various AQP knockout mice. The pleural surface fluorescence method has also been used to measure
Pd using an H2O/2H2O exchange
method and a 2H2O-sensitive fluorophore (7) and
can be used to study H+, Na+, and
Cl transport with suitable membrane-impermeant
fluorescent indicators added to the air space fluid.
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The air space-capillary osmotic water permeability measured by the approach above includes serial contributions from epithelial and endothelial barriers. A modified pleural surface fluorescence strategy was developed to directly measure microvascular endothelial water permeability in intact lung (8). The air space is filled with an inert, water-insoluble perfluorocarbon to restrict lung water to two compartments, the interstitium and capillaries, and thus establish a single rate-limiting permeability barrier, the capillary endothelium. The pulmonary artery is perfused with solutions of specified osmolalities containing identical concentrations of high-molecular-weight fluorescein-dextran. In response to a change in perfusate osmolality, water is osmotically driven into or out of the capillaries, resulting in fluorophore dilution or concentration, respectively. The change in fluorophore concentration is recorded as a prompt change (decrease for fluorophore dilution; Fig. 2C) in pleural surface fluorescence. The magnitude of the prompt deflection in fluorescence signal increases with increased water permeability or decreased pulmonary arterial flow. The prompt deflection is followed by a slower return of fluorescence signal to the original level as interstitial and capillary osmolalities equilibrate. With a three-compartment model to compute capillary Pf from the fluorescence data, microvascular Pf was ~0.03 cm/s, weakly temperature dependent, and inhibited by mercury compounds. To date, methods have not been developed to directly measure osmotic water permeability of the alveolar epithelial barrier in the intact lung.
Gravimetric method to measure osmotic water permeability and capillary filtration. Gravimetry is a classic method to study organ bed capillary filtration, which was introduced more than 50 years ago (58) and first used in the lung in 1962 (2). Gravimetry has been used to measure hydrostatic capillary filtration in dog lungs (15, 23, 59). Lung weight is the sum of solids and lung water in all compartments. For measurement of lung filtration in large animals, the pulmonary artery is generally perfused with a constant-flow infusion pump, and lung weight is measured in response to an increase in venous outflow pressure. The kinetics of lung weight increase consist of a relatively rapid phase corresponding to vascular engorgement, followed by a slower phase corresponding to extravascular fluid accumulation.
Song et al. (67) recently modified the classic gravimetric
method to measure both osmotically induced water transport and hydrostatic filtration in mouse lungs. In contrast to the pleural surface fluorescence approach, gravimetry measures water transport throughout the lung. Figure 3A
shows the apparatus in which lung weight is measured during perfusion.
When the air space compartment is filled with isosmolar saline, changes
in perfusate osmolality drive water movement across endothelial and
epithelial barriers, producing changes in the water content of the
interstitial and air space compartments observed as changes in lung
weight. Figure 3B shows representative gravimetric data in
which increasing the perfusate osmolality from 300 to 400, 500, and 600 mosM produced reversible decreases in lung weight as water was drawn
out of the air spaces of the lung. Figure 3C shows a filtration
study in which the pulmonary artery was continuously perfused with
saline and the pulmonary arterial pressure was increased from 8 to 18 cmH2O at a constant left atrial pressure of 5 cmH2O. The increased pressure produced a prompt increase in
lung weight due to vascular engorgement, followed by a further
approximately linear increase in lung weight due to fluid filtration.
In a variation of this approach, the air spaces were filled with an
inert perfluorocarbon to selectively probe microvascular permeability
properties. As described below, the gravimetry method has been used to
analyze lung water permeability in various AQP knockout mice.
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Water transport across isolated microperfused distal airways.
It is not possible to measure water permeability in small airways in
the intact lung because the alveolar epithelium comprises the majority
of the surface area. To measure the water transport properties of small
airways directly, a microperfusion technique was developed based on
methods developed originally for perfusion of isolated kidney tubules
(35). Small airways from the guinea pig were used because relatively
long segments of viable distal airway (up to 2 mm length, 0.1-0.2
mm diameter) without bifurcations could be microdissected (18). The
airway lumen was perfused at constant nanoliter per minute flow rates
with isosmolar saline containing a membrane-impermeant fluorophore. The
bath solution surrounding the airway was changed from isosmolar saline
to a solution of different osmolality. The anisosmolar bath drives water transport across the airway epithelium, resulting in a
steady-state profile of increasing fluorophore concentration (for a
hyperosmolar bath) along the axis of the airway. The fluorescence
signal at any point along the airway, proportional to lumen osmolality, is then related to transepithelial Pf, airway size,
and lumen flow rate. The Pf in guinea pig distal
airways was 4.5 × 103 cm/s at 23°C,
independent of the lumen flow rate and the magnitude and direction of
the osmotic gradient, and weakly temperature dependent
(Ea 4.4 kcal/mol). Unlike alveolar water
permeability, airway Pf was not inhibited by
HgCl2, consistent with a role for the mercurial-insensitive
water channel AQP4. The in vitro microperfusion method with
fluorescence detection was also used to measure diffusional water
transport by H2O/2H2O
exchange (18); however, unstirred layers preclude interpretation of
Pd values in terms of intrinsic membrane barrier properties.
Water permeability across plasma membranes of isolated lung and airway cells. The measurement of water permeability in isolated cells or plasma membrane vesicles can provide information about the contributions of specific cell types and individual plasma membranes in the overall water permeability. The stop-flow light-scattering technique has been used to measure water permeability in vesicles or cells in suspension. Cell or vesicle suspensions are subjected to an osmotic gradient by rapid mixture (generally <1 ms) with a solution of different osmolality in a stop-flow apparatus. Osmotic water movement across the cell plasma membrane produces a change in cell volume and an instantaneous change in the scattered light intensity. Osmotic water permeability is computed from the time course of the light-scattering signal and cell geometry. This approach was used to measure Pf in isolated type I and type II alveolar epithelial cells from the rat lung (13). In response to an increase in osmolality from 300 to 600 mosM, light scattering increased, with an apparent half-time of ~50 ms for freshly immunoisolated type I cells. The deduced Pf was exceptionally high (~0.07 cm/s), weakly temperature dependent, and inhibited by HgCl2. This high plasma membrane Pf in type I cells quantitatively accounts for the high air space-capillary Pf in intact lung.
