The Role Of Central Renin-angiotensin System In Progress Of Ischemic Acute Kidney Injury

BackgroundAcute kidney injury(AKI)is one of the most common critical diseases which may derive from various diseases.The ischemic AKI is the most common form in AKI,and the most common change in pathology and physiology.Ischemic AKI can be reversed,it can be prevent growing into kidney failure if realize and treat in time.Nowadays,though the level of medicine has been increased,the measure to prevent and protect the kidney from AKI is still unclear,the morbidity and the mortality is high.Because of the lack of specificity of target therapy,just depend on the dialysis,there will be a far distance to solve the question of high morbidity and mortality,and this question now become the worldwide challenge.Therefore,it's significant to realize the pathogenesis and explore the new treatment for AKI.The procedure of ischemic AKI is:in the early stage,renin-angiotensin system act as vasoconstrictor,and then come out endothelial cell injury,cells adhesion,therewith inflammation,result to cell injury,even cell apoptosis and necrosis.Renin-angiotensin system is the central link in regulating cardiovascular and renal function,via the classical angiotensin converting enzyme(ACE)-angiotensin ?-Ang ? type 1 receptor(AT1R)pathway,regulate the electrolyte,maintain the blood volume and angiotasis,regulate renal function.Ang ?,the most important bioactivator in kidney via the receptor.Ang ? receptor including two subtypes,AT1 receptor and AT2 receptor,according to the difference in pharmacological characteristics.Nowadays,as we all known that Ang ? works through AT1R.Oxidative stress reaction,a trigger of many chain reaction,acts as an important role in physiology and pathology,produce proinflammation cytokines and reactive oxygen species(ROS),which in turn trigger inflammation.Too much ROS will make the antioxidation mechanism overload,the lead to oxidative injury.Animal models of hypertension have been vital in detailing the contribution of various cardiovascular regulatory brain nuclei to neurogenic hypertension.Key nuclei that have been involved in this disorder include the organum subfornicale(SFO),paraventricular nucleus(PVN)of hypothalamus,rostral ventrolateral medulla(RVLM)and nucleus of the solitary tract(NTS)of the brain stem.The PVN plays an important role in regulating blood pressure in both the normotensive and hypertensive states.This nucleus receives neural input concerning cardiovascular status from regions such as the SFO and NTS,integrates the input and controls sympathetic outflow by sending projections directly to the intermediolateral cell column of the spinal cord,and by sending projections to other sympathetic regulatory regions including RVLM.PVN also regulates the production of hormones that influence cardiovascular functions including glucocorticoids and vasopressin.PVN and Hippocampus are the main position to oxidative stress reaction.According to some reports,PVN in the hypothalamus is the significant nucleus in renin-angiotensin system and inflammation.Some scholars found that AKI can lead Hippocampus to inflammation.Mortality during AKI is largely due to extrarenal manifestations.Central nervous system changes,the signs of which range from decreased mental status to obtundation and seizures,are one of the classic indications to begin dialysis during AKI.Other indications for renal re-placement therapy include pulmonary edema,hyperkalemia,pericarditis,and severe acidosis.Dialysis improves but does not fully correct central nervous system manifestations or other distant organ effects of renal failure,either acutely or chronically.In patients with AKI,the symptoms of encephalopathy are generally more pronounced and progress more rapidly than with chronic kidney disease or ESRD.Although neurologic sequelae of AKI are well established,the pathogenesis of acute uremic encephalopathy is poorly understood.A multitude of potential uremic toxins,including the nitric oxide synthase modulating guanidino compounds,have been implicated in uremic encephalopathy.Brain edema and alterations in water transport have also been implicated.Nowadays,lots of scholars have been making studies in pulmonary and cardiac inflammation in AKI model,and some set about to the brain inflammation in short time ischemic-reperfusion(IR).In order to explore the connection between AKI and the central nervous system(CNS),realize the changes in CNS in AKI model,we try to contrast the IR mice and the sham mice,observe RAS activity and inflammation and oxidative stress reaction in PVN and Hippocampus.In the basic of the study above,we try to inhibit the RAS,sympathetic nerve,and the oxidative stress reaction,observe the changes in PVN and Hippocampus,explain the mechanism between AKI and the brain changes.Materials and methods1.Preparation of Animal ModelAll animal procedures were approved by the Animal Experiment and Care Committee of the Southern Medical University.84 male C57 mice(initial weight,20-24g,from Southern Medical University Animal Experiment Center)were maintained under standardized conditions.The animals were subjected to two groups,one for sham,another one for ischemic reperfusion.Sham operation just expose the abdomen,separate the tissue around the renal pelvis,expose the renal artery.Another group goes through ischemic-reperfusion,the IR mice were randomized into subgroups,sacrifice after 1 hour,3hours,6hours,1 day,3days,7days.2.Experimental data and specimen collection2.1 Blood pressure and urine collectionThe blood pressure was detected by DSI system.the change in systolic blood pressure is used as the statistic.2.2 Blood collect and brain perfusionAt the completion of each protocol,rats were anesthetized with pentobarbital sodium(40mg/kg,i.p.),Trunk