Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice

Abstract

The mammalian lung expresses water channel aquaporin-1 (AQP1) in microvascular endothelia and aquaporin-4 (AQP4) in airway epithelia. To test whether these water channels facilitate fluid movement between airspace, interstitial, and capillary compartments, we measured passive and active fluid transport in AQP1 and AQP4 knockout mice. Airspace-capillary osmotic water permeability (Pf) was measured in isolated perfused lungs by a pleural surface fluorescence method. Pf was remarkably reduced in AQP1 (-/-) mice (measured in cm/s x 0.001, SE, n = 5-10: 17 +/- 2 [+/+]; 6.6 +/- 0.6 AQP1 [+/-]; 1.7 +/- 0.3 AQP1 [-/-]; 12 +/- 1 AQP4 [-/-]). Microvascular endothelial water permeability, measured by a related pleural surface fluorescence method in which the airspace was filled with inert perfluorocarbon, was reduced more than 10-fold in AQP1 (-/-) vs. (+/+) mice. Hydrostatically induced lung interstitial and alveolar edema was measured by a gravimetric method and by direct measurement of extravascular lung water. Both approaches indicated a more than twofold reduction in lung water accumulation in AQP1 (-/-) vs. (+/+) mice in response to a 5- to 10-cm H2O increase in pulmonary artery pressure for five minutes. Active, near-isosmolar alveolar fluid absorption (Jv) was measured in in situ perfused lungs using 125I-albumin as an airspace fluid volume marker. Jv (measured in percent fluid uptake at 30 min, n = 5) in (+/+) mice was 6.0 +/- 0.6 (37 degrees C), increased to 16 +/- 1 by beta-agonists, and inhibited to less than 2.0 by amiloride, ouabain, or cooling to 23 degrees C. Jv (with isoproterenol) was not affected by aquaporin deletion (18.9 +/- 2.2 [+/+]; 16.4 +/- 1.5 AQP1 [-/-]; 16.3 +/- 1.7 AQP4 [-/-]). These results indicate that osmotically driven water transport across microvessels in adult lung occurs by a transcellular route through AQP1 water channels and that the microvascular endothelium is a significant barrier for airspace-capillary osmotic water transport. AQP1 facilitates hydrostatically driven lung edema but is not required for active near-isosmolar absorption of alveolar fluid.

Figure 1

Airspace–capillary water permeability measured by a pleural surface fluorescence method. Isolated lungs were perfused continuously, and the airspace was filled with an isosmolar solution containing FITC-dextran (see Methods). (a) Representative time course data shown for lungs of mice of indicated genotypes. (b) Individual and averaged (mean ± SE) reciprocal half-time (1/t_1/2) for the fluorescence signal change with corresponding airspace–capillary osmotic water permeability coefficients (P_f). The investigator was blinded to genotype for all transport measurements. Each point is the averaged data for two to six sets of measurements on an individual mouse. *P < 0.005 vs. (+/+); **P < 0.0001 vs. (+/+).

Figure 2

Microvascular water permeability in (+/+) and AQP1 (–/–) mice. The airspace of a perfused lung was filled with an inert perfluorocarbon, and the pulmonary artery was perfused with solutions of indicated osmolalities containing FITC-dextran (see Methods). Representative time course data for lungs of (+/+) mice (left) and AQP1 (–/–) mice (right). Relative fluorescence changes (ΔF/F), which are related to microvascular osmotic water permeability, are summarized in the text.

Figure 3

Gravimetric measurement of hydrostatically driven lung edema. (a) Lung weight was monitored continuously in isolated lungs perfused with an isosmolar solution at hydrostatic pressures of 8 or 18 cm H₂O as set by adjusting reservoir height (see Methods). (b) Original records of the time course of lung weight increase in response to change in pulmonary artery pressure from 8 to 18 cm H₂O. Weight was calibrated in every experiment by briefly suspending a 100-mg weight standard from the lung.

Figure 4

Effect of AQP1 deletion on hydrostatically driven lung edema by measurement of extravascular lung water. Lungs were perfused for 10 min at 5 cm H₂O and then for 5 min at 5, 9, or 15 cm H₂O as described in Methods. Individual and averaged (mean ± SE) values of extravascular lung water (expressed as corrected wet/dry ratios) are shown for indicated genotypes.*P < 0.001 vs. (+/+).

Figure 5

Effect of aquaporin deletion on active isosmolar fluid transport. Measurements were done at 37°C (unless otherwise indicated) using an in situ perfused lung preparation in which the airspace was instilled with an isosmolar solution containing ¹²⁵I-albumin as a volume marker (see Methods). Percentage fluid absorption at 30 min was computed from the ratio of ¹²⁵I radioactivity in fluid samples obtained at 2 and 30 min after instillation. (Left) Percentage absorption rates shown for individual mice along with averaged values (mean ± SE). Where indicated, the instilled and perfused solutions contained isoproterenol (0.1 mM), terbutaline (10 mM), amiloride (1 mM), or ouabain (0.1 mM), or the measurement was done at 23°C instead of 37°C. (Right) Fluid absorption was measured at 37°C in the presence of 0.1 mM isoproterenol.*P < 0.01 compared with control; **P < 0.01 compared with isoproterenol condition.