Targeting HO-1 By EGCG Reduces Contrast-induced Renal Injury Via Anti-oxidative Stress And Anti-inflammation Pathways

Background:Contrast-induced nephropathy(CIN)is defined as an absolute(≥0.5mg/dl) or relative(≥ 25%) increase in serum creatinine value at 48~72h after exposure to contrast media(CM) compared to the baseline serum creatinine value, while alternative explanations for renal impairment have been excluded. CIN is considered a common complication after percutaneous coronary intervention(PCI) and a common cause of iatrogenic acute kidney dysfunction, especially in high-risk cohort of patients who suffered from diabetes mellitus, chronic heart failure, chronic kidney diseases and advanced age, the incidence of CIN may reach to 20%~30%. According to a risk scoring system to predict the incidence of CIN after PCI reported by Mehran et al, a risk score >6, 6~10, 11~16, or >16, predicts a CIN incidence of 7.5%, 14%, 26%, or 57%, respectively.Patients underwent PCI usually have several chronic cardiovascular diseases and are stratified as high-risk CIN candidates. The disease course of CIN may be short or long, the condition may be mild or severe, however at least 1% CIN patients needs hemodialysis. CIN is one important reason for the poor prognosis after PCI treatment. The outcome of CIN could not be overlooked. The occurrence of CIN can significantly increase the possibility of developing chronic kidney dysfunction in the long run, and chronic kidney dysfunction itself in turn becomes a strong prognostic factor for increased adverse cardiovascular events. Despite endeavors had been made to reduce the occurrence of CIN, including risk stratification for the individual patient, hydration, and newer and safer contrast media, the occurrence of CIN in high-risk patients still remains. So far, there still lacks evidence-prooved agent to treat CIN, including N-acetylcysteine, sodium bicarbonate, fenoldopam, statins, limb ischemic preconditioning, and preemptive hemodialysis. How to effectively treat CIN is always an issue concerned by all interventional cardiologists.Although the precise mechanisms that cause CIN are not fully understood, there seems to be a consensus that disturbance of oxygen homeostasis caused by CM and its toxicity per se, lead to the acute tubular necrosis.(1) Decreased oxygen supply: CM can induce vasoconstriction of the vasa recta, decreasing blood flow and oxygen delivery to tubular cells, especially in the Henle’s loops or medullary thick ascending limbs(mTAL)-located outer medulla, which is rich in mitochondria and oxygen demanding in its active re-obsorption action and is the most vulnerable portion in the acute insult. What makes the condition worse is the levels of regional nitric oxide(NO), prostanoids(PGs) and adenosine, which exert great modulatory effects on the vasorelaxation of microcirculation under physiological conditions, are reduced in high-risk CIN patients with chronic renal-impairment risks, leading to sustained intrarenal vasoconstriction.(2) Increased oxygen demand: The high-osmotic diuretic effect of CM adds the burden of the active re-obsorption to the mTAL, increasing oxygen demand, exacerbating the state of hypoxia and mitochondrial damages.(3) Direct cellular toxicity: CM can make the epithelial cells shrink, nuclei protrude; make the endothelial cell layer open windows, reduce the synthesis of NO, increase apoptosis and necrosis. Eventually, the state of renal hypoxia induced by CM triggers a series of reactions that lead to the release of free radicals, causing cellular damage and initiating the vicious cycle of oxidative stress and auto-inflammation, leading to severe acute kidney injury(AKI); self-amplifying uncontrolled inflammation response may further impair renal function, advancing the AKI into chronic kidney dysfunction. For renal cells, three blows, that are oxygen stress, toxicity of CM and sterile inflammation secondary to tissue necrosis, have to be underwent. Therefore, an effective treatment strategy should involve the use of medications that target the regulators of both renal oxydative stress and inflammation.(-)-epigallocatechin-3-gallate(EGCG) is a catechin monomer purified from green tea, a flavonoid classified under plant polyphenols, is the most abundant, active and widely studied catechin. Hitherto, a large amount of documents have demonstrated the antioxidant and anti-inflammatory features of EGCG in diseases of cardiovascular system, lung, liver and kidney. The protective effects of EGCG have been reported in various acute and chronic kidney diseases, including obstructive nephropathy, cisplatin nephrotoxicity, renal ischemia-reperfusion injury, kidney damage induced by extracorporeal circulation, diabetic nephropathy, lupus nephritis, and chronic glomerulonephritis. Whether or not EGCG has a protective effect on CIN is unknown.Objective:The present study is aimed to establish a model of contrast-induced renal injury in rat, and observe the therapeutic effect of intravenous EGCG, discuss the antioxidant and anti-inflammatory pathways influenced by