Airway surface fluid volume and Cl content in cystic fibrosis
and normal bronchial xenografts
Yulong
Zhang and
John F.
Engelhardt
Departments of Anatomy and Cell Biology and of Internal
Medicine, University of Iowa Medical School, Iowa City, Iowa 52242
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ABSTRACT |
We describe the
use of an in vivo human bronchial xenograft model of cystic fibrosis
(CF) and non-CF airways to investigate pathophysiological alterations
in airway surface fluid (ASF) volume (Vs) and Cl content.
Vs was calculated based on the
dilution of an impermeable marker,
[3H]inulin, during
harvesting of ASF from xenografts with an isosmotic Cl-free solution.
These calculations demonstrated that
Vs in CF xenographs (28 ± 3.0 µl/cm2;
n = 17) was significantly less than
that of non-CF xenografts (35 ± 2.4 µl/cm2;
n = 30). The Cl concentration of ASF
([Cl]s) was
determined using a solid-state AgCl electrode and adjusted for dilution
during harvesting using the impermeable
[3H]inulin marker.
Cumulative results demonstrate small but significant elevations
(P < 0.045) in
[Cl]s in CF (125 ± 4 mM; n = 27) compared with non-CF
(114 ± 4 mM; n = 48) xenografts.
To investigate potential mechanisms by which CF airways may facilitate
a higher level of fluid absorption yet retain slightly elevated levels
of Cl, we sought to evaluate the capacity of CF and non-CF airways to
absorb both 22Na and
36Cl. Two consistent findings were
evident from these studies. First, in both CF and non-CF xenografts,
22Na and
36Cl were always absorbed in an
equal molar ratio. Second, CF xenografts hyperabsorbed (~1.5-fold
higher) both 22Na and
36Cl compared with non-CF
xenografts. These results substantiate previously documented findings
of elevated Na absorption in CF airways and also suggest that the
slightly elevated
[Cl]s found in this
study of CF xenograft epithelia does not occur through a mechanism of
decreased apical permeability to Cl.
ion transport; electrolyte and water balance; lung epithelium; salt
absorption
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INTRODUCTION |
ANTIBACTERIAL DEFENSES IN the airway are likely
dependent on multifactorial influences that determine the composition
of both fluid and electrolytes at the surface of the airway, as well as the secretory products that aid in bacterial killing and clearance. In
cystic fibrosis (CF) these aspects of airway protection are defective,
leading to increased bacterial colonization by particular opportunistic
bacteria such as Pseudomonas
aeruginosa. Two central hypotheses
address the mechanisms relating improper fluid and electrolyte balance
to increased bacterial colonization in the CF airway. The most
traditional view has been that increased Na absorption and decreased Cl
secretion in CF lead to dehydration of the airway surface fluid (ASF)
layer. This may lead to thick mucus with altered biophysical
properties, resulting in impaired mucociliary clearance of inhaled
bacteria (1, 8, 18, 19). This mechanism suggests that the innate
antibacterial properties of ASF are unaltered in CF and that rather it
is the mechanical properties of clearance that are impaired. In
contrast, two recent studies comparing the innate defense mechanisms of
ASF from CF and non-CF epithelia have suggested that a higher
concentration of NaCl in CF may affect the antibacterial properties of
ASF (5, 16). Such studies have led to a resurgence of investigation comparing ASF from CF and non-CF airway epithelia in an effort 1) to better understand the
mechanisms underlying regulation of the NaCl concentration in the ASF
and 2) to characterize antibacterial substances in the airway that may have altered activity in CF.
The present understanding of airway pathogenesis in CF has suggested
that decreased airway surface liquid may in part, but not completely,
explain observations seen in CF-associated lung disease (3, 9, 14). The
most widely associated defect thought to be responsible for dehydration
of the airways in CF is Na hyperabsorption through defective regulation
of epithelial Na channels (ENaC) by the CF transmembrane conductance
regulator (CFTR) (7, 17). Several laboratories have investigated the rate of fluid transport in models of polarized CF and non-CF airway epithelia. In these studies, correlation of electrolyte and fluid transport using in vitro polarized nasal epithelia has supported the
notion that absorptive Na conductance is a primary driving force of
fluid absorption in airway epithelium (8, 15). Additionally, in vivo
studies using human bronchial xenografts demonstrate a fourfold higher
rate of fluid absorption in CF compared with non-CF airways under basal
conditions (23). Together, these in vitro and in vivo findings suggest
a mechanism by which increased fluid absorption in CF airway epithelia
leads to dehydration of mucus and impaired mucociliary clearance. Such
impaired clearance is hypothesized to lead to an increased bacterial
burden in the lungs of CF patients. Of importance to understanding the
implications of these studies is a better understanding of the
mechanism(s) by which polarized airway epithelia regulate ASF volume
(Vs). These mechanisms are
currently a source of wide debate in the field (12). For example,
findings that CF ASF has higher concentrations of Na and Cl than non-CF
ASF (4, 10) have suggested that the airway may be relatively
impermeable to Cl as a result of CFTR dysfunction. These findings are
inconsistent with the notion that CF airways actively hyperabsorb Na
and passively absorb Cl (through paracellular pathways) as the driving
force for salt and water movement out of the airways (8, 11). Such
discrepancies highlight the present lack of knowledge concerning
mechanisms of salt and fluid transport in the airways. Additionally,
model system dependencies for certain CF-associated phenotypes may also explain some of the variation in results seen between laboratories.
