Journal of Applied Physiology Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 87: 1852-1860, 1999;
8750-7587/99 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, Y.
Right arrow Articles by Matthay, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, Y.
Right arrow Articles by Matthay, M. A.
Vol. 87, Issue 5, 1852-1860, November 1999

Alveolar epithelial fluid transport can be simultaneously upregulated by both KGF and beta -agonist therapy

Yibing Wang, Hans G. Folkesson, Christian Jayr, Lorraine B. Ware, and Michael A. Matthay

Cardiovascular Research Institute and Departments of Medicine, Anesthesia, and Physiology, University of California, San Francisco, California 94143-0130


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although keratinocyte growth factor (KGF) protects against experimental acute lung injury, the mechanisms for the protective effect are incompletely understood. Therefore, the time-dependent effects of KGF on alveolar epithelial fluid transport were studied in rats 48-240 h after intratracheal administration of KGF (5 mg/kg). There was a marked proliferative response to KGF, measured both by in vivo bromodeoxyuridine staining and by staining with an antibody to a type II cell antigen. In controls, alveolar liquid clearance (ALC) was 23 ± 3%/h. After KGF pretreatment, ALC was significantly increased to 30 ± 2%/h at 48 h, to 39 ± 2%/h at 72 h, and to 36 ± 3%/h at 120 h compared with controls (P < 0.05). By 240 h, ALC had returned to near-control levels (26 ± 2%/h). The increase in ALC was explained primarily by the proliferation of alveolar type II cells, since there was a good correlation between the number of alveolar type II cells and the increase in ALC (r = 0.92, P = 0.02). The fraction of ALC inhibited by amiloride was similar in control rats (33%) as in 72-h KGF-pretreated rats (38%), indicating that there was probably no major change in the apical pathways for Na uptake in the KGF-pretreated rats at this time point. However, more rapid ALC at 120 h, compared with 48 h after KGF treatment, may be explained by greater maturation of alpha -epithelial Na channel, since its expression was greater at 120 than at 48 h, whereas the number of type II cells was the same at these two time points. beta -Adrenergic stimulation with terbutaline 72 h after KGF pretreatment further increased ALC to 50 ± 7%/h (P < 0.5). In summary, KGF induced a sustained increase over 120 h in the fluid transport capacity of the alveolar epithelium. This impressive upregulation in fluid transport was further enhanced with beta -adrenergic agonist therapy, thus providing evidence that two different treatments can simultaneously increase the fluid transport capacity of the alveolar epithelium.

keratinocyte growth factor; pulmonary edema; acute lung injury; lung fluid balance; lung fluid clearance


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DURING LUNG INJURY and normal cell turnover, the alveolar type II cell plays an important role in maintaining the normal architecture and function of the alveolar epithelium. Alveolar epithelial type II cells are progenitors of both alveolar type I and type II cells. In response to alveolar epithelial injury, the cuboidal type II cells can divide and differentiate into the flattened type I cell (43) that covers 95% of alveolar surface area (5) or proliferate to replenish the type II cell population. Type II cell hyperplasia is a common finding after acute lung injury (1, 15), but its physiological significance is not well understood.

In addition to its role as a progenitor cell and as a source of surfactant (8), another important function of the alveolar type II cell is active sodium transport (4, 25, 26), the principal driving force for vectorial fluid transport across the alveolar epithelium (12, 27, 28, 40). The ability to remove edema fluid from the alveolar space is critically important to the restoration of adequate gas exchange in the setting of alveolar flooding from several disorders, including congestive heart failure and the acute respiratory distress syndrome (29). For this reason, the factors responsible for inhibition and stimulation of alveolar liquid clearance have been extensively studied in the normal lung of many species as well as in a variety of models of acute lung injury (18, 23, 24, 27, 31, 33, 53). Important catecholamine-dependent and -independent mechanisms have been identified that can affect vectorial sodium transport and upregulate alveolar liquid clearance in the normal lung (2-4, 6, 13, 16, 27, 30, 42). Furthermore, alveolar liquid clearance has been observed to be upregulated in several models of acute lung injury including septic and hemorrhagic shock (36, 37), hyperoxia (31, 33, 53), and bleomycin lung injury (15).

Keratinocyte growth factor (KGF) is an epithelial cell-specific mitogen that promotes pronounced alveolar type II cell proliferation in vitro and in vivo (20, 35, 44). Several studies have indicated that pretreatment with KGF protects the lungs from hyperoxia, radiation, bleomycin, alpha -naphthylthiourea (ANTU), and acid instillation-induced injury (19, 20, 34, 41, 51, 52). Although one study of ANTU-induced lung injury reported that KGF increased the sodium transport capacity of the injured lung (19), there is very little in vivo information regarding the time-dependent effects of KGF on alveolar epithelial fluid transport in the lung. One possible mechanism for the protective effect of KGF may be related to its capacity to increase the number of alveolar epithelial type II cells (20, 35, 44) with an associated increase in net alveolar fluid transport capacity. On the other hand, a change in apical sodium uptake or sodium pump activity may also contribute to alterations in net sodium and fluid transport (4).

Therefore, the first objective of our study was to determine the effect of a single intratracheal dose of KGF (5 mg/kg body wt) on alveolar liquid clearance in rats at several time points over 240 h (10 days). Because KGF markedly increased alveolar liquid clearance, the second objective was to determine whether there was a relationship between alveolar epithelial type II cell proliferation and the measured increase in alveolar liquid clearance. The third objective was to study the alpha -subunit expression of the epithelial sodium channel (alpha -ENaC) at two time points (48 and 120 h), when the increase in the number of alveolar type II cells was similar but the clearance rates were different. Also, the amiloride sensitivity fraction of clearance was studied at the peak (72 h) of the KGF effect. Finally, the fourth objective was to determine whether alveolar liquid clearance in the KGF-pretreated lung could be further increased by beta -adrenergic stimulation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

For all experiments, male Sprague-Dawley rats (n = 55) weighing 180-300 g (Simonsen, Gilroy, CA) were used. The experimental protocol was approved by the University of California, San Francisco, Animal Research Committee.

General Experimental Protocol

Intratracheal KGF treatment. Rats were treated with recombinant human KGF (Amgen, Thousand Oaks, CA) by an intratracheal instillation, as described previously (15, 22), 48-240 h before the alveolar liquid clearance studies (see below). Control rats were given an intratracheal instillation of the same volume of sterile saline. During methoxyflurane (Mallinckrodt Veterinary, Mundelein, IL) anesthesia, the rats were placed on a slanted board (20° from vertical) hanging from their upper incisors. The KGF (5 mg/kg body wt) or saline was delivered via the mouth into the trachea by using a modified feeding needle in a volume of 2 ml/kg body wt followed by 0.6 ml air. After the instillation, the rats were allowed to recover in their cages, where they remained until the alveolar fluid clearance measurements were done at four different time intervals (see specific protocol below) over the next 240 h.

