The Role Of Complement In Acute Renal Injury Induced By Rhabdomyolysis And The Protective Effect Of Complement Inhibitor

Background High-intensity military training and earthquakes,landslides and other natural disasters can cause rhabdomyolysis,acute kidney injury was the main complication.Due to rhabdomyolysis caused by lack of full understanding of the pathogenesis of acute kidney injury,clinical without specific treatment,mortality is high.So,exploring the pathogenesis of striated muscle dissolution induced acute kidney injury,seeking effective preventive measures,have important military and social significance.Our results show that in the past,rhabdomyolysis induced renal oxidative stress,inflammatory response,and ultimately to apoptosis or necrosis,resulting in acute kidney injury.The complement system is an important immune disease prevention system,to maintaining the internal environment homeostasis plays an important role in whether it was involved in Striated Muscle dissolution induced acute kidney injury,and mechanisms,both at home and abroad are not reported.The complement system is activated by oxidative stress,plays an important role in the inflammatory response,but also regulatory role in apoptosis,is likely to participate in the injury process,this research and identify potential therapeutic targets,is of great significance.Objective The purpose of this study was to establish the animal model of rhabdomyolysis induced acute renal injury,rats in advance using complement depletion agent(CVF)on renal injury by depletion of complement,the observation and complement proteins,cytokines,apoptosis detection,judgment of complement is involved in rhabdomyolysis induced acute renal injury of the process.The activation of the pathway and the possible mechanism;at the same time to study the complement inhibitor on rat acute kidney injury induced by rhabdomyolysis may be the protective effect and mechanism.This study is divided into four parts:1.Striated Muscle dissolution induced acute renal injury in rats activation of complement;2.Striated Muscle dissolution induced acute renal injury in rats of complement,the role of inflammation and oxidative stress,3.Striated Muscle dissolution induced acute renal injury in rats the complement and cell apoptosis;4.Complement antagonist C1INH protective effects on Striated Muscle dissolution induced acute kidney injury research.Methods1.The activation of rhabdomyolysis induced acute renal injury in rats of complement.1)Male clean SD rats 72,weight 200?220 g,using random number tables are divided into 3 groups(n=24):the control group(GroupC),bilateral hindlimb muscle injection of saline 10 ml/kg;model group(AKI group),bilateral intramuscular injection of 50%glycerol saline solution 10 ml/kg;CVF+ AKI group,glycerol injection in 12 hours before the intravenous injection of CVF,50 ug/kg.And according to the sampling time of 2h,6h,24 h and 72 hours for 4 the sub group at each time point of venous blood and renal tissue samples were collected.2)The observation index:serum urea nitrogen(BUN),creatinine(CR),c-reactive protein(CRP)and complement reaction the total level of CH50;the kidney pathological changes;using immunofluorescence technique,RT-PCR technique,Western-blot detection of the expression of complement C3,C1q,B factor,MBL-A,DAF in kidney tissue.2.Rhabdomyolysis induced acute renal injury in rats complement and oxidative stress,inflammatory reaction1)The experimental group is the same as the first part.2)Observation index:the activity of serum MDA,SOD;the activity of MPO in renal tissue;expression of complement protein C5a,C5b-9,CD59 and TNF-? + IL-6 in renal tissue.3.Effect of complement and apoptosis in rats with acute renal injury induced by rhabdomyolysis.1)The experimental group is the same as the first part.2)Observation index:Tunel method was used to detect the apoptosis of renal cells in rats;Expression of apoptosis related protein bax,Bcl-2,Fas and caspase-12 in renal tissue4.Complement antagonist CIINH on injury induced by rhabdomyolysis protective effect on acute kidney.1)Male clean SD rats 54,weight 200?220 g,using random number tables are divided into 3 groups(n=24):the control group(Group c),double lateral hindlimb muscle injection of saline solution 10 ml/kg;model group(AKI),double lateral hindlimb muscle injection 50%10 ml/kg glycerol saline solution;C1INH+AKI Group 2 hours before glycerol injection intravenous CVF,50U/kg.And according to the sampling time of 6h,24 h and 72 h was divided into 3 subgroups,at each point in time taking kidney in venous blood and collect specimens.2)Observation index:Serum urea nitrogen,creatinine,CH50;activity of MPO in renal tissue;Detection of renal cell apoptosis in rats by Tunel method;expression of complement protein C5a,C5b-9,CD59 and TNF-a in renal tissue.Results1 The activation of rhabdomyolysis induced acute renal injury in rats of complement1)AKI group renal BUN,Cr and CRP were significantly higher than that of control group(P<0.01),the level of CH50 was significantly decreased(P<0.01);CVF+ AKI group renal function BUN,Cr and CRP were significantly lower than that in AKI group(P<0.01),CH50 remains at a very low level.2)Pathological observation of AKI group of renal injury was aggravated injury located in corticomedullary junction of renal tubules,24 hours of renal tubular epithelial cell shedding,necrosis,lumen broken cell and tube type,renal tubular injury score was significantly higher than the control group(P<0.01);CVF+AKI group injury was lighter than AKI group.Renal tubular injury score was significantly lower than AKI group(P<0.01).3)In group AKI,the expression of complement C3,C1q,Factor B,MBL-A,and DAF in renal tissue was stronger than that in control group at each time point,and was expressed in renal tubules.Among them,C3,MBL-A protein or mRNA expression level in 6 hours were the strongest;factor B,DAF expression enhanced;the expression of C1q was significantly increased at 2 hours,then decreased,but increased again at 72 hours;the expression of CVF+AKI group was lower than that of AKI group at each time point.2.Rhabdomyolysis induced acute renal injury in rats complement and oxidative stress,inflammatory reaction.1)AKI group of serum MDA activity