| PurposeEndotoxemia is often present in stress conditions such as severe trauma,burn, shock,severe infection,etc.In these conditions,plentiful LPS from the infective areas enter the body and cause the mediators of inflammationsynthesizing and releasing abundantly.Consequently,multiple organs including kidney are involved.Accordingly, endotoxemia plays an critical role in the onset and development in acute injury.Kidney is the major route for the excretion of metabolic product and toxin.The LPS reabsorbed by renal tubule can place the renal tubular epithelium cells in a high lipopolysaccharide(LPS) concentration microenvironment,which might have a great effect on the function and structure of renal tubular epithelium cells.It also acts on the onset and development of acute kidney injury(AKI).The characteristic change of acute kidney failure caused by AKI is the decline of glomerular filtration rate(GFR) and renal tubule dysfunction.The consummate renal tubular function including urine concentration and reabsorption of K+ depends on the involvement and functional expression of Aquaporin(AQP).AQPs distribute widely and possess various subtypes in many organs and tissues such as brain,lung,and kidney.AQPs play an important part in regulation of water and glycerol metabolism.They are vital substances for maintaining a stable internal environment,whose structure and function is influenced by many ingredients as well.AQPs can change water permeability and affect the metabolism of water and micromolecules.AQPs are also the key fact influencing cell apoptosis simultaneously. In 1992,Preston found out kidney AQP.This finding explained the problem of renal free water metabolism rationally.Kidney is the major organ of water metabolism.The research of AQPs in kidney operates a crucial fact on revealing renal physiological and pathological change.It is reported the expression of AQP is decreased apparently in renal failure caused by drugs,ischemia etc.This change of AQPs is close related to the injury degree of renal tubular epithelium cell,whose convalescence occurs earlier than morphological change.It has been proved thatAQP2 is the most important aquaporin which is unique regulated by AVP.It is distributed in cellular cell apicoplasmatic membrane and intracellular vesicle.There are two regulation ways of AVP to AQP2:(1) Short regulation action:253-256 sites in amino acid sequence of AQP2 contain protein kinase(PKA) phosphorylation position.The combination of AVP and V2R activates the AC-cAMP-PKA signal conduction pathway and causes the phosphorylation of AQP2.Then it makes intracellular vesicles containing AQP2 moving and fritting to cell apicoplasmatic membrane.Consequently,the permeability of cell membrane to water increases rapidly,which happens in several minutes.(2) Long regulation action:the stable activation of V2R can cause increased synthesis of AQP2.AVP phosphrylates intranuclear cAMP and stimulates the expression of cjun / cFos gene by V2RcAMP-PKA signal conduction pathway and then affects the RNA transcription and protein synthesis of AQF2.Accordingly,the total abundance of AQP2 is up-regulated and the permeability of renal collecting tube to water is changed.The alteration of AQP2 RNA can appear in several hours.Recent years' researches show that AQPs can improve micromolecule and water metabolism of lung,brain,kidney in some pathological process and relieve the edema and injury of these corresponding organs.The cell migration inedited by AQP is important for the recovery and reconstruction of renal tubular function in acute renal injury.It is close relative to the prophylaxis and treatment of these vital organs injury. Researching the changes of function and structure in these pathological processes can assist guiding drugs usage.Discovering and treating renal function disorder early can not only retrieve shock,but also protect viscera better. The researches shows that anesthetics such as isoflurane,sevoflurane,lidocaine, propofol,etc can protect heart,brain,kidney,lung from varied injuries.As a widely used clinical intravenous anesthetic,Propofol's function is sedative and hypnosis.It has been reported that propofol has its protective action through regulating AQP1 when confronting lung injury in endotoxemia,but still unsure whether it will go well for the rat's kidney injury in endotoxemia.Based on literature,this experiment imitates the observation of human endotoxemia in order to establish