In the present study, we seek to evaluate mechanisms of airway surface
electrolyte and fluid balance in CF and non-CF bronchial xenografts.
These studies provide another piece of key information using an animal
model of the intact human airway on which to build hypotheses for how
the airways facilitate the movement of Na, Cl, and water in the
baseline unstimulated condition. To this end, we have asked three
questions comparing CF and non-CF bronchial xenografts:
1) What is the equilibrated
Vs in air-exposed unstimulated xenograft airways, 2) What is the Cl
concentration in ASF
([Cl]s) of xenograft
airways, and 3) Is NaCl absorption
different in CF and non-CF xenograft airways?
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MATERIALS AND METHODS |
Generation of CF and non-CF human bronchial xenografts.
Human bronchial xenografts were generated from proximal non-CF and CF
bronchial tissue, harvested at the time of transplantation, as
previously described (23). Briefly, xenografts were generated by
seeding primary human airway cells into denuded rat tracheas. Efforts
were made to keep the dimensions of xenograft airways as constant as
possible. Typically, xenografts had dimensions of 2.5 mm (ID) × 1 cm (length), giving an approximate airway surface area of 1 cm2. These tracheas were ligated
to flexible plastic tubing and transplanted as a cassette
subcutaneously in nude mice. The ends of tubing exited the skin at the
back of the neck of mice such that the lumen of xenografts could be
accessed for functional studies. Grafts were analyzed 5 wk
posttransplantation and following bioelectric evaluation that
demonstrated transepithelial difference profiles indicative of fully
differentiated CF and non-CF xenograft epithelium (22, 23). All grafts
were analyzed by morphological criteria to confirm the integrity of the
epithelia following functional measurements. During functional
measurements, mice were anesthetized and xenografts remained
subcutaneous, and hence vascularized, while analyses were performed.
Bioelectric properties of xenografts.
Before functional analysis of Vs
and [Cl]s, the
bioelectric properties of each xenograft airway were evaluated by
transepithelial potential difference measurements. Methods of analysis
were identical to those previously described for this model system (22,
23). In brief, 4.5-wk-old xenografts were continuously perfused with a
Ringer solution containing the following series of buffer changes: 1) HEPES-phosphate-buffered Ringer
solution [HPBR; in mM: 10 HEPES (pH 7.4), 145 NaCl, 5 KCl, 1.2 MgSO4, 1.2 calcium gluconate, 2.4 K2HPO4,
and 0.4 KH2PO4],
2) HPBR with 100 µM amiloride,
3) HPBR and 100 µM amiloride, Cl
free (using gluconate in place of Cl), 4) HPBR and 100 µM amiloride, Cl
free, with 200 µM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and 10 µM
forskolin, 5) HPBR and 100 µM
amiloride, Cl free, with 200 µM CPT-cAMP, 10 µM forskolin, and 100 µM UTP, and 6) HPBR. Recordings
were taken by computer-assisted recording of millivolts every 5 s.
Measurement of Vs.
Fully differentiated 5-wk-old human bronchial xenografts (air filled)
were flushed with 1 ml of Ringer, followed by air, 48 h before
functional measurements. After 48 h of equilibration, the luminal
contents were collected by perfusion of the xenograft airways with 100 µl of a 5% (isosmotic) dextrose or mannitol solution containing a
known amount of
[3H]inulin. The
concentration of
[3H]inulin in the
perfusate was used to calculate dilution of ASF during harvesting.
