EGCG Prevents Adriamycin Nephropathy Development In Mice Via Reduces Oxidative Stress And Inhibiting NALP3Inflammasome

Background and ObjectiveFocal segmental glomerulosclerosis (FSGS) is one of the most common and seriouspathology of Nephrotic Syndrome (NS), as well as a major cause of end-stage renal disease(ESRD). So far, because of the mechanisms involved in the process of FSGS remainunknown and the related effective treatment methods are lacking, the prevention and cureof FSGS have became the urgent need. Inflammatory mechanisms and oxidative stress(OS)play a very important role in the progress of FSGS has been showed in recent reports.NALP3(NLR family pyrin domain containing-3protein，NRLP3, NALP3) inflammasome isone of the NLRs family members, is mainly expressed in macrophages, DCs and otherantigen-presenting cell, and play a role in the start of inflammasome assembly. Another side,NALP3is responsible for identifying the signal of danger, which activates caspase-1to theshearing precursors of IL-1family to be the activity proinflammatory cytokines such asIL-1β and IL-18, thereby causing a series of inflammatory responses. Study found thatNALP3inflammasome and its downstream inflammatory cytokines play an important rolein chronic kidney disease. In human renal tissue biopsies, the FSGS exhibited increasedexpression of NLRP3mRNA, which correlated with renal function. And on the other hand,the FSGS is reached that oxidatives stress can cause renal tissue necrosis, apoptosis,inflammation and fibrosis. The study on animal have found that the increase ROSproduction and reduce the antioxidases in FSGS mice is correlated with renal progression.Therefore, reducing the inflammation and the oxidative stress effectively is particularlyimportant with development of FSGS.The major and the most active component in green tea catechinsis(-)-epigallocatechin-3-gallate (EGCG). Studies have shown that EGCG protects cellulardamage by ant-oxidation, ant-infective, anti-inflammatory, inhibiting DNA damage and soon. To protect renal, EGCG focuses on the anti-oxidative and anti-inflammatory effects. Although there are no reports about EGCG treatment of FSGS, but we speculate that EGCGcan reduce the inflammation and inhibit the oxidative stress to slowing the process ofFSGS.In the study, we investigate the clinical value of renal NALP3inflammasome in FSGSpatients, and the effects of EGCG on Adriamycin treated mice and potential mechanisms bywhich it reduce oxidative stress and resolves inflammation to develop new strategies forNephrotic Syndrome.MethodClinical specimens examineTwenty-six paraffinaceous renal tissue samples from patients with clinical diagnosisNS and kidney biopsy confirmed FSGS in our department were collected as FSGS group,sixteen paraffinaceous renal tissue samples from renal hamartoma resection of thepathological diagnosis confirmed normal renal tissue as controls, immunofluorescencemethod was used to examine the macrophage marker F4/80to observe the amount ofmacrophage infiltration in the renal tissue. Immunohistochemistry was used to detectcontrol group and the FSGS groups NALP3, ASC, caspase-1, IL-1β, IL-18expression variation of renal tubular epithelial cells, The tubulointerstitial injury score andmacrophages in renal interstitium of FSGS patients and normal control were evaluated. Theserum creatinine, blood urea nitrogen, serum album,24hour urine protein and estimatedglomerular filtration rate (eGFR) were observed. The correlation of tubulointerstitial injurywith NALP3/ASC/caspase-1,IL-1β,IL-18respectively were analyzed.Study in animalA total of24healthy male BALB/C mice were randomly divided into four groups:①normal control group(N,n=6): Mice in the group were given gavage of0.2ml doubledistilled water on day-5(5days before injected intravenously) and continued untilsacrificed, and0.9%saline were injected intravenously twice on days1and14(10mg/kg).②Adriamycin nephtopathy model group(AN, n=6): Mice in the group were given gavageof0.2ml double distilled water on day-5(5days before injected intravenously) andcontinued until sacrificed, and adriamycin were injected intravenously twice on days1and14(10mg/kg).