rats endotoxemia model.Examine function and acquire kidney specimen after 12 hours.Find out renal injury condition by pathological examination and observe the alteration of ultramicrostructure in kidney.Detect the changes in expression of AQP2 mRNA,AQP2 protein,tumor necrosis factor-α(TNF-a), intercellar adhesion molecule-1(ICAM-1),B-cell lymphoma/leukemia-2(Bel2),Bax and Caspase3 and cell Apotosis Indexin in rats endotoxemia's kidney injury process by RT-PCR,Western-blotting hybridization skill,immuno-histology chemical staining skill,etc.Meanwhile,observe the effect of the well known anti-inflammation and anti-oxydation intravenous anesthetic--propofol in the rats endotoxemia's kidney injury. Take three different ways:pretreatment,use drugs simultaneously,aftertreatment.And then observe propofol's effect on rensl function,AQP2mRNA,AQP2protein,ICAM-1, Bcl2,Bax,Caspase3 and cell apoptosis Index in rats eodotoxemia's kidney.Observe and discuss the effects of different dosage time of propofol on rats endotoxemia's kidney injury from molecular biology aspect and provide experimental basis for guiding clinical dosage.The experiment includes three parts:1.the alteration of gene expression of AQP2,structure and function in endotoxemia rats' kidney.2.the effect of propofol on the gene expression of AQP2,structure and function in endotoxemia rats' kidney.3.the effect of propofol on cell apoptosis in endotoxemia rats' kidney. Materials1.Animals48 Wistar rats(body weight:200-300g),provided by Experimental Animal Center,Shenyang General Military Hospital2.Main reagents and apparatusaquaporin 2 antibody,propofol,Bcl-2/Bax multiclonal antibody, caspase-3 multiclonal antibody,PCR machine,stable temperature water bath, electrophoretic machine,eletroalance,liquid nitrogen tank,section machine,inverted phase contrast microscope,MDF-Ultra deep-freeze equipment,micro-image analysis system,etc.Methods1.Establishment of animal modelAnesthesia of the rats was induced and maintained by intraperitoneal injection of pentobarbital,LPS 5mg/kg intravenous injection was given to establish endotoxin model.2.Experiment design48 wistar rats were divided into 6 groups.Group C(sham group,n=8),Group Saline and Fat Milk(n=8),Group Endotoxin and Fat Milk(n=8),Group Propofol Predisposal (n=8,propofol infusion was given one hour before LPS injection),Group Propofol(n=8, propofol infusion began immediately after LPS injection),Group Propofol Postprocessing(n=8,ropofol infusion was given one hour after LPS injection).12 hours later,urine and 5ml of blood from ophthalmic artery were collected.After the collection of urine and blood,the rats were killed and their kidney medulla was sampled.3.Detection parameter(1) Morphological changes of renal collecting tube by light microscope (HE stain)(2) Ultrastructural morphological changes of kidney by electron microscope(3) Measurement of BUN,Cr and osmotic pressure of urine and blood(4) AQP2 mRNA,TNF-α,ICAM-1,caspase3,Bcl-2/Bax mRNA by RT-PCR and Western blot(5) Expression level of caspase3 protein,Bcl-2/Bax protein and AQP2, TNF-α,ICAM-1 by Immunocytoehemical stain(6) Renal apoptosis detection by TUNEL method4.Statistical analysisAll values were presented as mean SE and subjected to one-factor analysis of variance using SPSS 12.0 software.Homoscedasticity was checked by LSD method and Dunnett T3 method was adopted to check the heterogeneity of variance. Differences were considered significant at P＜0.05.Results1.compared with control group,in endotoxin group,the expression of rats' kidney AQP2,bcl-2 mRNA and protein was downregulated(P＜0.05),the expression of TNF, ICAM-1,caspase3,bax mRNA and protein was upregulated(P＜0.01),and propofol predisposal,coprocessing and postprocessing can retrieve those changes.2.Compared with control group,in endotoxin group,the urine volume decreas ed,the Cr,BUN and blood osmotic pressure increased significantly while the urine osmotic pressure decreased,propofol infusion can relieve those reactions.3.Compared with control group,the amount of apoptotic cell in endotoxin group increased significantly,propofol infusion can relieve those reactions.4.Ultrastructual morphological changes:The ultrastructure of nephridial tissue in control group was fundamentally normal, renal tubular epithelial cell and its organelle structures were normal.In endotoxin group, renal tubular cell demonstrated injury,nucleus degeneration and irregularity,proximal convoluted tubule microvillus