| Renal fibrosis is the most common pathological manifestation of CKD eventually leading to the end stage renal disease(ESRD).And it’s characterized by interstitial fibrosis and renal tubular expansion,it was found that renal interstitial fibrosis was associated with renal damage and the prognosis of patients with kidney diseases.But until now,the mechanisms of the progress of renal fibrosis are not fully understood.Thus,it’s important for further exploring the possible mechanism under the the pathological progress of renal fibrosis in clinical study.Renal fibrosis is related to various factors,such as inflammation,oxidative stress,those could lead to an increasing of fibrosis factor,and contributed to epithelial to mesenchymal transition(EMT)in tubuloepithelial cells and fibroblasts proliferation and activation.Uremia toxins accumulation is another hallmark feature of CKD.In recent years,many studies emphasized the role of uremic toxins,especially protein-bound toxins,such as indoxyl sulphate(IS),p-cresol(PCS)among the development of renal failure,which can’t be effectively remove by conventional dialysis,and exist in the circulation for a long time,then harmful to body.Over the years,study focused on the role of uremic toxins in the progression of renal fibrosis.Our and other scholars’ study found that protein bound toxins accumulation contribute to the process of renal fibrosis.However,the possible contribution of uremic toxins to renal fibrosis has not been raised concern until recent years.The activation of Wnt/β-catenin signaling play an important role inrenal fibrosis.Wnt signaling is silenced in adult renal,but reactivated after renal damage.Once Wnt signaling activated,abundant β-catenin translocate to the nucleus and combined with TCF/LEF,finally leading to activating the downstream target genes of transcription,such as plasminogen activator inhibitor-1(PAI-1),fibronectin,fibroblast-specific protein 1(Fsp1),Snail1 and MMP-7,which could contribute to the process of renal fibrosis.However,whether the Wnt signaling involved in IS-induced renal fribrosis is unclear.Previous studies showed that the activation of Wnt signaling is related to the endogenous regulation factors,including secreated frizzled-related proteins and Dickkopf.The s FRP family consist of five members,incuding s FRP1-5.The CRD of s FRPs share 30%-50% sequence with those of Fz receptors and comprises 10 conserved cysteine residues.Thus,the s FRPs can binds to Wnt and inhibits Wnt/β-catenin signaling.In contrast,a deficiency of s FRPs contributed to the activation of Wnt/β-catenin signaling pathway.The aberrant DNA methylations of s FRPs have been demonstrated to be involved in the activation of Wnt/β-catenin signaling,and thereafter promote the progress of various cancers.Interesting,in uremia state,protein toxin may regulate gene expression through a variety of epigenetic modifications,especially DNA methylation.Moreover,study has confirmed that IS could induce DNA hypermethylation of target gene.In this study,we analyzed the relationship between IS and the activation of Wnt/β-catenin signaling,addressed their associations with renal fibrosisand revealed the critical role of sFRP5 DNA hypermethylation in IS-induced renal fibrosis,to deeply explore the mechanism under the pathogenesis of renal fibrosis,and it can provide a new intervention target for prevention and treatment of CKD-associated renal fibrosis.In this study,we firstly observed the correlation among renal fibrosis,the serum IS concentration and the expression of β-catenin in patients with chronic kidney disease,then we used tubuloepithelial(HK-2)cells in vitro,investigate the relationship between IS and the activation of Wnt/β-catenin signaling and confirmed whether IS-induced sFRP5 DNA hypermethylation was association with the activation of Wnt/β-catenin;Then we employed CD-one mice to establishing a uninephrectomized mice model and intraperitoneal injection with IS,at the same time,injected exogenous 5Aza-2dc or sFRP5 to the mice,to study the inhibition of renal fibrosis by 5Aza-2dc or sFRP5.The results and conclusions are listed as follows:1.Serum IS level is positively correlated with the increased expression of β-catenin in kidney and renal fibrosis in CKD