Typically, the initial Cl-free perfusion solution contains 20,000 total
counts/min (cpm)
[3H]inulin in 100 µl. After the initial wash, xenografts were immediately perfused a
second time with 1 ml of Ringer, and the total cpm in this wash was
used to calculate the volume lost
(Vl; in µl) during the initial
harvest with Cl-free solution. The following formulas were used to
calculate the Vs of the xenograft,
where Vi is the input volume of
the first Cl-free wash (100 µl),
Ve is the volume (in µl) of
effluent collected from the first Cl-free wash,
Vf is the volume (in µl)
recovered from the final wash with 1 ml Ringer (directly measured),
[inulin]i is the
initial concentration (in cpm/µl) of [3H]inulin in the
first Cl-free wash,
[inulin]e is the final
concentration (in cpm/µl) of [3H]inulin in the effluent
of the first Cl-free wash, and
[inulin]f is the final
concentration (in cpm/µl) of [3H]inulin in the final
Ringer wash.
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Direct
measurement of Ve was difficult to
perform accurately due to the small volume (<130 µl). Therefore, we
chose to indirectly calculate this value by assessing the
Vl lost during effluent collection
of ASF and the dilution of cpm counts in the initial effluent. Because
Vl was calculated from the total
cpm counts recovered in a large volume (1 ml) of Ringer wash, this
measurement could be made with much higher accuracy than direct
measurement of Ve. Nonetheless,
because Vl typically represents
<2% of the Vi, corrections in
the derivative formulas were included for the sake of completeness.
These calculations were used to compare Vs among 17 CF xenografts (from 3 independent tissue samples, 2
F508/
F508 and 1 unknown/
F508)
and 30 non-CF xenografts (from 4 independent tissue samples).
Measurement of [Cl]s.
[Cl]s were determined
on duplicate 10-µl samples of ASF using a "flatbed" solid-state
(AgCl) Cl-specific electrode (Fisher no. 13-299-102) and calculated
against Cl standards in 5% dextrose or 5% mannitol, depending on the
isosmotic solution used for ASF collection. The linear range of these
calculations was from 1 to 200 mM NaCl, and standard curves
demonstrated consistent linearity (r > 0.99) over this range. The following formulas were used to calculate the final
[Cl]s, where the
concentration (in mM) of Cl in the effluent of the first wash
([Cl]e) was directly
measured using an AgCl electrode,
[Cl]w is the
concentration (in mM) of Cl from ASF in the effluent of the final
Ringer wash (assumed to be negligible, since the effluent wash is
vectorial and the highest concentrations of Cl will exit the xenograft
first), and Ve,
Vs,
Vi, and
Vl are calculated from the
equations above
Initial studies compared the
[Cl]s among 17 CF
xenografts (from 3 independent tissue samples, 2
F508/
F508 and 1 unknown/
F508) and 30 non-CF xenografts (from 4 independent tissue
samples) using isosmotic dextrose as the perfusate. Additional studies
evaluated the [Cl]s
using an alternative medium for collection (isosmotic mannitol), since
dextrose could affect glucose-Na transporters. In these studies, 10 CF
xenografts (from 2 independent tissue samples, 1
F508/
F508, 1 G551D/
F508) were compared with 18 non-CF xenografts (from 3 independent tissue samples). No significant differences in the ASF Cl
content were noted between experiments using mannitol or dextrose
perfusates. Cumulative data presented in this manuscript on ASF Cl
represent the mean values for points determined by both dextrose and
mannitol collection procedures.
Effects of mucin on Cl determinations with AgCl electrodes.
To determine whether mucin content in ASF may alter Cl measurements
with AgCl electrodes, several Cl standard curves were performed in
known concentrations of bovine submandibular gland mucin (0, 0.1, and
0.5 mg/ml). Mucin was first dissolved in 10 mM
dithiothreitol (DTT)-8 M urea and then diluted to the
appropriate concentration. Mock standards without mucin were also
generated with 10 mM DTT-8 M urea; 1 ml of each mucin or mock standard
was then dialyzed against four changes (2 liters total) of a known concentration of NaCl (0, 1, 2, 5, 10, 20, 50, 100, 150, and 200 mM) to
remove any DTT and urea. It should be noted that standards of identical
Cl concentration were grouped and dialyzed against the same pool of
NaCl to retain similar final Cl concentrations. Cl standards were then
analyzed using a solid-state AgCl electrode, and potentials (in mV)
were recorded. Standard curves generated in the presence of 0, 0.1, and
0.5 mg/ml mucin were used to calculate the predicted Cl concentration
for selected CF and non-CF ASF samples.
Measurements of 22Na and
36Cl absorption.
Rates of Na and Cl absorption in human bronchial CF and non-CF
xenografts were determined following in vivo luminal labeling with
200,000 cpm of each of 36Cl and
22Na in 5 µl of 5 mM HEPES-100
mM NaCl (pH 7.4). At the start of experiments, the luminal secretions
were removed by perfusion of the xenograft with 1 ml HPBR followed by
air. Radioisotope was then immediately loaded into the lumen of
xenografts through externalized tubing using a Hamilton syringe.