Water permeability measurements in intact epithelial cells in
suspension provide information about the composite water permeabilities of apical and basolateral membranes. However, measurements with suspended cells cannot resolve the individual permeabilities of the two
membranes that form serial barriers in the intact epithelium. To
measure Pf in individual cell membranes, either
purified membrane vesicles are studied or water permeability is
measured in polarized epithelial layers. To date, methods have not been
reported to isolate purified apical or basolateral membranes from lung
epithelial cells. However, several methods are suitable for the
measurement of cell membrane water permeability in epithelial cell
layers that are cultured on rigid supports or mounted in an Ussing-type chamber (reviewed in Ref. 75). A simple approach is spatial filtering
microscopy in which cell volume changes are recorded with transmitted
light as a probe of intracellular refractive index (16). Cell swelling
causes dilution of cytoplasmic contents and decreased intracellular
refractive index. Figure 4 shows the time
course of cell volume in primary cultures of human tracheal epithelial
cells grown on porous supports. The osmolalities of solutions perfusing
the apical and basolateral membranes were individually changed to give
information about the apical and basolateral membrane water
permeabilities. With appropriate modeling and assumptions
about cell surface geometry (16), the Pf in tracheal cell membranes was in the low-to-moderate range
(0.004-0.005 cm/s). Similar permeabilities have been measured in
cultured human nasal epithelial cells (82), where water permeability of
the apical membrane appeared to be greater than that of the basolateral membrane.
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Remaining questions. The functional studies indicate very high water permeabilities in cell membranes of the alveolar epithelium and microvascular endothelium, with lower water permeabilities in airway cells. The relative apical versus basolateral membrane permeabilities of alveolar and small-airway epithelial cells, which may be important in cell volume regulatory mechanisms, are not known. Such measurements in type I alveolar epithelial cells will be particularly challenging because of their high water permeabilities. As described below, water permeability measurements in lung tissues from mice lacking specific AQPs have provided clear-cut information about the role of transcellular water movement through molecular water channels. Transgenic mice also permit evaluation of the more important issue of whether high lung water permeabilities are required for the functioning of normal and diseased lungs.
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AQP WATER CHANNELS IN THE LUNG |
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Family of AQP water channels. There are at least 10 AQP-type water-transporting proteins identified in mammalian tissues, at least 4 of which are expressed in lung. The AQPs are small (28-31 kDa) hydrophobic proteins that are homologous to the major intrinsic protein of lens fiber. The AQPs contain several conserved amino acid sequences, including two asparagine-proline-alanine (NPA) motifs, with overall amino acid identities of 22-60% after sequence alignment. In addition, there is considerable homology in the amino acid sequence between the first and second halves of each protein, suggesting an early gene duplication event (60). Mutagenesis and topological and crystallographic analyses of AQP1, an erythrocyte water channel that is easily purified, indicated that AQP1 monomers contain water pores but associate in the membrane as tetramers. Each AQP1 monomer consists of six membrane-spanning helical domains forming a tilted helical barrier surrounding a putative aqueous pore. The reader is referred to recent reviews for details about AQP structure and topology (29, 76). Although the only crystallographic information about mammalian AQPs is available for AQP1, it is likely that the general features such as tetrameric association and the tilted helical barrel motif apply to the other AQPs as well.
Descriptions of the genetics, functional analysis, and expression patterns of the mammalian AQPs are provided in numerous recent reviews (12, 54, 76). Briefly, major intrinsic protein (or AQP0) is expressed only in lens fibers and may function as a weak and possibly pH-regulated water transporter. AQP1 is widely expressed in the epithelia and endothelia of the kidney (proximal tubule, thin descending limb of Henle, vasa recta), lung (microvascular endothelia), choroid plexus, ciliary body, and other sites. AQP2 is the vasopressin-regulated water channel expressed primarily in epithelial cells of the kidney collecting duct. AQP3 is expressed most strongly in the kidney collecting duct but also in the gastrointestinal tract, large airways, skin, and urinary bladder. AQP4 (the mercurial-insensitive water channel) was originally cloned from the lung (26) but is most strongly expressed in astroglia of the central nervous system and, to a lesser extent, in the kidney collecting duct, skeletal muscle, and colon. AQP5 is expressed in alveolar epithelial cells as well as in salivary and lacrimal glandular epithelia. AQP6 is expressed only in the kidney. AQP7 is expressed primarily in the testis but is found as well in fat cells and, to a lesser extent, in a subsegment of the kidney proximal tubule. AQP8 is localized primarily to sites in the gastrointestinal tract including liver, pancreas, colon, and salivary gland. AQP9 is also strongly expressed in the liver as well as in peripheral leukocytes and the testis. In general, the AQPs are expressed at sites where rapid vectorial fluid transport is thought to occur. However, there are a number of exceptions such as AQP1 expression in erythrocytes, AQP3 in the skin and urinary bladder, AQP4 in skeletal muscle, AQP7 in fat cells, and AQP9 in leukocytes.
The AQPs function primarily as water-transporting pores in which intrinsic water permeability appears not to be subject to regulation by posttranslational modulation. AQP1, AQP2, AQP4, and AQP8 appear to be selective for the passage of water. AQP1 has also been proposed to transport CO2 (52), glycerol (1), and cations (88) under some conditions, but these results have been questioned (3, 77, 84, 86). Interestingly, despite its efficient water-transporting ability, AQP1 does not transport protons or small monovalent anions (73, 89). AQP3 and AQP7 also transport glycerol and possibly urea as well as water; however, it is not clear whether glycerol and water share a common aqueous pore. AQP9 appears to be a unique, relatively nonselective solute transporter with the ability to carry a variety of small polar solutes including some monosaccharides (70).
AQP expression in the lung. Four members of the AQP protein
family are expressed in the airways and lung. The first localization of
an AQP in the lung was reported by in situ hybridization showing AQP1
transcript expression in alveoli (28). Subsequently, AQP1 was
immunolocalized on the plasma membrane of microvascular endothelial cells and, to a lesser extent, on some pneumocytes (19, 25, 55, 63).
AQP3 is expressed in basal epithelial cells in large airways and
throughout the nasopharnyx (20, 33, 53). AQP4 is expressed at the
basolateral plasma membrane surface in epithelial cells throughout the
trachea and the small and large airways (20, 53). AQP5 is expressed at
the apical membrane of type I alveolar epithelial cells (22, 53). Type
I alveolar epithelial cells also express the T1 protein, which has
been proposed to be involved in water transport (81); however, an
expression study (43) has indicated that T1
does not appear to
transport water itself or to regulate AQP-type water channels (43). Of
note, water channels have not yet been identified at the basolateral
surface of alveolar epithelial cells nor at the apical surface of
airway epithelial cells. Small quantities of transcripts encoding AQP8, AQP9, and AQP9L appear to be expressed in the lung, but these proteins
have not yet been immunolocalized.