patients.Thirty-four patients with Primary chronic renal diseases stages1-5 were enrolled in our study.And there estimated glomerular filtration rate(e GFR)rang from 6.92-148.42 ml/min/1.73 m2.Kidney paraffin sections and serum samples were obtained from these patients for subsequent immunohistochemistry,masson and IS measurement.It was shown that serum level of IS,the level of β-catenin positive-staining and the ratio of renal fibrosis area increased gradually.The serum IS level and β-catenin positive-staining arae were negatively related to e GFR,but the increase of serum IS level was positivy related to the level of β-catenin positive-staining and the ratio of renal fibrosis.This data indicate that an intreractive relationship may exist between IS and the activation of wnt/β-catenin pathway during the progression of renal fibrosis in CKD patients.2.IS injection-induced renal fibrosis is accompanied with the activation of Wnt/β-catenin signaling pathway in CKD mice.To further investigate the role of wnt/β-catenin pathway in IS-induced renal fibrosis,we treated uninephrectomized CD-1 mice with IS(100mg/kg per day)by intraperitioneal injection for 4 weeks and found that IS injection induced a 5.0-fold increase of IS serum level.Moreover,the significant increase of β-catenin and α-SMA expression and collagen deposition were observed in the kidneys of CKD mice,as assessed by Massons trichrome staining,western blot and immumohistochemical staining.These results indicate that IS-induced renal fibrosis was accompanied by the activation of wnt/β-catenin signalling.3.IS stimulates Wnt/β-catenin signalling IS related to the decreasing of the expression of sFRP5.To further confirmed whether IS induced the activation of wnt/β-catenin,we examined the expression of β-catenin in IS-treated HK-2 cells and found that the β-catenin expression was up-regulated by IS in a dose-dependent manner.Because secreted Frizzled-related proteins(s FRPs)are Wnt antagonists and the suppression of s FRPs genes can lead to Wnt/β-catenin signallingactivation,we then detected the m RNA expressionof all s FRP members in IS-treated HK-2 cells by semiquantitative PCR.The results indicated that the m RNA expression of s FRP1 and s FRP2 were very weak in HK-2 cells,in addition,s FRP3 and s FRP4 m RNA expression did not affect by IS,but IS dose dependently suppressed sFRP5 expression in HK-2 cells.Moreover,the reduction of sFRP5 protein expression induced by IS was also observed.These data hint that IS may activate the wnt/β-catenin signalling pathway through the inhibition of sFRP5 expression.Moreover,we further explored the expression of sFRP5 in CKD patients and mice.As revealed by immumohistochemical staining,the expression levels of sFRP5 expression in renal tubular cells was decreased in kidney biopsy sample from patients with CKD and renal fibrosis,as compared with the normal kidney sample from renal carcinoma patients.In IS-injected CKD mice,the significant reduction of sFRP5 m RNA and protein expression was also found by immumohistochemical staining,RT-PCR and western blot analysis.These results further suggest that the decrease of sFRP5 expression may be involved in IS-induced Wnt/β-catenin activation and renal fibrosis.4.IS stimulates Wnt/β-catenin signaling in HK-2 cells by inducing sFRP5 DNA hypermethylation.Because DNA hypermethylation is the main cause of down-regulation of gene expression and IS has the potential ability to induce gene DNA hypermethylation,we further revealed whether IS stimulates Wnt/β-catenin signaling in HK-2 cells by inducing sFRP5 DNA hypermethylation.The methylation analysis indicated that the DNA methylation level of sFRP5 gene was markedly increased in IS-treated HK-2 cells,and DNMT1,DNMT3 a,DNMT3b m RNA expression were significantly increased in IS-treated HK-2 cells at concentrations of 10 and 50 mg/L as assed by semiquantitative PCR analysis.Moreover,western blot analysis showed that the protein expression of DNMT1 was also increased after treatment with 10 and 50 mg/L IS.To determine the promoter DNA methylation status at multiple Cp G sites across the promoter of the sFRP5 in IS-treated HK-2 cells,BSP analysis of Cp G islands in sFRP5 promoters was performed,as compared with 6.2%±5.7 of control