Preparation of instillates. A 5% bovine serum albumin (Sigma Chemical, St. Louis, MO) solution was prepared in Ringer lactate to be isosmolar with plasma. 125I-labeled human serum albumin (1.5 µCi, Draximage, Kirkland, Quebec, Canada) was added to the instillate solution and used as an alveolar protein tracer. Anhydrous Evan's blue dye (1 mg) (Aldrich Chemical, Milwaukee, WI) was added to the instillate for postmortem examination of fluid localization in the lungs. In the studies of beta -adrenergic stimulation, 10-4 M terbutaline (Sigma Chemical) was added to the 5% albumin instillate solution. In the studies of the fractional inhibition by amiloride on alveolar liquid clearance, 10-3 M amiloride (Sigma Chemical) was added to the 5% albumin solution, as previously described (16, 22, 38).

Anesthesia, surgical preparation, and ventilation. The rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg body wt; Nembutal, Abbott Laboratories, Chicago, IL) at 48, 72, 120, and 240 h after the initial KGF instillation. A 0.2-mm ID endotracheal tube (PE-240; Clay Adams, Becton Dickinson, Parsippany, NJ) was inserted through a tracheostomy. A PE-50 catheter (Clay Adams, Becton Dickinson) was inserted into the right carotid artery to monitor systemic blood pressure and to obtain blood samples. Airway pressures, heart rate, and systemic arterial pressure were measured by using calibrated pressure transducers (PD 23ID, Gould, Oxnard, CA) and recorded continuously on a Grass polygraph (Grass model 7 Polygraph, Grass Instruments, Quincy, MA). The mean systemic arterial pressure was calculated. Arterial blood gases and pH were measured at 30-min intervals.

Pancuronium bromide (0.3 mg/kg body wt; Pavulon, Organon, West Orange, NJ) was given hourly for neuromuscular blockade. The rats were maintained in the right lateral decubitus position during the experiments and ventilated with a constant-volume piston pump (Harvard Apparatus, Dover, MA) with an inspired oxygen fraction of 1.0 and with peak airway pressures of 7-9 cmH2O during the baseline period. A positive end-expiratory pressure of 4 cmH2O was maintained throughout the experimental period. The respiratory rate was adjusted to maintain arterial PCO2 between 35-40 Torr during the baseline period.

Fluid instillation and alveolar liquid clearance measurements. In all experiments, after the surgical preparation, a 1-h baseline of stable heart rate and blood pressure was recorded before the fluid instillation. Fifteen minutes before the end of the baseline period, 1.5 µCi of 131I-albumin were given via the arterial catheter as a vascular tracer. Blood samples for radioactivity counts and arterial blood gases were obtained every 30 min during the baseline period.

Fluid instillation was done by disconnecting the rat briefly from the ventilator, instilling 12 ml/kg body wt of fluid followed by 0.5 ml air intratracheally, and then immediately reconnecting the rat to the ventilator. After instillation, blood was sampled hourly for 125I-albumin and 131I-albumin activity. Arterial blood gases were measured every 30 min. At the end of the experiment, the abdomen was opened, and the rats were exsanguinated by transection of the abdominal aorta. The lungs were removed through a median sternotomy. An alveolar fluid sample (0.1-0.2 ml) was obtained by gently passing the sampling catheter (PE-50 catheter; Clay Adams, Becton Dickinson) into a wedged position in the instilled area of the lungs. Tracer concentrations in the alveolar fluid samples as well as in the plasma samples were measured by radiometric analysis. We have previously reported that the protein concentration of liquid aspirated with a catheter wedged into the distal air spaces is a good reflection of the alveolar liquid clearance (2). Protein concentrations in plasma samples were measured by the Biuret method (15,16). The right and left lungs were homogenized separately for wet-to-dry weight measurement and radioactivity counts.

Specific Experimental Protocol

Effect of KGF treatment on alveolar liquid clearance at 48, 72, 120, and 240 h. SALINE CONTROLS (N = 11). After intratracheal instillation of 0.4 ml 0.9% NaCl, alveolar liquid clearance was measured over a 1-h period at 48, 72, 120, and 240 h after saline instillation. Because there were no differences in the rate of alveolar liquid clearance among the different pretreatment periods, all saline control data were combined into one group.

ALVEOLAR LIQUID CLEARANCE AT 48 (N = 6), 72 (N = 4), 120 (N = 5), AND 240 (N = 4) H AFTER KGF TREATMENT. KGF (5 mg/kg body wt) in 0.4 ml was instilled in the trachea. Then, alveolar liquid clearance was measured at 48, 72, 120, and 240 h after the KGF instillation. For all studies, the rats were anesthetized and instilled with the albumin solution, and alveolar liquid clearance was measured over 1 h. In four preliminary studies, acute administration of KGF had no effect on alveolar liquid clearance. To study a lower dose of KGF, 1 mg/kg KGF was instilled in rats (n = 4). Alveolar liquid clearance was then measured at 72 h.

Effect of sodium-channel inhibition on alveolar liquid clearance after KGF pretreatment. AMILORIDE CONTROLS (N = 6). The rats were instilled with an albumin solution containing 10-3 M amiloride, and alveolar liquid clearance was measured over 1 h at 72 h after the intratracheal instillation of 0.4 ml saline.

AMILORIDE STUDIES 72 H AFTER KGF PRETREATMENT (N = 4). The rats were instilled with an albumin solution containing 10-3 M amiloride, and alveolar liquid clearance was measured over 1 h at 72 h after the intratracheal KGF instillation.

Effect of beta -adrenergic stimulation on alveolar liquid clearance after KGF pretreatment. TERBUTALINE CONTROLS (N = 4). The rats were instilled with an albumin solution containing 10-4 M terbutaline, and alveolar liquid clearance was measured over 1 h at 72 h after the intratracheal instillation of 0.4 ml saline.

TERBUTALINE STUDIES 72 H AFTER KGF PRETREATMENT (N = 6). The rats were instilled with an albumin solution containing 10-4 M terbutaline, and alveolar liquid clearance was measured over 1 h 72 h after the intratracheal administration of KGF.

