Role Of ACE/ACE2 In Kidney Injury Mediated By Mesenteric Lymph Following Hemorrhagic Shock

Hemorrhagic shock caused by trauma,surgical bleeding is a common clinical critical disease,acute kidney injury?AKI?following hemorrhagic shock is one of the common complications.Previous studies showed that post-hemorrhagic shock mesenteric lymph?PHSML?reflux plays an important role in the process of kidney injury and block PHSML backflow to circulation may alleviates kidney injury,its mechanism may be related to alleviation of renal oxidative stress,inflammation and so on.Renin-angiotensin system?RAS?is one of the important endocrine system in the body,it includes two key axises,one is angiotensin– converting enzyme?ACE?–angiotensin?Ang?II– Ang II type 1 receptor?AT1R?axis and another isACE2–Ang?1-7?– Mas-related G-proteincoupled receptor?MasR?axis,previous studies showed that the expression of ACE/ACE2 was unbalance after hemorrhagic shock,and applying with drainage of mesenteric lymph could improve the unbalance of ACE/ACE2,and these results indicated that the unbalance of ACE/ACE2 involved in AKI mediated by PHSML,but its mechanism is needed further study.In this experiment,we take wild and ACE2 gene knockout?ACE2-/-?C57 male as object of study,regulating the balance of ACE/ACE2 by using tool agents in two axises of RAS to investigate the role of ACE/ACE2 balance in the AKI mediated by PHSML.First,18 mice were randomly divided into three groups: sham group,shock group and shock+ligaion group.As shock group and shock+ligaion group mice,after anesthetizing we found mesenteric lymphatic duct and threaded silk without ligation.After the separation of the left femoral artery,the femoral artery was connected to biological signal acquisition system monitoring blood pressure of mice,while the right femoral artery was connected to push injection machine for bleeding and resuscitation.Thirty minutes after the operation,the mean blood pressure decreased to 40 mmHg within 10 minutes and maintained blood pressure at 40±2 mmHg by bleeding and administrating,and then resuscitated the mice with blood mixed with salinger liquid ratio 1:1.The mesenteric lymphatic duct of Shock + Ligation group mice was ligated after resuscitation,except Sham group and Shock group mice.4 hours later,we collected the kidneys and plasma for further experiment.Then,the another 18 micewere randomly divided into three groups: Shock⁺Enalapril group,Shock⁺Losartan group and Shock⁺Ang?1-7?group.We duplicated the animal model of hemorrhagic shock according to the method above in the other 18 mice,and adminstrated Enalapril?10 mg/kg?,Losartan?50 mg/kg?and Ang?1-7?(1×10^-7 mol/kg)when conducting fluid resuscitation,respectively,the mesenteric lymphatic duct was not ligated after resuscitation.Four hours later,we collected the kidneys and plasma for further experiment.Another 12 mice were randomly divided into two groups: Shock⁺Ligation+Ang II group and Shock⁺Ligation+A-779 group.We duplicated the animal model of hemorrhagic shock according to the method above,the two groups mice,respectively,adminstrated MasR inhibitor A-779?0.1 mg/kg?and Angiotensin II(1×10^-7 mol/kg)when conducting fluid resuscitation,liagted the mesenteric lymphatic duct after resuscitation.4 hours later,we collected the kidneys and plasma for further experiment.At last,6 ACE2-/-mice were conducted the same operation above.The mesenteric lymphatic duct was ligated after resuscitation.4 hours later,we collected the kidneys and plasma for further experiment.Then we observed kidney tissue pathological change by using hematoxylin–eosin staining.Plasma Ang II and Ang?1-7?and kidney tissue Ang II and Ang?1-7?levels were measured by ELISA method according to the manufacturer's protocol.The expressions of ACE,ACE2,AT1 R and MasR were measured by the immunohistochemistry method.The results showed that the organizational structure of sham group was basic normal,Shock group kidney pathological changes were more dramatically worse than Sham group,mesenteric lymphatic ligation,Enalapril,Losartan,Ang?1-7?treatment alleviated the renal tissue injury,ACE2-/-,Ang II,A-779 aggravated the injury degree of organizational structure in the Shock⁺ligation group.The expression of ACE,AT1 R,Ang II in kidney tissue in the shock group were upregulated compared to Sham group,while the expression of ACE,AT1 R,Ang II in the Shock⁺Enalapril?Shock⁺Ang?1-7??Shock⁺Losartan ?Shock⁺Ligation group were dramatically downrugulated compared to Shock group.The Ang II content in renal tissue in the Shock⁺Ligation+Ang II group increased compared to Shock⁺Ligation group,while there is no significantly statistical difference between Shock⁺Ligation+ACE2-/-,Shock⁺Ligation+A-779 and Shock⁺Ligation group.The plasma Ang II in Shock group elevated dramatically compared to Sham group,the plasma Ang II in the Shock + Enalapril group and Shock⁺Ang?1-7?group decreased significantly compared to Shock group,while the plasma Ang II in the Shock⁺Ligation+Ang II group increased compared to Shock⁺Ligation group.The expression of renal ACE2,MasR and renal Ang?1-7?content in the Shock group decreased dramatically compared to Sham group,while the renal ACE2,MasR expression and renal Ang?1-7?content in the Shock⁺Ang?1-7?and Shock⁺Ligation group significantly elevated compared to Shock group and there is no statistical difference between Shock⁺Enalapril?Shock⁺Losartan group and Shock group.The renal ACE2 expression and renal Ang?1-7?content in the Shock + Ligation + ACE2-/-group were significantly decreased compared to Shock + Ligation group.The renal MasR expression in the Shock + Ligation + Ang II group significantly decreased compared to Shock + Ligation group.The renal ACE2,MasR expressions and renal Ang?1-7?content in the Shock + Ligation + A-779 group significantly decreased compared to Shock + Ligation group.The plasma Ang?1-7?levels in the Shock group decreased significantly compared to Sham group,while plasma Ang?1-7?increased dramatically in the Shock⁺Enalapri,Shock⁺Ang?1-7?,Shock⁺Losartan and Shock⁺Ligation compared to Shock group.The plasma Ang?1-7?level in the Shock⁺Ligation+ACE2-/-group,Shock + Ligation + Ang II group and Shock + Ligation + A-779 group decreased dramatically compared to Shock + Ligation group.These results showed that PHSML-mediated AKI is related to the hyper-expression of ACE-Ang II-AT1 R and hypo-expression of ACE2-Ang?1-7?-Mas1 R,and the unbalance of ACE/ACE2 involved in AKI mediated by PHSML,take RAS as a target point can contribute to improve the AKI following hemorrhagic shock.