| BACKGROUNDContrast-induced nephropathy(CIN)is an iatrogenic disease induced by injection of iodinated radiographic contrast media.CIN is defined as an ARF that occurs within 24～72 hours of exposure to iodinated contrast media injection except for the setting of other causes of renal injury.The renal injury is defined as any of the following:increase in serum creatinine by ≥ 0.5 mg/dl(≥ 44.2μmol/l);serum creatitine to 1.25 times baseline;estimated glomerular filtration rate(eGFR)less than 60 mL/min;or decreased in eGFR more than 30 ML/min · m3.The pathophysiology of CIN may be attributed to following mechanisms:①renal hemodynamic changes induced by contrast agents ② directly nephrotoxity induced by contrast agents ③ blockage tubular ④Contrast agents may also cause osmotic diuresis which might increase energy demand and oxygen consumption.These reactions might lead to a worsening of medullary hypoxia,and further renal injury.Medullary hypoxia increseaing the production of reactive oxygen species(ROS)that may lead to tubular and vascular endothelial injury and might further intensify renal parenchymal hypoxia.It is reported by previous studies that hydration therapy is effective in preventing CIN,.The potential mechanism is reducing contraction of the renal arteries by decreasing the concentration of contrast agent in the renal arteries.,increasing renal blood flow and increasing the excretion of contrast agent.Previous studies have reported that antioxidant drugs may be useful for preventing CIN.Firstly,N-acetylcysteine,a free-radical scavenger and a drug able to increase the vasodilating effect of NO,was used to prevent CIN.However,in clinical trials found that the renal protective effect of N-acetylcysteine,in patients undregoing contrast agent injection.It is an urgent need to explore effective methods to prevent contrast media-nephrotoxicity.Severe hyperuricemia can lead to both AKI and CKD.However,whether the long-term mildly elevated serum uric acid levels can lead to kidney injury is unclear.In the past,soluble uric acid was regarded as a physiologically unactive substance.The general view was that the mildly to moderate increase in serum uric acid levels without gout did not play a pathogenic role.Therefore,in clinical practice,hyperuricemia was generally considered as a sign of renal insufficiency but not a risk factor of CKD progression.Recent studies have confirmed that hyperuricemia is an independent risk factor for kidney disease,however,whether hyperuricemia is an independent factor for kidney disease is not confirmed.To explore the association between hyperuricemia and CKD,recognizing the pathogenic role of mildly elevation of serum uric acid in the development and progression of renal disease is one of key roles.Generally,it is thought that mildly to moderate elevated serum uric acid without gout does not have a pathogenic role for renal disease.That is another key issue need to be addressed in the current studyNrf2-ARE(antioxidant response element),an important antioxidant pathway in body,is an antioxidant enzymes and a common promoter sequence of detoxification enzyme.In vivo,Nrf2 is an important nuclear transcription factors.It also can combine ARE in vivo.Growing literatures suggest that the activation of Nrf2-ARE signaling pathway is effective in anti-oxidative stress induced-injury.Nrf2,an important transcription factor,is often called heme-binding protein 1(HEBP1).In vivo,it can up-regulate cytoprotective genes expression when oxidative stress injury or external stimuli damage.Nrf2 is also a potent transcriptional activator.Many studies suggest that activation of Nrf2-ARE signaling pathway is effective in againsting oxidative stress injury.HO-1 is also an important antioxidant enzyme protein and has a strong antioxidant effect when the organisms are undergoing external stimuli damage or oxidative stress.Sulforaphane(SFN),a potential antioxidant,is abundant in cruciferous vegetables.It has transcriptional activation Nrf2 which can activate downstream reaction of antioxidant response.It is a new promising agent for the prevention of a range of diseases related to oxidative action.The antioxidant effect of SFN has been investigated in acute liver injury,acute kidney injury and chronic kidney injury.In our study,the aim of the first part was to investigate the antioxidant effects of sulforaphane(SFN)on CIN in a rat experiment including rat renal function,pathology,oxidative stress and other related indicators.The aim of the second part was to investigate the antioxidant effects of sulforaphane(SFN)on the hyperuricemia in the progression of chronic kinder disease in rat models.Part I The role of Nrf2/HO signaling pathway in the pathogenesisof Contrast-induced nephropathyObjectiveThe aim of this srudy was to investigate the antioxidant effects of sulforaphane(SFN)on CIN in a rat model of and the oxidative stress model in HK2 cells induced by H202 or contrast agent,and to observe the SFN effect on rat kidney function,pathology,oxidative stress and other related indicators.Methods:Twenty-four Male Sprague-Dawley rats were randomized into four groups(n=6).The control group received intravenous saline injection.The contrast media(Ioversol)group received