Expression of alpha -ENaC after KGF pretreatment. Alveolar epithelial type II cells were isolated from the lung of control (saline-instilled; n = 3) and 48-h (n = 3) and 120-h (n = 3) KGF-pretreated rats by elastase digestion (7,8). Total cellular RNA was extracted from aliquots of 3 × 106 cells immediately after isolation with TRIZOL reagent (GIBCO BRL, Gaithersburg, MD), as previously described (32). For Northern blot analysis, total RNA from equal number of cells was denatured in sample loading buffer (Sigma Chemical), size fractionated on 1% agarose containing 2.2 M formaldehyde, and transfixed to nylon membranes. Membranes were hybridized to a cDNA probe for the alpha -subunit of rat ENaC, as previously described (15). The rat alpha -ENaC probe was kindly provided by Dr. Yves Berthiaume (University of Montreal, Montreal, Canada) and consisted of a 446-bp fragment from nucleotide 985 to nucleotide 1,431 of the rat alpha -ENaC sequence. After hybridization, membranes were exposed to an autoradiographic film at -80°C for 5 days. Equivalent loading of the RNA was verified by hybridization to a beta -actin probe (Oncor, Gaithersburg, MD).

Measurements

Lung endothelial and epithelial barrier protein permeability. The permeability of the alveolar epithelium to albumin was measured by two methods. First, residual 125I-albumin (the alveolar protein tracer) in the lung was measured. Second, the accumulation of the alveolar protein tracer in the blood was measured. For measuring clearance of the alveolar protein tracer from the lung, several measurements were necessary. First, the total radioactivity instilled into the lungs (125I-albumin, counts · min-1 · g-1) was calculated by multiplying the radioactivity in aliquots of the instillate by the volume instilled. To calculate the quantity of 125I-albumin in the lungs at the end of the experiment, the averages of duplicate radioactivity counts from the lung homogenates were multiplied by lung homogenate volumes. To measure the accumulation of 125I-albumin from the alveolar spaces into the blood, radioactivity counts in the plasma samples were multiplied by the estimated plasma volume. The plasma volume was estimated by the following Eq. 1
Plasma volume (ml) = body weight (g) × 0.07 

× (1 − hematocrit) (1)
To estimate the clearance of the vascular tracer protein, 131I-albumin, into the lung extravascular compartments (lung interstitium and air spaces), the total extravascular 131I-albumin accumulation in alveolar liquid recovered from the air spaces and in the lung homogenate was measured and expressed as extravascular plasma equivalents. The extravascular 131I-albumin was calculated by Eq. 2
<SUP>131</SUP>I-albumin<SUB>extravascular,lung</SUB> =<SUP> 131</SUP>I-albumin<SUB>total,lung</SUB> 

−<SUP> 131</SUP>I-albumin<SUB>vascular space,lung</SUB> (2)
To calculate 131I-albuminvascular space,lung, 131I-albumin counts in the last arterial blood sample were multiplied by the lung blood volume, corrected for hematocrit. Because the amount of 131I-albumin in 1 ml of plasma was known, the extravascular 131I-albumin represents the volume of plasma that had leaked out from the blood vessels during the experiment.

Alveolar liquid clearance. The change in concentration of instilled 125I-labeled albumin over 1 h was used to quantify alveolar liquid clearance from the distal air spaces (15, 16, 17, 22). Because rats that received intratracheal saline before the alveolar liquid clearance studies had the same progressive increase in alveolar 125I-labeled albumin concentration as did normal noninstilled rats (16, 22), an earlier intratracheal liquid instillation per se did not seem to change the capacity to transport fluid clearance from the air spaces of the lung.

Alveolar liquid clearance was calculated by the following Eq. 3 from the counts of labeled albumin in the instillate and the aspirate
ALC (%instilled volume) = [(V<SUB>i</SUB> − V<SUB>f</SUB>)/V<SUB>i</SUB>] × 100 (3)
where Vi is volume of instillate and Vf is final volume left in the alveoli at the end of the experiment. Vi was measured from weighing the syringe with instillate before and after instillation. Vf was calculated as follows
V<SUB>f</SUB> = V<SUB>i</SUB> × C<SUB>i</SUB>/C<SUB>f</SUB> (4)
where Ci and Cf are initial and final concentrations, respectively.

Immunohistochemistry Studies on KGF-Treated Rat Lungs

Preparation for proliferation studies with bromodeoxyuridine (BrdU). For BrdU studies, four rats were instilled intratracheally with KGF (5 mg/kg body wt), and one control rat received 0.9% NaCl as described above. At 30, 54, 102, and 222 h after the intratracheal KGF instillation (18 h before death), 100 mg/kg body wt BrdU (Boehringer Mannheim, Germany) were injected intraperitoneally. The control rat received the same dose of BrdU at 54 h, and was killed at 72 h after instillation of saline.

Histology fixation. At 48, 72, 120, and 240 h after KGF pretreatment, the rats were anesthetized with pentobarbital sodium (100 mg/kg body wt ip). The chest was opened through a median sternotomy. After 10 mg heparin had been injected into the right ventricle to prevent coagulation, the lungs were fixed with 4% paraformaldehyde by perfusion through the pulmonary artery, with a perfusion pressure <7-10 cmH2O. Immediately after surgical removal of the lungs, 4% paraformaldehyde was instilled intratracheally by using <10 cmH2O pressure. The lungs were removed and cut into 1-cm3 cubes, which were immersed in 4% paraformaldehyde solution for 4 h, followed by an exchange into a 30% sucrose solution. The sucrose-immersed tissue cubes were embedded in O.C.T. compound (Miles, Indiana), and the tissue cubes were snap-frozen in liquid nitrogen. After being frozen, 4- to 5-µm sections were cut and mounted on 3-aminopropyl-triethoxysilane-coated slides (Fisher Scientific, Pittsburgh, PA). The slides were stored at -70°C.

Immunohistochemistry. The number of alveolar type II cells was determined by using a specific monoclonal antibody directed against an alveolar type II cell surface antigen (9). After a wash in 50 mM Tris-buffered saline (TBS), the slides were incubated in 3% horse serum (GIBCO BRL Life Technologies, Gaithersburg, MD) in PBS for 30 min at room temperature. Then, the sections were exposed to the alveolar type II cell antibody (gift from Dr. L. Dobbs, University of California, San Francisco), diluted 1:100 in 3% horse serum for 20-40 min at room temperature, and washed again in PBS. The sections were incubated with a secondary antibody, horseradish peroxidase-conjugated goat anti-mouse IgG3 (Boehringer Mannheim, Indianapolis, IN), diluted 1:300 in 3% horse serum for 1 h at room temperature. After being rinsed with PBS, the sections were incubated with a diaminobenzidine (Sigma Chemical) solution (6 mg diaminobenzidine in 10 ml of 50 mM Tris · HCl buffer, pH 7.6, and 0.1 ml of 3% H2O2), and the color development was monitored. When a desired intensity was reached (usually within 10 s to 10 min), the slides were immediately washed in PBS.

