Protective Effect Of ATRA On The Acute Renal Failure Induced By High-dose Cisplatin In Mice

Background:Malignant tumor is one of the common diseases that which seriously endanger the health of human being.Now surgery,chemotherapy,radiotherapy,etc.are the clinically comprehensive treatement for malignant tumor,among which,chemotherapy is the most effective treatment for advanced cancer.Cisplatin(DDP)is one of the nonspecific cell cycle chemotherapy drugs and it is commonly used for the treatment of solid renal tumors such as lung cancer,testicular cancer,ovarian cancer,bladder cancer,head and neck cancer,etc.Clinical trials found that the antitumor effect of DDP was positivaly correlated with its dosage.However,DDP also produced dose-dependent side effects accompanied by its antitumor effects,for example,gastrointestinal tract reaction,liver and kidney injury.It was reported that 60 to 80%of the patients would have acute renal failure when the patients received high-dose DDP treatment,which greatly limited DDP's clinical application.lt was demonstrated that high-dose DDP could induce tubular epithelial cell apoptosis and the mechanisms of DDP-induced nephrotoxicity was thought to be related with oxidative stress.But till now,the exact mechanism of nephrotoxicity induced by DDP remains unclear and there is no reliable and specific treatment methods in clinic.Recent studies have shown that inflammation and renal hemodynamic changes might play important roles in DDP-induced nephrotoxicity.On the basis of previous works,we are to explore the mechanism of DDP-induced nephrotoxicity,aiming at establishing the theoretical and experimental foundation for DDP-induced nephrotoxicity control in clinic.Objective:To explore the mechanism and changes in early stage of high dose DDP-induced nephrotoxicity,especially renal glomerulus,by using animal model of high dose cisplatin with acute renal failure and for futher understand of the protective effects of all trans-retinoic acid(ATRA)on high dose DDP-induced nephrotoxicity.Methods:C57/BL6 mice were randomly divided into two groups by weights,half male and female:DDP treatment group(DDP group)and normal saline control(Control group).5?45 mg/kg DDP was administrated with single intraperitoneal injection and same dosage of NS was used as control.Toxic effects and survival time of mice after DDP injection were observed subsequently.The lowest dosage of DDP that could induce most mice died would be considered as the dose used in the experiment for renal failure.Then the mice used were randomly divided into groups by weights and genders:DDP treatment group(DDP group),DDP+ATRA treatment group(ATRA group),DDP+CXC receptor antagonist G31P treatment group(G31P group)and DDP+NF-?B inhibitors PDTC treatment group(PDTC group).15 mg/kg DDP was administrated with single intraperitoneal injection,20 mg/kg ATRA was administrated with single intraperitoneal injection 0.5 h before DDP use,500 mg/kg G3lP was injected subcutaneously 0.5 h before DDP use per day for 3 days,50mg/kg PDTC was injected subcutaneously 0.5 h before DDP use per day for 3 days and the same doase of NS was used as control.Each group was evaluated after drug treatment for 6h,24h,48h and 72h,respectively.Then we observed the mice's general life status.Injection with DDP,abdominal anaesthesia by 0.5%pentobarbital natrium,blood was extracted from mice,s heart.The content of the blood urea nitrogen(Bun)and serum creatinine(Cre)were detected by biochemical methods.Meanwhile xanthine oxidase(XOD),uric acid(UA),MPO and MDAD in 10%renal cortex homogenate were detected by biochemical method.Thirdly take 5 mice respectively in a metabolic cages,every other 24h beginning with sixth hour,respectively to collect urine,with lowery methods to calculate urine protein quantitative and qualitative detection,further to identify whether there exists expression of albumin.At the same time,take a small amount of kidney,CD31 changes in kidneycortex tissue were analysed by Western Blot.Collagen type IV(COL-IV)and fiber connection(FN-1)were detected by immunohistochemical method.Then morphological characteristics of glomerular mesangial cells and renal tubular basement membrane were evaluated by H&E staining and electron microscope.The expression of KC,TNF-? and IFN-? in the mRNA level were detected via real-time qPCR.Results:Compared with the control,after intraperitoneal injection of different doses of DDP,they appeared sleepiness,always crowded together,also the effect led to loss for activities,appetite and weight,etc.and survival rate in DDP(15mg/kg)deceased within 1 week to 30%.Therefore,we choose 15 mg/kg as preparation of acute renal failure induced by DDP.Further we found that after injection of DDP 48h blood Bun and Cre significantly higher than control group in mice,confirmed 15 mg/kgDDP can cause acute renal failure in mice.HE testing identified that DDP group within 48 h of renal tubule has no obvious morphological change,but significantly increased glomerular cell number in the control group,difference has statistical significance(p<0.05);DDP medication after 48h,renal tubules appeared obvious morphological changes.As a result,this research mainly explores large dose DDP within 48h of glomerular function and the mechanism of action of ATRA intervention.Then morphological characteristics of glomerular mesangial cells and renal tubular basement membrane were evaluated by H&E staining and electron microscope.By experiments of Electron microscop,Western Blot detection,immunohistochemical and biochemical detection experiments,these results showed that high doses of DDP not only could cause glomerular endothelial cell damage,activation of neutrophils,but also could cause damage of glomerular basement rYlembrane.As a result,it produced large molecular weight proteinuria.20 mg/kg of ATRA could lighten the DDP in toxic effects of the medicine,effectively prolong the survival time of mice after DDP medication,make DDP lw survival rate up to 90%of the medicine.It was found that there was no significant difference with control group(p<0.05).Electron microscopy(sem)test confirmed that glomerular endothelial cell apoptosis in mice after ATRA drug reducing,basement membrane and reduce podocyte damage,and neutrophils.Western Blot test proved that after ATRA treatment,expression of renal cortex CD31 in the DDP group significantly increased,but still slightly lower than control group.Immunohistochemical detection showed that expression of COL-IV,and FN-1 decreased significantly,but it 1s still more than the control group.Biochemical tests of serum UA and XOD cortex in after ATRA treatment,results were significantly higher than that in mice,and there was no statistically significant difference compared with control group(P>0.05);MPO and MDA content of renal cortical in ATRA group decreased significantly and there was no statistically significant difference compared with the control mice(p>0.05).In addition,the urine protein of DDP group was obviously increased,and the molecular of urine protein excretion were mainly large molecular weight likely ALB.The expression of TNF-? and IFN-? in the mRNA level were detected via real-time qPCR,we found that the expression of TNF-? significiently within six hours after ATRA treatment compared with DDP group(P<0.05).Conclusion:1.15 mg/kg DDP medication after 1 w can make 60%of the C57/BL6 mice died.DDP group within 48h,blood BUN and CRE significantly increased,prompt the mice of acute renal failure.Morphological study showed that DDP groups within 48h,there was no obvious morphological change of glomerular,but number of cells in glomerula increased obviously,we also found mitochondria edema in epithelial cells of renal tubular.2.ATRA treatment can obviously reduce the side effects of renal caused by high doses of DDP-induced nephrotoxicity,and improve the function of kidney,prolonged survival rate after DDP to 90%within 1 week.ATRA can significantly reduce activation of neutrophils,glomerular filtration membrane damage,and that caused by DDP,and reduce the.Conlusionally,possible mechisms of prevention of ATRA treatment may through inhibiting Europhile activation and reducing release of inflammatory mediators caused by high doses of DDP-induced nephrotoxicity.