AQP expression and water transport in lung development. The lung at birth undergoes a dramatic transition from a fluid-secreting to a fluid-reabsorbing organ in preparation for alveolar respiration (5, 56). Several studies (32, 61, 72, 87) have addressed the issue of AQP developmental expression in lung. AQP1 transcript expression increases slowly throughout fetal life and then strongly just after birth. Quantitative immunoblot analysis showed a progressive increase in AQP1 protein expression over the first week of life. Steroid treatment was associated with increased AQP1 transcript expression. AQP4 transcript expression sharply increases near the time of birth. In contrast, AQP5 transcript expression increases slowly during the first week of life. To address the functional consequences of these observations, the pleural surface fluorescence method was used to measure air space-capillary osmotic water permeability in the developing lung (9). A significant increase in osmotic water permeability in the first 24 h after birth was found, consistent with a role for AQPs in the maintenance of a dry air space during the first few days of postnatal life.
Indirect evidence of a role for AQPs in lung physiology. The specific expression pattern of AQPs in the lung suggests that they play a role in water movement between the air space, interstitial, and capillary compartments. The increased AQP expression and air space-capillary water permeability near the time of birth supports a role for AQPs in neonatal lung fluid clearance. In addition, there is evidence that the expression of various lung AQPs can be regulated by steroids, keratinocyte growth factor, and possibly other hormonal factors (6, 32, 78). Together, these results provide indirect evidence of a role for AQPs in lung physiology. Direct investigation of AQPs in lung physiology requires the development of specific AQP blockers or phenotype analysis of animal models with defined AQP deficiencies. To date, the only AQP inhibitors are mercury compounds that are too toxic for in vivo use. Our laboratory has studied in vivo AQP function by the generation and phenotype analysis of transgenic knockout mice deficient in specific AQPs. Each of the four major airway or lung AQPs has been deleted by targeted gene disruption procedures. In addition, several double-knockout mice have been generated in which pairs of AQPs (AQP1 and AQP3, AQP1 and AQP4, AQP1 and AQP5, AQP3 and AQP4) have been deleted by cross-breeding of single-knockout mice.
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FUNCTIONAL ANALYSIS OF AQP KNOCKOUT MICE |
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Role of AQPs in extrapulmonary fluid secretion and absorption. Phenotype studies in AQPdeficient mice have indicated multiple physiological abnormalities. In the kidney, deletion of AQP1 produces a severe defect in urinary-concentrating ability, resulting in profound fluid loss when mice are deprived of water (42). Mechanistic analysis revealed that the urinary-concentrating defect results from a combination of defective proximal tubule fluid absorption (62) and defective countercurrent exchange because of low water permeability in the thin descending limb of Henle (10) and outer medullary descending vasa recta (57). The decreased active fluid absorption in the proximal tubule after AQP1 deletion supports the general paradigm that high epithelial cell water permeability is required for active, near-isosmolar fluid transport. Mice lacking AQP3, a collecting duct water channel, manifest nephrogenic diabetes insipidus with polyuria, polydipsia, and urinary hypoosmolality (38). The decreased basolateral membrane water permeability impairs the osmotic extraction of water from the collecting duct lumen. Mice lacking AQP4, a basolateral membrane water channel expressed in the inner medullary collecting duct, have a mildly impaired maximal urinary-concentrating ability (41) despite a fourfold decrease in transepithelial water permeability in the inner medullary collecting duct (11). Phenotype studies in AQP knockout mice have thus been informative in elucidating basic mechanisms of renal physiology (74).
The salivary gland provides another example where deletion of an AQP is associated with defective active transepithelial fluid transport. Saliva is produced by active salt pumping into the acinar lumen driving osmotic water transport, followed by expulsion through the watertight salivary duct. AQP5 is expressed at the apical membrane of acinar epithelial cells. Whereas wild-type mice produce large amounts of clear, nonviscous saliva after pilocarpine injection, AQP5 knockout mice produce relatively little highly viscous fluid (37). Fluid analysis indicated that the AQP5 knockout mice produced a hyperosmolar hypernatremic saliva, suggesting that AQP5 null mice are able to pump salt actively into the acinar lumen but that water permeability is too low to permit osmotic equilibration. These observations support the paradigm that high epithelial water permeability facilitates active fluid transport.
A recent study of brain edema provides an example of the importance of an AQP in tissue swelling (44). AQP4 is most strongly expressed in the brain at the blood-brain and brain-cerebrospinal fluid barriers. AQP4 protein is found in glial cells lining the ependyma and pial surfaces in contact with the cerebrospinal fluid. Initial evaluation of AQP4 null mice showed no overt neurological abnormalities or defects in osmoregulation (41). However, based on the specific AQP4 expression pattern, the hypothesis that AQP4 plays a role in producing brain edema in response to two established neurological insults, acute water intoxication, producing serum hyponatremia and cellular brain edema, and ischemic stroke, producing a combination of cellular and vasogenic edema, was tested (44). AQP4 deletion conferred remarkable protection from brain edema in these models, with improved mouse survival and clinical outcome as well as reduced brain swelling. In other recent studies, significant phenotype differences were found in peritoneal fluid transport (83) and dietary fat processing (39) in AQP1 null mice and colonic water transport in AQP4 null mice (39).
Organ-specific AQP expression does not indicate physiological significance. The phenotype studies described above provide clear-cut examples of the importance of AQPs in organ function. However, does the tissue-specific expression of an AQP imply a role in normal organ physiology or an adaptation to pathophysiological stress? Recent studies suggest that this is not the case. For example, AQP4 is expressed at the plasmalemma of fast-twitch skeletal muscle fibers and has been suggested to play a role in muscle physiology and in the pathophysiology of hereditary muscular dystrophies (21). However, analysis of skeletal muscle water permeability and contractile function, including treadmill performance, and muscle force generation and swelling showed no difference between wild-type and AQP4 knockout mice (85). AQP4 is also expressed at the basolateral membrane of gastric parietal cells (21) and has been proposed to play a role in gastric acid secretion (49). However, measurements of basal and agonist-stimulated gastric acidification and serum gastrin levels showed no differences between wild-type and AQP4 knockout mice (78a). As mentioned above, AQP5 deletion caused defective salivary gland fluid secretion; however, deletions of AQP1 and AQP4, which are also expressed in the salivary gland, did not affect saliva production (37). Deletions of the lacrimal gland AQPs (AQP1, AQP3, AQP4, and AQP5) did not affect basal or agonist-stimulated tear production (50). From these and additional examples, we conclude that tissue-specific AQP expression does not imply physiological significance. Therefore, the evaluation of AQP function in organ physiology needs to be done on a case-by-case basis. Lung phenotype studies on aquaporin knockout mice are described in the next section.
