| PART 1Effects of repeating intravenous-iron administration on oxidative stress in chronic renal failure of subtotal nephrectomized ratsBACKGROUNDAnemia is a common complication of chronic kidney disease(CKD) and was associated with increased risk of cardiovascular disease(CVD),morbidity,and mortality,particularly in high-risk populations.Anemia of CKD generally is attributed to absolute or relative erythropoietin deficiency.However,other factors,such as iron deficiency However,other factors,such as iron deficiency,blood loss,shortened red blood cell life span,and inflammation,may contributeto its development.Iron deficency was a common mobidity of CKD,especially during the erythropoiesis stimulating agents(ESA) treatment.Now,intravenous iron preparations had been widely used associated with ESA administration.The efficacy and short-term safety of parenteral Fe administration has been amply documented.However,Fe also is a potent oxidant that is capable of redox cycling.This results in the production of oxygen-based free radicals that can damage tissue proteins,lipids,and RNA/DNA. Oxidative stress produces compounds,such as oxidized low-density lipoprotein (OxLDL),may mediate the development of atherosclerosis by enhancing foam cell formation,fatty streak development,and endothelial lesion progression.As far,Fe supplementation now was almost routinely performed by intravenous administration of Fe-carbohydrate(CHO) complexes(dextran,sucrose,or gluconate polymers). Further more,low weight iron dextran and iron sucrose were commercially available in our country.Intravenous iron therapy increased both plasma and urinary malondialdehyde(MDA) as markers of lipid peroxidation.The elevated MDA levels were transient,and completely resolved in 24 hours.Data had proved that different parenteral iron formulations contained different peroxidative characteristics.There were few data comparing the peroxidative stress differences in these iron preparations with repeated intravenous injection therapy.In this part of our experiment,we compared the effects of repeated low dose infusion of iron dextran and iron sucrose on oxidative stress in CRF rats to get some data about this issue. METHODSMale Sprague-Dawley rats weighing 200-230g(6 weeks age) were used in this study.The chronic renal failure model was established with 5/6 subtotal nephrectomy (5/6 NX) operation.Four weeks after removing the right kidney,rats met the enrolled criteria were randomly divided into low molecular weight iron dextran group,sucrose iron group,CRF control group.We also set up the sham group(Sham group),in which only laparotomy and separation of the kidney peripheral fat vesicle were performed.There were 6 rats in each group.Animals were observed for 6 weeks,the blood,urine and tissue samples were collected,indexes of renal function,anemia,iron status and oxidative stress were investigated.RESULTSIn all three groups of 5/6 NX rats,baseline characteristics such as the proportion of NX,levels of serum creatinine(Scr),blood urea nitrogen(BUN),ratio of urinary protein and creatinine(Upro/cr),hemoglobulin(Hb),hematocrit(Hct),systolic blood pressure(SBP) and bodyweight(BW) had no significant differences.At the end of experiment,there was no difference of BW,SBP,Upro/cr,Scr and BUN among the NX groups.The amount of iron administrated in each rat was similar in iron dextran group and sucrose iron group.The serum iron,ferritin and transferrin were obviously lower in CRF control group than in Sham group(P＜0.05).Serum iron,ferritin and transferrin were significantly higher in the two iron groups than CRF control group group(P＜0.01).There was no significant difference between the iron dextran group and sucrose iron group on iron status indexes(P＞0.05).Compared with CRF control group,the Hb and Hct were significantly improved in iron dextran and sucrose iron group(P＜0.05),but not different between the two iron groups(P＞0.05).The serum superoxide dismutase(SOD),Catalase(CAT) and total anti-oxidant capacity(TAOC) were obviously elevated in all three NX groups comparing the Sham group(P＜0.05). The serum SOD and TAOC in two iron groups was not different with those in CRF control group,and serum CAT was higher in two iron groups than it in CRF control group(P＜0.05).The concentration of plasma Glutathione peroxidase(GSH-Px) was significantly reduced in all NX groups(P＜0.05),plasma GSH-Px was futher lower in sucrose iron group than iron dextran and the CRF control group(P＜0.05).For the concentration of plasma advanced oxidation protein products(AOPP),it was obviously higher in NX groups than it in Sham group,and also higher in two iron groups than in CRF control group(P＜0.05).The plasma malonaldehyde(MDA) was significantly higher in NX groups than