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


    ABSTRACT
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Abstract
Introduction
Materials and methods
Results
Discussion
References

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


    INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

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?


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

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.
V<SUB>l</SUB> = [([inulin]<SUB>f</SUB> × V<SUB>f</SUB>)/([inulin]<SUB>i</SUB> × V<SUB>i</SUB>)] × V<SUB>i</SUB>
V<SUB>l</SUB> = (Total cpm in final Ringer wash/
total cpm of input first Cl-free wash) × V<SUB>i</SUB>
V<SUB>l</SUB> = (Fraction of counts lost following first wash) × V<SUB>i</SUB>
V<SUB>e</SUB> = [([inulin]<SUB>i</SUB>/[inulin]<SUB>e</SUB>) × (V<SUB>i</SUB> − V<SUB>l</SUB>)]
V<SUB>s</SUB> = V<SUB>e</SUB> − V<SUB>i</SUB> + V<SUB>l</SUB>
V<SUB>s</SUB> = [([inulin]<SUB>l</SUB>/[inulin]<SUB>e</SUB>) × (V<SUB>i</SUB> − V<SUB>l</SUB>)] 
− V<SUB>i</SUB> + {[([inulin]<SUB>f</SUB> × V<SUB>f</SUB>)/([inulin]<SUB>i</SUB> × V<SUB>i</SUB>)] × V<SUB>i</SUB>}
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, Delta F508/Delta F508 and 1 unknown/Delta 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
[Cl]<SUB>s</SUB> × V<SUB>s</SUB> = ([Cl]<SUB>e</SUB> × V<SUB>e</SUB>) + ([Cl]<SUB>w</SUB> × V<SUB>l</SUB>)
[Cl]<SUB>s</SUB> × V<SUB>s</SUB> = [Cl]<SUB>e</SUB> × V<SUB>e</SUB> where [Cl]<SUB>w</SUB> → 0
[Cl]<SUB>s</SUB> × V<SUB>s</SUB> = [Cl]<SUB>e</SUB> × (V<SUB>s</SUB> + V<SUB>i</SUB> − V<SUB>l</SUB>)
[Cl]<SUB>s</SUB> = {[Cl]<SUB>e</SUB> × (V<SUB>s</SUB> + V<SUB>i</SUB> − V<SUB>l</SUB>)}/V<SUB>s</SUB>
Initial studies compared the [Cl]s among 17 CF xenografts (from 3 independent tissue samples, 2 Delta F508/Delta F508 and 1 unknown/Delta 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 Delta F508/Delta F508, 1 G551D/Delta 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.


    RESULTS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

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.

                              
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Table 1.   Accuracy of proposed methods for determining Vs and [Cl]s in a tubular system

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).

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.

                              
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Table 2.   Effect of mucin on Cl determinations with AgCl electrodes

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).

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).


    DISCUSSION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

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.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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Am J Physiol Cell Physiol 276(2):C469-C476
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