Role of AQPs in lung water permeability. Pleural surface
fluorescence and gravimetric methods were used to quantify the role of
AQPs in lung water permeability (4, 36, 67). Figure 5A shows pleural surface recordings
of air space-capillary osmotic water permeability in perfused lungs.
Deletion of AQP1 or AQP5 separately produced a remarkable decrease in
water permeability. Therefore, AQP1 provides the major route for
osmotically driven water movement across lung microvascular endothelia
and AQP5 provides the major route for water movement across the
alveolar epithelium. Because AQPs are expressed in cell plasma
membranes, osmotically driven water transport occurs mainly by a
transcellular rather than a paracellular pathway. These findings
support the notion that the primary route for water movement between
the capillary and air space compartments is by serial passage across
alveolar epithelial and microvascular endothelial barriers. The minimal residual water permeability across the alveolar apical surface after
AQP5 deletion may involve type II alveolar epithelial cells, the apical
membrane lipid of type I cells, and possibly as yet unidentified water
transporters. The residual osmotic water permeability across the
microvessels probably represents a combination of lipid-mediated and
paracellular transport.
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Figure 5B summarizes air space-capillary Pf values for AQP single- and double-knockout mice. Pf was greatly reduced when AQP1 and AQP5 were deleted together. AQP4 deletion by itself had little effect on Pf, but deletion of AQP4 together with AQP1 resulted in a decreased Pf compared with deletion of AQP1 alone. It was reasoned that the relatively low lung water permeability in AQP1 null mice might permit the detection of a small incremental effect of AQP4 deletion. The 1.4- to 1.6-fold effect of AQP4 deletion on water permeability in the AQP1 null mice suggests an overall contribution of AQP4 to lung water permeability of ~4% (taking into account the 10-fold effect of AQP1 deletion alone). Although this represents a relatively small contribution, it is greater than that predicted based on estimates that the airways comprise only 1.4% of the total air space epithelial surface area (30, 80) and that transepithelial water permeability in the airways is 5- to 10-fold lower than across the alveoli (18). Thus the airways may have a greater role in net lung fluid transport than previously anticipated.
The effect of AQP deletion on hydrostatically driven lung fluid accumulation was also studied. AQP1 deletion produced a small but significant 1.4-fold decrease in lung fluid accumulation in response to a hydrostatic stress despite a 10-fold reduction in osmotically driven air space-capillary water permeability (67). Therefore AQP1-independent pathways, probably involving paracellular water transport, play the major role in hydrostatically driven transcapillary water movement. Paracellular water permeability also dominates over transcellular water permeability in the renal microvasculature (57), where water movement driven by hydrostatic pressure differences and osmotic gradients of large solutes is substantially greater than that driven by small solutes like NaCl. In lung, renal, and other microvascular beds, transvascular water movement is governed by classic Starling forces. Because AQP1-independent pathways account for the vast majority of total water permeability in lung microvessels, it is unlikely that AQP1 would play an important role in hydrostatically driven lung edema in conditions such as congestive heart failure.
AQP5 deletion did not impair the accumulation of lung edema driven by increased hydrostatic pressure (36). Mechanistically, it is understandable why AQP5 should not facilitate hydrostatically driven fluid movement across the relatively tight alveolar epithelial barrier. Because AQP5 is a water-selective transporter with a unity reflection coefficient for solutes, solute-free water driven into the air space compartment by hydrostatic forces, unless accompanied by solute entry, would be opposed by strong osmotic driving forces. The accumulation of alveolar edema probably involves transient breakdown of the integrity of the alveolar barrier, permitting the entry of solutes and protein (47). Such bulk movement of fluid through rifts in the alveolar barrier would not be expected to involve AQP5 as was found experimentally.
Role of AQPs in alveolar epithelial fluid transport. An
important function of the alveolar epithelium is the active clearance of fluid from the air spaces, which is responsible for the resolution of pulmonary edema (45). Fluid absorption across the alveolar epithelium involves active salt transport, which is energized by
basolateral membrane Na+-K+-ATPase and requires
apical membrane (epithelial) Na+ channels (ENaC and
non-ENaC proteins) and possibly Cl transporters.
Water transport is driven by osmotic gradients created by salt
transport by a presumed near-isosmolar transport mechanism in which
small osmotic gradients drive water movement across the highly
water-permeable alveolar epithelium. As described above, other
near-isosmolar transport mechanisms require AQPs, including AQP1 in the
kidney proximal tubule (62) and AQP5 in salivary gland (37). It was
thus postulated that high alveolar epithelial water permeability would
facilitate active fluid absorption from the air spaces across the
alveolar epithelium.
Alveolar fluid clearance was studied in wild-type mice and mice lacking
various AQPs (4, 36). Several experimental strategies were used,
including the isolated perfused lung, the in situ perfused and
nonperfused lungs, and the in situ ventilated lung (models reviewed in
Refs. 24, 46). Interestingly, results from all models indicated that
AQP deletion did not affect alveolar fluid clearance. Figure
6 shows original data from a study in which the air space was filled with an isosmolar solution containing radiolabeled albumin as a volume marker. The core temperature was
maintained at 37°C, and albumin radioactivity was determined at 15 min as a measure of alveolar fluid clearance. Alveolar fluid clearance
was inhibited by the sodium inhibitor amiloride. Clearance was enhanced
by -agonists or by pretreatment of mice with keratinocyte growth
factor for 3 days to induce alveolar type II cell proliferation (71,
78). Deletion of AQP1 or AQP5, which decreased air space-capillary water permeability 10-fold, did not effect alveolar fluid clearance even under maximal keratinocyte growth factor induction and
-agonist stimulation.
|
The insensitivity of alveolar fluid clearance to AQP deletion is
probably the consequence of the substantially lower rate of active
fluid absorption (per unit surface area) in the lung compared with the
proximal tubule or salivary gland. In the latter systems, active fluid
transport exceeds 0.5 µl · min1 · µm
2
epithelial surface area. With a mouse lung epithelial surface area of
600 cm2, an active fluid absorption rate of 10 µl/min
(equivalent to 30% absorption in 15 min) gives a rate of 0.016 µl · min
1 · µm
2,
substantially lower than that in the kidney proximal tubule and
salivary gland. Slower rates of active fluid transport probably do not
require very high cell membrane water permeabilities (68). Another
consideration is that in the alveolus, unlike in the kidney proximal
tubule and salivary gland, salt and water move primarily through
different cells, type II and type I cells, respectively. Thus
three-compartment models of solute-solvent coupling to accomplish isosmolar fluid transport may not apply to the alveolar epithelium. Another difference between active fluid transport in the alveolus versus the kidney proximal tubule and salivary gland is that fluid is
rapidly cleared on both sides of the epithelium in the kidney (by lumen
and capillary flow) and salivary gland (by capillary flow and saliva
expulsion from the acinus), whereas fluid moves relatively slowly in
the alveolar air spaces. Whatever the mechanism, the lack of effect of
AQP deletion on alveolar fluid clearance raises doubt about the
physiological role of AQPs and high water permeability in lung.