cells,the frequency of DNA methylation showed that sFRP5 gene exhibit hypermethylation in 40.3%±7.3 of the IS-treated HK-2 cells.These data firmly confirm that IS induces sFRP5 DNA hypermethylation in HK-2 cells.To further determined the sFRP5 DNA hypermethylation was involved in IS-induced Wnt/β-catenin signalling activation,HK-2 cells were pertreated with DNA methylation inhibitor 5Aza-2dc(0.1,1,10μM)before incubated with 50 mg/L IS.As expected,IS-induced the increase of DNA methylation status and DNA hypermethylation of sFRP5 gene in HK-2 cells were significantly inhibited by 5Aza-2dc in a dose-dependent manner.The RT-PCR and western blot results exhibited that IS-induced the decreased expression of sFRP5 and the increased expression of β-catenin were also suppressed by 5Aza-2dc.Furthermore,we assessed the effect of IS on the ability of sFRP5 binding to Wnt5 a in HK-2 cells by immunoprecipitation using Wnt5 a antibody,and found that IS obvious suppressed sFRP5 binding to Wnt5 a which was reversed by 5Aza-2dc.Therefore,IS-induced the activation of Wnt/β-catenin is primarily due to sFRP5 DNA hypermethylation.5.IS induces sFRP5 hypermethylation through ROS/ERK1/2 signallingIt is well known that IS is liable to increase oxidative stress in various cells and this was associated with abnormal DNA methylation.Thus,we investigated whether IS-stimulated oxidative stress and its downstream signalling play a key role in sFRP5 hypermethylation.DCFH-DA analysis revealed that IS significantly increase ROS production in dose-dependent manner,IS also promoted activation ERK1/2 as assessed by western blot analysis.In contrast,pretreatment with 5mmol/L NAC(a ROS scavenger)and 10μmol/L U0126(ERK1/2 inhibitor)significantly suppressed the phosphorylation of ERK1/2 and DNA methylation of sFRP5 gene,as assayed by western blotting and MSP.Furthermore,the results of western blotting found that the increase of DNMT1 and the decrease of sFRP5 expression induced by IS were inhibited by pretreatment with NAC and U0126.6.5Aza-2dc inhibits the DNAhypermethylation of sFRP5 gene and up-regulates sFRP5 expression in IS-injected mice.In uninephrectomized mice,IS injection increase the DNA methylation status of the kidneys,and 5Aza-2dc(0.35 mg/kg/48 h,intraperitoneal injection)treatment significantly decreased the DNA methylation of sFRP5 gene,as assayed by MSP.Moreover,RT-PCR,western blotting and immumohistochemical staining showed that treatment with5Aza-2dc evidently reversed the reduction of sFRP5 expression in the kidneys of IS-injected mice.Hence,we conclude that IS definitely induce DNA hypermethylation of sFRP5 gene,and thereafter led to the dwon-regulated sFRP5 expression.7.Recombinant sFRP5 protein and 5Aza-2dc amelioraterenal fibrosis through inhibiting Wnt/β-catenin signalling in IS-injected mice.To definitely confirm whether DNA hypermethylation of sFRP5 gene contribute to IS-induced Wnt/β-catenin activation and renal fibrosis,we treated IS-injected CKD mice with recombinant sFRP5 protein(0.01 or 0.02 mg/kg/48h)or 5Aza-2dc(0.35 mg/kg/48h)immediately after uninephrectomized.sFRP5,especially using high dose sFRP5 protein and 5Aza-2dc treatment significantly ameliorated renal fibrosis in IS-injected mice,as detected by Massons trichrome staining,and inhibited α-SMA expression,as revealed by immumohistochemical staining.Noticeably,sFRP5 and 5Aza-2dc treatment reduced β-catenin expression in the kidneys of IS-injected mice.These data indicate that both exogenous supplement sFRP5 and up-regulated sFRP5 expression by the inhibition of its DNA hypermethylation attenuate renal fibrosis through the suppression of Wnt/β-catenin activation.Therefore,promoter hypermethylation of sFRP5 is a key mechanism of IS-induced renal fibrosis.In conclusion,our results suggested that IS aggravate renal fibrosis through the activation of Wnt/β-catenin signaling,the potential mechanism is IS promoted the sFRP5 DNA methylation by activating ROS/ERK1/2 signal pathways,which could down-regulated sFRP5 expression,ultimately promote renal interstitial fibrosis through activating the Wnt/β-catenin signaling.And both exogenous supplement sFRP5 or 5Aza-2dc attenuate ISinduced renal interstitial fibrosis. |
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