| Part One: Changes of iron metabolism in rats after renal ischemia/reperfusion injuryObjective: To investigate iron metabolism in rats after renal ischemia/reperfusion injury(IRI),probing into the change and significance of hepcidin and iron metabolism after IRI in rat.Methods: We constructed a rat IRI model,and a total of 48 male Sprague-Dawley rats were randomly divided into IRI group(n=42)and control group(n=6).The rats in IRI group were divided into seven subgroups(n=6 each):IRI 1h,IRI 4 h,IRI 8 h,IRI 12 h,IRI 16 h,IRI 20 h,IRI 24 h.Once the rats were under anesthesia,the right kidney of rats was removed and a non-traumatic vascular clamp was applied to the left renal pedicle for 45 min.Then,the clamp was removed for renal reperfusion.The rats in the control group underwent right kidney excision without clamping of the left renal pedicles.Samples were collected at 1 h,4 h,8 h,12 h,16 h,20 h and 24 h after renal reperfusion in the IRI rats and at 1 h after a 45-min exposure of the left kidney in the control rats.After we successful established the IRI model,we measured and analyzed the serum creatinine(Scr),blood urea nitrogen(BUN),hepcidin,serum iron(SI)and serum ferritin(SF)in blood;the iron content in liver,kidneys,spleen and duodenum;the pathological change in kidneys;the expression of hepcidin mRNA and protein in liver,and the expression of ferroportin 1(FPN1)mRNA and protein in kidneys.Results: Scr and BUN were time-dependent increased in the IRI rats than rats in the control group(P<0.05).Compared with rats in the control group,renal iron content,SI and sSF in the IRI rats increased early after renal ischemia reperfusion(P<0.05),and then declined.In the IRI rats,the splenic iron content decreased significantly in the early stage after reperfusion(P<0.05)and then increased time-dependently with increasing reperfusion time,the hepatic iron content showed a decrease in the early stage after reperfusion,but had no significance difference compared with rats in the control group(P>0.05).In addition,the duodenal iron content in the IRI rats showed time-dependently decreased since 12 h after reperfusion in the IRI groups compared to the control group.The expression level of hepcidin mRNA in the liver significantly increased early after renal reperfusion,and the serum concentrations of hepcidin increased obviously since 8h after renal ischemia reperfusion in the IRI rats(P<0.05).The expression level of FPN1 protein in the kidney declined obviously after renal ischemia reperfusion in the IRI rats(P<0.05).Conclusion: The changes in iron metabolism indexes observed in our study demonstrate an iron metabolism disorder in renal IRI,and hepcidin might be involved in maintaining iron homeostasis in renal IRI.Part Two: The role of hepcidin in iron homeostasis regulation and kidney protection in rats during renal ischemia/reperfusion injuryObjective: To explore the role of hepcidin in iron homeostasis regulation and its relationship with kidney injury during renal ischemia/reperfusion injury(IRI).Methods: A total of 48 male Sprague-Dawley rats were randomly divided into hepcidin group(n=24)and normal saline group(normal)(n=24),and then the rats in each group were randomly divided into control,IRI 4h,IRI 12 h,IRI 24 h groups respectively by differences in ways of model construction and renal reperfusion time,n=6 each group.Rats in the hepcidin group were respectively given synthesized hepcidin by intraperitoneal injection at 24 h and 16 h before model construction,while rats in the normal group were given 1.0 ml normal saline instead in the same way.Once the rats were under anesthesia,the right kidney of the IRI rats was removed and a non-traumatic vascular clamp was applied to the left renal pedicle for 45 min.Then,the clamp was removed for renal reperfusion.The rats in the control group underwent right kidney excision without clamping of the left renal pedicles.Samples were collected at 4 h,12 h and 24 h after renal reperfusion in the IRI rats,and after a 45-min exposure of the left kidney in the control rats.After we successful established the IRI model,we measured and analyzed the serum creatinine(Scr),blood urea nitrogen(BUN),hepcidin,serum iron(SI)and serum ferritin(SF)in blood;the iron content in liver,kidneys,spleen and duodenum;the pathological change in kidneys;the expression of hepcidin mRNA and protein in liver,and the expression of ferroportin 1(FPN1)mRNA and protein in kidneys.Results: Comparison between the two groups of the hepcidin and normal at the same time,the level of Scr,BUN in blood,scores of kidney injury and the renal iron content were significantly decreased at 4h and 12 h after reperfusion in the hepcidin group(P<0.05).The concentration of serum hepcidin and the splenic iron content were significantly increased,while duodenal iron content was significantly decreased in the hepcidin group(P<0.05).However,there was no statistical difference of hepatic iron content between the hepcidin group and the normal group(P>0.05).The hepcidin mRNA and protein expression in liver and FPN1 protein expression in kidney were significantly decreased in the hepcidin group(P<0.05).There was no statistical difference in FPN1 mRNA in kidney between the two groups of the hepcidin and normal(P>0.05).Conclusion: Hepcidin can stimulate the iron intake in spleen,and inhibit the iron absorption and exportation in duodenum.Besides,hepcidin can inhibit the iron release of liver and spleen,and reduce the degree of SI during renal IRI.The intervention of synthesized hepcidin could down-regulate endogenous liver hepcidin expression,but has no influence in the up-regulation of iron-mediated liver hepcidin expression in renal IRI.The iron metabolism regulation of hepcidin may have a protective effect on kidney in renal IRI. |
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