A recent study (66) was done to investigate whether air space fluid clearance in the neonatal period requires AQPs. In lungs from wild-type mice harvested at specified times after spontaneous delivery, lung water content (assessed by wet-to-dry weight ratios) decreased from 7.9 at birth to 5.3 at 24 h after birth, with a 50% reduction at ~25 min. At 45 min after birth, wet-to-dry weight ratios were similar for wild-type mice and mice lacking AQP1, AQP4, or AQP5. It thus appears that lung AQPs are not required for fluid clearance in the neonatal period or for maintenance of a dry air space. This result contrasts sharply with the finding that ENaC-deficient mice die within 40 h after birth because of defective fluid clearance from the air spaces (31).
Role of AQPs in acute lung injury. An initial study was done to test the hypothesis that AQPs play a role in lung edema in response to various forms of acute lung injury. Three established models of lung injury were studied: hyperoxia, acid aspiration, and thiourea administration (66). Experiments were done with wild-type mice and mice lacking AQP1, AQP4, or AQP5. In mice exposed to >95% oxygen, mean survival was not affected by AQP deletion nor was the wet-to-dry ratio after 65 h of hyperoxia. In mice undergoing intratracheal acid instillation to produce epithelial cell injury, AQP deletion did not affect mean wet-to-dry weight ratios at 2 h. In mice receiving intraperitoneal thiourea to produce endothelial lung injury, AQP deletion did not affect mean wet-to-dry weight ratios or pleural fluid volume at 3 h. Together, these data suggest that despite their major role in osmotically driven lung water transport, the known lung AQPs have little importance in the physiological absorption of lung water or in the accumulation of fluid in the injured lung. Although not yet tested, the results so far would suggest that AQPs are not important in the pathophysiology of other clinically relevant causes of acute lung injury such as pneumonia, sepsis, or the resolution of pulmonary edema from cardiac failure.
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SUMMARY AND DIRECTIONS |
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The findings reviewed here indicate that water permeabilities across epithelial and endothelial barriers in the lung are in general high and facilitated by AQP-type water channels. Specific deletions of AQPs in mice resulted in remarkably decreased water permeabilities across air space-capillary barriers. However, physiologically important processes such as alveolar fluid clearance and edema accumulation in response to lung injury do not appear to require high lung water permeabilities or AQPs. The substantially lower rates of fluid movement in the lung compared with those in other organs such as the kidney and salivary gland probably account for the findings. Our results thus suggest that modulation of lung AQP expression and function by pharmacological or genetic means would not have clinical utility.
However, a number of issues remain unresolved. It is not clear whether the high level of specific expression of AQPs in the lung represents a vestigial remnant from ancestral mammals or whether AQPs are advantageous in some situations. High basolateral membrane water permeability in lung epithelia might facilitate cell volume regulation during physiological variations in airway fluid osmolality resulting from changes in hydration state or aspiration of anisosmolar fluids. Based on its expression in pleural membranes, AQP1 may play a role in pleural fluid accumulation as was found for water transport in the peritoneal cavity (83). Phenotype studies of airway physiology in AQP knockout mice are needed to define the role of AQPs in air space humidification and airway surface liquid properties in normal and diseased lungs. The possible role of airway AQPs in the pathophysiology of cystic fibrosis warrants examination. There is evidence that the water permeability of airway AQP3 is regulated by pH (90) and interactions with the cystic fibrosis conductance transmembrane regulator protein (64). The cystic fibrosis conductance transmembrane regulator protein itself has been shown to transport water when expressed at a high density in a heterologous expression system (27). Studies in AQP3 and AQP4 null mice and in recently generated AQP3 and AQP4 double-knockout mice (38) may provide evidence of an important role for AQPs in airway physiology. The identification and physiological analysis of novel lung water transporters is needed, particularly the water transporter responsible for high basolateral membrane water permeability in type I alveolar epithelial cells. Last, possible non-water-transporting roles of AQPs in the lung merit consideration. Although AQP1-mediated carbon dioxide transport in the lung does not appear to be physiologically important (84), other lung AQPs might facilitate gas transport under some conditions. The lung AQPs might also be involved in functions unrelated to membrane transport, such as lung growth and angiogenesis.
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ACKNOWLEDGEMENTS |
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We thank Drs. Chunxue Bai, Hans Folkesson, Norimasa Fukuda, and Tonghui Ma for continued collaboration on lung and airway phenotype studies in aquaporin null mice and for critical review of this manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-60288, HL-51854, and HL-42368; National Institute of Diabetes and Kidney and Digestive Diseases Grants DK-35124 and DK-43840; and National Cystic Fibrosis Foundation Grant R613.
Address for reprint requests and other correspondence: A. S. Verkman, 1246 Health Sciences East Tower, Cardiovascular Research Institute, Univ. of California, San Francisco, CA 94143-0521 (E-mail: verkman{at}itsa.ucsf.edu; website: http://www.ucsf.edu/verklab).
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abrami, L,
Berthonaud V,
Deen PM,
Rousselet G,
Tacnet F,
and
Ripoche P.
Glycerol permeability of mutant aquaporin 1 and other AQPMIP proteins: inhibition studies.
Pflügers Arch
431:
408-414,
1996[ISI][Medline].
2.
Agostoni, E,
and
Piiper J.
Capillary pressure and distribution of vascular resistance in isolated lung.
Am J Physiol
202:
1033-1036,
1962[ISI].
3.
Agre, P,
Lee MD,
Devidas S,
and
Guggino WB.
Aquaporins and ion conductance.
Science
275:
1490,
1997
4.
Bai, C,
Fukuda N,
Song Y,
Ma T,
Matthay MA,
and
Verkman AS.
Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice.
J Clin Invest
103:
555-561,
1999
5.
Bland, RD.
Lung epithelial ion transport and fluid movement during the perinatal period.
Am J Physiol Lung Cell Mol Physiol
259:
L30-L37,
1990
6.
Borok, Z,
Lubman RL,
Danto SI,
Zhang XL,
Zabski SM,
King LS,
Lee DM,
Agre P,
and
Crandall ED.
Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression of aquaporin 5.
Am J Respir Cell Mol Biol
18:
554-561,
1998
7.
Carter, EP,
Matthay MA,
Farinas J,
and
Verkman AS.