For BrdU analysis, the sections were incubated in 1% H2O2 (Sigma Chemical), dissolved in 0.1% Triton X-100 for 5 min, and washed in PBS. After incubation in 100 µg/ml pronase E (Sigma Chemical) in 20 mM Tris · HCl, 20 mM CaCl2, pH 7.6 for 10-30 min, the sections were rinsed again with the pronase buffer. To remove DNA-binding proteins, the sections were incubated in ice-cold 0.1 N HCl for 10 min; DNA was denatured by incubating in 2 N HCl for 30 min at 37°C, followed by acid neutralization with 0.1 M borax (Sigma Chemical), pH 8.5, for 5-10 min. After being washed in PBS, the sections were incubated in 3% horse serum in PBS for 30 min at room temperature. An anti-BrdU monoclonal antibody (DAKO, Glostrup, Denmark), diluted 1:50 in 3% horse serum, was then applied for 1 h at room temperature. After the slides were washed in PBS, the sections were incubated with biotinylated goat anti-mouse IgG Fc (Sigma Chemical) diluted 1:400 in 3% horse serum in PBS for 1 h at room temperature, and washed in 50 mM TBS. A streptavidin-alkaline phosphate conjugate (Sigma Chemical), diluted 1:20 in TBS, was added to the slides for 30-60 min. After a wash in 0.1 M Tris · HCl (pH 9.5), 0.1 M NaCl, 50 mM MgCl2, the sections were incubated in the freshly prepared substrate solution [44 µl of NBT to 10 ml of 0.1 M Tris · HCl (pH 9.5), 0.1 M NaCl, 50 mM MgCl2, and 33 µl of BCIP] (GIBCO BRL Life Technologies) at room temperature for 1-10 min in the dark. The slides were washed in double-distilled water. After dehydration with alcohol, the sections were mounted with Canada balsam (Sigma Chemical).

Cell counting. At each time point after KGF instillation, two random sections from different rats were selected. For each section, five random fields were selected. The number of cells per high-power field staining positive for BrdU or alveolar type II cell antigen were counted for each field by observers blinded to the treatment group. In addition, we also counted the number of BrdU-positive cells and the type II cell antigen-positive cells minus the number of double-labeled cells to avoid duplicate counting.

Statistical Analysis

All data are shown as means ± SD. Alveolar liquid clearance and cell counts were analyzed by ANOVA with the Student-Newman-Keuls test for multiple comparisons. The correlation between alveolar liquid clearance and the number of alveolar type II cells was analyzed by simple linear regression. Statistical significance was defined as P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of KGF on Alveolar Liquid Clearance at 48, 72, 120, and 240 h after Treatment

Acute administration of KGF had no effect on alveolar liquid clearance (data not shown). However, 48, 72, and 120 h after pretreatment, KGF (5 mg/kg) increased alveolar liquid clearance by 30, 70, and 54%, respectively, over control levels (Fig. 1). At 240 h after KGF pretreatment, alveolar liquid clearance had returned to near-control levels (Fig. 1). Interestingly, four rats that were studied 72 h after pretreatment with a lower dose of KGF (1 mg/kg) had an increase in alveolar liquid clearance to the same level (41 ± 4.1%/h) as the rats pretreated with 5 mg/kg KGF.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 1.   Time-dependent effect of keratinocyte growth factor (KGF) on alveolar liquid clearance in anesthetized ventilated control rats, and in KGF-pretreated rats at 4 time points. Data are means ± SD. * P < 0.05 compared with control group; dagger  P < 0.05 compared with 48 h after KGF treatment; # P < 0.05 compared with 240 h after KGF treatment. Top inset shows %stimulation (means ± SD) by KGF at each time point. Note that 1 mg/kg of KGF had the same effect as 5 mg/kg at 72 h; see text for data.

Quantification of Alveolar Type II Cells and Relationship to Alveolar Liquid Clearance

The number of BrdU antibody-stained cells, representing newly proliferated cells, had increased by 48 h after KGF pretreatment; the peak level was reached at 72 h after KGF pretreatment, with a return to near-control levels at 120 h after KGF pretreatment (Table 1). The number of alveolar type II cells, as measured by the alveolar type II cell antigen, was increased significantly at 72 and 120 h after KGF pretreatment and then decreased to near-normal levels at 240 h after KGF pretreatment (Figs. 2 and 3). There was a good correlation (r = 0.85) between the number of type II cells and alveolar liquid clearance, although the correlation did not quite reach statistical significance (P = 0.07). When the combination of both BrdU-positive stained cells and alveolar type II cell antibody-positive stained cells was counted (minus the double-labeled cells), the correlation between alveolar liquid clearance and the number of proliferating and alveolar type II cells was excellent (r = 0.92, P = 0.02) (Fig. 4). Interestingly, the dry weight of the lung normalized to body weight increased in parallel to the observed increase in alveolar epithelial type II cells, peaking at 1.12 ± 0.33 mg/body wt 72 h after KGF pretreatment, compared with 0.73 ± 0.11 mg/body wt in controls (P < 0.05).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   KGF-induced cell proliferation in the lung



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 2.   No. of alveolar epithelial type II cells (as measured by alveolar type II cell antigen) at 4 time points after instillation of KGF (5 mg/kg it). Data are means ± SD. * P < 0.05 compared with controls.



View larger version (46K):
[in this window]
[in a new window]
  		Fig. 3.   Light micrographs of lung sections from control and KGF-pretreated rats, stained with alveolar type II cell antibody. White arrows show positive-staining alveolar type II cells. Magnification ×40.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 4.   Correlation between alveolar liquid clearance on y-axis and total no. of alveolar type II cells, antibody-positive cells plus bromodeoxyuridine (BrdU)-positive cells minus no. of double-labeled cells, on x-axis in control and at 4 different time points (r = 0.92, P = 0.02).

Effect of Sodium-Channel Inhibitor on Alveolar Liquid Clearance After KGF Pretreatment

In rats that were pretreated 72 h earlier with KGF, amiloride inhibited 38% of alveolar liquid clearance. The magnitude of the inhibition was similar to the inhibitory effect of amiloride in control rats (Fig. 5). Thus both the amiloride-sensitive and the amiloride-insensitive fractions of alveolar liquid clearance were increased by KGF in the 72-h KGF-pretreated rats.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 5.   Effect of amiloride on alveolar liquid clearance over 1 h in rats 72 h after KGF treatment. Data are also shown for alveolar liquid clearance over 1 h in control rats and in control rats treated with amiloride (10-3 M). Data are means ± SD. * P < 0.05 compared with control group. # P < 0.05 compared with 72 h after KGF treatment. Top inset shows that %inhibition (means ± SD) by amiloride was similar in control and in KGF-pretreated rats.