Initial studies evaluated the time necessary for non-CF xenograft
epithelia to absorb 50% of 22Na.
In these experiments, 3 h postloading was determined to be the optimal
time point at which ~50% of counts remained in the lumen of
xenografts. Hence, for all absorption studies, the percentages of
absorbed 36Cl and
22Na were calculated by irrigating
xenografts with 1 ml Ringer at 3 h postlabeling and assessing the
remaining content of radiolabel in the effluent. This assay was used to
compare absorption rates between non-CF
(n = 29) and CF
(n = 18) xenografts.
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RESULTS |
Accuracy of proposed methods for calculating
Vs and
[Cl]s.
To evaluate the proposed methods for calculating
Vs and
[Cl]s in xenograft
airways, we generated a tubular setup that mirrored collection of
xenograft ASF. In these studies, a defined volume of fluid (1, 2, 4, or
10 µl) containing 50 mM NaCl was loaded into the center of plastic
tubing with a diameter similar to that of a bronchial xenograft.
Perfusions were performed as described for xenograft airways, and
effluent concentrations of
[3H]inulin were used
to calculate the starting volume of fluid loaded into the lumen of the
tubing. Furthermore, the dilution of
[3H]inulin counts was
also used with direct AgCl electrode determinations to calculate the
starting concentration of Cl in luminal fluid. These studies
demonstrated an average error of 4.8% and 1.8% for calculations of
Vs and
[Cl]s, respectively,
when 1-10 µl of fluid was loaded into the lumen (Table
1). In summary, these studies confirm the
accuracy of our proposed methods for determining small volumes and Cl
concentrations in a tubular airway.
Measurements of steady-state
[Cl]s by direct assessment with
AgCl electrodes.
Fully differentiated 5-wk-old xenografts, which demonstrated
characteristic bioelectric properties for CF and non-CF xenografts (Fig. 1), were used for
analyses of Vs and Cl content. As
shown in Fig. 1, the main differences in bioelectric properties seen in
CF xenografts included a slight elevation in amiloride-sensitive Na
permeability and a complete absence of cAMP-forskolin-inducible changes
in Cl permeability. The means ± SE of potential change in response
to amiloride were 4.0 ± 0.5 and 5.6 ± 0.5 mV for non-CF and CF
xenografts, respectively (P < 0.03, Student's t-test). [Cl]s were assessed in
air-exposed xenografts following a 48-h postirrigation equilibration
period. Initial efforts attempted to harvest ASF directly from
xenografts in a 100-µl volume of Cl-free isosmotic dextrose. Cl
concentrations and Vs were then assessed using AgCl electrodes and direct pipetting, respectively. However, we found it difficult to obtain reproducible
Vs measurements due to retention
of irrigation solution within the xenograft. To this end, we adapted an
alternative protocol in which an impermeable radiolabeled marker
([3H]inulin) was added
to a Cl-free isosmotic perfusate solution used to collect ASF (Fig.
2A).
In these studies, the dilution of a known concentration of
[3H]inulin in the
perfusate was used to back calculate the dilution of ASF during
harvesting. Initial experiments evaluated
[Cl]s using isosmotic
Cl-free dextrose solution as the perfusate. However, because the
potential presence of Na-glucose cotransporters in the apical membrane
might alter ion transport, we also evaluated Cl concentrations using a
isosmotic mannitol solution as the perfusate (Fig.
2B). No differences were seen in
matched sets of xenografts evaluated with these two perfusate
solutions. These results suggest that, within the time course of
irrigation (<10 s), ion transport by the Na-glucose cotransporter
does not significantly influence the Cl composition in the airway. The
combined data (Fig. 2C) using both
mannitol and dextrose perfusate solutions demonstrate a slight trend
toward higher concentrations of
[Cl]s in CF ASF (125 ± 4 mM; n = 27) compared with
non-CF (114 ± 4 mM; n = 48; P < 0.045). However, these trends
required a large sample population to reach significance, as is seen by
the lack of significance between CF and non-CF under the two individual
perfusate conditions (Fig. 2B). In
selected initial experiments,
[Cl]s were also
determined 24 h after irrigation of xenograft airways (data not shown).
These values were not significantly different from the values from
48-h-equilibrated airways presented in this paper. However, we chose to
use 48 h as a standard for analysis to assure that adequate time was
allowed for ions to equilibrate in the airway postirrigation. In
summary, our findings suggest that, in the xenograft model system,
differences between CF and non-CF
[Cl]s are small.

