Effect Of Inhaled Nitric Oxide Or Ethyl Nitrite On Aquaporin (AQP) 1 And AQP5 In Neonatal Rats With Acute Hyperoxic Lung Injury

BackgroundHyperoxic lung injury often occurs in the newborn with inspiration of high O2 concentrations. The pathologic changes after hyperoxic exposure occur in two sequential phases: an early acute-exudative to form pulmonary edema and a late proliferative-reparation leading to fibrosis. Reabsorption of alveolar edema fluid is essential for the resolution of pulmonary edema and may have significant impact on the prognosis of patients with pulmonary edema. The aquaporins (AQPs) are a family of water-selective channels that function to increase membrane water permeability and provide a route for rapid fluid movement under many pathologic conditions. .The specific localization of AQP1 (in alveolar endothelial cells) and AQP5 (at the apical membrane of type I epithelial cells) support a role for AQPs in this process. Fluid movement through AQPs is governed by an osmotic gradient that is generated by transcellular ion movement through epithelial sodium channel (ENaC) and Na, K-adenosine triphosphatase (NKA).Inhaled NO (iNO), as a selective pulmonary vasodilator, has been successfullyused in a wide variety of clinical situations especially in the management of persistent pulmonary hypertension in the newborn. NO can briefly reverses pulmonary vasoconstriction and improve arterial oxygenation. However, when delivered simultaneously with a high inspired concentration of oxygen, iNO has been shown to either aggravate or protect against oxidant stress depending on experimental context. Little is know about its effect on lung fluid clearance.Unlike inhaled nitric oxide, endogenous nitric oxide synthase is resistant to potentially toxic reaction with O2/O2" and its endogenous NO is reacted with thiols to form S-nitrosothiols (SNOs). Realizing the difference between inhaled nitric oxide and endogenous NO and the advantages of SNOs, researchers have been exploring to find an ideal drug which would closely simulate endogenous NO by transforming NO into SNOs. Among them, Stamler research group selected ethyl nitrite (ENO) as a candidate gas. ENO fulfils following criteria: (1) resistance to decomposition in the presence of O2;(2) reaction with glutathione to form GSNO;(3) limited ability to oxidize hemoglobin and (4) biocompatibility.Up to present, there are a few medical reports about ENO. These reports indicated that ENO can effectively attenuate pneumoperitoneum-induced blood flow decrease within the peritoneal cavity during laparoscopic procedures, they also indicate that inhalation of ENO can produce sustained improvements in arterial oxygenation in PPHN, and in a porcine model of lung injury, ENO inhalation provided immediate relief for hypoxic pulmonary vasoconstriction without affecting systemic hemodynamics. Furthermore, in these studies, there was no rebound in cardiopulmonary hemodynamics or fall in oxygenation on withdrawing the drug, and there was no NOx detectable. All of these studies suggest ENO a promising drug in medicine.In present study, we explored the mRNA expression of AQP1, AQP5, a-ENaC and oti-NKA in the context of pulmonary edema resulting from acute hyperxic lung injury and the combined exposure of nitric oxide and hyperoxia or ethyl nitrite and hyperoxia in newborn rats. Immunohistochemistry study for AQP1 and AQP5 protein expression in lungs were also done in all groups. By this study, we aimed to providesome clues to further investigation on ENO and AQPs in acute lung injury.Part 1 Effect of inhaled nitric oxide on aquaporins (AQPs) in neonatal rats with acute hyperoxic lung injuryObjectives:To investigate the impact of hyperoxia on AQP1 and AQP5 in newborn rats with acute hyperoxic lung injury and explore the effect of inhaled nitric oxide on lung edema, AQP1 and 5 expressions and its possible mechanism in this animal model. Methods:1. Animal Treatment and Group32 newborn SD rats were randomly divided into 4 groups (n=8).