at each time point were significantly higher than the control group(P<0.01),reached the peak in 24 hours,the activity of SOD decreased significantly compared with the control group(P<0.01),in 24 hours minimum;MDA activity in CVF+AKI group was significantly lower than that in AKI group(P<0.01),the activity of SOD obviously increased(P<0.01).2)the activity of MPO in renal tissue was significantly higher in AKI group than in control group(P<0.01).The MPO activity was significantly lower in CVF+ AKI group than in AKI group(P<0.01)3)the expression of C5a,C5b-9,CD59 and TNF-?,IL-6 in renal tissue in second group were higher than the control group at different time points,including C5a,C5b-9,IL-6 was the strongest in 24 hours;CD59,TNF-a were enhanced;while the expression in CVF +AKI groups were significantly decreased compared with AKI group.3.Effect of complement and apoptosis in rats with acute renal injury induced by rhabdomyolysis.1)The second group apoptosis rate was higher than that of the first group(P<0.01)at each time point,and reached the peak at 24 hours.The apoptosis rate of CVF+AKI group was significantly lower than that of AKI group(P<0.01).2)The expression of Bax,Fas and caspase-12 in renal cells of AKI group was significantly higher than that of control group,and reached the peak at 24 hours;The expression of them in CVF+ AKI group was significantly lower than that of AKI group;the expression of Bcl-2 in AKI group was decreased,and the expression of CVF+AKI group was stronger than that of AKI group.4..Complement antagonist C1INH on protective effect on acute kidney injury induced by rhabdomyolysis.1)The renal function of C1INH+AKI group was significantly improved(P<0.01)compared with AKI group(P<0.01),and the decrease of CH50 was lower than that of group AKI(P<0.01).2)The activity of MPO in renal tissue of C1INH +AKI group was significantly lower than that of AKI group(P<0.01)3)The apoptosis rate of renal tissue of C1INH group AKI was significantly lower than that of AKI group(P<0.01)4)The expression of C5a,C5b-9,CD59 and TNF-a in renal tissue of C1INH AKI group was significantly lower than that of AKI group.Conclusion1.The activation of rhabdomyolysis induced acute renal injury in rats of complement.1)Rhabdomyolysis can cause acute kidney injury,including morphology and function.2)Complement to play an important role in acute kidney injury in rhabdomyolysis,complement depletion cause renal injury significantly reduced;3)Striated Muscle dissolution induced acute kidney injury,the complement system can be activated through three ways,classical pathway of complement may play in early organ protective effects,alternative pathways for the amplification and increased late injury.4)The enhanced expression of complement regulatory protein DAF partially antagonized the activation of complement and played a protective role.2 Rhabdomyolysis induced acute renal injury in rats complement and oxidative stress,inflammatory reaction.1)Rhabdomyolysis induced acute renal injury in the pathogenesis of active oxygen generation,which can activate complement,complement activation and many complement proteins produced during oxidative stress and reinforcement.2)Complement play "cytolytic" function through MAC,besides,it can also complement the chemotaxis protein leads to inflammatory cells to collect,and releasing a variety of cytokines,Chemokines,inflammatory mediators,inflammatory response,complement and continue in the process is activated by Inflammatory Cytokines,ultimately increases rhabdomyolysis-induced acute kidney injury.3)During the course of the injury,the expression of complement regulatory protein was up-regulated in preliminary stage,after that,the activation of complement was over stimulated by many factors.3.Effect of complement and apoptosis in rats with acute renal injury induced by rhabdomyolysis.1)After the activation,complement can affect the apoptosis of acute renal injury induced by rhabdomyolysis,it can play dual roles in inhibiting the apoptosis of inflammatory cells,promote cell apoptosis in renal tissue,cause and aggravate renal injury.2)The mitochondrial pathway,death receptor pathway and endoplasmic reticulum pathway in different degrees in rhabdomyolysis induced acute renal injury apoptosis signal transduction.4..Complement antagonist C1INH on injury induced by rhabdomyolysis protective effect on acute kidney1)C1INH can reduce the injury of rat kidney complement activation induced by rhabdomyolysis.2)C1INH can inhibit the activation of complement through three major pathways,and reduce C5a production,reduce inflammatory response and tissue apoptosis,thus play a role in organ protection.Innovation:1.For the first time,it's approved that complement induced play an important role in acute kidney injury in rhabdomyolysis.2.It's approved that complement and oxidative stress,inflammatory reaction interaction aggravate rhabdomyolysis induced acute renal injury,3.Complement activation increased the acute kidney injury in renal cell apoptosis induced by rhabdomyolysis4.Complement antagonists can inhibit inflammatory reaction,reduce the acute renal injury and apoptosis protection caused by rhabdomyolysis.Limitations1.This research is to study the role of complement in rhabdomyolysis induced acute renal injury.It belongs to the general research,involving a wide range,but not in-depth study2.Without using single complement proteins gene defects animals or transfection studies,this study may have relative lack of conviction.We have the following considerations:first of all,many complement protein,critical study complement proteins only about funding and workload are maximum requirements;second,complement interaction between each complement proteins is too complecated to be distinguished definitely.3.The fourth part of the research maybe superfluous,the space is small,but also very important.Study on the final serve for the clinic.CVF has been used as a tool for medicine,but has not got approval for clinical use;C1INH has been used for many years;CVF for the purpose of scientific research,almost exhausts the complement,C1INH partially inhibited,and the mechanism is different,the results may not be the same,and the latter are more valuable for the clinic.