Angiotensin-Converting Enzyme (ACE) And ACE2 In The Lungs And Kidneys Of Adult Rats After Smoke Inhalation Injury

Objective:To establish an animal model with stability and repeatability used to investigate smoke inhalation lung injury. To observe the changes of pathomorphism and water contents in the rats'lungs, then to assess the reliability and degree of lung injury of the smoke inhalation models. To observe the changes of ACE and ACE2 with injury time course in the lungs and kidneys of smoke inhalation rats, so to analysis the participation mechanism of ACE and ACE2 in lungs and kidneys of smoke inhalation injury rats.Methods:1. Establish the model of smoke inhalation lung injury in rats Thirty male Sprague-Dawley rats of clean grade were used to experiment after one week of adaptive feeding in our laboratory, weighing 220±20 g. According to the serial number, we used the completely randomized group division naming that normal control group (N), 1 h after injury (A), 4 h after injury (B), 10 h after injury (C), and 24 h after injury (D). Each group had 6 rats. The models of smoke inhalation lung injury were established according by the methods reported by Erfan Xie et al. in Burn Institute of the Third Military Medical University. The injury groups (A, B, C, and D) were exposed to dense smoke for 10 minutes and then removed from the holder for 5 minutes. This cycle was repeated three times, and the lungs and kidneys were removed at the indicated times. We measured the rats'breathing rates immediately after finishing the models. And then the rats whose breathing rates were greater than 30/min had been taken to the following experiment.2. Assess the rats'lung injury after smoke inhalationThe rats in injury groups and normal group were killed by decapitation at the indicated time points (1 hour, 4 hours, 10 hours and 24 hour after smoke inhalation). The rats'entire lungs were taken and marked, then frozen in the refrigerator at -80℃. Take the left top part and left lower part of rats'lung tissue in different groups to measure wet-to-dry weight ratio and to observe lung histopathologic changes by hematoxylin and eosin staining (HE staining). Then assess the degree of rats'lung injury after smoke inhalation.3. The expression of ACE and ACE2 in rats'lungs and kidneysThe rats in injury groups and normal group were killed by decapitation at the indicated time points (1 hour, 4 hours, 10 hours and 24 hour after smoke inhalation). Once taken their lungs, we revealed their abdominal cavity and took their entire kidney tissues. Then these tissues were marked and taken into the refrigerator at -80℃. We took the rest lungs and kidneys divided to two parts to semi-determinate the expression of ACE and ACE2 in rats'lungs and kidneys of every groups by mean of Immunohistochemical staining and western-blot analyses.Result:1. The rats'general state of healthThe injury rats'breathing rates were all quicker than 30/min, and they all occurred that light and fast breathing, oral respiration, wheezing rale, accompany by depression and sluggish action. All rats were involved in the continued experiments.2. Assessment of the smoke inhalation rat modelsThe rats'lung tissue wet-to-dry weight ratio in injury groups all higher than that in normal group significantly (P<0.05, or P<0.01). As seen through a light microscope, the normal lung tissue displayed a distinct framework, complete alveolar walls, and an interstitium without exfiltration. The lungs from the smoke inhalation injury groups had some common characteristics: small vessels in the lung and the micrangium in the interalveolar septum were stretched and hyperemic, the alveolar walls were fractured, the alveolar space was filled with erythrocytes, the alveoli were collapsed, and the lungs exhibited interstitial edema. The number of monocytes and macrophages surroundings the small vessels, in the interalveolar septum, and in the alveolar space was increased. Neutrophils infiltrate in the alveoli and the mesenchyme. With the time course the lung injury became worse. Lungs from rats in the 10 h and 24 h groups were in worse condition than the lungs from rats in the 1 h and 4 h groups in terms of tissue hyperemia, neutrophil infiltration, and interstitial edema.3. The change of ACE and ACE2 in rats'lungs The Immunohistochemical staining assay showed that ACE and ACE2 were expressed on the cellular membranes of normal lung cells. The major sites of ACE and ACE2 protein accumulation were in the alveolar type I epithelium, the alveolar type II epithelium, the bronchiolar epithelium, the endothelium, and the smooth muscle cells of the pulmonary vascular structure. The ACE2 intensity in the bronchiolar epithelium was similar to the intensity in the alveolar epithelium and in the endothelium of the vascular structure.The ACE and ACE2 content in the lungs was significantly different among the different experimental groups, The ACE content was dramatically increased in rat lungs immediately after smoke inhalation injury as compared to normal lungs (P <0.05; N: 1.00, A: 1.37, B: 1.51, C: 1.73, D: 2.04 ). The distinction of ACE2 expression between normal lungs and lungs from rats sacrificed 1 h after smoke inhalation has no statistical significant (A: 1.10, N: 1.00, P>0.05). But the ACE2 content was dramatically distinct between the normal lungs and the lungs of rats sacrificed 4 h to 24 h after injury N: 1.00, A: 1.10, B: 1.38, C: 1.59, D: 1.85, P<0.05). So the ACE2 contents in rats'lungs became obviously increased during 1 h to 4 h after smoke inhalation. The change in ACE and ACE2 protein content was also roughly shown by the immunohistochemical analysis.4. The change of ACE and ACE2 in rats'kidneysIn the rats'kidneys, ACE and ACE2 were localized to the tubules, strongly in the apical brush border, and both were also present in the cytoplasm in all subjects. Both ACE and ACE2 proteins were also present in the glomeruli, albeit at a lesser amount than in the tubules.The ACE expression in rats'kidney was not significantly different between all the five groups (N: 9%, A: 10%, B: 12%,C: 15%, D: 16%, P>0.05). And what's the same, the ACE2 expression in rats'kidney was not significantly different between all the five groups either (N: 14%, A: 13%, B: 15%, C: 17%, D: 20%, P>0.05). So there were no distinct differences in kidney ACE and ACE2 expression among the five groups. The change in ACE and ACE2 protein content was same to the results above, but the data was not shown.Conclusion:Smoke inhalation repeatedly can induce rats to emerge typical symptoms of lung injury, such as light and fast breathing, oral respiration, wheezing rale, depression, sluggish action and so on, and which can display lung injury pathology, such as visible lung edema. Therefore this method can finish stable and reliable smoke inhalation animal models.In 24 hours after smoke inhalation lung injury, lung ACE expression started to increase at once after injury, but lung ACE2 expression started between 1 h and 4 h after injury. The changes of ACE and ACE2 expression and protein content were no significant in the rats'injury kidneys compared with normal ones. These results suggest ACE and ACE2 have tissue specificity, which effects in tissues are not same as in circulation systems. Lung ACE activity may be taken as the signal which can reflex endothelium damage degree of lung tissues. The protect mechanism of lung ACE2 activity started slightly later than the ACE activity increasing, and the imbalance between ACE and ACE2 was involved in the pathogenesis of smoke inhalation. To decrease the level of ACE or elevate the level of ACE2 in lungs may be good for the process of smoke inhalation lung injury.