EGCG, and investigate the key molecule target of EGCG.Methods:1. Firstly, establish the model of contrast-induced renal injury in rat: Male Sprague-Dawley(SD) rats, weighing 220~250g, were used for the experiments. Indomethacin, Nω-nitro-L-arginine methyl ester hydrochloride(L-NAME) and iopromide, were sequentially intravenously injected to establish the model. Rats were devided into three groups: the vehicle group, CM group and CM+EGCG group(N=5). Serum creatinine(Cr), blood urea nitrogen(BUN) were measured by an automatic biochemistry analyzer. The natural disease course of 72 hours in this renal injury model in rat was observed, to choose the best observing time-point in reflecting the renal injury.2. Then, evaluate the protective effects of different EGCG interventions: Varying dosages of EGCG(5, 10, 20mg/kg body wt) were administered intravenously before the establishment of CIN to observe the effect on serum Cr, BUN and creatinine clearance(CrCL). The best concentration was chosen for the subsequent interventional experiments. Thereafter, EGCG was administered intravenously at different time-points(before or after the establishment of model) to observe the effect on renal function, histological injury site, pathological scoring(H&E staining), and cell apoptosis(TUNEL),so as to comprehensively evaluate the therapeutic effect of EGCG on the contrast-induced renal injury.3. Next, evaluate the antioxidant effect of EGCG and investigate its antioxidant molecular mechanism: Markers of oxidative stress: the marker of lipid peroxidation--malondialdehyde(MDA) and the antioxidant enzyme─superoxide dismutase(SOD) in the renal homogenates were determined by colorimetric method according to their respective assay kit instructions to evaluate the antioxidant effect of EGCG on the contrast-induced renal injury. Furthermore, the nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1) antioxidant signaling pathway was investigated. Renal protein expression and histological distribution of HO-1 were detected by western blotting and immunofluorenscence staining. After nuclear protein extraction was prepared according to the extraction kit instruction, protein expression of Nrf2 was detected by western blotting.4. Evaluate the anti-inflammatory effect of EGCG and investigate its anti-inflammatory molecular mechanism: Markers of inflammation: the marker of neutrophil activation─myeloperoxidase(MPO) and the pro-inflammatory cytokine─interleukin-1β(IL-1β) in the renal homogenates were determined by colorimetric method and enzyme-linked immunosorbent assay(ELISA) respectively according to their assay kit instructions to evaluate the anti-inflammatory effect of EGCG on the contrast-induced renal injury. Furthermore, the NLRP3(nucleotide-binding oligomerization domain-like receptor protein 3) inflammasone/IL-1β anto-inflammatory signaling pathway was investigated. Renal protein expression of NLRP3 was detected by western blotting and demonstrated by immunofluorenscence staining.5. Furthermore, investigate the key molecule target in the antioxidant and anti-inflammatory actions of EGCG, observe the influence of HO-1 inhibitor on the renal protective effect of EGCG: Inhibition of the systematic HO-1 beforehand was performed by intraperitoneal injection of protoporphyrin IX zinc(II)(ZnPP) or tin protoporphyrin IX dichloride(SnPP), designated as ZnPP+EGCG+CM group and SnPP+EGCG+CM group respectively. Renal function, i.e. serum Cr and BUN, was determined to observe the role of HO-1 in the protective effect of EGCG on the contrast-induced renal injury. Markers of oxidative stress(MDA and SOD), markers of inflammation(MPO and IL-1β) in kidney were measured to observe the inhibition of HO-1 on the antioxidant and anti-inflammatroy effects of EGCG in preventing against contrast-induced renal injury. To investigate the role of HO-1 in the anti-inflammatroy signaling pathway of EGCG, NLRP3 protein expression was detected under the condition of HO-1 inhibition6.6. All data were analyzed by SPSS 13.0 and presented as mean ± SEM. Data were compared by one-way ANOVA with Bonferroni post-hoc test for multiple comparisons, after checking for normality(Kolmogorov-Smirnov) and homogeneity(Levene). The H&E scores among groups were compared by the nonparametric Kruskal-Wallis test. P<0.05 was considered statistically significant.Results:1. Evaluation of the model of contrast-induced renal injury in ratSerum Cr and BUN were measured at 24 h, 48 h and 72 h to observe the natural disease course of the model. The levels of serum Cr and BUN were found to peak at 24 h after the establishment of the model, decrease at 48 h, and almost recede back to normal at 72 h. Compared with the vehicle group, the differences at time-points 24 h and 48 h have statistical significances(P<0.05). The time-point at 24 h was chosen as the optimal time-point to evaluate renal function in this contrast-induced AKI model and subsequent interventional experiments.2. Evaluation of the protective effects of EGCG interventions(1) Varying dosages of EGCG(5, 10, 20mg/kg body wt) were administered intravenously before the establishment of CIN to observe the protective effects on the contrast-induced renal injury. All three dosages of EGCG were found able to reduce the extent of CIN(P<0.05). As compared with the CM group, serum Cr was reduced 12.8%, 47% and 52% in three dosage groups respectively, serum BUN was reduced 26%, 36% and 43%, CrCl increased 0.5 fold,2 fold and 1.8 fold at 24 h after the injury. The optimal effect of EGCG was found at the dosage of 10mg/kg body wt, which was chosen for the subsequent experiments.