intravenous loversol injection to induce CIN.The loversol+SFN group received intraperitoneal SFN for 5 days before the induction of CIN.The SFN group was given an equal amount of SFN for 5 days before the induction of CIN.The effect of SFN was analyzed on loversol—induced injury in HK2 cells.Renal function,malondialdehyde(MDA)and reactive oxygen species(ROS),were measured.Renal injury was also messureed by histologic examination.Westernblot,real-time polymerase chain reaction analysis and immunohistochemical analysis were performedto detect nuclear factor erythroid-derived 2-like 2(Nrf2)and heme oxygenase-1(HO-1).To explore the contribution of Nrf2 signaling to the renoprotective effect of SFN in HK2 cells undergoing Ioversol-induced injury,the survival and viability of HK2 cells transiently transfected with Nrf2 siRNA were assessed after loversol treatmentResults1.SFN ameliorated CIN-Associated Renal Dysfunction in CIN rats.Both the serum creatinine and BUN levels were significantly increased in the Ioversol group,compared with the control group(P<0.001).In the Ioversol +SFN group,the administration of SFN significantly decreased the serum creatinine and BUN levels,compared with Ioversol group(P<0.001).2.SFN ameliorated renal histological damage.The kidney sections of the control group did not exhibit marked histological changes.The kidney sections of the Ioversol group exhibited severe damage,consisting of tubular lesions,tubular necrosis,and hemorrhagic casts.In the Ioversol +SFN group,pretreatment with SFN significantly attenuated the development of tissue damage.Nrf2 immunopositivity was especially marked in the glomeruli and tubular epithelium.Strong positive Nrf2 staining was detected in the tissue of CIN rats treated with SFN.Due to severe oxidative stress in CIN rats,Nrf2/HO-1 signaling was significantly activated(P<0.05,Ioversol vs.control group).3.SFN decreased MDA and increased SOD in renal tissues.The levels of MDA in the renal tissue in the Ioversol group were significantly higher than those in the control group(P<0.05).SFN significantly attenuated these increases in the MDA levels in the Ioversol + SFN group,and the MDA levels did not significantly differ between the control and Ioversol +SFN groups.Moreover,contrast media decreased the SOD activities,but this effect was inhibited by pretreatment with SFN.4.SFN pretreatment enhances Nrf2 target gene expression.The administration of SFN significantly increased the gene expression ofNrf2,NQO-1,and HO-1 compared to other groups.5.SFN pretreatment enhances Nrf2 nuclear translocation and increases NQO-1and HO-1 protein expression.To confirm that SFN treatment activates the Nrf2/HO-1 pathway,the protein levels of Nrf2,HO-1,and NQO-1 were measured by western blot analysis.SFN treatment significantly increased Nrf2 nuclear translocation Moreover,SFN treatment also increased the HO-1 and NQO-1 protein levels.6.SFN protects against oxidative stress and increased cell viability in vitro.The effects of SFN on the cellular ROS levels induced by H2O2(500 μ mol/L)or 50 mg/mL Ioversol in vitro were measured using proximal tubule(HK2)cells.H202 or Ioversol significantly increased the ROS levels in HK2 cells.However,the H2O2-or Ioversol-induced ROS increase was significantly inhibited by pretreatment with SFN in a dose-dependent manner.SFN pretreatment protected against Ioversol-induced cytotoxicity in MTT assays.the administration of SFN significantly increased Nrf2,NQO-1,and HO-1 gene expression in Ioversol-injured cells.7.SFN increased Nrf2,NQO-1,and HO-1 gene expression in Ioversol-injured cells.The administration of SFN significantly increased Nrf2,NQO-1,and HO-1 gene expression in Ioversol-injured cells.8.SFN exerts its renoprotective role via the activation of Nrf2 in HK2 cells.we investigated the effects of SFN on Nrf2 and HO-1 expression in Nrf2-deficient cells.SFN did not increase the cell viability of Nrf2-deficientcells.However,the knockdown of Nrf2 increased the ROS level.CDDO-ME also attenuated ROS in a dose-dependent manner.ConclusionOur study demonstrated that SFN ameliorates CIN,as measured by renal function and kidney pathology.Moreover,SFN increased Nrf2 nuclear translation,the HO-1 protein level,cell viability,and expression of Nrf2,HO-1,and NQO-1in Ioversol-injured HK2 cells.These beneficial effects are mainly due to the improved antioxidant defense in the kidney and the activation of the Nrf2 pathway.Part ⅡThe role of Nrf2/HO signaling pathway in the pathogenesis of mild hyperuricemia in the progression of chronic kidney disease ObjectiveIn this study,by constructing a mildly high uric acid chronic renal failure rat model,and to investigate the antioxidant effects of sulforaphane(SFN)on this model,to observe the SFN effect on rat kidney function,pathology,oxidative stress,Nrf2/HO-1 pathway and other related indicators.MethodsForty Male Sprague-Dawley rats were randomized into five groups(n =8).The control group had a bilateral renal capsule excision to discharge the impact caused by surgery.The renal failure(RK)group has both sides 5/6 nephrectomy.The oxonic acid group had a bilateral renal capsule excision and administrated 750mg/kg/d oxonic acid for 6 month.The renal failure plus mildly uric acid