The Effects of Aquaporin-1 in Pulmonary Edema Induced by Fat Embolism Syndrome

Abstract

This study was designed to investigate the role of aquaporin1 (AQP1) in the pathologic process of pulmonary edema induced by fat embolism syndrome (FES) and the effects of a free fatty acid (FFA) mixture on AQP1 expression in pulmonary microvascular endothelial cells (PMVECs). In vivo, edema was more serious in FES mice compared with the control group. The expression of AQP1 and the wet-to-dry lung weight ratio (W/D) in the FES group were significantly increased compared with the control group. At the same time, inhibition of AQP1 decreased the pathological damage resulting from pulmonary edema. Then we performed a study in vitro to investigate whether AQP1 was induced by FFA release in FES. The mRNA and protein level of AQP1 were increased by FFAs in a dose- and time-dependent manner in PMVECs. In addition, the up-regulation of AQP1 was blocked by the inhibitor of p38 kinase, implicating the p38 MAPK pathway as involved in the FFA-induced AQP1 up-regulation in PMVECs. Our results demonstrate that AQP1 may play important roles in pulmonary edema induced by FES and can be regarded as a new therapy target for treatment of pulmonary edema induced by FES.

Keywords: AQP1; fat embolism syndrome; free fatty acid; p38 MAPK signaling pathway; pulmonary edema.

Figure 1

Pulmonary edema occurs in FES mice. (A) Gross morphology of the lungs in the control and FES (fat embolism syndrome) groups. Blue arrow, infarction tissues; (B) Lung sections from the control and FES groups were stained with Oil Red O; (C) Lung sections from the FES group at different time points after fat injection were stained with H & E. Blue arrow, ruptured alveolar wall; (D) W/D ratio in the FES group at different time points after fat injection. * p < 0.05, the statistics were made by comparing with Ctrl group, respectively.

Figure 2

AQP1 is increased in lung of FES mice and inhibition of AQP1 reverses pulmonary edema in FES mice. (A) Western blot and (B) immunohistochemical analyses of AQP1 expression in the FES group at different time points after fat injection. Staining score was shown on the right; (C) Lung sections from the control, FES and FES + AQP1 inhibitors (bumetanideand acetazolamide, respectively) groups were stained with H&E. Blue arrow, ruptured alveolar wall, infiltration of red blood cells, and widened alveolar septa; (D) W/D ratio of the control, FES, FES + bumetanide and FES + acetazolamide groups. * p < 0.05; ** p < 0.001, the statistics were made by comparing with Ctrl group, respectively. ^# p < 0.05, the statistic was made by comparing with FES group.

Figure 3

FFA induced up-regulation of AQP1 expression in PMVECs. (A) Primary cultured PMVECs obtained from normal rats. The PMVECs were polygonal or fusiform with a uniform size and displayed a similar and typical cobblestone-like morphology. Magnification 200×; (B) Fluorescence microscopy showed that the PMVECs exhibited green fluorescence after staining with FITC-BSI. The nuclei were stained blue by DAPI. Magnification 200×; (C,D) The protein and mRNA levels of AQP1 in PMVECs stimulated by 500 μM FFAs for different times; (E,F) The protein and mRNA levels of AQP1 in PMVECs stimulated by different concentrations of FFAs for 6 h. ** p < 0.01, the statistics were made by comparing with Ctrl group, respectively.

Figure 4

Activation of phosphorylation of MAPK by FFAs and effects of p38 inhibition on FFA-induced up-regulation of AQP1 in PMVECs. (A) Western blot analysis of p-ERK, p-p38 kinase, p-JNK expression in PMVECs stimulated by FFAs. P-p38 kinase was significantly activated; (B) SB203580 was confirmed to inhibit the expression of p-p38 kinase in PMVECs stimulated by FFAs; (C) Expression of AQP1 was reduced by SB203080 in PMVECs stimulated by FFAs. * p < 0.05, the statistics were made by comparing with Ctrl group, respectively. ^# p < 0.05, the statistics were made by comparing with FES group.