(2) EGCG(10mg/kg body wt) was given intravenously 15 min before or after the establishment of CIN(pre-EGCG+CM group or post-EGCG+CM group) to observe the protective effects of EGCG interventions at different peri-operative time-points. It resulted that EGCG given just after the establishment of CIN model could equivalently ameliorated the CIN-associated increase in serum Cr and BUN(P>0.05).The major damages were in the tubules of the outer medulla(location of mTAL) by histopathological observation(H&E staining). Grading assessment of the renal medullary damage including tubular vacuolar degeneration/necrosis, tubular casts and interstitial congestion, indicated a significant increase of the histopathologic score after contrast-induced renal injury, and both EGCG-treated groups ameliorated the extent of medullary damages in CM group(P<0.05). The number of TUNEL-positive nuclei, reflecting the cell apoptosis, was substantially increased after contrast-induced AKI, and reduced by EGCG either pre- or post-treatment(P<0.05).3. Changes of oxidative stress in kidney(1) MDA content and SOD activity were determined in the renal homogenates, reflecting the extent of lipid peroxidation and the level of antioxidant enzyme, respectively. Renal MDA was significantly increased and activity of SOD was decreased after contrast-induced renal injury(P<0.05). Compared with the CM group, EGCG treatment evidently decreased the renal MDA content and increased the activity of SOD, ameliorating the state of renal oxidative stress(P<0.05).(2) Changes of the Nrf2/HO-1 antioxidant signaling pathway in kidney were detected by Western blot after contrast-induced renal injury and EGCG intervention. The renal protein expression of HO-1 was increased after contrast-induced renal injury, and EGCG treatment increased HO-1 expression to an even higher level(P<0.05). Immunofluorescence staining of HO-1 demonstrated that in vehicle group HO-1 was mainly expressed in tubules, rather than in glomeruli; HO-1 was increased in the tubules both in cortex and medulla either after CM-induced renal injury or EGCG treatment, only with different high levels. Nrf2 protein expression in the nuclei, the upper-stream antioxidant nuclear transcription factor of HO-1, was evidently reduced after contrast-induced renal injury(P<0.05), EGCG treatment could partially increase the expression of Nrf2(P<0.05).4. Changes of inflammation in kidney(1) MPO activity and IL-1β level were determined in the renal homogenates, reflecting the activation of neutrophils and the level of pro-inflammatory cytokine, respectively. It resulted that renal MPO activity and expression of IL-1β were significantly increased after contrast-induced renal injury(P<0.05). EGCG treatment evidently decreased the renal MPO activity and content of IL-1β, ameliorating the state of renal inflammation(P<0.05).(2) Further, changes of the NLRP3/IL-lβ anti-inflammatory signaling pathway in kidney were detected by Western blot after contrast-induced renal injury and EGCG intervention. The renal protein expression of NLRP3 was significantly increased after contrast-induced renal injury, and EGCG treatment markedly down-regulated the NLRP3 expression(P<0.05).5. The role of HO-1 in the antioxidant and anti-inflammatory effects of EGCGTo investigate the key molecule target in the antioxidant and anti-inflammatory effects of EGCG, two inhibitors of HO-1, ZnPP and SnPP, were used to observe the effect of HO-1 inhibition on the EGCG treatment in contrast-induced renal injury. Compared with the HO-1 un-inhibition group(i.e. CM+EGCG group), both serum Cr and BUN were significantly increased in the inhibition groups(ZnPP+EGCG+CM group or SnPP+EGCG+CM group). HO-1 inhibition offset the protective effect of EGCG. Further study found that renal levels of MDA、MPO activity and IL-1β all were elevated, and SOD activity was decreased, protein expression of NLRP3 was significantly increased in the ZnPP+EGCG+CM group compared with the CM+EGCG group(P<0.05), with no statistic difference compared with CM group(P>0.05), indicating the pivotal role of HO-1 in the antioxidant effect and anti-inflammatory signaling of EGCG.Conclusion:The peri-operative intravenous administration of EGCG could protect against contrast-induced renal injury. EGCG could ameliorate the renal state of oxidative stress and inflammation via up-regulating the Nrf2/HO-1 antioxidant signaling pathway and down-regulating the NLRP3/IL-1β inflammatory signaling pathway, where the HO-1 is the key molecule target of EGCG in the EGCG-mediated protection.