American Journal of Respiratory and Critical Care Medicine

Mucociliary clearance is determined by ciliary activity and rheology of airway surface liquid. To test the hypothesis that mucociliary clearance would increase after inhalation of an osmotically active agent that would increase the volume of airway surface liquid, we measured mucociliary clearance in 16 normal subjects after inhalation of varying tonicities of saline alone, and after pretreatment with a Na+ channel blocker (amiloride). Subjects inhaled vehicle (0.12% saline) or amiloride, followed by inhalation of 0.12, 0.9, or 7% saline. Subsequently, mucociliary clearance rates were measured by γ scintigraphy of inhaled 99mTc Fe2O3. Mucociliary clearance of whole and peripheral lung was increased (approximately twofold) after inhalation of increasing concentrations of saline (p < 0.04). Pretreatment with amiloride increased mucociliary clearance rates (approximately twofold) after inhalation of 0.12 and 0.9% saline (p < 0.05), but not 7% saline. The rates of mucociliary clearance by pretreatment with amiloride and 7% saline alone (approximately 1.4% per minute) approached the rapid mucociliary clearance rates (approximately 2.0% per minute) reported in systemic pseudohypoaldosteronism, which has loss-of-function mutations of the epithelial Na+ channel and an increased volume of airway surface liquid. We conclude that maneuvers that increase the volume of airway surface liquid are associated with increased rates of mucociliary clearance in normal subjects.

Mucociliary clearance (MCC) rates depend on ciliary activity coupled to the volume and rheological properties of airway surface liquid (ASL) (1), and rates of MCC can be upregulated for airway host defense. Under normal circumstances, there is cephalad movement of the ASL secondary to ciliary activity (2). The volume (depth) of ASL is regulated by isotonic volume transport, with active Na+ absorption as the dominant basal ion transport activity of the airway surface epithelia (28). In vitro, the acute addition of NaCl expands ASL height (9), improves mucus rheologic properties (elasticity and viscosity), and accelerates mucus transport rates (9, 10).

In cystic fibrosis there is accelerated absorption of NaCl from airway surfaces, which leads to reduced volume (depth) of ASL, concentration of mucin macromolecules, and slowing of MCC (7, 8). In vivo, aerosolized hypertonic saline or mannitol increases the rate of mucus clearance in patients with cystic fibrosis, perhaps reflecting partial restoration of ASL volume (11, 12). In contrast to cystic fibrosis, patients with systemic pseudohypoaldosteronism (PHA) have loss-of-function mutations in the epithelial Na+ channel (ENaC) and an absence of airway epithelial Na+ absorption, which is associated with an increased volume of isotonic ASL (13, 14). Adults with PHA appear to “compensate” for the excess ASL by accelerated rates of clearance of liquid (MCC) from the airway surface (14).

No previous study has systematically tested the relationship between an acute increase in ASL volume and the rate of MCC in normal subjects. The goal of the current study was to study the effect of maneuvers predicted to acutely increase the volume of ASL on MCC rates in normal subjects. Our strategy was to measure MCC rates with isotopic markers delivered after airway surfaces were dosed by inhalation with increasing amounts of NaCl in aerosolized 0.12, 0.9, and 7% saline. We further tested the hypothesis that pretreatment with inhaled amiloride, an Na+ channel blocker, might increase the magnitude of inhaled NaCl–induced changes in MCC by preventing Na+ (and ASL volume) absorption over the MCC measurement interval (4, 15).

Subjects

Sixteen normal subjects completed this protocol. The first cohort (Group 1; Table 1)

TABLE 1. Aerosolized dosing regimens


Group 1* (n = 7)

Group 2 (n = 9)
0.12% saline/0.12% salineND
Amiloride/0.12% salineND
0.12% saline/0.9% saline0.12% saline/0.9% saline
Amiloride/0.9% salineAmiloride/0.9% saline
ND0.12% saline/7% saline
ND
Amiloride/7% saline

*C/P = 1.40 ± 0.08.

C/P = 1.51 ± 0.06.

Definition of abbreviation: ND = not done.