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Fig. 1.
Bioelectric properties of cystic fibrosis (CF) and non-CF xenograft
airways. Transepithelial potential difference (PD) measurements were
used to confirm differentiated bioelectric properties of airway
epithelium in xenografts before functional analyses of airway surface
fluid (ASF) volume (Vs) and ASF
Cl concentration
([Cl]s). Typically,
these measurements were performed 4.5 wk after transplantation of
xenografts. Assays were performed as previously described (22, 23).
Arrows mark order of buffer changes from starting
HEPES-phosphate-buffered Ringer (HPBR) solution, including:
1) HPBR with 100 µM amiloride
(Amil); 2) Cl-free HPBR with 100 µM amiloride (marked Cl-free); 3)
Cl-free HPBR with 100 µM amiloride, 200 µM CPT-cAMP, and 10 µM
forskolin (marked cAMP); 4) Cl-free
HPBR with 100 µM amiloride, 200 µM CPT-cAMP, 10 µM forskolin, and
100 µM UTP (marked UTP); and 5)
HPBR. Means ± SE (n = 26) of
transepithelial PD under baseline and amiloride-treated conditions were
5.3 ± 0.9 and 1.3 ± 0.7 mV, respectively, for non-CF
xenografts and 6.6 ± 0.9 and 1.0 ± 0.6 mV, respectively,
for CF xenografts. Changes in PD with amiloride were 4.0 ± 0.5 and
5.6 ± 0.5 mV for non-CF and CF xenografts, respectively. This
difference in amiloride-induced change in PD was significant
(P < 0.03, Student's
t-test).
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Fig. 2.
[Cl]s in CF and non-CF
xenografts. A: experimental time line
for evaluating [Cl]s
in xenografts. Xenografts were transplanted on
day
1, and experimental analyses began at
day
35 posttransplant. At this time,
xenograft epithelium was fully differentiated and secreting mucus.
Xenografts were exposed to air at all times during analyses except when
ASF was harvested. ASF was harvested in 100 µl of isosmotic dextrose
or mannitol with inclusion of 20,000 counts/min (cpm) of
[3H]inulin as an
internal impermeable marker for dilutional calculations. Counts lost
during ASF harvest were determined by perfusing graft with a large
excess (1 ml) of Ringer;
[cpm]e, cpm in
effluent of 1st wash;
[cpm]f, cpm in final
wash. B: Cl concentrations determined
for n independent xenografts in which
ASF was harvested with either isosmotic dextrose or mannitol.
Calculations of total Cl were made as described in
MATERIALS AND METHODS, using a
solid-state AgCl electrode. C:
individual cumulative data points from both dextrose and mannitol
perfusate experiments (means ± SE;
P determined by Student's
t-test).
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Effect of mucin on Cl concentration measurements using AgCl
electrodes.
Non-Cl anionic compounds have the potential to interfere with AgCl
electrode determinations of
[Cl]s. One of the most
abundant anionic macromolecules in ASF is mucin. Mucin contains several highly negatively charged modifications, including sulfate and sialic
acid (13). For this reason, we used reconstitution experiments to
evaluate whether bovine submandibular gland mucin would affect Cl
determinations using AgCl electrodes. At concentrations of 0.5 mg/ml,
the addition of mucin to Cl standards generated a 12% increase in
potential from AgCl electrodes (Table
2). This effect demonstrated a
dose response in that higher mucin concentration had a greater effect
on potential. In the present study, because standards to which mucin
had not been added were used to calculate the
[Cl]s of the samples
containing mucin, these calculated values will be 12% too high if 0.5 mg/ml mucin is present in ASF. Because the exact concentration and
composition of mucin in the ASF is unknown, we do not know the exact
magnitude of this elevation. However, we have previously shown that CF
and non-CF xenografts secrete similar levels of mucin using
[3H]glucosamine
labeling (21). Therefore, we would predict that, although mucin present
in the ASF would artificially elevate the absolute value of Cl,
differences between CF and non-CF should be directly comparable.
Measurements of Vs in the airways of CF
and non-CF xenografts.
