| At present,the most effective and successful treatment for end-stage renal disease is kidney transplantation.However,when after kidney transplantation maybe lead to acute renal function injury and renal function loss.However,there are many factors leading to acute graft dysfunction.The most important reason is renal ischemiareperfusion injury.How to prevent renal ischemia-reperfusion injury has become an important link in the prevention of acute kidney injury after renal transplantation.Renal ischemia-reperfusion(I/R)is more sensitive to ischemia-reperfusion injury due to increased vascular fragility and high metabolic rate after transplantation,which can lead to acute kidney injury,graft recovery delay and chronic graft dysfunction.Therefore,IRI is also one of the important causes of death after renal transplantation.The pathophysiology of ischemia-reperfusion is complex,including metabolic changes,oxidative free radicals,inflammatory factors,congenital factors,activation of adaptive immune response,programmed cell death,complement system,platelet and coagulation cascade reaction.At present,drugs,physical or surgical pretreatment are mainly used to prevent kidney from I/R injury,but the best treatment is still unclear.Itaconic acid has been considered to play an important role in industrial synthesis.It's considered that Itaconic acid act very important role in chemicals,but now reserch have found that itaconic acid is also an anti-inflammatory metabolite,which can play an important role in anti-inflammatory and anti-oxidation.In mammalian macrophages Immune response gene 1(Irg1)is expressed highly,which can plays an active role in anti-inflammatory process.Irg1 can regulate the gene encoding CIS aconitic acid decarboxylase to produce itaconic acid.In mammalian and human immune cells,the expression level of irg1 is related to the amount of itaconic acid.Irg1 plays very important role in regulate the expression of itaconic acid.Whether irg1 can inhibit renal ischemia-reperfusion injury by regulating itaconic acid has not been reported.This study is mainly carried out in two parts.Firstly,the correlation between itaconic acid concentration and delayed graft function and inflammatory response.Inflammatory many factors caused by renal IR injury were detected by constructing a mouse ischemia-reperfusion model.Secondly,by activating Nrf2 pathway to explore the mechanism of Irgl protecting acute kidney injury through dimethyl itaconic acid,and to find the role of Irg1 regulating dimethyl itaconic acid in restrainting AKI induced by ischemia reperfusion after renal transplantation.ObjectiveThe blood samples of patients on the 10th day after operation in Linyi people's hospital renal transplantation center were collected,and the correlation between itaconic acid(IA)and renal function and inflammatory factors was detected by biological methods.O bjective to investigate the protective effect of itaconic acid(IA)on acute renal injury induced by ischemia-reperfusion in C57BL/6 mice.Methods(1)We selected 40 patients who underwent kidney transplantation in the people's hospital of Linyi from December 2017 to December 2020.We collected blood samples on the tenth day postoperative to detect the expression relationship between itaconic acid with SCr,BUN,TNF-? and IL-1?.(2)We divided into three groups which the weighing 23-25g C57BL/6 mice:including Sham operation group(Sham),Ischemia-reperfusion group(IR)and Ischemia-reperfusion group(IR+IA).(3)In IR group,clamped bilateral renal pedicles for 30 minutes before reperfusion.The rats in the treatment group were injected with itaconic acid(500mg/mice)intraperitoneally 2 hours before clamping the renal pedicle.we injected with same volume water in other groups.(4)After 24 hours of reperfusion to collect blood and renal tissue samples.Oxidative stress factors were detected in blood and hematoxylin eosin staining in renal tissue.(5)Using ELISA kit to detect the inflammatory factors.Results(1)By GC-MS/MS method,it was found that the concentration of serum itaconic acid was correlated with SCr,BUN,TNF-? and IL-1? on the tenth day after renal transplantation.We found a relativity between them.(2)With Sham group,the expression of MDA in IR group was visibly higher.Compared with IR group,the MDA expression in IR+IA group was significantly decreased.(3)Compared with Sham group,the ratio of GSH/GSSG in IR group and IR+IA group decreased significantly.Compared with IR,the ratio of GSH/GSSG in IR+IA group was higher.the SOD activity in IR group was lower than Sham group,and that of IR+IA group was also slightly decreased.The SOD activity in IR+IA group was higher than that in IR group.Compared with Sham group,the catalase activity of IR group and IR+IA group was also significantly decreased.The catalase activity of IR+IA group was higher than that of IR group.(4)The expression of TNF-?,IL-1? and iNOS,which in IR group IR+IA group were higher than Sham group,but IR+IA group decreased in varying degrees in comparison with IR group,there was statistical difference.