The Role Of HMGB1/RAGE In Acute Lung Injury Induced By Renal Ischemia-reperfusion In Neonatal Rats

Background and objective:Acute kidney injury(AKI)is a common clinical syndrome in critically ill patients,which is closely related to the death and prognosis of critically ill patients.Acute kidney injury combined with other organ dysfunction is an important cause of increased mortality risk.Because of the abundant pulmonary capillaries,cytokines and inflammatory molecules are easy to accumulate in the lungs.Acute lung injury induced by acute kidney injury is common in clinic,and the mortality rate is sharply increased.As a key pro-inflammatory factor,high mobility group protein B1(HMGB1)can be released by necrotic cells to the outside of cells at the earliest stage after injury,triggering systemic inflammatory response and mediating inflammation damage in many organs.HMGB1 is closely related to kidney and lung injury.Antagonism of HMGB1 can reduce the degree of renal injury and mortality in rats with ischemia-reperfusion injury.Activation of HMGB1 signal also affects the development of respiratory diseases such as acute lung injury.Injection of anti-HMGB1 antibody or the gene knockout of its specific receptor late glycation end product receptor(RAGE)can alleviate lung injury in mice induced by infection of staphylococcus aureus.However,the role of HMGB1/RAGE in acute lung injury induced by renal ischemia-reperfusion in neonatal rats remains unclear.The purpose of this study was to investigate the role of HMGB1,binding to its specific receptor RAGE,in acute lung injury induced by renal ischemia-reperfusion in neonatal rats,so as to provide experimental basis and possible therapeutic targets for clinical intervention of neonatal patients with AKI and ALI.The purpose of this study was to investigate the role of HMGB1 in acute lung injury induced by renal ischemia-reperfusion in neonatal rats,and to understand the process of HMGB1 binding to its specific receptor RAGE,so as to provide experimental basis and possible therapeutic targets for clinical intervention of neonatal patients with AKI and ALI.Methods:1.Establish a neonatal rat model of renal ischemia-reperfusion:Sprague-Dawley neonatal rats aged 10 days were used as experimental subjects,and their body weight was measured and recorded.Rats were randomly divided into normal group(Control group),ischemia reperfusion group(IR group)and sham group(Sham group).The IR group rats were divided into five subgroups according to the time of reperfusion:IR-3h group,IR-6h group,IR-12h group,IR-24h group and IR-48h group.The Sham group was divided into Sham-12h group and Sham-24h group according to the time of sham operation.Neonatal rat models of ischemia-reperfusion renal injury were established by ligating bilateral renal pedicles for 45 minutes and restoring renal perfusion of newborn rats.Sham-operated group models were established by only dissociating bilateral renal pedicles of rats,but not ligating the renal pedicles of rats.2.Histopathological changes of kidneys and lungs were detected by HE staining,and tissue damage scores were scored;serum creatinine(Scr)concentration was detected by biochemical method to evaluate renal function;myeloperoxidase(MPO)concentration of kidneys and lungs was detected by ELISA to determine the degree of inflammation of tissues;alveolar lavage fluid of rats was collected,protein content of lavage fluid was detected and cell smears were made,and cells were observed under microscopy.The number of neutrophils was measured by Giemsa staining and the percentage of neutrophils was calculated to evaluate the degree of alveolar neutrophils infiltration.3.The concentration of HMGB1 in plasma,kidney and lung tissues of neonatal rats was detected by ELISA,and the differences of HMGB1 levels in kidney,lung and plasma respectively,were compared among groups.Cytoplasmic and nuclear proteins were isolated from kidney tissues.The protein expression of HMGB1 andtissue inhibitor of Metalloproteinase-2(TIMP-2)in kidney and lung tissues were detected by Western blot,and the p expression of HMGB1,RAGE,Nuclear factor kappa B(NF-kappa B,P65),Phosphorylated NF-kappa B,(P-NF-kappa B,P-P65)and TIMP-2 in lung tissues were detected.Result:1.Successful establishment of rat model of AKIThe Scr level increased significantly at 12 h after reperfusion in IR group,reaching its peak at 24 h after reperfusion.HE staining showed that there were obvious pathological injuries in the kidney and lung tissues of IR-24h group,and the pathological scores of the kidney and lung injuries of rats were significantly higher than those of control group.2.Comparison of MPO concentrations in kidney and lung tissuesThe concentration of MPO in kidney and lung tissue of rats in IR-3h group was significantly higher than that in control group(P<0.05).The concentration of MPO reached its maximum at 24 hours of ischemia-reperfusion in rats.3.Comparison of components of rat alveolar lavage fluidThe alveolar lavage fluid protein concentration and lung permeability index in IR-24h group were significantly higher than those in control group.Compared with the control group,the number of the cells and the percentage of neutrophils in the alveolar lavage fluid staining with Giemsa in IR-24h group rats increased significantly(P<0.05).4.The results of elisaThe concentration of HMGB1 in kidney and plasma of IR rats increased significantly from 3 hours after ischemia-reperfusion(P<0.05).The concentration of HMGB1 in lung began to increase from 3 hours after ischemia-reperfusion;however did not reach significance.There was a significant difference between IR and Control rats at 6 hours after reperfusion(P<0.05).5.The results of western blotThe prtein expression of HMGB1 in renal cytoplasm in IR-24h group rats was significantly higher than that in control group(P<0.001),and The ratio of cytoplasmic to nuclear HMGB1 in kidney in IR-24h group was significantly higher than that in control group(P<0.001);the expression of HMGB1,RAGE,NF-κB,P-NF-κB and TIMP-2 in lung of rats in1 IR group was significantly higher than that in normal group(P<0.05).Conclusion1.Renal ischemia-reperfusion can induce acute injury of distal lung tissue in neonatal rats.2.HMGB1 may play an important role in acute lung injury induced by renal ischemia-reperfusion in neonatal rats.3.HMGB1 may regulate acute lung injury in neonatal rats with renal ischemia-reperfusion by binding to RAGE,a specific receptor highly expressed in lung tissue.