This perfused human lung model was developed to address three main objectives. The first objective was to determine whether AFC could be measured in the presence of pulmonary vascular perfusion in human lungs rejected for transplantation. Such a model would allow future studies incorporating the delivery of test substances through the vasculature. The second objective was to evaluate the contributions of epithelial fluid transport and epithelial protein permeability to lung fluid balance in the model. The final objective was to determine whether biological markers of alveolar epithelial injury were associated with alveolar epithelial fluid transport rates in the perfused human lung model.
Previous studies have found that most human lungs rejected for transplantation maintain alveolar epithelial fluid transport capacity (39
). However, the measured basal AFC rates in these previous studies and in lung lobes resected from lung cancer patients were only 4%/h (24
). These previous studies were done in ex vivo lungs and lung segments in the absence of pulmonary vascular perfusion that were preserved under hypothermic conditions for at least 12–24 h. The present study confirmed that, in passively rewarmed lungs, AFC was 5 ± 2%/h in the absence of perfusion. Compared with nonperfused conditions, perfusion resulted in markedly higher basal AFC rates (). Because vascular perfusion may affect both AFC and pulmonary edema formation, we simultaneously measured alveolar epithelial fluid transport, pulmonary vascular permeability to albumin, and lung fluid balance in these experiments. Measurable basal AFC was present in 79% (19
) of the lungs studied. The mean basal AFC rate for lungs in which AFC was present was 19 ± 10%/h (). This AFC rate is comparable to that in other species commonly used in experimental studies such as rats and mice (20
). The rate of AFC determined in this model is also comparable to estimated AFC rates in our previous clinical studies of patients with pulmonary edema (34
). It is also notable that because the intratracheal instillate and the perfusate have identical protein concentrations at the start of the experiment, increased albumin flux could potentially result in an underestimation of AFC in this model.
Why does pulmonary vascular perfusion significantly increase AFC in the ex vivo human lung model? In previous animal studies, AFC measured in situ immediately after the cessation of perfusion was comparable to AFC measured during perfusion. There are important differences between the present study and those prior animal studies. In the animal studies, lung temperature is held constant at 37°C, and AFC is immediately measured. However, donor human lungs are removed and then undergo cold preservation for several hours. Because temperature is inversely associated with AFC (29
), it is reasonable to assume that temperature must be restored to normal for AFC in the ex vivo lung to be comparable to the in situ conditions used in previous experimental studies. The addition of perfusion to the human lung model may have resulted in more uniform and complete rewarming of the lung. Therefore, it is possible that in the perfused lung, the temperature in the interior of the lung is higher than in the ex vivo, nonperfused human lungs in which the passive rewarming protocol involves submerging the lungs in a water bath for 2 h. Without perfusion, it is difficult to determine the internal temperature of the passively rewarmed lungs. In addition, the surface area for fluid transport may be greater in the perfused lung preparation compared with the ex vivo protocol without perfusion. The surface area for fluid transport could be increased by the addition of perfusion to the pulmonary vasculature or the suspension of the lungs from the bronchus in an air-filled chamber, which may result in more uniform inflation of the lung. Finally, in the absence of functioning lymphatics and vascular perfusion, the capacity for accumulation of pulmonary interstitial space for fluid is fixed. Therefore, the measured AFC rate could be affected by preexisting air space and interstitial pulmonary edema, the volume of fluid instilled for the measurement, and the duration of the measurement period (12
). The addition of perfusion could have increased the clearance of fluid from the interstitial space, potentially allowing for greater removal of fluid from the air spaces. The later explanation is least likely, but remains a possibility.