(5)We found that difference Sham group by HE staining,the renal injury in IR group was obvious,at the same time the injure of renal tissue damage in IR+IA group was reduced,also observed the changes of renal tissue injury.ConclusionsIt was found that there was a correlation between serum itaconic acid concentration and inflammatory state and delayed recovery of renal function in renal transplant patients.Itaconic acid can activate the related antioxidant factors and reduce the damage to the kidney.After treatment with itaconic acid,the indexes of related inflammatory factors in renal ischemia-reperfusion mice increased,which stimulated the systemic inflammatory regulatory system and played an anti-inflammatory and protective role in the kidney.Part? Irgl-Dimethyl ItaconateAxisProtects Against Acute Kidney Injury ViaActivation of Nrf2ObjectiveObjective to explore the mechanism of immune response gene1-dimethyl itaconate in protecting renal cells from oxidative stress and preventing macrophage activation by activating Nrf2 transport to the nucleus through establishing renal ischemia-reperfusion model in mice and various biological methods.Methods(1)C57BL/6 mice were used to establish renal IRI model by clamping bilateral renal pedicle for 20 or 40 minutes.The mouse model of kidney transplantation by ransplanteing left kidney t to the recipient.(2)Rat kidney cells were obtained from the kidneys of lactating mice and cultured in vitro with RAW264.7 cells.The expression was detected by WB and RT-PCR.HE staining technique was used to detect the degree of renal ischemia-reperfusion injury.(3)We used ELISA to detect the TNF-? and IL-1? expression levels in serum and culture supernatant.Serum creatinine and urea nitrogen levels were measured by bun or creatinine test kit.(4)siRNA was used to transfect RAW264.7 cells and renal cells.The expression of Irfl and Cebp? were detected by RT-PCR and Western blot.Results(1)Compared with Sham group,the mRNA and protein levels of Irgl in PBMC and kidney tissue of IR group were up-regulated at 0-36 h after ischemia-reperfusion.The difference was statistically significant at 12 h in PBMC and 6 h in kidney tissue.The expression of TNF-? and IL-1? in serum increased continuously after 30 min ischemia and 0-36 h reperfusion,but with decreased at 12 h.(2)After ischemia-reperfusion,the survival rate of Irg1-/-mice or wild-type mice decreased significantly(P=0.0004;Kaplan Meier test).Compared with Sham group,showed that renal cells of Irgl-/-mice were severely damaged after IR.There was difference in Scr between Irg1+/+and Irgl-/-mice at 24 h after 30 min ischemia and 0-36 h reperfusion.The difference of urea nitrogen was statistically significant at 12h.The TNF-? and IL-1? expression between Irgl+/+and Irg1-/-mice after 30 min ischemia and 0-36 h reperfusion has significant differences.(3)After bilateral renal ischemia-reperfusion in Irg1+/+ and Irg1-/-mice,DI treatment significantly prolonged the postoperative survival time.After DI treatment,can significantly reduce the TNF-? and IL-1? expression.(4)After hypoxia treatment,the expression of LDH decreased significantly.The activation of Caspase-3 was reduced by treatment with dimethyl DI under hypoxia by WB.And the expression of Nrf2 and its downstream molecule HMOX1 increased.After H/R treatment,the expression of LDH in DI group decreased significantly after Nrf2 gene knockout mediated by shRNA.(5)The TNF-? and IL-1? expression in macrophages pretreatment DI and in the supernatant of cultured neonatal mouse renal cells in H/R state decreased significantly.The TNF-? and IL-1? expression in the supernatant of macrophages without shNrf2 treated with itaconic acid decreased.Western blot analysis showed that p-p38,p38,pp65,p65,ERK,p-ERK,JNK and p-JNK decreased after treatment with DI.(6)After siRNA interference compared with NC,Irfl and Cebp? were knocked out led to the decrease of Irgl mRNA level.SiCebp?-1 had the highest transfection efficiency in RAW264.7 cells and renal cells.The levels of Irgl mRNA in silrfl and siCebp? transfected RAW264.7 cells were decreased after H/R treatment;However,in renal cells,the levels of Irgl mRNA in siIrfl,siCebp? and PDTC transfected groups were decreased,but the difference were statistically significant in siCebb? group and PDTC treated group.ConclusionsThe Irgl expression was negatively correlated with infl ammatory cytokines in IR injury.DI treatment can significantly improve the survival time of Irg1+/+mice after renal ischemia-reperfusion injury,which also can protect renal ischemia-reperfusion injury and systemic inflammation.At the same time,DI may prevent macrophage activation by promoting Nrf2 transport to the nucleus,thus protecting renal cells from oxidative stress.This study found the mechanism of DI and Irgl in the protection of AKI induced through ischemia-reperfusion,and provided a new therapeutic target for clinical prevention of acute kidney injury. |
PDF Full Text Request