| Objective:Acute kidney injury(AKI)and its developed renal interstitial fibrosis are global clinical syndromes associated with high morbidity and mortality.Renal ischemiareperfusion(I/R)injury which commonly occurs during surgical operations,is one of the major causes of AKI.Nevertheless,to date,efficient therapeutic approach for AKI and its developed renal interstitial fibrosis is still lacking due to its elusive pathogenetic mechanism.Chitosan oligosaccharide(COS)is a natural oligosaccharide derived from the degradation of chitosan.The renoprotective effect of COS has been widely studied,but its effect on I/R injury-induced AKI and its development of renal interstitial fibrosis has not been reported.Therefore,this work aims to investigate the protective effect of COS against AKI and subsequent chronic kidney disease(CKD)induced by I/R.Methods:1.AKI and subsequent CKD models were established using male C57BL/6J mice aged 8-10 weeks by unilateral renal I/R injury and contralateral nephrectomy.In AKI experiment,all mice were randomly divided into five groups: Sham,I/R,I/R-COS100,I/R-COS200 and I/R-NAC group.N-acetylcysteine(NAC),an inhibitor of reactive oxygen species(ROS),is a potent antioxidant which was applied as a positive control in AKI experiment.In AKI-CKD transition experiment,all mice were randomly divided into four groups: Sham,I/R,I/R-COS200 and I/R-VAL group.Valsartan(VAL),an angiotensin Ⅱ receptor antagonist widely used to treat hypertension,is known to slow progression of CKD,and served as a positive control in this CKD experiment.The protective effect of COS on renal I/R-induced AKI was evaluated by H&E staining,serum creatinine concentration detection and immunohistochemical staining.Furthermore,the underlying mechanism by which COS alleviates I/R-induced AKI was dissected by analyzing the expression of endoplasmic reticulum(ER)stress-related proteins glucoseregulated protein(GRP78)and C/EBP homologous protein(CHOP)using immunohistochemical staining and Western blot.The protective effect of COS on renal interstitial fibrosis was evaluated by H&E staining,Masson staining and immunohistochemical staining.2.Oxidative stress model was established using mouse renal tubule epithelial cell line TCMK-1 treated with tert-butylhydrogen peroxide(TBHP).The cells were divided into four groups: control,TBHP,TBHP+NAC and TBHP+COS group.The effect of COS on the proliferative activity of TCMK-1 cells treated with TBHP was measured by CCK-8assay.The level of intracellular ROS was detected by fluorescent probe DCFH-DA.The expression of ER stress-related proteins GRP78,CHOP and activating transcription factor4(ATF-4)were examined using immunofluorescence staining and Western blot.The change of mitochondrial membrane potential was measured using mitochondrial membrane potential detection assay.Transcriptome sequencing analysis was performed to analyze the relevant signaling pathways and genes products involved in this protective process,and to explore the molecular mechanism of COS against TBHP-induced oxidative stress in renal tubule epithelial cell.Results:1.In AKI mice induced by kidney I/R,compared with I/R group,COS treatment can reduce kidney tissue injury,decrease serum creatinine concentration,and inhibit the expression of kidney injury molecule-1(KIM-1)and ER stress-related proteins GRP78 and CHOP.2.In AKI-CKD transition experiment,COS treatment significantly reduced renal interstitial fibrosis and decreased the expression of alpha-smooth muscle actin(α-SMA)in renal tubules,compared with I/R group.3.In TCMK-1 oxidative stress experiment induced by TBHP,the proliferative activity of TCMK-1 cells in TBHP group was significantly decreased comparing with the control group,and COS treatment could markedly recover the TCMK-1 cell proliferation with the treatment of TBHP.DCFH-DA staining showed that the intracellular ROS level in the TBHP group was markedly higher than that in control group,and COS treatment could significantly inhibit TBHP-induced ROS production in TCMK-1 cells.Western blot and immunofluorescence results showed that the expression of ER stress-related proteins GRP78,CHOP and ATF-4 increased in TBHP group compared with the control group,and COS treatment inhibited the expression of these ER stress-related proteins.JC-1staining analysis showed that,mitochondrial membrane potential in the TBHP group was significantly reduced compared with the control group.However,mitochondrial membrane potential was significantly increased after COS or NAC treatment compared with the TBHP group.4.Transcriptome sequencing analysis showed that 746 genes were up-regulated and996 genes were down-regulated in the TBHP group compared with the control group.After COS treatment,37 of 746 up-regulated genes and 13 of 996 down-regulated genes were reversed.GO enrichment analysis of these genes showed that COS may play a protective role in kidney injury by regulating genes Pam16,Trem2 and Esrra involved in metabolic and REDOX biological processes.Conclusion:Taken together,these in vitro and in vivo data demonstrates that COS can significantly reduce I/R-induced AKI and maintain glomerular filtration function.This protective effect of COS is associated with inhibition of oxidative stress,mitochondrial damage and excessive ER stress.In addition,this study also demonstrated that long-term COS administration can alleviate renal interstitial fibrosis caused by renal I/R injury. |