Previous results assessing in vivo fluid transport in bronchial
xenografts have demonstrated a fourfold higher rate of fluid absorption
in CF than in non-CF xenografts (23). These studies evaluated the
kinetics of fluid transport in fluid-filled xenografts linked to a
microcapillary apparatus capable of assessing small changes in lumen
fluid volume (10 nl/min). However, it is recognized that the physiology
of fluid transport in liquid-filled airways may be substantially
different from that of air-exposed epithelia. Therefore, in the present
study, we sought to assess the 48-h-equilibrated fluid volume
(including sol and gel) on the airway surface
(Vs). Analysis of
Vs utilized an internal marker,
[3H]inulin, for
assessment of dilution during the collection of ASF. This approach
afforded the added sensitivity and accuracy of determining the extent
of fluid volume loss (Vl) during
the collection period, which was then used to correct the final
determination of Vs. As shown in
Fig. 3, 48-h-equilibrated
Vs in CF (28 ± 3.0 µl/cm2) was significantly
smaller (P < 0.05) than the
Vs seen in non-CF (35 ± 2.4 µl/cm2) xenografts. Although
these results are consistent with the previously reported fourfold
higher baseline fluid absorption rates in CF xenografts (Fig. 3), the
magnitude of differences between CF and non-CF was significantly less
in air-exposed epithelia. These findings suggest that differences in
the physiology of fluid transport in air- and liquid-exposed epithelia
likely exist.

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Fig. 3.
Vs determinations in CF and non-CF
xenografts. Vs determinations were
performed on CF and non-CF xenografts as described in
MATERIALS AND METHODS. ASF
(equilibrated 48 h) was harvested by perfusion with isosmotic dextrose
containing a known concentration of
[3H]inulin. Dilution
of 3H cpm was used to calculate
starting Vs. Counts lost during
perfusion were used to correct for volume lost during perfusion. Solid
bars, results of Vs determinations
performed in air-exposed xenografts as described in this paper. Open
bars, data from a previous study (23) by this laboratory that
kinetically assessed fluid transport in fluid-filled human bronchial CF
and non-CF xenografts. That study evaluated baseline rate (nl/min) of
fluid absorption using a microcapillary apparatus linked to one end of
xenograft. All values are normalized to average area of a xenograft
(1.0 cm2). Results are means ± SE for n independent xenografts.
In all cases, comparisons between CF and non-CF reached statistical
significance (P < 0.05, Student's
t-test).
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Both Na and Cl are hyperabsorbed in CF bronchial xenografts.
To investigate potential mechanisms by which fluid absorption is
increased in CF xenografts, we evaluated the extent of
22Na and
36Cl absorption following luminal
labeling. Although limitations in the model system do not allow for
true kinetic assessment of net radioisotopic flux, we reasoned that the
percent absorption would allow for a comparative assessment of
differences in the rates of absorption between CF and non-CF
xenografts. Results from these studies demonstrate a significant
(P < 0.02) elevation in percent
absorption of 36Cl in CF (74.3 ± 5.3%; n = 18) compared with
non-CF (53.7 ± 5.7%; n = 29)
xenografts following 3-h luminal exposure to radioisotope (Fig.
4). Furthermore, simultaneous luminal
labeling with 22Na demonstrated
absorptive rates similar to those seen with
36Cl for all CF (72.6 ± 5.6%;
n = 18) and non-CF (51.5 ± 5.7%;
n = 29) xenografts analyzed. These
data suggest that Na and Cl are absorbed in an equal molar ratio. In
summary, results from this analysis suggest that CF xenograft epithelia
hyperabsorb NaCl and may explain the decreased
Vs and increased fluid absorptive rates observed in CF xenografts.

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Fig. 4.
Relative efficiency of 22Na and
36Cl absorption in CF and non-CF
xenografts. A: fully differentiated
xenografts were perfused with 1 ml Ringer followed by air to remove
excess mucus at 48 h before and again immediately prior to functional
measurements of 22Na and
36Cl absorption. With a Hamilton
syringe, a 5-µl volume containing 200,000 cpm each of
22Na and
36Cl was injected directly into
the lumen of CF and non-CF xenografts. Counts remaining in xenograft
were harvested by perfusion with 1 ml Ringer at 3 h postlabeling, and
percent absorption was calculated for each isotope.
B: means ± SE for
n independent xenografts. Comparisons
between CF and non-CF reached statistical significance
(P < 0.002, Student's
t-test).
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DISCUSSION |
With the recent findings that the ionic composition can affect the
antibacterial properties of ASF (5, 16), the debate regarding the exact
[Cl]s has become a
central aspect of understanding pathophysiology in CF. Several reports
have supported differences in the ionic composition of ASF in CF and
non-CF airways (4, 6, 10), whereas others have been unable to
demonstrate these differences (12). Furthermore, among the groups that
have demonstrated differences in the ASF ionic composition in CF, some
have found ASF Cl to be higher (4, 10) and others have found it to be significantly lower (6) than in nondiseased healthy subjects. Discrepancies in the values obtained for ASF Cl measurements between these various laboratories are likely dependent on the model system and
techniques used for analysis and may also be affected by the state of
inflammation (6). Although in vivo analysis of ASF in patients is
without a doubt the best model system, this approach is hindered by the
technical hurdles of collection and assessment of relatively small
volumes of fluid. In the present study, we have attempted to use a
sensitive technique incorporating radioactive tracers to assess ASF Cl
and fluid volumes in the airways of human bronchial xenografts, a close
in vivo correlate model of the CF and non-CF airway.