③Small doses of EGCG-treated group (EGCG A, n=6), Mice in the groupwere given gavage of50mg/kg.bw EGCG on day-5(5days before ADM injected intravenously) and continued until sacrificed, and modeling method according the ANgroup.④large doses of EGCG-treated group (EGCG A, n=6), Mice in the group weregiven gavage of100mg/kg.bw EGCG on day-5(5days before ADM injected intravenously)and continued until sacrificed, and modeling method according thr AN group. At days28,all rats were sacrificed. Blood samples were taken and the kidneys were removed. Periodicacid-Schiff stain observes the degree of the renal injury and interstitial phlogocytesinfiltration and biochemistry index observes serum creatinine, blood urea nitrogen, serumalbumin and24h urine protein. MDA, CAT, GPx and SOD levels in mice kidneyhomogenate were measured by commercial kits. Immunofluorescence is used to detectNALP3and HO-1. Finally, western blot is used for the protein expression of NALP3、ASC、caspase-1、IL-1β、IL-18、Nrf2、HO-1. The entire indexes were compared and analyzedby SPSS16.0.ResultsClinical specimens examine1. The expression of NALP3/ASC/caspase-1, IL-1β, IL-18in FSGS patients’ renaltissue increased significantly compared with those in the control group (P<0.01)2. NALP3/ASC/capspase-1expression was positively correlated with the expression ofIL-1β, IL-18(P<0.01). NALP3/ASC/caspase-1,IL-1β,IL-18expression was positivelycorrelated with renal tubulointerstitial injury and macrophages marked as F4/80(P<0.01).NALP3/ASC/caspase-1, IL-1β, IL-18was significantly positively correlated with24hoururine protein, Scr, and negatively with the eGFR(P<0.05), but was not correlated with BUN,serum total protein and albumin.Animal examine1. Effect of EGCG on AN condition in mice.The serum creatinine, blood urea nitrogen, serum albumin and24h urine proteinsignificantly increased the serum albumin levels significantly decrease in AN groupcompared with that of N group (P<0.05). With oral administration of EGCG (both50and100mg/kg), serum creatinine, serum urea nitrogen and urinary protein declinedsignificantly compared with that of AN group(P<0.05), and the serum albumin levels weresignificantly improved compared with that of AN group(P<0.05). The mice in AN group,renal injury was characterized by severe glomerulosclerosis(GSI2.10±0.19), tubulointerstitial injury(TILI2.70±0.34), and Neutrophil Infiltration(2.70±0.34)comparedwith N group (P<0.05), and these renal lesions were greatly reduced in50mg/kg b.w./d and100mg/kg b.w./d EGCG-treated mice on dose depentded(P<0.05), although the mice stillshowed mild glomerular proliferation and mild interstitial inflammation.2. Effect of EGCG on inflammatoty factors of AN mice renal.The level of renal NALP3, ASC, caspase-1, IL-1β and IL-18were determined byWestern blot. The expression of NALP3, ASC, caspase-1, IL-1β and IL-18in kidney wasincreased in AN group compared with the expression in the N group (P<0.05). Theincreased NALP3, ASC, caspase-1, IL-1β and IL-18expressions in kidney were decreasedby the EGCG treatment (P<0.05), and100mg/kg EGCG treated group was lower than50mg/kg EGCG treated group.3. Effect of EGCG on oxidative stress of AN mice renalActivities of SOD, CAT,and GPx in the kidneys of the AN group mice were decreasedcompared to those of the N group. However, the activities of SOD, CAT,and GPx in thekidneys of the EGCG B group were significantly increased when compared to those of theAN group (p <0.05). The kidney MDA activities of AN group was significantly greaterthan those of N group, whereas the MDA activities of EGCG(50and100mg)-treated groupwas lower than in the AN group. The accumulation of Nrf2in the nuclear fraction andHO-1was lower in the kidney of AN group mice than that of the N group mice. The Nrf2and HO-1accumulation in the kidney was significantly increased by treatment withEGCG(both50mg/kg and100mg/kg) mice, as compared with AN group (P<0.05).Conclusion1. The NALP3inflammasome might play important role in the pathogenic mechanismof FSGS.2. EGCG attenuated the kidney disease of adriamycin nephropathy with the lightenedkidney dysfunction, renal tissue injury, and reduced oxidative stressand the levels ofNLRP3/ASC/Caspase-1inflammasome, IL-1β, IL-18in renal tissue, and increased theexpression of nuclear factor E2-related factor2(Nrf2)/HO-1.