blood was collected in chilled vacuum tubes,and plasma samples were separated and stored at-80 ? until assayed.Part of the rat brain tissue were collected for western blot studies,and the others were used for immunohistochemical studies.2.3 Blood collect and brain perfusionAt the completion of each protocol,mice were anesthetized with pentobarbital sodium(35mg/kg,i.p.),Blood was collected in right ventricle to chilled vacuum tubes,and plasma samples were separated and stored at-80? until assayed.The blood were used for renal function and Ang II,NE test.Part of the mice brain tissue were collected for western blotting studies and PCR technology,and the others were used for immunohistochemical studies.2.4 In all IR mice,we found that in IR 6 hours,the RAS activity in PVN and Hippocampus increased the most significant.Therefore,we choose IR 6h to inhibit.AKI mice randomize into 10 groups(n=12 in each group)and treated as follows,respectively:(1)Losartan Omg/kg/d by intragastric gavage(IG):Mice were admini-strated with daily intragastric of d2H20;(2)Losartan lmg/kg/d IG:Mice were administrated with daily intragastric of losartan(lmg/kg per day,Sigma Chemical,St Louis,MO,USA);(3)Losartan 75mg/kg/d IG:Mice were administrated with daily intra-gastric of losartan(75mg/kg per day);(4)Losartan Omg/kg/d by intracerebroventricular injection(ICV):Mice were administrated with daily intracerebroventricular injection of artificial cerebrospinal fluid(aCSF);(5)Losartan lmg/kg/d ICV:Mice were administrated with daily intracere-broventricular injection of losartan(lmg/kg per day);(6)ICV Clonidine:Mice were administrated with daily intracerebro-ventricular injection of clonidine(5.76?g/kg per day,Sigma,USA);(7)Renal denervation(RDX);(8)ICV Tempol:Mice were administrated with daily intracerebroven-tricular injection of tempol(9)Tempol 30mg/kg/d IG:Mice were administrated with daily intragastric of tempol(3Umg/kg per day,Sigma5 USA).(10)Hydralazine IG:Mice were administrated with daily intragastric of Hydralazine.(30mg/kg per day,Sigma,USA).After stimulation above,urine and Blood were collected for biochemical indicators test,the brain tissue were used for brain RAS and TH,Nox4 and c-fos detect by immunohistochemistry or western blotting.3.Statistical AnalysesAll data are presented as mean±SD.Continuous variables between groups were compared using one-way ANOVA,followed by LSD method when P<0.05.Nonparametric test is used when heterogeneity of variance.Statistical analyses were conducted with SPSS 16.0 for Windows(SPSS,Chicago,IL).Significance was defined as P<0.05.Results1.AKI mice blood pressure and renal functionIR mice blood pressure is increased in 3h,6h and 24h compared to sham(P<0.05),as well as the serum creatinine levels and 24h urine protein,which mean that the AKI model is succeed.2.Changes in general characteristics in AKI miceThe NE level,Ang ? level,and the sodium level in serum and 24h urine protein are increased in AKI mice compared to sham mice.3.Immunohistochemical and PCR technology detect the RAS activity in PVN and Hippocampus in AKI miceWe found that RAS activity increased in PVN and Hippocampus in AKI mice brain via immunohistochemical and PCR technology,there are statistical differences in 3h,6h and 24h in gene and protein level compare to sham.4.Cellular localization of AGT or AT1R in PVN and Hippocampus in 5/6Nx ratsAGT or AT1R immunoreactivity is colocalized on neurons within PVN and Hippocampus.5.Blood brain barrier permeability in AKI miceThrough the Evans blue experiment,we found that the BBB permeability increased in AKI mice compare to sham.6.Inflammation in PVN and Hippocampus in AKI miceWe found that GFAP and Ibal increased in PVN and Hippocampus in AKI mice brain via immunohistochemical technology,there are statistical differences in 3h,6h and 24h in gene and protein level compare to sham.7.Oxidative stress reaction in PVN and Hippocampus in AKI miceWe found that Nox4 increased in PVN and Hippocampus in AKI mice brain via western blotting technology,there are statistical differences in 3h,6h and 24h in gene and protein level compare to sham.8.Activity of RAS in AKI to different inhibition in PVN and HippocampusWe found that AT1R decreased in IG losartan 75mg/kg.d and RDX compared to IG losartan 0 mg/kg.d(p<0.05),but no difference in IG tempol and IG Hydralazine through immunohistochemical technology.AT1R decreased in ICV losartan lmg/kg.d and ICV tempol compared to ICV losartan 0 mg/kg.d(p<0.05),but no difference in ICV Clonidine.9.Inflammation in AKI to different inhibition in PVN and Hippocampus We found that GFAP and Ibal decreased in IG losartan 75mg/kg.d and RDX compared to IG losartan 0 mg/kg.d(p<0.05),but no difference in IG tempol and IG Hydralazine through immunohistochemical technology.GFAP and Ibal decreased in ICV losartan lmg/kg.d and ICV tempol compared to ICV losartan 0 mg/kg.d(p<0.05),but no difference in ICV Clonidine.10.Oxidative stress reaction in AKI to different inhibition in PVN and HippocampusWe found that Nox4 decreased in IG losartan 50mg/kg.d and RDX compared to IG losartan 0 mg/kg.d(p<0.05),but no difference in IG tempol and IG Hydralazine through western blotting.Nox4 decreased in ICV losartan lmg/kg.d and ICV tempol compared to ICV losartan 0 mg/kg.d(p<0.05),but no difference in ICV Clonidine.Conclusion1.RAS activity increased in PVN and Hippocampus in AKI mice.2.The permeability in blood brain barrier increased,in other word,BBB injury in AKI mice.3.Inflammation increased in PVN and Hippocampus in AKI mice.4.Oxidative stress reaction increased in PVN and Hippocampus in AKI mice.5.Large gose in peripheral inhibition(IG losartan 75mg/kg.d)and RDX decrease the activity of RAS,inflammation and oxidative stress reaction in PVN and Hippocampus,but no difference in IG tempol and IG Hydralazine.