Transalveolar osmotic and diffusional water permeability in intact mouse lung measured by a novel surface fluorescence method.
J Gen Physiol
108:
133-142,
1996[Abstract].
8.
Carter, EP,
Ölveczky BP,
Matthay MA,
and
Verkman AS.
High microvascular endothelial water permeability in mouse lung measured by a pleural surface fluorescence method.
Biophys J
74:
2121-2128,
1998
9.
Carter, EP,
Umenishi F,
Matthay MA,
and
Verkman AS.
Developmental changes in alveolar water permeability in perinatal rabbit lung.
J Clin Invest
100:
1071-1078,
1997
10.
Chou, CL,
Knepper MA,
van Hoek AN,
Brown D,
Yang B,
Ma T,
and
Verkman AS.
Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice.
J Clin Invest
103:
491-496,
1999
11.
Chou, CL,
Ma T,
Yang B,
Knepper MA,
and
Verkman AS.
Four-fold reduction in water permeability in inner medullary collecting duct of aquaporin-4 knockout mice.
Am J Physiol Cell Physiol
274:
C549-C554,
1998
12.
Deen, PM,
and
van Os CH.
Epithelial aquaporins.
Curr Opin Cell Biol
10:
435-442,
1998[ISI][Medline].
13.
Dobbs, L,
Gonzalez R,
Matthay MA,
Carter EP,
Allen L,
and
Verkman AS.
Highly water-permeable type I alveolar epithelial cells confer high water permeability between the air space and vasculature in rat lung.
Proc Natl Acad Sci USA
95:
2991-2996,
1998
14.
Effros, RM,
Darin C,
Jacobs ER,
Rogers RA,
Krenz G,
and
Schneeberger EE.
Water transport and distribution of aquaporin-1 in the pulmonary air spaces.
J Appl Physiol
83:
1002-1016,
1997
15.
Ehrhard, JC,
Granger WM,
and
Hofman WF.
Filtration coefficient obtained by stepwise pressure elevation in isolated dog lung.
J Appl Physiol
56:
862-867,
1984
16.
Farinas, J,
Kneen M,
Moore M,
and
Verkman AS.
Plasma membrane water permeability of cultured cells and epithelia measured by light microscopy with spatial filtering.
J Gen Physiol
110:
283-296,
1997
17.
Finkelstein, A.
Water Movement Through Lipid Bilayers, Pores, and Plasma Membranes: Theory and Reality. New York: Wiley, 1987.
18.
Folkesson, HG,
Matthay M,
Frigeri A,
and
Verkman AS.
Transepithelial water permeability in microperfused distal airways. Evidence for channel-mediated water transport.
J Clin Invest
97:
664-671,
1996
19.
Folkesson, HG,
Matthay MA,
Hasegawa H,
Kheradmand F,
and
Verkman AS.
Transcellular water transport in lung alveolar epithelium through mercury-sensitive water channels.
Proc Natl Acad Sci USA
91:
4970-4974,
1994[Abstract].
20.
Frigeri, A,
Gropper M,
Turck CW,
and
Verkman AS.
Immunolocalization of the mercurial-insensitive water channel and glycerol intrinsic protein in epithelial cell plasma membranes.
Proc Natl Acad Sci USA
92:
4328-4331,
1995[Abstract].
21.
Frigeri, A,
Gropper M,
Umenishi F,
Kawahima M,
Brown D,
and
Verkman AS.
Localization of MIWC and GLIP water channel homologs in neuromuscular, epithelial and glandular tissues.
J Cell Sci
108:
2993-3002,
1995
22.
Funaki, H,
Yamamoto T,
Koyama Y,
Kondo D,
Yaoita E,
Kawasaki K,
Kobayashi H,
Sawaguchi S,
Abe H,
and
Kihara I.
Localization and expression of AQP5 in cornea, serous salivary glands, and pulmonary epithelial cells.
Am J Physiol Cell Physiol
275:
C1151-C1157,
1998
23.
Gaar, KA,
Taylor AE,
Owens LJ,
and
Guyton AC.
Pulmonary capillary pressure and filtration coefficient in the isolated perfused lung.
Am J Physiol
213:
910-914,
1967[ISI][Medline].
24.
Garat, C,
Carter E,
and
Matthay MA.
New in situ mouse model to quantify alveolar epithelial fluid clearance.
J Appl Physiol
84:
1763-1767,
1998
25.
Hasegawa, H,
Lian SC,
Finkbeiner WE,
and
Verkman AS.
Extrarenal tissue distribution of CHIP28 water channels by in situ hybridization and antibody staining.
Am J Physiol Cell Physiol
266:
C893-C903,
1994
26.
Hasegawa, H,
Ma T,
Skach W,
Matthay M,
and
Verkman AS.
Molecular cloning of a mercurial-insensitive water channel expressed in selected water transporting tissues.
J Biol Chem
269:
5497-5500,
1994
27.
Hasegawa, H,
Skach W,
Baker O,
Calayag MC,
Lingappa V,
and
Verkman AS.
A multi-functional aqueous channel formed by CFTR.
Science
258:
1477-1479,
1992[ISI][Medline].
28.
Hasegawa, H,
Zhang R,
Dohrman A,
and
Verkman AS.
Tissue-specific expression of mRNA encoding the rat kidney water channel CHIP28k by in situ hybridization.
Am J Physiol Cell Physiol
264:
C237-C245,
1993
29.
Heymann, JB,
Agre P,
and
Engel A.
Progress on the structure and function of aquaporin 1.
J Struct Biol
121:
196-206,
1998.
30.
Horsfield, K.
The structure of the tracheobronchial tree.
In: Scientific Foundations of Respiratory Medicine, edited by Scadding JG,
and Cumming G.. Philadelphia, PA: Saunders, 1981, p. 54-70.
31.
Hummler, E,
Barker P,
Gatzy J,
Beermann F,
Verdumo C,
Schmidt A,
Boucher R,
and
Rossier BC.
Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice.
Nat Genet
12:
325-328,
1996[ISI][Medline].
32.
King, LS,
Nielsen S,
and
Agre P.
Aquaporin-1 water channel protein in lung-ontogeny, steroid-induced expression, and distribution in rat.
J Clin Invest
97:
2183-2191,
1996
33.
King, LS,
Nielsen S,
and
Agre P.
Aquaporins in complex tissues. I. Developmental patterns in respiratory and glandular tissues of rat.
Am J Physiol Cell Physiol
273:
C1541-C1548,
1997[ISI][Medline].
35.
Kuwahara, M,
Berry CA,
and
Verkman AS.
Rapid development of vasopressin-induced hydroosmosis in kidney collecting tubules measured by a new fluorescence technique.