Expression of alpha -ENaC in Alveolar Epithelial Type II Cells Isolated 48 and 120 h After KGF Pretreatment

Because the number of alveolar epithelial type II cells measured at 48 and 120 h after KGF treatment was similar (Fig. 2), but alveolar liquid clearance was higher at 120 than at 48 h, we measured the expression of alpha -ENaC in alveolar epithelial type II cells at these two time points. Compared with control rats, mRNA expression of the alpha -subunit of rat ENaC decreased at 48 h after KGF pretreatment (Fig. 6). However, at 120 h after KGF pretreatment, expression of alpha -ENaC was slightly increased compared with control levels. When the same blots were probed for beta -actin mRNA, there was an equal level of expression in all lanes.


View larger version (66K):
[in this window]
[in a new window]
  		Fig. 6.   Northern blot analysis of alpha -subunit of rat epithelial Na channel (alpha -ENaC) expression in isolated alveolar epithelial type II cells from rats treated with intratracheal saline [C (control)] or 48 or 120 h after KGF treatment. Expression of alpha -ENaC was markedly decreased at 48 h compared with control, whereas it increased at 120 h compared with 48 h or control. Same blots were probed for beta -actin mRNA to control for loading of equal amounts of mRNA.

Effect of beta -Adrenergic Stimulation on Alveolar Liquid Clearance After KGF Pretreatment

By 72 h after KGF pretreatment, alveolar liquid clearance had reached its highest level. At this time point, terbutaline treatment resulted in a significant additional stimulation of alveolar liquid clearance by 30% over the maximal KGF-pretreated level (Fig. 7).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 7.   Effect of terbutaline on alveolar liquid clearance over 1 h in rats 72 h after KGF treatment. Data are also shown for alveolar liquid clearance over 1 h in control rats and in control rats treated with terbutaline. Data are means ± SD. * P < 0.05 compared with control group. dagger  P < 0.05 compared with 72 h after KGF treatment and P < 0.05 compared with terbutaline control. Top inset shows %stimulation (means ± SD) by terbutaline with and without KGF.

There was no effect of KGF pretreatment on alveolar epithelial or lung endothelial permeability to the labeled protein tracers, 131I-albumin and 125I-albumin (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There are three remarkable findings from this experimental study in rats. First, KGF induced a marked increase in alveolar liquid clearance to a peak level that was 66% above baseline levels. This effect is similar in magnitude to the short-term upregulation of alveolar epithelial fluid clearance that we have previously reported with beta -adrenergic agonist therapy in rats (22, 39). Second, the effect of KGF was sustained for 5 days (120 h). No other pharmacological agent has been reported to upregulate alveolar epithelial fluid transport with a sustained effect for several days after administration of a single dose. Third, it was possible to augment the rate of alveolar epithelial fluid clearance to an even higher level by administering the beta -adrenergic agonist terbutaline to KGF-pretreated rats. Thus, even though KGF pretreatment had already substantially increased alveolar fluid clearance, there was an additive effect of beta -adrenergic treatment in KGF-treated rats. The combined effect of KGF pretreatment and the acute administration of terbutaline increased alveolar liquid clearance by >100% over baseline levels.

What are the mechanisms that account for the impressive increase in alveolar liquid clearance in KGF-treated rats? KGF is known to be a potent mitogen for epithelial cells in general and for alveolar epithelial cells in particular. Several prior studies (20, 35, 44) have demonstrated that intratracheal KGF induces a brisk increase in the number of alveolar epithelial type II cells. The proliferative response was impressive in prior studies as well as in our own experiments (Table 1). Quantitation of the number of alveolar epithelial type II cells was initially accomplished in this study by staining with an alveolar epithelial type II-specific antibody. The increase in alveolar type II cells was statistically significant at 72 and 120 h. There was a good correlation between the increase in alveolar liquid clearance and the increase in the number of alveolar type II cells (r = 0.85), although the correlation did not quite reach statistical significance (P = 0.07). However, some investigators have observed that there may be a lag in the expression of the alveolar type II cell phenotype after KGF treatment in vivo. For example, in one study (20), expression of surfactant proteins did not mature until 3 days after intratracheal administration of KGF in rats. Therefore, we also analyzed the proliferative effect of KGF by quantifying both the type II cell antigen-positive cells and the BrdU-positive cells with subtraction of the double-labeled cells. This approach allows quantification of all alveolar type II cells, including newly proliferated alveolar type II cells that may not yet express the type II cell antigen. This method of analysis is supported by the fact that only epithelial cells express the KGF receptor (21). Thus it is probable that the majority of the newly proliferated, BrdU-positive cells are alveolar type II cells. Interestingly, when using this approach, the correlation between alveolar liquid clearance and the total number of alveolar type II cells was even stronger (r = 0.92, P = 0.02) (Fig. 4). It is likely, therefore, that alveolar epithelial type II cell hyperplasia accounts for most of the KGF effect on alveolar liquid clearance.

However, alveolar type II cell hyperplasia alone cannot fully account for all of the observed change in the rate of alveolar liquid clearance. For example, although the number of type II cells measured by either method (Fig. 4) was similar at 120 and 48 h, alveolar liquid clearance was higher at 120 than at 48 h (Fig. 1). Therefore, we hypothesized that expression of the epithelial sodium channel might not yet be mature at 48 h in the newly proliferated alveolar type II cells. Interestingly, alpha -ENaC expression was less at 48 h than in controls, whereas alpha -ENaC was slightly increased at 120 h compared with controls and markedly increased compared with the 48-h KGF-pretreated rats (Fig. 6). Although we did not study the protein content of alpha -ENaC in these rats, the results suggest that the modestly slower alveolar liquid clearance at 48 h compared with 120 h may be due to immature apical sodium channels at the earlier time point. Because the functional studies at 72 h with amiloride show no difference in amiloride sensitivity (Fig. 5), it is likely that alpha -ENaC expression and function were mature by 72 h after KGF pretreatment. This interpretation also fits well with the sharp increase in the alveolar type II cell antigen at 72 h (Figs. 2 and 3) as well as with data from other investigators, in which surfactant protein expression was mature 72 h after intratracheal KGF treatment (20). An in vitro study by Borok et al. (4) reported that KGF increases active sodium transport across alveolar epithelial type II cell monolayers by inducing an increase in sodium pump capacity, primarily due to an increase in the Na-K-ATPase alpha -subunit. Thus KGF may work by both increasing the total number of alveolar type II cells as well as by potentially upregulating the transport capacity of individual type II cells. The studies in these experiments did not address the role of sodium pump activity.