that in Sham group(P＜0.05),also higher in sucrose iron group than in CRF control group and iron dextran group(P＜0.05).The renal pathologic changes were similar among all three CRF groups.There were no significant difference in the glomerular sclerosis indexes and the tubular interstitial fibrosis scores.For the affects of intravenous iron injection on the oxidative stress in liver,kidney and cardio tissues,it was more complicated.The concentration of MDA in kidney and liver tissues were higher in two iron groups than in other groups (P＜0.05) and MDA in cardio tissues was significantly higher in Sucrose iron group than Iron dextran group.The SOD in kidney and liver was similar and obviously lower in all NX groups than Sham group(P＜0.01).The level of GSH-Px in liver was significant lower in sucrose iron group than the iron dextran group(P＜0.05).CONCLUSIONSIn the period of four to ten weeks after removing the right kidney,the 5/6 NX rats appear obvious renal insufficiency,anemia and hypertension.The renal dysfunction and the amount of urinary protein excretion keep relative steady.The 5/6 NX CRF rats can develop iron deficiency anemia.Injection of intravenous iron alone can partially correct the anemia state and improve the iron status indexes.Both iron dextran and iron sucrose can increase plasma MDA and AOPP as markers of lipid and protein peroxidation.Intravenous administration of iron can also reduce the plasma GSH-Px as marker of anti-oxidant enzymes.We observe that Iron dextran has some advantages to Sucrose iron on the adverse effects of MDA,GSH-px in palasma and liver tissue.Furthermore,intravenous injection of iron did not adversely affect kidney function and kidney pathologic changes in a relative long period. PART 2Comparison of different intravenous-iron treatment regimens on oxidative stress in chronic renal failure of subtotal nephrectomized ratsBACKGROUNDThere are two intravenous iron treatment regimens,total dose infusion(TDI) therapy and repeated low dose infusion,in clinical practice.We have confirmed that repeated low dose intravenous injection of iron resulted in a significant rise in the lipid and albumin peroxidation products in 5/6 NX rats.It isn't clear if there is any different effect on oxidative stress between the TDI and the repeated low dose infusion therapy in CRF.Is it possible that the TDI therapy has any advantage over the repeated low dose infusion regimen on the oxidative stress in CRF stage? As far, K/DOQI recommends that only iron dextran could be used in total dose infusion.So, in this part of our experiment,we compared the effects of TDI and repeated low dose infusion of iron on oxidative stress in CRF rats to get some data about this issue.METHODSMale Sprague-Dawley rats weighing 200-230g(6 weeks age) were used in this study.The chronic renal failure model was established with 5/6 subtotal nephrectomy (5/6 NX) operation.Four weeks after removing the right kidney,rats met the enrolled criteria were randomly divided into total dose intravenous iron group(TDI group), repeated low dose intravenous iron group(rLDI group),CRF control group.We also set up the sham group(Sham group),in which only laparotomy and separation of the kidney peripheral fat vesicle were performed.There were 6 rats in each group. Animals were observed for 6 weeks,the blood,urine and tissue samples were collected,indexes of renal function,anemia,iron status and oxidative stress were investigated.RESULTSAmong three groups of 5/6 NX rats,there were no difference in baseline characteristics such as the proportion of NX,levels of serum creatinine(Scr),blood urea nitrogen(BUN),ratio of urinary protein and creatinine(Upro/cr),hemoglobulin (Hb),hematocrit(Hct),systolic blood pressure(SBP) and bodyweight(BW).At the end of experiment,there was no difference of BW,SBP,Upro/cr,Scr and BUN among the NX groups.The amount of iron administrated in each rat was similar in TDI group and rLDL group.The serum iron,ferritin and transferrin saturation were obviously lower in CRF control group than Sham group(P＜0.05).Serum iron,ferritin and transferrin saturation were significantly higher in the two iron groups than in CRF control group group(P＜0.01).There was no significant difference between the rLDI group and TDI group on iron status indexes(P＞0.05).Compared with CRF control group,Hb and Hct were significantly improved in TDI group and rLDI group (P＜0.05),but not different between the TDI group and rLDI group(P＞0.05). Compared to Sham group,the serum level of superoxide dismutase(SOD),Catalase (CAT) and total anti-oxidant capacity(TAOC) was obviously elevated in all three NX groups(P＜0.05).The serum level of SOD and TAOC in two iron groups was not different to those in CRF control group,and the