Biophys J
54:
595-602,
1988[Abstract].
36.
Ma, T,
Fukuda N,
Song Y,
Matthay MA,
and
Verkman AS.
Lung fluid transport in aquaporin-5 knockout mice.
J Clin Invest
105:
93-100,
2000
37.
Ma, T,
Song Y,
Gillespie A,
Carlson EJ,
Epstein CJ,
and
Verkman AS.
Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels.
J Biol Chem
274:
20071-20074,
1999
38.
Ma, T,
Song Y,
Yang B,
Gillespie A,
Carlson EJ,
Epstein CJ,
and
Verkman AS.
Nephrogenic diabetes insipidus in mice deficient in aquaporin-3 water channels.
Proc Natl Acad Sci USA.
97:
4386-4391,
2000
39.
Ma, T,
and
Verkman AS.
Aquaporin water channels in gastrointestinal physiology.
J Physiol (Lond)
517:
317-326,
1999
41.
Ma, T,
Yang B,
Gillespie A,
Carlson EJ,
Epstein CJ,
and
Verkman AS.
Generation and phenotype of a transgenic knock-out mouse lacking the mercurial-insensitive water channel aquaporin-4.
J Clin Invest
100:
957-962,
1997
42.
Ma, T,
Yang B,
Gillespie A,
Carlson EJ,
Epstein CJ,
and
Verkman AS.
Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels.
J Biol Chem
273:
4296-4299,
1998
43.
Ma, T,
Yang B,
Matthay MA,
and
Verkman AS.
Evidence against a role for mouse, rat and cloned human T1 isoforms as a water channel or regulator of aquaporin-type water channels.
Am J Respir Cell Mol Biol
19:
143-149,
1998
44.
Manley, GT,
Fujimura M,
Ma T,
Filiz F,
Bollen A,
Chan P,
and
Verkman AS.
Aquaporin-4 deletion in mice reduces brain edema following acute water intoxication and ischemic stroke.
Nat Med
6:
159-163,
2000[ISI][Medline].
45.
Matthay, MA,
Flori HR,
Conner ER,
and
Ware LB.
Alveolar epithelial fluid transport: basic mechanisms and clinical relevance.
Proc Assoc Am Physicians
110:
496-505,
1998[ISI][Medline].
46.
Matthay, MA,
Folkesson H,
and
Verkman AS.
Salt and water transport across alveolar and distal airway epithelia in the adult lung.
Am J Physiol Lung Cell Mol Physiol
270:
L487-L503,
1996
47.
Matthay, MA,
and
Wiener-Kronish JP.
Intact epithelial barrier function is critical for the resolution of alveolar edema in humans.
Am Rev Respir Dis
142:
1250-1257,
1990[ISI][Medline].
48.
Maya, S.
Lung endothelium: structure-function correlates.
In: The Lung: Scientific Foundations, edited by Crystal RG,
and West JB.. New York: Raven, 1991, p. 301-312.
49.
Misaka, T,
Abe K,
Iwabuchi K,
Kusakabe Y,
Ichinose M,
Miki K,
Emori Y,
and
Arai S.
A water channel closely related to brain aquaporin 4 is expressed in acid- and pepsinogen-secretory cells of human stomach.
FEBS Lett
381:
208-212,
1996[ISI][Medline].
50.
Moore M, Ma T, Yang B, and Verkman AS. Tear secretion by
lacrimal glands in mice is not affected by deletion of water channels
AQP1, AQP3, AQP4 or AQP5. Exp Eye Res. In
press.
51.
Murry, JF.
The Normal Lung. Philadelphia, PA: Saunders, 1986, p. 23-59.
52.
Nakhoul, NL,
Davis BA,
Romero MF,
and
Boron WF.
Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes.
Am J Physiol Cell Physiol
274:
C543-C548,
1998
53.
Nielsen, S,
King LS,
Christensen BM,
and
Agre P.
Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat.
Am J Physiol Cell Physiol
273:
C1549-C1561,
1997[ISI][Medline].
54.
Nielsen, S,
Kwon TH,
Christensen BM,
Promeneur D,
Frokiaer J,
and
Marples D.
Physiology and pathophysiology of renal aquaporins.
J Am Soc Nephrol
10:
647-663,
1999
55.
Nielsen, S,
Smith BL,
Christensen EI,
and
Agre P.
Distribution of the aquaporin CHIP in secretory and resorptive epithelia and capillary endothelia.
Proc Natl Acad Sci USA
90:
7275-7279,
1993[Abstract].
56.
Olver, RE.
Fluid secretion and absorption in the fetus.
In: Fluid and Solute Transport in the Airspaces of the Lung, edited by Effros RM,
and Chang HK.. New York: Dekker, 1993, vol. 70, p. 281-302. (Lung Biol Health Dis Ser)
57.
Pallone, TL,
Edwards A,
Ma T,
Silldorff E,
and
Verkman AS.
Requirement of aquaporin-1 for NaCl driven water transport across descending vasa recta.
J Clin Invest
105:
215-222,
2000
58.
Pappenheimer, JR,
and
Soto-Rivera A.
Effective osmotic pressure of the plasma proteins and other quantities associated with the capillary circulation in the hindlimbs of cats and dogs.
Am J Physiol
152:
471-490,
1948[ISI].
59.
Parker, JC,
Prasad R,
Allison RA,
Wojchiechowski WV,
and
Martin SL.
Capillary filtration coefficients using laser densitometry and gravimetry in isolated dog lungs.
J Appl Physiol
74:
1981-1987,
1993[Abstract].
60.
Reizer, J,
Reizer A,
and
Saier MH.
The MIP family of integral membrane channel proteins: sequence comparisons, evolutional relationships, reconstructed pathway of evolution, and proposed functional differentiation of two repeated halves of the protein.
Crit Rev Biochem Mol Biol
28:
235-257,
1993[Abstract].
61.
Ruddy, MK,
Drazen JM,
Pitkanan OM,
Rafii B,
and
Harris HW.
Modulation of aquaporin 4 and the amiloride-inhibitable sodium channel in perinatal rat lung epithelial cells.
Am J Physiol Lung Cell Mol Physiol
274:
L1066-L1072,
1998
62.
Schnermann, J,
Chou J,
Ma T,
Knepper MA,
and
Verkman AS.
Defective proximal tubule reabsorption in transgenic aquaporin-1 null mice.
Proc Natl Acad Sci USA
95:
9660-9664,
1998
63.
Schnitzer, JE,
and
Oh P.
Aquaporin-1 in plasma membrane and caveolae provide mercury-sensitive water channels across lung endothelium.
Am J Physiol Heart Circ Physiol
270:
H416-H422,
1996
64.