group(RK+OA)had 5/6 nephrectomy and administrated 750mg/kg/d oxonic acid for 6 month.The renal failure plus mildly uric acid and SFN group(RK+OA+SFN)had 5/6 nephrectomy and administrated 750mg/kg/d oxonic acid and received intraperitoneal SFN 5mg/kg/d for 6 month.Six month later the rat was sacricefyied.After 6 weeks rats were killed specimens of blood serum uric acid,urea nitrogen and serum creatinine,24-hour urine specimens 24-hour urine protein excretion,and renal tissue specimens observed renal pathological changes,MDA,SOD vitality case,immunohistochemistry observation of kidney tissues of Nrf2 and HO-1 immunohistochemical expression situation,Westernblot method of detecting tissue Nrf2,HO-1,Keap-1,GSK/pGSK,P38/p-P38,ERK/p-ERK protein expression.The above data were statistically analyzed to P<0.05 was considered statistically significant..Renal function tests,malondialdehyde(MDA)and reactive oxygen species(ROS),24-hour urine protein excretion,was measured.Histologic examination was carried out for renal injury.Westernblot and immunohistochemical analysis were performed for Nrf2,HO-1,Keap-1,GSK/pGSK,P38/p-P38,ERK/p-ERK protein expression detection.Results1.SFN attenuated renal dysfunction in RK+OA ratsCompared with the control group,uric acid was significantly increased in OA group and RK+OA group,with significant statistically difference(P<0.05),but no significant changes between RK group(P>0.05);Compared with RK group,uric acid was increased significantly in RK + OA group;compared with RK+OA group,RK +OA + SFN group significantly lower uric acid((P<0.05),the difference was statistically significant.Compared with the control group,serum creatinine,blood urea nitrogen,24-hour urinary protein excretion were no significant difference in OA group(P>0.05),those indicators were significantly increased in RK+OA group(P<0.05);compared with RK group,serum creatinine,blood urea nitrogen,24-hour urinary protein excretion was significantly increased in RK+OA group(P<0.05);compared with RK+OA group,serum creatinine,blood urea nitrogen,24-hour urinary protein excretion was significantly lower in RK+OA + SFN group((P<0.05).2.SFN ameliorated renal histological damage.The kidney sections of the control group and OA group animals did not exhibit marked histological changes.The kidney sections of the RK group and RK+OA group animals exhibited glomerulosclerosis and hypertrophic,tubular atrophy and/or compensatory expansion,renal interstitial inflammatory cell infiltration,interstitial fibrosiss.The changes was obviously in the RK+OA group.In the RK + OA + SFN group group,treatment with SFN significantly attenuated the development of these lesions and tissue damage.3.SFN attenuated MDA levels and increased SOD levels in renal tissues.The levels of MDA in the renal tissue in the RK group and RK+OA group were significantly higher than those in the control group(P<0.001),and the SOD activity was lower in the RK group and RK+OA group compared with the control group(P<0.05).The MDA level and SOD activity were no significant difference between OA group and control group(p>0.05).SFN significantly attenuated these increases in the MDA levels in the RK+OA group,and the MDA levels did not significantly differ between the control and RK + OA + SFN groups.Moreover,SFN increased the SOD activity in the RK+OA+SFN group compared with those in the RK+OA group.The MDA level was no significant difference between control group and RK+OA +SFN group.4.SFN pretreatment enhances Nrf2 target gene expression.Due to the combination effects of oxidative stress and inflammation existing,Nrf2 was significantly damaged in the RK group and RK+OA group,Nrf2 protein levels and HO-1 protein were lower in RK and RK+OA group than the control group by IHC analysis and westernblot analysis,Nrf2 and HO-1 protein expression were higher in the OA group than the control group.In the RK+OA+SFN treatment group,SFN significantly increased the expression of Nrf2 nuclear protein and HO-1 levels.5.SFN treatment enhanced protein phosphorylation p-GSK,reducing the phosphorylated P-p38 protein phosphorylation p-ERK.phosphorylated p-GSK rarely expressed in the control group,phosphorylated GSK reduced in the RK + OA group,treatment with SFN P-GSK was significantly enhanced phosphorylation GSK expression in the RK+OA+SFN group(P<0.05).Total P38,ERK protein level in each group had no significant difference.the phosphorylation p-P38 and the phosphorylation of p-ERK were rarely expressed in the control group,p-P38,p-ERK expression was significantly increased in RK group and RK + OA group.Treatment with SFN significantly reduced the expression of p-P38 and p-ERK in the RK+OA+SFN group(P<0.05)Conclusion1.slightly mildly high uric acid accelerated the progression of chronic kidney disease.2.Nrf2/HO-1 attenuated the progression of mild hyperuricemia chronic kidney disease.3.SFN attenuated chronic renal failure and high uric acid induced renal injury:reduce serum uric acid,improve renal function and reduce proteinuria and mitigate fibrosis.4.SFN plays a renal protective effect byactiving Nrf2/HO-1 pathway,enhancing GSK phosphorylation,inhibition of P38 phosphorylation and ERK phosphorylation... |
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