serum CAT concentration was higher in two iron groups than that in CRF control group(P＜0.05).As for the serum SOD, CAT and TAOC,there was no significant difference between TDI group and rLDI group(P＞0.05).The concentration of plasma Glutathione peroxidase(GSH-Px) was significantly reduced in All NX groups(P＜0.05),but there has no difference between the two iron groups and the CRF control group(P＞0.05).For the concentration of plasma advanced oxidation protein products(AOPP),it was obviously higher in NX groups than that in Sham group,and also higher in rLDI group than in TDI group(P＜0.05).The plasma malonaldehyde(MDA) level was significantly higher in NX groups than that in Sham group(P＜0.05),while it was also higher in rLDI group than in CRF control group and TDI group(P＜0.05),and had no difference between TDI group and CRF control group(P＞0.05).The renal pathologic changes were similar among the three CRF groups,there were no significant difference in the glomerular sclerosis indexes and the tubular interstitial fibrosis scores.For the effects of intravenous iron injection on the oxidative stress in liver,kidney and cardio tissues, it was more complicated.The level of SOD,GSH-Px and MDA in heart tissues had no significant difference among all four groups.The level of SOD in kidney and liver was similar and obviously lower in CRF groups than in Sham group(P＜0.01).The concentration of MDA in renal tissue was higher in rLDI group than in other groups (p＜0.05).The level of MDA in liver was significant higher in the two intravenous-iron groups than the Sham group and CRF control group.CONCLUSIONSBoth TDI and LDI therapy can correct the anemia and the iron status indexes in 5/6 NX CRF Sprague-Dawley rats.Intravenous-iron can aggravate oxidative stress rather the lipid peroxidation of lipid and albumin than reducing the anti-oxidant enzymes in chronic renal failure rats.Repeated low dose intravenous iron administration resulted in a more significant rise in plasma concentration of albumin and lipid peroxidation product,malondialdehyde and AOPP than total dose intravenous iron administration in the CRF rats.Different regimen of iron dextran intravenous injection has different effects on oxidative stress in kidney,liver and the cardiac tissues in the CRF rats.Furthermore,intravenous injection of iron dextran do not adversely affect kidney function and kidney pathologic changes in a relative long period. PART 3Effects of repeating intravenous-iron administration on oxidative stress in chronic renal failure of subtotal nephrectomized ratsBACKGROUNDThe oxidative stress contributed to the pathogenesis of atherosclerosis,dialysis related amyloidosis and anemia in chronic kidney disease.Antioxidant therapy was one of important approaches for halting progression of chronic kidney disease.It had been mentioned that intravenous injection of routine dose iron resulted in transient rise of lipid peroxidation products as MDA,and transient elevation of urinary NAG enzyme or protein excretion,which might reverse within 24h.In the first and second part of our experiment,we observed that repeated low dose iron infusions or one total dose iron infusion intravenously induced obvious elevation of biomarkers for oxidative stress as MDA and AOPP in a relative long period.These findings indicated that it is necessary to give antioxidant therapy when repeated low dose iron infusions or high dose iron infusion were performed in CKD.High dose infusion of iron had some conveniences in clinical practice for correcting iron deficiency anemia and had been proved to be safe and efficient.While,there hardly had data about the oxidative stress and tissue injuries following with high dose infusion of iron in CKD stages. Would the new found early biomarkers for acute kidney injury as interleukin-18 (il-18),kidney injury molecular 1(KIM-1) and neutrophil gelatinase-associated lipocalin(NGAL) be useful to recognize the high dose iron related acute kidney injury? Antioxidant complement was one usual strategy to alleviate the oxidative stress in CKD.Alpha-tocopherol(Vit E) andα-lipoic acid(α-LA) were used as antioxidant for many years.So,in this part of our experiment,we studied the changes of oxidative stress,inflammatory factors and some biomarker of acute kidney injury in 5/6 subtotal neprhectomy rats when high dose iron(iron element 50mg/kg) was administrated intravenously alone or companied with Vit E orα-LA.METHODSMale Sprague-Dawley rats weighing 200-230g(6 weeks age) were used in this study.The chronic renal failure model was established with 5/6 subtotal nephrectomy (5/6 NX) operation.Four weeks after removing the right kidney,rats met the enrolled criteria were randomly divided into low molecular weight iron dextran