The findings in this study have several implications for the potential effect of KGF on the resolution of alveolar edema. In one short-term experimental study in rats that were injured with ANTU, Guery et al. (19) reported that KGF-treated rats had an increased capacity to reabsorb alveolar edema fluid after lung injury. Based on their short-term experimental studies as well as the longer-term studies in our present experiments, it is likely that the protective effect observed with KGF in several experimental models including bleomycin (52), hyperoxia (34), and acid-induced lung injury (51) is explained, in part, by the ability of KGF to upregulate net alveolar epithelial fluid transport. Of course, KGF may have other beneficial effects on attenuating the degree of lung injury. For example, KGF may increase surfactant production (50), may have antioxidant effects (47), and, based on work in two different epithelial systems (46, 49), KGF may accelerate the capacity of the epithelium to repair itself in terms of reforming a tight barrier.

What are the implications of the findings in these experimental studies for clinical acute lung injury? Although the prior experimental studies reported a protective, rather than a therapeutic, effect of KGF in experimental lung injury, the data in this study indicate that the KGF-induced upregulation of alveolar epithelial fluid transport capacity may be sustained for several days. Because most patients with increased permeability pulmonary edema from acute lung injury survive the initial few days in the intensive care unit (11), it is conceivable that there might be enough time for KGF to be administered and for its therapeutic effect to occur before the ultimate outcome of the patient is determined.

In addition, a recent clinical study from our own institution (45) indicated that KGF levels are low in pulmonary edema fluid from patients with either hydrostatic or increased pulmonary edema, potentially suggesting that pharmacological doses of KGF might have a beneficial effect. In fact, in this recent clinical study, the median level of soluble KGF in alveolar edema fluid was 0.5 ng/ml. If we assume that patients in that study had ~400 ml of alveolar edema fluid, a reasonable assumption based on an estimated wet-to-dry ratio of 8-10:1, then the total amount of KGF present in the alveolar space would be equal to ~200 ng of KGF. If we divide 200 ng by an estimated body weight of 60 kg, then the quantity of KGF in the lung would be 3.3 ng/kg, a concentration that is six orders of magnitude below the pharmacological dose given in this study (5 mg/kg). Our analysis of the soluble concentration of KGF does not account for KGF that might be tissue bound and thus not measured in pulmonary edema fluid. Nevertheless, this analysis does suggest that the quantities of KGF present in the human lung after acute lung injury are several orders of magnitude below the doses that are needed experimentally for a mitogenic effect. In prior studies with KGF, other investigators have found that the mitogenic effect of KGF is reduced if the dose is reduced from 5 to 1 mg/kg (20, 34). Perhaps because of the heparin-binding characteristics of KGF (48), relatively high doses are needed to achieve a pharmacological effect. However, we did find that a dose of 1 mg/kg intratracheal KGF had a comparable effect in these studies to the dose of 5 mg/kg in upregulating alveolar liquid clearance at 72 h.

Another remarkable finding in this set of experiments was the additive effect of beta -adrenergic therapy on alveolar liquid clearance in the KGF-treated rats. Short-term administration of terbutaline augmented alveolar epithelial fluid clearance by 30% above the maximal effect of KGF. The cumulative effect of KGF and terbutaline resulted in a >100% increase in alveolar epithelial fluid transport capacity. In our prior experiments, when alveolar epithelial fluid clearance has been upregulated by a catecholamine-independent mechanism, it has not been possible to achieve an additive effect with beta -adrenergic therapy. For example, we previously reported (16) that transforming growth factor-alpha increased alveolar epithelial fluid clearance in rats, but there was no additive effect when beta -adrenergic therapy was administered with transforming growth factor-alpha . Also, preliminary experiments indicated that dexamethasone upregulated alveolar epithelial fluid clearance in rats, but terbutaline had no additive effect (14).

In fact, one might have expected that the increase in alveolar liquid clearance from beta 2-agonist therapy should have been greater. Terbutaline increased clearance by 80% in control rats and by only 30% in KGF-treated rats. Conceivably, beta 2-receptors or their signaling properties are reduced in KGF-treated alveolar type II cells. Alternatively, the alveolar liquid clearance of 50% in 1 h, achieved with both KGF and terbutaline (Fig. 7), may represent a maximal clearance capacity because the rapid transport of a large volume of alveolar fluid to the lung interstitial space may show fluid clearance. In preliminary unpublished studies in mice, we found evidence that interstitial fluid volume may slow alveolar fluid transport under some conditions.

In conclusion, intratracheal KGF treatment results in a sustained stimulation of alveolar liquid clearance in rats over a 120-h period (5 days). The increase in alveolar fluid transport capacity was primarily accounted for by the mitogenic effect of KGF, although there was evidence that delayed maturation of alpha -ENaC may limit the upregulation of transport at 48 h, whereas later upregulation of alpha -ENaC may help to sustain transport at a later time point (120 h), when the mitogenic effect of KGF begins to wane. When instilled into KGF-pretreated rats, terbutaline had an additive stimulatory effect on increasing alveolar liquid clearance to a rate of 50%/h. Thus these results indicate that alveolar liquid clearance can be simultaneously upregulated in the normal lung by two different treatment strategies, one a catecholamine-independent and the other a catecholamine-dependent mechanism. Because a single dose of KGF produced sustained upregulation of alveolar liquid clearance, KGF should be considered as a potential treatment for patients with acute respiratory failure from pulmonary edema and acute lung injury.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Oscar Osorio and Dr. Boxue Yang.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-51854.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: Y. Wang, CVRI, HSW-825, Univ. of California, 505 Parnassus Ave., San Francisco, CA 94143-0130 (E-mail: ybwang{at}itsa.ucsf.edu).

Received 7 December 1998; accepted in final form 2 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bachofen, M., and E. R. Weibel. Alterations of the gas exchange apparatus in adult respiratory insufficiency associated with septicemia. Am. Rev. Respir. Dis. 116: 589-615, 1977[Medline].

2.   Berthiaume, Y., V. C. Broaddus, M. A. Gropper, T. Tanita, and M. A. Matthay. Alveolar liquid and protein clearance from normal dog lungs. J. Appl. Physiol. 65: 585-93, 1988[Abstract/Free Full Text].

3.   Berthiaume, Y., N. C. Staub, and M. A. Matthay. Beta-adrenergic agonists increase lung liquid clearance in anesthetized sheep. J. Clin. Invest. 79: 335-343, 1987.

4.   Borok, Z., S. I. Danto, L. L. Dimen, X. L. Zhang, and R. L. Lubman. Na+-K+-ATPase expression in alveolar epithelial cells: upregulation of active ion transport by KGF. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L149-L158, 1998[Abstract/Free Full Text].

5.   Crapo, J. D., B. E. Barry, P. Gehr, M. Bachofen, and E. R. Weibel. Cell number and cell characteristics of the normal human lung. Am. Rev. Respir. Dis. 126: 332-337, 1982[Medline].

6.   Danto, S. I., Z. Borok, X. L. Zhang, M. Z. Lopez, P. Patel, E. D. Crandall, and R. L. Lubman. Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells. Am. J. Physiol. 275 (Cell Physiol. 44): C82-C92, 1998[Abstract/Free Full Text].