Schreiber, R,
Nitschke R,
Greger R,
and
Kunzelmann K.
The cystic fibrosis conductance regulator activates aquaporin 3 in airway epithelial cells.
J Biol Chem
274:
11811-11816,
1999
65.
Simionescu, M.
Lung endothelium: structure-function correlates.
In: The Lung: Scientific Foundations, edited by Crystal RG,
and West JB.. New York: Raven, 1991, vol. 1, p. 301-312.
66.
Song Y, Fukuda N, Bai CX, Ma T, Matthay MA, and Verkman AS.
Role of aquaporins in alveolar fluid clearance in neonatal and
adult lung, and in edema formation following lung injury. J Physiol
(Lond). In press.
67.
Song, Y,
Ma T,
Matthay MA,
and
Verkman AS.
Role of aquaporin-4 in air space-to-capillary water permeability in intact mouse lung measured by a novel gravimetric method.
J Gen Physiol
115:
17-27,
2000
68.
Spring, KR.
Routes and mechanism of fluid transport by epithelia.
Annu Rev Physiol
60:
105-119,
1998[ISI][Medline].
69.
Stone, KC,
Mercer RR,
Gehr P,
Stockstill B,
and
Crapo JD.
Distribution of cell numbers and volumes between alveolar and nonalveolar tissue.
Am J Respir Cell Mol Biol
6:
235-243,
1992[ISI][Medline].
70.
Tsukuguchi, H,
Shayakul C,
Berger UV,
Mackenzie B,
Devidas S,
Guggino WB,
and
van Hoek AN
Hediger MA. Molecular characterization of a broad selectivity neural solute channel.
J Biol Chem
273:
24737-24743,
1998
71.
Ulich, TR,
Yi ES,
Longmuir K,
Yin S,
Blitz R,
Morris CF,
Housley RM,
and
Pierce GF.
Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo.
J Clin Invest
93:
1298-1306,
1994[ISI][Medline].
72.
Umenishi, F,
Carter EP,
Yang B,
Oliver B,
Matthay MA,
and
Verkman AS.
Sharp increase in rat lung water channel expression in the perinatal period.
Am J Respir Cell Mol Biol
15:
673-679,
1996[Abstract].
73.
Van Hoek, AN,
and
Verkman AS.
Functional reconstitution of the isolated erythrocyte water channel CHIP28.
J Biol Chem
267:
18267-18269,
1992
74.
Verkman, AS.
Lessons on renal physiology from transgenic mice lacking aquaporin water channels.
J Am Soc Nephrol
10:
1126-1135,
1999
75.
Verkman, AS.
Water permeability measurements in living cells and complex tissues.
J Membr Biol
173:
73-87,
2000[ISI][Medline].
76.
Verkman, AS,
and
Mitra AK.
Structure and function of aquaporin water channels.
Am J Physiol Renal Physiol
278:
F13-F28,
2000
77.
Verkman, AS,
and
Yang B.
Aquaporin and ion conductance.
Science
271:
1491,
1997[Medline].
78.
Wang, Y,
Folkesson HG,
Jayr C,
Ware LB,
and
Matthay MA.
Alveolar epithelial fluid transport can be simultaneously upregulated by both KGF and -agonist therapy.
J Appl Physiol
87:
1852-1860,
1999
78a.
Wang K, Komar KS, Ma T, Filiz F, McLeroy J, Verkman
AS, and Bastidas JA. Gastric acid secretion in aquaporin-4
knock-out mice. Am J Physiol Gastrointest Liver Physiol. In
press.
79.
Wangensteen, OD.
Nonselective solute transport across the pulmonary epithelium.
In: Fluid and Solute Transport in the Airspaces of the Lungs, edited by Effros RM,
and Chang HK.. New York: Dekker, 1993, vol. 70, p. 374-397. (Lung Biol Health Dis Ser)
80.
Weibel, ER.
Lung morphometry and models in respiratory physiology.
In: Respiratory Physiology, edited by Chang HK,
and Pavia M.. New York: Dekker, 1989, vol. 40, p. 1-56. (Lung Biol Health Dis Ser)
81.
Williams, MC,
Cao Y,
Hinds A,
Rishi AK,
and
Wetterweld A.
T1 alpha protein is developmentally regulated and expressed by alveolar type I cells, choroid plexus, and ciliary epithelia of adult rat.
Am J Respir Cell Mol Biol
14:
577-585,
1996[Abstract].
82.
Willumsen, WJ,
Davis CW,
and
Boucher RC.
Selective response of human airway epithelia to luminal but not serosal solution hypertonicity. Possible role for proximal airway epithelia as an osmolality transducer.
J Clin Invest
94:
779-787,
1994[ISI][Medline].
83.
Yang, B,
Folkesson HG,
Yang J,
Matthay MA,
Ma T,
and
Verkman AS.
Reduced water permeability of the peritoneal barrier in aquaporin-1 knockout mice.
Am J Physiol Cell Physiol
276:
C76-C81,
1999
84.
Yang, B,
Fukuda N,
van Hoek AN,
Matthay MA,
Ma T,
and
Verkman AS.
Carbon dioxide permeability of aquaporin-1 measured in erythrocytes and lung of aquaporin-1 null mice and in reconstituted proteoliposomes.
J Biol Chem
275:
2686-2692,
2000
85.
Yang B, Verbavatz JM, Song Y, Manley G, Vetrivel L, Kao WM, Ma T,
and Verkman AS. Skeletal muscle function and water transport in
aquaporin-4 deficient mice. Am J Physiol Cell Physiol. In
press.
86.
Yang, B,
and
Verkman AS.
Water and glycerol permeability of aquaporins 1-5 and MIP determined quantitatively by expression of epitope-tagged constructs in Xenopus oocytes.
J Biol Chem
272:
16140-16146,
1997
87.
Yasui, M,
Serlachius E,
Lofgren M,
Belusa R,
Nielsen S,
and
Aperia A.
Perinatal changes in expression of aquaporin-4 and other water and ion transporters in rat lung.
J Physiol (Lond)
505:
3-11,
1997[Abstract].
88.
Yool, AJ,
Stamer WD,
and
Regan RW.
Forskolin stimulation of water and cation permeability in aquaporin 1 water channels.
Science
273:
1216-1218,
1996[Abstract].
89.
Zeidel, ML,
Ambudkar SV,
Smith BL,
and
Agre P.
Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein.
Biochemistry
31:
7436-7440,
1992[ISI][Medline].
90.
Zeuthen, T,
and
Klaerke DA.
Transport of water and glycerol in aquaporin 3 is gated by H+.
J Biol Chem
274:
21631-21636,
1999