damaged,mitochondfia swelled,broken,some of the cristae disappeared,PER dilated.In Group P1 and P2,most of renal tubular cell structure showed normal or essentially normal,tubular cell nucleus showed normal morph and structure,nucleoli were clear,mitochondria cristae swelled slightly,RER and microvillus dilated slightly,ultrastructural morphologh improved significantly.In Group P3,renal tubule damage was slightly severer than that of endotoxin group.1.Kidney histological morphologic changes:There was no manifest morphologic abnormality in control group.In endotoxin group,the renal tubular cells showed degeneration,necrosis and falling off. The lumens of renal tubule were narrowed,renal interstitial showedobvious edema,congestion and infiltration of inflammatory cell.The alignment of renal tubule in Group P1 and P2 were fundamentally normal,morphologic changes consisted of swell and small amounts of congestion and infiltration of inflammatory cell.Morphologic improvement of renal tubule was also observed in Group P3.Under light microscope,the Paller's Score of renal tubule damage in Group P1 and P2 were significantly lower than that of Group L(P＜0.01) and Group P3(P＜0.05).DiscussionBased on literature,this experiment imitates the observation of human endotoxemia in order to establish rats endotoxemia model.Research the effect of different dosage time of propofol on renal function and acquire kidney specimen.Operate pathological examination and ultramicrostrcture observation as well as immuno-histology chemical skill and western-blotting skill to detect the change of aquaporin,TNF-a,ICAM-1, bcl2/bax,caspase3,etc,aiming to discuss propofors kidney protection and mechanism and provide experiment basis for guiding clinical anesthetic dosage.Compared to control group,the result shows that endotoxemia rats' blood Cr,BUN increase significantly.Serum osmolarity(Sosm) also increases while Urine osmolarity(Uosm) decreases.AKI's negative water equilibrium and high osmotic pressure are present. Observe ultramicrostructure of renal tubular Through transmission electron microscope we can find that endotoxemia group encounter severe injury of renal tubular nucleus, mitochondria,RER,microvillus,etc.Through light microscope we can find that endotoxemia group encounter renal tubular epithelium cell's degeneration,necrosis, defluxion,the stenosis of renal tubular lumen,the edema of renal interstitium and congestion with inflammatory cell infiltration apparently.The immuno-histology chemical examination finds that the content of aquaporin in endotoxemia group declines significantly while TNF-a,ICAM-,caspase3 express increasely.The ratio of Bcl2/Bax decreases and the apoptosis of cell increase.After utilizing propofol,all kinds of functional examination and ultramicrostructure as well as pathological change get great improvement,the expression of aquaporin increases.The expression of anti-apoptosis gene Bcl-2mRNA is up-regulated while that of caspase3 is down-regulated.The apoptosis of renal tubular cells decrease.From the results mentioned above,we can see that different dosage time of utilizing propofol can antagonize kidney injury in endotoxemia and assist recovering renal structure and function.Endotoxemia is often present in stress conditions such as severe trauma,burn, shock,severe infection,etc.In these conditions,plentiful LPS from the infective areas enter the body and cause the mediators of inflammation synthesizing and releasing abundantly,which causes cell degeneration and necrosis.These manifestations are expressed by the following symptoms:the impairment of tissue and structure the deterioration of organ function;the blood redistribution of kidney resulting from renal glomerulus especially renal arteriole contraction caused by LPS,which causes the decline of renal blood flow and GFR,hence,inducing renal ischemia-reperfusion injury. It also plays a vital role in the onset and development of AKI.Kidney is the major route for the excretion of metabolic product and toxin.The LPS reabsorbed by renal tubule can place the renal tubular epithelium cells in a high lipopolysaecharide(LPS) concentration microenvironment,which might have a great effect on the function and structure of renal tubular epithelium cells.It also acts on the onset and development of acute kidney injury(AKI).Propofol is a common clinical intravenous anesthetic,whose unique function and structure enable it possess the ability that inhibits