Aerosolized dosing regimens were administered in randomized and blinded fashion on four separate days to two groups of study subjects.

had seven subjects (six females) with a mean age of 25 years. The second cohort (Group 2) had 10 subjects (5 females) with a mean age of 29.9 years; 1 subject (female, aged 22 years) was excluded based on predetermined criteria because of variation in ratio of central to peripheral (C/P) radionuclide counts. The study was approved by the University of North Carolina IRB and informed consent was obtained.

Techniques
Drug preparation, storage, and administration.

Dry powder amiloride (Merck, West Point, PA) was dissolved in 0.12% saline as a stock (10−2 M).

Radioisotopic aerosol labeling, generation, and administration.

The aerosol (4 μm mass median aerodynamic diameter [MMAD]) was formed from 2% (by weight) colloidal suspension of 99mTc-Fe2O3 by a spinning disk aerosol generator to produce dry iron oxide particles (16).

Protocol

Subjects were studied over 4 days in a randomized, double-blind, crossover design. To limit isotope exposure, two groups were studied and exposed to a common treatment (0.9% saline). Before isotope was administered, subjects inhaled solutions of vehicle or amiloride, then different concentrations of saline, for up to 15 minutes (or until “sputter”; see Figure 1)

, using a PARI LC Star nebulizer with an MMAD of approximately 4 μm (Figure 1). The lowest concentration of saline (0.12%; hypotonic) was selected because the rate of MCC is not stimulated in normal subjects after inhalation of 0.12% saline, i.e., the rate of MCC after hypotonic saline (alone) is the “basal” rate of MCC in healthy subjects (17, 18). Subsequently, subjects inhaled an aerosol (4 μm MMAD; geometric SD = 1.25) of insoluble 99mTc-Fe2O3 (19, 20). An initial deposition scan was recorded and γ scanning was performed for 120 minutes (2-minute images every 10 minutes) to measure particle clearance. Subjects returned at 24 hours after deposition for a 30 minute scan.

Data Analysis

The approach for measurement and analysis is well-used in our labs (17, 21). In brief, particle clearance after inhalation is initially rapid, but slows after the first 10–30 minutes (Figure 2)

. This pattern of clearance (initially rapid, with later slowing) is also seen in measures of “baseline” (no treatment) MCC (22). Initial comparisons between different aerosol treatments were made by repeated-measures analysis of variance of clearance over the initial 20 minutes for each day. Comparisons between doses of saline (0.12% versus 0.9% saline) were assessed by nonpaired t tests, whereas comparisons of pretreatment with amiloride were assessed by two-tailed paired t tests. Significance was set at p < 0.05 for two-tailed analysis. Unless otherwise specified, data are presented as means ± SD.

Modeling (Estimating) the Relative Volume (Depth) of ASL after Aerosolized Saline

These estimates used a model that has been validated by comparison with experimental (total and regional deposition) lung deposition measurements in humans over a large range of particle sizes (2326).

Whole lung clearances of 99mTc iron oxide particles are shown for different aerosolized solutions in the first group (n = 7; Figure 2A) and the second group (n = 9; Figure 2B) of subjects. The pattern and rate of clearance after inhalation of 0.12/0.12% saline is similar to “basal” (no treatment) clearance in healthy subjects, and does not represent a stimulated rate of MCC (17, 18). In general, the whole lung (Figure 2) and peripheral lung (data not shown) MCC curves revealed an early period of rapid clearance, which then plateaued to a slower rate. This change in rate could reflect either a true slowing of clearance, and/or reduced availability of clearable isotopic marker in conducting airways, over time.

To quantitate the initial rate of rapid isotope clearance, we calculated the breakpoint in the rate of clearance, which reflects the time-shift from a phase of rapid to apparent “slower” clearance (see Methods). The breakpoint in the rate of whole and peripheral lung MCC for all study days was determined to be 20 minutes. This analysis indicated that the predominant effect of the different aerosolized solutions on rates of MCC occurred within the first 20 minutes after deposition of isotope-labeled iron oxide particles.

