Klotho Prevents Tubulointerstitial Fibrosis In Diabetic Kidney Disease By Down-regulation Of MiR-21 And MiR-34

BackroundTubulointerstitial fibrosis(TIF)is crucial in the development of renal fibrosis in diabetic kidney disease(DKD).Previous data shows that Klotho and many miRNAs are involved in TIF,but little is known regarding to the relationship between Klotho and miRNAs.In this study,we first explored whether Klotho had any connection with miR-21,miR-34 and underlying mechanisms in TIF.Furthermore,we investigated whether the miR-34/SIRT1 pathway affects TIF.Methods1.Animal experimentMale C57BL/6J mice induced by HFD/STZ was set into DM model group,and homologous normal mice were defined as control group.(n=6)DM mice which were 12w after successful DM model were injected with empty vector pCMV or pCMV-Klotho.Gene expression in vivo was performed by a hydrodynamic-based gene delivery approach.(n=6)Blood and urine samples were collected for biochemical indexes test respectively.Masson's trichrome staining was used to observe morphological changes of Kidney and immunohistochemistry stainning was used to observe Klotho,?SMA,FN and COL1 protein expression.2.Cell culture and transfectionHuman renal tubular epithelial cell line(HK2)were grown in DMEM with 1.0 g/L glucose containing 10%FBS.HK2 cells were seeded in 12-well plates,when the cell density reached about 70%,the plamid?siRNA?mimic?inhibitor and the appropriate negative controls were transfected at a final concentration of 50-100nM in cells.All transfections were performed using LipofectamineTM 3000 reagent.3.RNA extraction and quantitative real-time polymerase chain reaction(qRT-PCR)Total RNA from renal tissues and HK2 cells was extracted using TRIzol reagent.NanoDrop ND-1000 spectrophotometer was used to determine RNA quality.Reverse transcription was carried out using an M-MLV Kit and miRcute miRNA cDNA first strand synthesis kit,respectively.RNA and miRNA expression levels were measured by qRT-PCR with Roche 480 Thermal Cycler using SYBR Green qPCR Kit and miRcute miRNA qPCR Detection Kit respectively.The 2-??Ct method was used to calculate the each gene mRNA and target miRNA quantification using ?-actin and U6 as internal controls.4.Western BlotTotal protein from HK2 cells and renal tissues was extracted using RIP A Lysis Buffer and protein concentration was assessed using a BCA assay.Same amount of protein extracted from mouse kidney cortices and HK2 cells,were electrophoresed on 10%SDS-PAGE gels,transferred to PVDF and blocked and then incubated with primary antibodies.And then fluorescent secondary antibody was then added and incubated with the blots.The images were acquired using an Odyssey infrared imaging system.Gel-Pro analyzer software was used for the densitometry analysis.5.Luciferase activity assaymiR-34 mimics or miRNA negative control,and with lug reporter plasmid and 100nM miR-34 inhibitor or miRNA inhibitor negative control in 24-well plates,respectively.After 48 h transfection,the luciferase activity was analyzed using luciferase assay kits(Promega).The data were expressed as the quotient of Renilla luciferase activities.6.Chromatin immunoprecipitation(ChIP)ChIP was performed using the motif-ChIP Kit according to the operation guide.Briefly,HK2 were cross-linked,chromatin was isolated,and genomic DNA was sheared by sonication into fragments of 500-1000bp.Cross-linked protein-DNA was immunoprecipitated overnight with 1 ?g antibody or negative control IgG.The immunoprecipitated ChIP product was washed,de-cross-linked,treated with proteinase K,and eluted to obtain ChIP-enriched DNA.PCR reactions contained the ChIP-enriched DNA,primers to amplify NF-?B binding sites in the miR-21 and miR-34 promoter or a binding site in the promoter of the gene encoding platelet-derived growth factor(PDGF)-B as a positive control,and the SYBR Premix Ex Taq ? kit.PCR products were analyzed on a 2%agarose gel.7.Statistical AnalysisAll results are presented as mean± SD.A two-tailed Student's t-test was used for comparisons of two independent groups.One-way ANOVA was used to compare three or more independent groups.P values less than 0.05 were considered significant.Results1.Klotho level is associated with expression of miR-21 and miR-34 in the DM mice and HG induced HK2.Klotho gene and protein levels were markedly down-regulated and the expression of miR-21,miR-34 was up-regulated in the DN mice at 12 weeks after HFD/STZ compared with the control mice.The same situation appeared time-dependently in high glucose in HK2 with TIF markers increasing.2.Overexpression of Klotho reverses HG-induced nuclear NF-?B p65,miR-21,miR-34 and consequently TIF factor expression in HK-2 cells and DM mice.Overexpression of Klotho brought increase of nuclear NF-?B p65?miR-21 and miR-34 expression back in high glucose-induced HK2.The upregulation effect of si-Klotho on NF-?B p65,miR-21,miR-34 could be reversed by PDTC.Klotho inhibits HG-induced recruitment of NF-?B p65 on miR-21 and miR-34 promoters.At last,groups of mice were administered pCMV-Klotho or pCMV by rapid injection of a large volume of DNA solution through the tail vein.The result indicated that expression of Klotho in vivo is able to ameliorate renal fibrosis in diabetic nephropathy models through NF-?B canonical pathway.3.SIRT1 level is associated with expression of miR-34 in the DM mice and HG induced HK2.SIRT1 gene and protein levels were markedly down-regulated and the level of miR-34 is up-regulated in the DN mice at 12 weeks after HFD/STZ compared with the control mice.The same situation appeared time-dependently in high glucose in HK2 with TIF markers increasing.4.MiR-34 aggravate fibrosis in HK2 cells via downregulating SIRT1.HK2 cells transfected with mimic revealed remarkably increased miR-34 expression,FN,COL1 and TGF-?1 mRNA and protein levels and decreased expressions of SIRT1.HK2 cells transfected with inhibitor revealed the opposite result.The data from Dual-luciferase activity assay demonstrated that miR-34 can directly target the 3'UTR of SIRT1 mRNA.We treated HK2 cells with si-SIRT1 first and then with miR-34 inhibitor.The results revealed that the increased expression of TGF-?1,FN,and COL1 was falled down when SIRT1-silenced cells were treated with miR-34 inhibitor.Conclusions1 Klotho expression decreases significantly and miR-21,miR-34 expression increased significantly in renal cortex of early DN mice and HG induced HK2.Klotho could reduce the level of miR-21 and miR-34 via inhibiting NF-?B p65 nuclear translocation in HG stimulated HK2 and DN mice.2 The expression of SIRT1 is declined and the level of miR-34 is up-regulated in the kidneys of DN mice and HG induced HK2.MiR-34 directly targets SIRT1 3'UTR.MiR-34 aggravate fibrosis through suppressing SIRT1 in HK2 cells.