inflammatory reaction,relieves inflammatory ingredients releasing,inhibits inflammatory cell congregation and activation to relieve inflammatory intensity,potentiates anti-inflammatory function and reduces necrosis as well as apoptosis,protects organs,etc.There are researches showing that propofol can relieve alveolar epithelium injury in endotoxemia'lung injury by regulating AQP1 and reducing local TNE In this experiment,we can see that the groups utilizing proprofol all manifest relieving injury symptoms to different extent in endotoxemia's AKI.However,the groups using lipid solvent don't have the same effect. Consequently,propofol might protect kidney by regulating local aquaporin expression and relative facts as well as renal perfusion per se.The characteristic change of acute renal failure caused by kidney injury of endotoxemia is the decline of GFR and renal tubular dysfunction.The consummate renal tubular function including urine concentration and reabsorption of K+ depends on the involvement and functional expression of Aquaporin(AQP) and other ion transportation protein.AQPs distribute widely and possess various subtypes in many organs and tissues such as brain,lung,and kidney.AQPs play an important part in regulation of water and glycerol metabolism.They are vital substances for maintaining a stable internal environment,whose structure and function is influenced by many ingredients as well.Because AQPs are sensitive to multiple pathological process and celluar inflammatory ingredients,AQPs can change water permeability and affect the metabolism of water and micromolecules.AQPs are also the key fact influencing cell apoptosis simultaneously.The discovery of kidney AQP explained the problem of renal free water metabolism rationally.Kidney is the major organ of water metabolism.The research of AQPs in kidney operates a crucial fact on revealing renal physiological and pathological change.It is recently reported the expression of AQP is decreased apparently in renal failure caused by drugs,ischemia etc.This change of AQPs is close related to the injury degree of renal tubular epithelium cell,whose convalescence occurs earlier than morphological change.It has been proved thatAQP2 is the most important aquaporin which is distributed in cellular cell apicoplasmatic membrane and intracellular vesicle.Its abundance influences the permeability of collecting tube to water,which can happen in several minutes.Recent years' researches show that AQPs can improve micromolecule and water metabolism of lung,brain,kidney in some pathological process and relieve the edema and injury of these corresponding organs. The cell migration medited by AQP is important for the recovery and reconstruction of renal tubular function in acute renal injury.It is close relative to the prophylaxis and treatment of these vital organs injury.Researching the changes of function and structure in these pathological processes can assist guiding drugs usage.In this experiment,compared to control groups,the groups injecting lipid and endotoxin encounter the decline of aquaporin,which indicates that the decline of aquaporin might be a fact of AKI.However,the content of AQP2 in the groups utilizing propofol all increase to different extent,which indicates that propofol possess direct or indirect action on regulating AQP2 itself to protect kidney.Of course,local inflammatory reaction other aquaporin and ion channels are also involved.In all,the definite mechanism is still to be researched.There are researches showing that plentiful cytokines participate the endotoxemian process,which includes IL-1β,IL-6,ICAM-1,VCAM-1 and TNF-a.These inflammatory induce and amplify inflammatory cascade reaction.TNF-a is a 17kd polypeptide which is considered as the priming and key fact for systematic inflammatiry reaction.It is reported that TNF-a has strong toxicity to kidney.This cytokine possesses direct cytotoxicity and induces cell necrosis directly.TNF-a also can cause cell necrosis by microcirculation disorder.Stimulated by endotoxin,TNF-a and other inflammatory medium activate each other,inducing epithelium cell and macrophage cell releasing IL-1.IL-1 stimulates the biological synthesis of other cytokines.For example,intensify the expression of ICAM-1mRNA to amplify inflammatory signal,which enhance s the sensitiveness of tissue to TNF-a in turn. Injecting appropriate TNF-αto animals can cause AKI.If given TNF-αantibody or take measures to reduce