(1) Control group (C group): room air for 48h(2) Hyperoxic group (O group): >90%O2 for 48h(3) Hyperoxia+NO group (NO group) : >90%O2 for 48h + lOppm NO (NO only in the first 12 h)(4)NO group (CN group): room air for 48h + lOppm NO (NO only in the first 12h)2. Laboratory studiesAfter the rats were sacrificed, we measured the lung wet-to-dry weight ratio (Qw/Qd) and AQP1, AQP5, cn-NKA, a-ENaC mRNA expressions. The lung pathology was oberserved and immunohistochemistry was used for the localization of expressions of AQP1 and AQP5. Results: 1. The Qw/Qd RatioThe Qw/Qd ratio in O group were significantly increased than in Control group (P<0.01). Compared with O group, the Qw/Qd of NO group were significantly decreased (PO.05). But no significant difference was found between the CN group and C group (P>0.05).2. The Lung PathologyThere were no histopathological changes in C group. Alveolar septal thickening, edema, and neutrophil infiltration were found in O group, which were not obvious in NO or CN group.3. The mRNA Expressions of AQP1, AQP5, arNKA and a-ENaCCompared with Control group, the expression of AQP1 mRNA was reduced significantly (P<0.01) and the expression of AQP5 mRNA was also decreased (P<0.05) while the mRNA expressions of ai-NKA, a-ENaC were significantly increased (P<0.05, respectively). Compared with O group, the mRNA expressions of AQP1 or AQP5 in NO group were significantly increased (jP<0.05, respectively). No significant differences were found between CN and C group (P>0.05, respectively).4. The AQP1 and AQP5 Expression in Immunohistochemistry studyIn the immunohistochemistry study, AQP1 was expressed in capillary endothelium and AQP5 at the apical membrane of type I epithelial cells in the lungs. There were less AQP1 positive endothelial cells and less AQP5 positive epithelial cells in O group. More AQP1 or AQP5 positive cells were found in NO group than in O group.The numbers of AQP1 or AQP5 positive cells between CN and C group had no significant difference. Conclusions:1. Newborn rats with acute hyperoxic lung injury demonstrated increased permeability, decreased expression of AQP1 and AQP5 and the formation of lung edema.2. Inhaled lOppm nitric oxide for 12h may attenuate lung edema, increase AQP1 and AQP5 expressions, suggesting that inhaled lOppm nitric oxide for 12h may protect the AQP1 and AQP5 in lung in this model of acute lung injury.Part 2 Effect of inhaled ethyl nitrite on aquaporins (AQPs) in neonatal rats with acute hyperoxic lung injuryObjectives:To investigate the effect of inhaled ethyl nitrite on alveolar epithelial fluid transport and AQPl and AQP5 expressions in newborn rats with acute hyperoxic lung injury. Methods:1. ENO SynthesisENO was synthesized by dissolving of sodium nitrite and ethanol in milli-Q water and carefully titrating 6M sulphuric acid into the mixture. When the ENO gas was produced, it was cooled into liquid phase and collected into flask. ENO was purified by gasifing and cooling repeatedly, and analyzed by mass spectrum and gas chromatography system.Nitrogen gas (1 liter/min) was passed through a gas washer containing 0.125%ENO (dissolved in ethanol) and then blended with a room air gas flow (10 liters/min).2. Animal Treatment and Group32 newborn SD rats were randomly divided into 4 groups (n=8).(1) Control group (C group): Same as Part one.(2) Hyperoxic group (O group): Same as Part one.(3) Hyperoxia+ENO group (ENO group) : >90%O2 for 48h + 0.125%ENO (2ppin) (ENO only in the first 12 h)(4)ENO group (EN group): room air for 48h + 0.125%ENO (2ppm) (NO only in the first 12 h)3. Laboratory studies The same as part one.Results:1. The Qw/Qd RatioCompared with O group, the Qw/Qd ratio of ENO group were significantly decreased (P<0.05). No significant differences were found between the EN group and C group, ENO group and EN group (PX3.O5, respectively).2. The Lung PathologyThere were mild alveolar septal thickening and no obvious edema in ENO group. No obvious pathological changes were found in EN group.3. The mRNA Expressions of AQP1, AQP5, arNKA and a-ENaCCompared with O group, the mRNA expressions of AQP1 or AQP5 in NO group were significantly increased (P<0.05, respectively) while the mRNA expressions of ai-NKA, a-ENaC did not change significantly (P>0.05, respectively). No significant differences were found between EN and C group, ENO and EN group (P>0.05, respectively).4. The AQP1 and AQP5 Expressions in Immunohistochemistry study Increased AQP1 or AQP5 positive cells were found in ENO group than in Ogroup. AQP1 or AQP5 positive cells count between EN and C group, EN and ENO group had no significant differences.5. Comparsion of the effect of inhaled NO and ENOCompared with NO group, ENO^group did not show significant differences in terms of lung pathology, histochemistry and AQP1/AQP5 expressions (P>0.05, respectively). There were also no significant differences between CN and EN group in laboratory studies (P>0.05, respectively). Conclusions:1. Inhaled 2ppm ENO for 12h may attenuate lung edema, increase AQP1 and AQP5 mRNA expression, suggesting that inhaled 2ppm ENO for 12h may protect the AQP1 and AQP5 in lung in this model of acute lung injury.2. Inhaled 2ppm ENO for 12h in normal newborn rats had no significant impact on AQP1 and AQP5 in lungs.3. Inhaled lOppram NO or 2ppm ENO had similar effect on AQP1 and AQP5 in lungs in the rat model of acute lung injury.