The whole lung clearance rates (%/minute) as a function of concentration of inhaled saline with or without amiloride pretreatment over the first 20 minutes for subjects from both groups are summarized in Figure 2C. For inhaled saline without amiloride pretreatment, increasing “masses” of NaCl, delivered by aerosols with varying concentrations of NaCl, increased the rate of MCC when tested for all study subjects (p = 0.04, analysis of variance). There was little effect for 0.9% saline to increase MCC as compared with 0.12% saline (p = 0.19, nonpaired t test), whereas 7% saline clearly increased MCC as compared with 0.12 saline (p = 0.03). Pretreatment with amiloride increased the rates of MCC observed after inhalation of 0.12% (p < 0.02) or 0.9% saline (p < 0.0001), but not 7% saline. Further, with amiloride pretreatment, the rates of MCC after inhalation of 0.12 and 0.9% saline (i.e., 1.1–1.3%/minute) were similar to that observed for 7% saline alone.

The peripheral lung clearance rates (%/minute) over the first 20 minutes for all subjects are shown in Figure 2D. For inhaled saline aerosols without amiloride pretreatment, there was a tendency for increasing “masses” of inhaled NaCl, i.e., increased concentrations of saline, to increase the rate of MCC (p = 0.12, analysis of variance). Comparisons of MCC after inhalation of different concentrations of saline revealed no effect of 0.9% saline to increase MCC as compared with 0.12% saline, but 7% saline increased peripheral MCC compared with 0.12% saline. Again, pretreatment with amiloride increased peripheral MCC rates after inhalation of 0.12 and 0.9% saline (p < 0.05), but not 7% saline. After pretreatment with amiloride, the rates of MCC (approximately 1%/minute) were similar for all concentrations of inhaled saline.

We also analyzed the MCC data over longer-term intervals (0 to 60 and 120 minutes). For whole lung clearance, pretreatment with inhaled amiloride systematically increased the fraction of isotope cleared after inhalation of 0.12% saline (p < 0.001 for 60 and 120 minutes) and 0.9% saline (p < 0.004 for 120 minutes), when compared with pretreatment with inhalation of vehicle (Figure 2A). In contrast, amiloride pretreatment did not increase the “net clearance” induced by 7% saline when compared with pretreatment with vehicle (Figure 2B). Similar patterns were seen for net clearance over 60 and 120 minutes from the peripheral lung regions (data not shown).

At 24 hours (Figures 2A and 2B), pretreatment with amiloride tended to increase net clearance when compared with 0.12 and 0.9% saline, alone (p = 0.09 for whole lung; p = 0.001 for peripheral lung), but amiloride had no effect on net clearance as compared with 7% saline aerosols with vehicle pretreatment for either whole or peripheral lung.

The aerosol inhalations were well tolerated by all subjects, and there were no episodes of bronchospasm or other untoward effects. To assess the effect of study design on the results, we determined the role of several covariates to any observed differences in clearance for the different treatment study days. Overall, only subject and treatment were significant (p < 0.05) predictors of the variation in 20-minute clearance rate. For 0.9 and 7% saline, treatment interaction with C/P ratio was significant (p = 0.03) for predicting 20-minute clearance rate, which implies that the difference in 20-minute clearance rate between treatments was greatest as C/P increased. There was no evidence for a role for cough frequency over the period of interest, treatment order, or interactions between these two variables and treatment (p = 0.4).

To approximate the relative change in depth of ASL along the tracheobronchial tree, we estimated the increase in the depth of ASL after acute inhalation of saline from modeling (see Methods). In Figure 3A

, we plotted estimates of particle deposition fraction after inhalation of 0.9% saline (right ordinate) based on the parameters of the aerosol droplet size and respiratory flow rates. We then estimated the volume of ASL “added” from the volume of 0.9% saline aerosol deposited and the estimated surface area of each airway generation (left ordinate). Although the deposition fraction of particles is relatively similar along different generations of the conducting airways, the model suggests that the greatest relative increase in the depth of ASL occurs in proximal (third generation) bronchi, whereas the smallest change is in more distal (12th–16th generation) bronchioles. These differences in estimated depth of ASL added largely reflect the relative surface areas of proximal (small surface) versus distal (large surface) conducting airways. In Figure 3B, we plotted the relative estimated change in added ASL depth as a function of the concentration of inhaled saline. As can be seen, the change in ASL depth is not a function of the fold-change in saline concentration (i.e., eightfold for 0.9 versus 0.12%, and 7 versus 0.9%, respectively), but rather a direct function of the mass of NaCl added to the airway surface by the aerosols of different concentrations of NaCl (see Figure 3B). We recognize that saline is deposited over 15 minutes in our research protocol in humans, and absorption of Na+ (and volume) is taking place over that interval, in the absence of amiloride. Thus, these modeling estimates may be better correlated with conditions after pretreatment with amiloride, which blocks Na+ (and volume) absorption.