TNF-αproduction,the injury caused by eodotoxin can be blocked.It is demonstrated in this experiment that the expression of TNF-αmRNA increased sharply in renal cortex after endotoxemia.This phenomenon shows that TNF-αparticipate the pathological process of AKI.However,giving propofol can reduce the expression of TNF-αmRNA and TNF-αprotein.Conaequently,the content of TNF-αin kidney decreased significantly and relieve renal injury.ICAM-1 is a glycoprotein on cell surface as a part of epithelium cellular structure. The stimulation off LPS and organism stress reaction engender abundant inflammatory mediums and cytokines after endotoxemia.These substances activate WBC as well as blood vessel endothelium(BVE) cell.WBC express CD11/CD18 glycoprotein adhering complex on the cell surface and BVE cell express ICAM-1.These expression products combine with each other,which induce WBC and epithelium cell adhering and promote WBC congregating,activating and releasing inflammatory medium.Normal renal tissue lack the expression of ICAM-1.LPS and cytokines both can up-regulate the expression of ICAM-1mRNA,causing WBC in circulation adhering at local sites.This adhesion forms obstruction in outer medulla and increase the permeability of epithelium.Moreover,these infiltrated and activated WBC can also induce direct tissue injury.The congregation of RBC leads to absent perfusion of outer medulla after reperfusion and aggravates the ischemia-reperfusion injury in the pathological process of endotoxemia.However,reducing local and circulated ICAM-1 can reduce the activity and injury of epithelium cell,whose content can reflect injury condition well. Kelly,etc researched and found that ICAM-I lacking rats can relieve renal ischemia-repeffusion injury obviously.Giving rats ICAM-1 antibody will also go well for that injury.The result of RT-PCR in this experiment shows that the expression of ICAM-1 in renal cortex increased 12 hours after endotoxemia onsets,which illustrates that ICAM-1 is a fact influencing AKI as well.Different dosage of propofol can reduce the expression of ICAM-1mRNA and ICAM-1 protein in kidney by different degrees to relieve the kidney injury.Kidney is an important target organof endotoxemia.Its injury mechanism is still not definite for many ingredients participating the process.As a kind of cell death form distinct from necrosis,whether apoptosis participates and induce sinjury needs researched.Cell apoptosis is a special cell deathform distinct from necrosis,referring to aging,useless,injured cell death in the organism.Cell apoptosis has special degradation,whose morphology appears that karyopycnosis,cell membrane effervesce, form apoptosis body.This process is regulated by special gene without definite cell dissolving process.Caspase3 is an important member of cycteine proteinase group.It is the most important terminal executing enzyme in the cell apoptosis.Inhibiting Caspase3's activity may impede apoptosis.Bcl-2mRNA is an important anti-apoptpsis gene in this gene group.The equilibrium between promoting apoptosis influences the onset of apoptosis and antagonizes the apoptosis dependent or independent on Caspase. Researches shows that the obvious Increase of apoptosis cell in LPS group indicates that apoptosis participates AKI process compared t o control group.The different dosage of propofol can increase the expression of bcl-2 and decrease that of Caspase3 to reduce cell apoptosis.Consequently,the protection of propofol to renal cell is partly through regulating the expression of bcl-2 and Caspase3 to impede cell apoptosis and antagonize injury.From all mentioned above,this research find that propofol can protect kidney from AKI in endotoxemia.The possible mechanism might be through regulating aquaporin, down-regulating adhering molecule and TNF,influencing apoptosis,etc.Compared to preadministration and simultaneous administration,delay-administration can also work but weak.This result shows that early aftertreatment(in an hour) possesses some protect action,providing theory basis for clinical anesthetic administration.ConclusionThe change of renal function,morphology and molecular biology of rats in endotoxemia indicates that AKI exits in endotoxemia.In this process,different dosage of propofol can relieve inflammatory reaction by different degrees and impede renal function and morphology change,which protects the kidney to some extent. |
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