We also found that a β2
-selective adrenergic agonist (terbutaline, 10−5
M) significantly increased basal AFC rates more than twofold in this model. The mean stimulated AFC rate was 43 ± 13%/h in the presence of perfusion (). Previous studies of nonperfused ex vivo human lungs have reported stimulated fluid clearance rates of ~7%/h (24
). Of the lungs studied, 70% (12
) demonstrated a significant increase in AFC rate with terbutaline (). These data confirm that the alveolar epithelium remains responsive to β2
-adrenergic stimulation hours after organ procurement and that stimulated fluid transport rates in human lungs are comparable to those in commonly used experimental animal models. Experimental data in animal models indicate that increased alveolar epithelial fluid transport rates result in less pulmonary edema and more rapid improvement in oxygenation during hydrostatic pulmonary edema and acute lung injury (2
). Previous clinical data indicate that intact AFC is associated with improved survival in patients with acute lung injury and acute respiratory distress syndrome and with more rapid recovery from reperfusion pulmonary edema after lung transplantation (21
) or hydrostatic pulmonary edema (34
). Most of the lungs used in this study were rejected in part due to concern for acute lung injury, but a minority (7/24) of donors met criteria for acute lung injury or acute respiratory distress syndrome. However, these data support the plausibility of β2
-adrenergic agonist therapy targeted at increasing AFC rates and effecting a more favorable lung fluid balance in patients with acute lung injury and in patients with posttransplant reperfusion pulmonary edema and primary graft failure. Because previous clinical studies have shown that AFC may be impaired in acute lung injury and acute respiratory distress syndrome patients (38
), more studies are needed to determine if β2
-adrenergic agonists increase AFC in this patient population.
To determine the contribution of AFC to lung fluid balance in the perfused human lung, we simultaneously measured epithelial albumin flux, AFC, and lung weight gain (as a measure of lung fluid balance) in 10 lungs. There was a strong inverse association between AFC rate and lung weight gain (). There was a weaker association between AFC and lung albumin flux. Multivariable regression analysis showed that the association between AFC and lung weight gain was largely independent of epithelial albumin flux in the model. Therefore, consistent with a previous study in rats (10
), AFC can be preserved or increased in the perfused human lung in the presence of increased epithelial permeability. These results emphasize the critical contribution of AFC to favorable lung fluid balance and further highlight alveolar epithelial fluid transport as a potential target of pharmacological therapy in patients with pulmonary edema and acute lung injury.
Assessment of lung function before transplantation remains a challenge. A previous study found that many lungs rejected for transplantation based on conventional clinical criteria were without significant histological or functional abnormalities (39
). Those data, along with patient outcome data from recipients of lungs not meeting all of the usual pretransplant clinical criteria (4
), call into question the validity of certain clinical criteria used for screening donor organs. To investigate whether air space or circulating levels of biological markers of alveolar epithelial cell injury were associated with measures of epithelial barrier function, we also measured levels of RAGE in the perfusate and air spaces in these donor lungs. RAGE is made by alveolar epithelial cells and other epithelial cells. A recent report found that air space and plasma levels of RAGE were associated with lung injury severity in rats and in patients with acute lung injury (33
). Although RAGE is also produced by epithelial cells in other organs, it has been used recently as a type I pneumocyte marker (33
). In this isolated lung model, type I pneumocytes are probably the only source of RAGE. In the present study, alveolar epithelial injury as measured by baseline perfusate and air space levels of RAGE were significantly greater in lungs without AFC (). There was a significant inverse association between perfusate levels of RAGE and AFC rate. The average RAGE level in lungs without basal AFC was 544 ± 70 ng/ml compared with 76 ± 44 ng/ml in lungs with measurable AFC (P
< 0.00001, ). There was a weaker association between perfusate RAGE levels and albumin flux in this model, suggesting that epithelial permeability may be influenced by factors other than epithelial cell injury alone. However, because of the new evidence that alveolar epithelial type I cells transport sodium and contribute to AFC (5
), a marker of type I cell injury could be an especially useful marker of intact AFC. These results suggest that RAGE may be a useful biological marker of epithelial injury and barrier dysfunction clinically, although additional prospective validation is necessary. These data also raise the possibility that physiological and biochemical markers of alveolar epithelial injury could have predictive value in the assessment of lungs before transplantation.
Data from this new experimental model have potentially important clinical implications. First, these data demonstrate that β2-adrenergic agonists significantly increase AFC rates in perfused human lungs, raising the possibility that pharmacological therapy directed at augmenting AFC is plausible. Second, these data confirm previous reports that many donor lungs rejected for transplantation due to concern for lung injury maintain adequate epithelial barrier function. We also show for the first time that preserved AFC is associated with more favorable lung fluid balance in the perfused human lung. Finally, measurement of biological markers of alveolar epithelial injury may be useful in predicting the presence or absence of intact alveolar fluid clearance in donor lungs and perhaps in patients with acute lung injury. Therefore, functional and biochemical assessment of alveolar epithelial injury in donor lungs before lung transplantation may be useful clinically.