This study demonstrated that maneuvers designed to acutely increase the volume (depth) of ASL are associated with increased rates of MCC in normal subjects (Figure 2). In general, increasing the amount (mass) of salt deposited on airway surfaces by increasing the concentration of NaCl in aerosols increased MCC. Similarly, pretreatment with amiloride to promote retention of inhaled NaCl on airway surfaces increased the effect of saline aerosols to raise MCC rates.

We designed our protocols to focus on the effects of added salt on ciliary-dependent, rather than cough-dependent, Fe2O3 (mucus) clearance. To remove the potential contribution of cough clearance in response to the different saline ± amiloride aerosol solutions, subjects were exposed to the different aerosol solutions before radiolabeled isotopic marker was given. With this protocol, there were no significant episodes of coughing after the inhalation of the tracer. Only four subjects experienced any cough and a multivariate analysis failed to show any effect of cough on MCC. There also appeared to be no change in airway caliber after delivery of the test aerosol that would bias estimation of MCC based on tracer deposition, as a comparable deposition of the inhaled radiolabel was achieved over the four study days as reflected by relatively uniform C/P ratios. Thus, there were no artifacts of cough-clearance or regional differences in tracer deposition inherent in this protocol design, which may have limited quantitative interpretation of the effects of saline aerosols on MCC in other studies, where cough occurred during nebulization of (hypertonic) saline or amiloride (11, 12).

In this study, there was little effect of inhaled isotonic (0.9%) saline to stimulate MCC as compared with a hypotonic saline (0.12). This result may reflect the fact that the difference in inhaled salt mass between a 0.12% saline aerosol (1.2 mg) and a 0.9% saline aerosol (9 mg) is relatively small (approximately 8 mg). In contrast, the mass of delivered salt is relatively large with 7% saline (70 mg), perhaps accounting for the large increase in MCC after inhalation of 7% as compared with 0.12 and 0.9% saline. Indeed, this hypothesis is consistent with the model analyses comparing salt deposition with increased ASL height (Figure 3B).

Interestingly, amiloride administered before inhalation of smaller doses of saline (0.12 or 0.9%) induced twofold increases in rates of MCC over the comparable saline doses with vehicle pretreatment for whole and peripheral lung (see Figures 2C and 2D). The effect of amiloride pretreatment to increase the MCC response to 0.12 and 0.9% saline (Figure 2) could reflect the capacity of amiloride to conserve all salt added to airway surfaces before and during the interval of the MCC measurement. However, because the quantity of added salt with 0.12% saline is relatively small, and there is little predicted effect on ASL height (Figure 3), the increase in MCC rates effected by amiloride may reflect the drug's effect to block basal ASL volume absorption (4, 27). The failure of amiloride to potentiate the effect of 7% saline inhalation on MCC could reflect the fact that the block of basal absorption is small relative to the very large mass of salt, and consequently, ASL volume, which is added to the airway surface by the concentrated (7%) salt solution. It is also likely that the addition of such a large mass of salt led to an acute increase in the ASL, resulting in a clearance of most of the radioisotope by 20 minutes.