7.   Dobbs, L. G., R. Gonzales, and M. C. Williams. An improved method for isolating type II cells in high yield and purity. Am. Rev. Respir. Dis. 134: 141-145, 1986[Medline].

8.   Dobbs, L. G., R. J. Mason, M. C. Williams, B. J. Benson, and K. Sueshi. Secretion of surfactant by primary cultures of alveolar type II cells isolated from rats. Biochim. Biophys. Acta 713: 118-127, 1982[Medline].

9.   Dobbs, L. G., M. Pian, S. Dumars, M. Maglio, and L. Allen. Maintenance of the differentiated type II cell phenotype by culture with an apical air surface. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L347-L354, 1997[Abstract/Free Full Text].

10.   Doumas, B. T. A candidate reference method for determination of total protein in serum. Clin. Chem. 27: 1642-1650, 1981[Abstract/Free Full Text].

11.   Doyle, R., N. Szlafarski, G. Modin, J. P. Weiner-Kronish, and M. A. Matthay. Identification of patients with acute lung injury: predictors of mortality. Am. J. Respir. Crit. Care Med. 152: 1854-1859, 1995[Abstract].

12.   Effros, R. M., G. R. Mason, H. Hukkanen, and P. Silverman. New evidence for active sodium transport from fluid-filled rat lungs. J. Appl. Physiol. 66: 906-919, 1989[Abstract/Free Full Text].

13.   Factor, P., F. Saldias, K. Ridge, V. Dumasius, J. Zabner, H. A. Jaffe, G. Blanco, M. Barnard, R. Mercer, R. Perrin, and J. I. Sznajder. Augmentation of lung liquid clearance via adenovirus-mediated transfer of a Na,K-ATPase 1 subunit gene. J. Clin. Invest. 102: 1421-1430, 1998[Medline].

14.   Folkesson, H. G., and M. A. Matthay. Dexamethasone upregulates alveolar epithelial liquid clearance in anesthetized ventilated rats (Abstract). FASEB J. 11: A561, 1997.

15.   Folkesson, H. G., G. Nitenberg, B. L. Oliver, C. Jayr, K. H. Albertine, and M. A. Matthay. Upregulation of alveolar epithelial fluid transport after subacute lung injury in rats from bleomycin. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L478-L490, 1998[Abstract/Free Full Text].

16.   Folkesson, H. G., J. F. Pittet, G. Nitenberg, and M. A. Matthay. Transforming growth factor-alpha increases alveolar liquid clearance in anesthetized ventilated rats. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L236-L244, 1996[Abstract/Free Full Text].

17.   Garat, C., E. P. Carter, and M. A. Matthay. New in situ mouse model to quantify alveolar epithelial fluid clearance. J. Appl. Physiol. 84: 1763-1767, 1998[Abstract/Free Full Text].

18.   Garat, C., M. Meignan, M. A. Matthay, D. F. Luo, and C. Jayr. Alveolar epithelial fluid clearance mechanisms are intact after moderate hyperoxic lung injury in rats. Chest 111: 1381-1388, 1997[Abstract/Free Full Text].

19.   Guery, B. P. H., C. M. Mason, E. P. Dobard, G. Beaucaire, W. R. Summer, and S. Nelson. Keratinocyte growth factor increases transalveolar sodium reabsorption in normal and injured rat lungs. Am. J. Respir. Crit. Care Med. 155: 1777-1784, 1997[Abstract].

20.   Guo, J., E. S. Yi, A. M. Havill, I. Sarosi, L. Whitcomb, S. Yin, S. C. Middleton, P. Piguet, and T. R. Ulich. Intravenous keratinocyte growth factor protects against experimental pulmonary injury. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L800-L805, 1998[Abstract/Free Full Text].

21.   Igarashi, M., P. W. Finch, and S. A. Aaronson. Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7). J. Biol. Chem. 273: 13230-13235, 1998[Abstract/Free Full Text].

22.   Jayr, C., C. Garat, M. Meignan, J.-F. Pittet, M. Zelter, and M. A. Matthay. Alveolar liquid and protein clearance in anesthetized ventilated rats. J. Appl. Physiol. 76: 2636-2642, 1994[Abstract/Free Full Text].

23.   Lane, S. M., K. C. Maender, N. E. Awender, and M. B. Maron. Adrenal epinephrine increases alveolar liquid clearance in a canine model of neurogenic pulmonary edema. Am. J. Respir. Crit. Care Med. 158: 760-768, 1998[Abstract/Free Full Text].

24.   Lasnier, J., O. D. Wangensteen, L. S. Schmitz, C. R. Gross, and D. H. Ingbar. Terbutaline stimulates alveolar fluid resorption in hyperoxic lung injury. J. Appl. Physiol. 81: 1723-1729, 1996[Abstract/Free Full Text].

25.   Mason, R. J., M. C. Williams, J. H. Widdicombe, M. J. Sanders, D. S. Misfeldt, and L. C. J. Berry. Transepithelial transport by pulmonary alveolar type II cells in primary culture. Proc. Natl. Acad. Sci. USA 79: 6033-6037, 1982[Abstract/Free Full Text].

26.   Matalon, S., D. J. Benos, and R. M. Jackson. Biophysical and molecular properties of amiloride-inhibitable sodium channel in alveolar epithelial cells. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L1-L22, 1996[Abstract/Free Full Text].

27.   Matthay, M. A., H. G. Folkesson, and A. S. Verkman. Salt and water transport across alveolar and distal airway epithelia in the adult lung. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L487-L503, 1996[Abstract/Free Full Text].

28.   Matthay, M. A., C. C. Landolt, and N. C. Staub. Differential liquid and protein clearance from the alveoli of anesthetized sheep. J. Appl. Physiol. 53: 96-104, 1982[Abstract/Free Full Text].

29.   Matthay, M. A., and J. P. Wiener-Kronish. Intact epithelial barrier function is critical for the resolution of alveolar edema in humans. Am. Rev. Respir. Dis. 142: 1250-1257, 1990[Medline].

30.   Minataka, Y., S. Suzuki, C. Grygorczyk, A. Dagenais, and Y. Berthiaume. Impact of beta -adrenergic agonist on Na+ channel and Na+-K+-ATPase expression in alveolar type II cells. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L414-L422, 1998[Abstract/Free Full Text].

31.   Nici, L., R. Dowin, M. Gilmore-Hebert, J. D. Jamieson, and D. H. Ingbar. Upregulation of rat lung Na-K-ATPase during hyperoxic injury. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L307-L314, 1991[Abstract/Free Full Text].