group(Fe group),low molecular weight iron dextran withα-tocopherol group(Fe+Vit E group),low molecular weight iron dextran withα-lipoic acid group(Fe+α-LA group) and CRF control group.Vit E andα-LA were administrated intragastrically by gavage with a dose of 100mg/kg respectively 5 days before intravenously injecting high dose iron dextran and lasted until sacrificing the rats.Iron dextran was injected through tail veins at dose of 50mg/kg.Rats in control group were correspondently treated with normal saline.Animals were killed 24h and 72h after injecting iron.There were 6 rats in each time point in the four groups.The blood,urine and tissue samples were collected,indexes of oxidative stress,inflammation and the mRNA expression of early biomarkers for acute kidney injury as KIM-1,IL-18,and NGAL in renal cortex were investigated.RESULTSIn all four groups of 5/6 NX rats,at the time of 24h and 72h after intravenously injecting iron,characteristics such as levels of serum creatinine,blood urea nitrogen, hemoglobulin,systolic blood pressure and bodyweight had no significant differences (P＞0.05).Serum SOD,GSH-Px of rats in Fe group,Fe with Vit E group and Fe withα-LA group were significant lower than CRF control group 24hs and 72hs after injecting iron dextran(P＜0.05).At the time of 24hs after iron administration,serum CAT and TAOC of rats in Fe group have no difference with CRF control group (P＞0.05),serum CAT and TAOC of rats in Fe withα-LA group were obvious higher than Fe group and CRF control group(P＜0.05).72hs after iron injection,serum CAT and TAOC of rats in Fe group rised to a significantly higher level than CRF control group(P＜0.05).Serum CAT and TAOC of rats in Fe withα-LA group and Fe with Vit E group were rather higher than Fe group and CRF control group(P＜0.05).At the time of 72hs after iron treatment,rats' plasma AOPP were not different among the four CRF groups(CRF control group Vs Fe group Vs Fe withα-LA group Vs Fe with Vit E group,51.31±9.56 Vs 66.83±18.61 Vs 67.81±11.38 Vs 63.97±19.19μmol/L, P＞0.05).High dose infusion of iron dextran resulted in a obvious rise of serum TNF-αand MCP-1 at the first 24hs after iron treatment(P＜0.05),and there were no difference about serum TNF-αand MCP-1 among the three groups(P＞0.05).72hs after iron injection,serum TNF-αof rats in Fe group,Fe with Vit E group and Fe withα-LA group declined obviously than 24hs'(P＜0.05),but the serum MCP-1 still kept higher than CRF control group except in Fe with Vit E group.For the affects of intravenous iron injection on the oxidative stress in liver,kidney and cardio tissues,it was more complicated.The concentration of MDA,SOD,GSH-Px in kidney and GSH-Px in liver had no difference both at 24hs and 72hs after iron injection in all group(P＞0.05).The level of MDA in liver in Fe withα-LA group was significant lower than Fe group and Fe with Vit E group either at 24hs or 72hs after giving iron (P＜0.05),while liver MDA in Fe group and Fe with Vit E group was higher than CRF control group(P＜0.05).The SOD,GSH-Px in cardio tissue were obvious lower in Fe group and Fe with Vit E group than in CRF control and Fe withα-LA group(P＜0.05). Compared to rats in CRF control group,Expression of KIM-1 mRNA in renal cortex in Fe group down-regulated significantly 24hs after iron administration(P=0.001), and reversed at the time 72hs.While expression of KIM-1 mRNA in renal cortex in Fe with Vit E group or Fe withα-LA group had no difference with CRF control either at 24hs or 72hs after iron treatment.The IL-18 and NGAL mRNA in renal cortex expressed obvious up-regulated in Fe group 24hs after injecting iron(P＜0.05).On the contrary,IL-18 and NGAL mRNA in renal cortex expressed obvious down-regulated in Fe with Vit E group or Fe withα-LA group than in Fe group and CRF control group (P＜0.05).For the 72hs after iron treatment,Expression of IL-18 and NGAL mRNA in renal cortex had no difference in all group(P＞0.05).CONCLUSIONSHigh dose intravenous-iron administration of low weight molecular iron dextran (50mg/kg) results in significant oxidative stress and inflammation with declining of serum antioxidant enzymes SOD,GSH-Px and raising serum level of TNF-αand MCP-1 in a short-term period.Oral administration ofα-tocopherol andα-lipoic acid can partially alleviate this oxidative stress,but has no protective effect on iron induced inflammation.At the time of 24h and 72h after high dose infusion of iron dextran,early biomarkers for acute kidney injury as IL-18 and NGAL mRNA express transiently upregulated in renal cortex of CRF rats,which can be prevented by Vit E andα-LA.Vit E andα-LA may have some renal protective effects when giving intravenous injection of high dose iron in CRF rats.
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