From the MCC protocol used in this study, it is difficult to clearly define the duration of the effect on MCC after acute addition of salt ± amiloride to airway surfaces (Figure 2). The effect of inhaled saline to rapidly accelerate MCC appeared to last only 20 minutes, which (if true) could reflect rapid absorption of Na+ (and volume) and a short duration of effect on MCC. This interpretation is compatible with studies of well-differentiated human bronchial epithelial cell cultures that observed rapid (< 12 min) absorption of an equivalent quantity of saline (9%) (9, 15). However, the strategy to pretreat with amiloride and to block volume absorption would be predicted to extend the duration of effect at least 30 to 60 minutes beyond the 20 minutes observed here, as demonstrated previously in vitro (9). Interestingly, in vivo measurements in patients with PHA also demonstrated that the duration of rapid radiotracer clearance, i.e., “MCC,” lasted only 20 minutes (14). Because patients with PHA have a genetic loss-of-function defect of Na+ channels with a constitutive defect in volume absorption, the duration (20 minutes) of rapid rate of particle clearance (MCC) in these patients cannot reflect the variables in this study (i.e., retention time of salt or amiloride on airway surfaces), but likely reflects a technical limitation of the MCC protocol. The simplest hypothesis is that there is reduced availability of clearable isotopic marker over time, related in part to inhaled Fe2O3 particles entering a second compartment on airway surfaces (e.g., macrophages) that is not cleared with cephalad movement of ASL. Future studies will focus on aerosol pretreatment with saline ± amiloride, followed by a delay (30–60 minutes) before isotopic marker is administered, to improve the ability to measure more accurately the duration of effect of salt/drugs on MCC rates.

Although maneuvers predicted to acutely add volume to ASL (Figure 3) are associated with increased rates of MCC, the maximal rates of MCC (approximately 1.4 and 1.1% per minute for whole and peripheral lung) after 7% saline and amiloride pretreatment of inhaled 0.9% saline fell short of those seen in PHA (approximately 2.0 and 1.6% per minute, respectively) (14). The failure to achieve MCC rates in our normal subjects as fast as those observed in PHA could reflect failure to deliver a maximal dose of salt and/or drug (amiloride). This possibility seems unlikely, because maximal MCC rates in these normal subjects appeared to plateau with delivery of 0.9 or 7% saline when pretreated with amiloride (Figures 2C and 2D). It is also unlikely that the higher rates of MCC achieved in patients with PHA reflect the slightly more central deposition pattern in subjects with PHA or a short duration of effect of amiloride in normal subjects (14, 27). It seems more likely that the higher rates of MCC in PHA reflect an adaptive response to raised ASL volume, perhaps related to ciliary function or activity, type of mucins secreted onto airway surfaces, or the fact that the limited duration of action of amiloride and salt did not induce the magnitude of “excess” ASL observed in patients with PHA, who have persistent (genetic) blockade of Na+/ASL volume absorption.

Understanding the link between the increase in rates of MCC after maneuvers to increase the volume of ASL requires detailed studies of the interactions between ciliary beat activity/efficiency and the properties of both the periciliary liquid and mucus layers. In vitro studies of well-differentiated airway epithelial cultures that exhibit ASL with well-defined periciliary liquid and mucus layers and rotational (surface) mucus transport have revealed that addition of NaCl (dry or in solution) increased mucus transport rates (15). This effect appears to reflect the fact that the added volume was exclusively accommodated by the mucus layer, leaving intact normal ciliary-mucus layer interactions, but perhaps making these interactions more efficient due to more favorable viscoelastic properties in either or both the periciliary liquid and mucus layers (9). We speculate that the increased volume of ASL from aerosol dosing of NaCl (and amiloride) to the normal subjects in the study led to ASL volume addition with selective hydration/swelling of the mucus layer and an acceleration of MCC rates, although the precise mechanism to link these events is not known. The greater change in rates of MCC in whole lung versus peripheral regions after similar aerosol treatments (Figures 2C and 2D), which parallel the predicted regional changes in ASL volume (Figure 3B), are also congruent with this hypothesis.

In summary, maneuvers that are predicted to acutely increase the volume of ASL are associated with acceleration of MCC rates in normal subjects. These findings are consistent with previous studies that demonstrated the short-term effect of inhaled hypertonic saline and/or amiloride to increase mucociliary clearance in patients with cystic fibrosis (4, 11, 12, 28, 29). The development of long-acting osmotic agents, and/or drugs that block volume absorption (or stimulate liquid secretion) in the airways, has therapeutic potential for the treatment of patients with reduced volume of ASL, including cystic fibrosis.

The authors express their appreciation to Lisa Brown for graphical and editorial assistance.

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Correspondence and requests for reprints should be addressed to Namita Sood, M.D., Division of Pulmonary and Critical Care Medicine, Ohio State University, 201, Heart & Lung Research Institute, 473 West 12th Avenue, Columbus, OH 43210. E-mail:

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