Several aspects of the bronchial xenograft model system make it
attractive for such studies. First, genetically defined CF and non-CF
airways can be generated in the absence of bacterial infection. Second,
this model represents a fully differentiated mucus-secreting,
air-exposed epithelium. Third, these xenografted airways are
vascularized and mirror tubular airways seen in vivo. Although no model
system can completely reconstitute all aspects of the in vivo lung, we
feel that this bronchial xenograft model is an attractive alternative
to other model systems.
Results from ASF analysis of human bronchial xenografts demonstrate
small but detectable differences in
[Cl]s between CF and non-CF ASF. The values obtained in this study (CF, 125 ± 4 mM Cl; non-CF, 114 ± 4 mM Cl) closely mirror those found
for both CF and non-CF nasal epithelia in a recent clinical study
(~125 mM) (12). However, they are also divergent from results of
other clinical methods used for assessing ASF (4, 10), which have suggested that CF ASF is significantly higher in Cl than non-CF ASF.
Important to our model system, and to others using AgCl electrodes, is
the finding that mucin content in the ASF can affect the absolute [Cl]s value obtained.
These results suggest that the data obtained using AgCl electrodes may
overestimate the absolute level of Cl in ASF, since the non-Cl
composition (i.e., mucin levels) could not be accurately controlled for
in the Cl standard curves. If the level of mucin is elevated in CF ASF,
as in the clinical condition, this effect would potentially lead to an
artifactually elevated ASF Cl. However, the human bronchial xenograft
model has been demonstrated to reverse the goblet cell hyperplasia in
the CF airways, since environmental influences of bacterial infection have been removed (21). Furthermore, CF and non-CF xenografts secrete
similar levels of
[3H]glucosamine-labeled
mucin (21). Hence, although it is unlikely that the slight elevation in
ASF Cl seen in CF xenografts is due to increased mucin production, it
remains plausible that the absolute values obtained for ASF Cl in both
CF and non-CF are higher than in other model systems using detection
techniques for which mucin does not interfere. Nonetheless, because
mucin secretion is similar for CF and non-CF xenografts, relative
analyses of the ASF Cl content can be accurately performed.
Another feature of our methodology for assessing ASF Cl also allows for
accurate determination of Vs. In
these studies, Vs in CF xenografts
were significantly reduced by 20% (P < 0.05) compared with non-CF xenografts. Conclusions from these
studies suggest that fluid is hyperabsorbed in the CF airway,
substantiating other in vitro models in this regard (8). The fact that
these differences were smaller than kinetic measurements of fluid
absorption (CF 400% greater than non-CF xenografts) likely reflects
alterations in the physiology of fluid movement in air- vs.
liquid-filled airways. Additionally, one important caveat is that our
measurements of Vs assess the
total secretory volume (both sol and gel) in the xenograft;
measurements of Vs included the
volume contributions of mucin and other high-molecular-weight
aggregates. For example, in CF xenografts, a mean
Vs of 30 µl would generate a
layer of fluid on a 1.0-cm2
xenograft epithelial surface of 300 µm, if evenly distributed. This
value is quite a bit larger than what has traditionally been thought to
be the height (5-10 µm) of the fluid that bathes the cilia
(called "sol") but is closer to the estimates of combined height
of both sol and gel layers (~25-100 µm) that contain both fluid and mucus (18, 20). Hence, we feel that our measurements reflect
the total secretory volume in the airways, including mucin as well as
sol. Because large aggregates of mucin are characteristic of ASF from
both CF and non-CF xenografts, the contribution of mucin to the total
volume may be quite large. However, mucin content in ASF appears to be
identical between CF and non-CF xenografts (21). Additionally, it is
likely that mucous secretions may accumulate in connecting tubing at
the end of the xenograft due to ciliary clearance and capillary action
of the tubing. Hence, it is plausible that the total secretory volume
of xenografts may not be evenly distributed. Taken together, our data
demonstrate that the total secretory volume at the airway surface in CF
xenografts is reduced and suggest that hydration in CF airways may be
impaired. The mechanisms of this alteration in hydration of the CF
airway surface have been previously suggested to result from defects in
Na permeability, which is the likely driving force for fluid movement
in the airway.