32.   Oliver, B. L., R. I. Sha'afi, and J.-J. Hahhar. Transforming growth factor-alpha and epidermal growth factor activate mitogen-activated protein kinase and its substrates in intestinal epithelial cells. Proc. Soc. Exp. Biol. Med. 210: 162-170, 1995[Abstract].

33.   Olivera, W. G., K. M. Ridge, and J. I. Sznajder. Lung liquid clearance and Na,K-ATPase during acute hyperoxia and recovery in rats. Am. J. Respir. Crit. Care Med. 152: 1229-1234, 1995[Abstract].

34.   Panos, R. J., P. M. Bak, W. S. Simone, J. S. Rubin, and L. J. Smith. Intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced mortality in rats. J. Clin. Invest. 96: 2026-2033, 1995.

35.   Panos, R. J., J. S. Rubin, S. A. Aaronson, and R. J. Mason. Keratinocyte growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium. J. Clin. Invest. 92: 969-977, 1993.

36.   Pittet, J. F., T. J. Brenner, K. Modelska, and M. A. Matthay. Alveolar liquid clearance is increased by endogenous catecholamines in hemorrhagic shock in rats. J. Appl. Physiol. 81: 830-837, 1996[Abstract/Free Full Text].

37.   Pittet, J. F., J. P. Wiener-Kronish, M. C. McElroy, H. G. Folkesson, and M. A. Matthay. Stimulation of lung epithelial liquid clearance by endogenous release of catecholamines in septic shock in anesthetized rats. J. Clin. Invest. 94: 663-671, 1994.

38.   Rezaiguia, S., C. Garat, C. Delclaux, M. Meignan, J. Fleury, P. Legrand, M. A. Matthay, and C. Jayr. Acute bacterial pneumonia in rats increases alveolar epithelial fluid clearance by a tumor necrosis factor-alpha-dependent mechanism. J. Clin. Invest. 99: 325-335, 1997[Medline].

39.   Sakuma, T., H. G. Folkesson, S. Suzuki, G. Okaniwa, S. Fujimura, and M. A. Matthay. Beta-adrenergic agonist stimulated alveolar fluid clearance in ex vivo human and rat lungs. Am. J. Respir. Crit. Care Med. 155: 506-512, 1997[Abstract].

40.   Saumon, G., and G. Bassett. Electrolyte and fluid transport across the mature alveolar epithelium. J. Appl. Physiol. 74: 1-15, 1993[Abstract/Free Full Text].

41.   Savla, U., and C. M. Waters. Barrier function of airway epithelium: effects of radiation and protection by keratinocyte growth factor. Radiat. Res. 150: 195-203, 1998[Medline].

42.   Sznajder, J. I., K. M. Ridge, D. B. Yeates, J. Ilekis, and W. Olivera. Epidermal growth factor increases lung liquid clearance in rat lungs. J. Appl. Physiol. 85: 1004-1010, 1998[Abstract/Free Full Text].

43.   Uhal, B. D. Cell cycle kinetics in the alveolar epithelium. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L1031-L1045, 1997[Abstract/Free Full Text].

44.   Ulich, T. R., E. S. Yi, K. Longmuir, S. Yin, R. Blitz, C. F. Morris, R. M. Housley, and G. F. Pierce. Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo. J. Clin. Invest. 93: 1298-1306, 1994.

45.   Verghese, G. M., K. McCormick-Shannon, R. J. Mason, and M. A. Matthay. Hepatocyte growth factor and keratinocyte growth factor in the pulmonary edema fluid of patients with acute lung injury. Am. J. Respir. Crit. Care Med. 158: 386-394, 1998[Abstract/Free Full Text].

46.   Ware, L. B., H. G. Folkesson, and M. A. Matthay. Keratinocyte growth factor increases alveolar epithelial wound healing in vitro (Abstract). FASEB J. 12: A778, 1998.

47.   Waters, C. M., U. Savla, and R. Panos. Keratinocyte growth factor prevents hydrogen peroxide-induced increases in airway epithelial cell permeability Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L681-L689, 1997[Abstract/Free Full Text].

48.   Wen, J., E. Hsu, W. C. Kenney, J. S. Philo, C. F. Morris, and T. Arakawa. Characterization of keratinocyte growth factor binding to heparin and dextran sulfate. Arch. Biochem. Biophys. 332: 41-46, 1996[Medline].

49.   Werner, S., H. Smola, X. Liao, M. T. Longaker, T. Krieg, P. H. Hofschneider, and L. T. Williams. The function of KGF in morphogenesis of epithelium and reepithelialization of wounds. Science 266: 819-822, 1994[Abstract/Free Full Text].

50.   Xu, X., K. McCormick-Shannon, D. R. Voelker, and R. J. Mason. KGF increases SP-A and SP-D mRNA levels and secretion in cultured rat alveolar type II cells. Am. J. Respir. Cell Mol. Biol. 18: 168-178, 1998[Abstract/Free Full Text].

51.   Yano, T., R. R. Deterding, W. S. Simonet, J. M. Shannon, and R. J. Mason. Keratinocyte growth factor reduces lung damage due to acid instillation in rats. Am. J. Respir. Cell Mol. Biol. 15: 433-442, 1996[Abstract].

52.   Yi, E. S., S. T. Williams, H. Lee, D. M. Malicki, E. M. Chin, S. Yin, J. Tarpley, and T. R. Ulich. Keratinocyte growth factor ameliorates radiation- and bleomycin-induced lung injury and mortality. Am. J. Pathol. 149: 1963-1970, 1996[Abstract].

53.   Yue, G., W. J. Russell, D. J. Benos, R. M. Jackson, M. A. Olman, and S. Matalon. Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats. Proc. Natl. Acad. Sci. USA 92: 8418-8422, 1995[Abstract/Free Full Text].

J APPL PHYSIOL 87(5):1852-1860
8570-7587/99 $5.00 Copyright © 1999 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
H. G. Folkesson and M. A. Matthay
Alveolar Epithelial Ion and Fluid Transport: Recent Progress
Am. J. Respir. Cell Mol. Biol., July 1, 2006; 35(1): 10 - 19.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
G. M. Mutlu and J. I. Sznajder
Mechanisms of pulmonary edema clearance
Am J Physiol Lung Cell Mol Physiol, November 1, 2005; 289(5): L685 - L695.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
L. B. Ware
Modulation of Alveolar Fluid Clearance by Acute Inflammation: The Plot Thickens
Am. J. Respir. Crit. Care Med., February 1, 2004; 169(3): 332 - 333.
[Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
Y. Berthiaume, H. G. Folkesson, and M. A. Matthay
Lung Edema Clearance: 20 Years of Progress: Invited Review: Alveolar edema fluid clearance in the injured lung
J Appl Physiol, December 1, 2002; 93(6): 2207 - 2213.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page