In CF airways, Na has long been suggested to be hyperabsorbed in
comparison to non-CF airways (2). Central to the debate concerning the
ionic composition of ASF in CF is how CF airway epithelia can
hyperabsorb Na and retain a higher
[Cl]s than non-CF airway epithelia. Given the observed differences in fluid absorption between CF and non-CF xenografts in air- vs. fluid-filled airways, we
hypothesized that the air-liquid interface in air-exposed xenografts may also be critical for regulating ion movement. Because all previous
Na and Cl flux data have been obtained from model systems in which the
epithelium was exposed to an excess reservoir of fluid, we sought to
use a modified protocol to look at Na and Cl absorption in air-exposed
xenografts. Because the fluid used for radioisotope loading was <17%
of the Vs, we feel that these conditions closely mirror air-exposed epithelia in vivo. Although, in
the present model, efflux rates cannot be determined because of limited
access to the serosal side of the epithelium, we can determine
differences in the absolute movement of Na and Cl out of the airways
between CF and non-CF.
Assessment of 22Na and
36Cl absorption suggests that CF
xenograft epithelia hyperabsorb NaCl. However, because the net rate of absorption will be determined by both the absorptive and backflux secretion rates, it is important to understand the limitations of these
in vivo analyses. Given the technical limitations in assessing true
kinetic fluxes in the xenograft model, there remains a possibility that
CF xenograft epithelium is impaired in secretion of NaCl, while
absorption is equivalent to that of non-CF epithelium. However, because
a relatively small radioisotope load was used and because absorbed
radioisotope would be diluted into the total body water of mice, we
anticipate that an increased backflux of radioisotope secretion in
non-CF would be minimal even if higher than in CF. Nonetheless, these
findings support the conclusion that in CF net removal of NaCl from the
airway is increased. This is consistent with results demonstrating an
increase in net fluid movement out of the CF airway (8, 23). Hypotheses
that CF airways hyperabsorb fluid are not new, but, given the current debate on how a higher Cl gradient in CF can be maintained in the
presence of hyperabsorption of NaCl, these issues are central to our
understanding of ion transport physiology in the airway.
The human bronchial xenograft model has provided another perspective on
ion transport physiology in the airway and how it may be defective in
CF. No single model system of the airway can completely address all
aspects of the debate on the ionic composition of CF ASF and how these
alterations may influence the pathophysiology of CF airway disease.
However, the human bronchial xenograft model has several attractive
advantages that make possible an alternative viewpoint to those
provided by previously studied model systems. Conclusions
from this model suggest that differences in the ASF Cl content between
CF and non-CF airways are minimal. On the basis of previous studies of
defensin activity, one would anticipate that alterations in ASF NaCl
concentrations between non-CF (~114 mM) and CF (~125 mM) human
bronchial xenografts would not lead to alterations in defensin activity
(5). These results contradict a previous report evaluating Cl content
in xenograft secretions between CF and non-CF (5). Potential reasons
for this discrepancy are unclear but may reflect differences in the
methodologies used for harvesting secretions and/or detection.
However, we feel that the methods presented in this report, using an
internal radioisotopic marker, provide an added level of accuracy in
the ASF Cl determinations. Data evaluating ASF fluid volumes and
22Na36Cl
absorption suggest that CF xenografts hyperabsorb salt and fluid,
leading to more dehydrated airways. Taken together, these data
substantiate findings that defective regulation of ENaC by CFTR leads
to higher levels of salt absorption and fluid movement out of the CF
airways. Furthermore, the steady-state movement of Cl though
paracellular pathways and/or alternative Cl channels does not
appear to be impaired in the CF airway. The fact that ASF Cl may be
slightly higher in CF remains a somewhat paradoxical phenomenon, given
the fact that absorption of Cl and Na appears to be elevated in
comparison with non-CF airway. Such a conundrum highlights the present
lack of understanding in the physiology of ion and fluid movement in
the airway.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the support of the Cystic Fibrosis
Foundation, National Heart, Lung, and Blood Institute Specialized Center of Research Grant HL-61234, and National Institute of Diabetes and Digestive and Kidney Diseases Gene Therapy Center Animal Models Core Grant DK-54759.
 |
FOOTNOTES |
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: J. F. Engelhardt, Dept. of Anatomy and
Cell Biology, University of Iowa School of Medicine, 51 Newton Rd., Rm.
1-111 BSB, Iowa City, IA 52242.
Received 29 June 1998; accepted in final form 16 November 1998.
 |
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