Klotho Prevents Epithelial-to-mesenchymal Transition In Diabetic Kidney Disease By Down-regulation Of Egr-1

BackgroundDiabetic kidney disease(DKD)has become the leading cause of end-stage renal disease(ESRD).It was thought that the glomerulus is mainly involved in the early lesion of DKD,and the tubulointerstitial fibrosis is the pathological feature in the late stage of DKD.However,in recent years,it has been found that the degree of tubulointerstitial damage is the most important clinical indicator to reflect the severity and prognosis of renal dysfunction.The epithelial-to-mesenchymal transition(EMT)is a key process in renal tubulointerstitial fibrosis.Klotho is an anti-aging factor.The plasma soluble-Klotho deficiency is an early event and a novel biomarker of the early stage of DKD.Klotho prevents the progression of DKD partially by inhibition of TGF-β1/Smad3 and ERK1/2 signaling pathways.Early growth response protein 1(Egr-1),a transcription factor with zinc finger structure,promotes the progression of DKD in part by facilitating the formation of a positive feedback loop involving TGF-β1 and ERK1/2.Moreover,it has been reported TGF-β1 promotes renal fibrosis partially by activating ERK1/2 signaling pathway.By now,there is no report on the interaction between Klotho and Egr-1 in the pathogenesis of DKD.The aim of this study was to investigate the effects of Klotho and Egr-1 on EMT in DKD,and to illustrate whether Klotho down-regulates Egr-1 by inhibition of TGF-β1/Smad3-ERKl/2 signaling pathway in progression of DKD,which can provide a theoretical basis for understanding the molecular mechanism of DKD pathogenesis,as well as finding new therapeutic targets.Methods1 Animal experimentC57BL/6J mice with hyperlipidemia induced by STZ(n=12)was set into DM model group,and homologous normal mice(n=12)were defined as control group.Blood and urine samples were collected for biochemical indexes test respectively at 6w and 12w after successful DM model.Then all animals were sacrificed and the kidney tissue was frozen in the refrigerator at-80℃,or was fixed in 4%paraformaldehyde.PAS and Masson’s trichrome stainning was used to observe morphological changes of Kidney.Immunohistochemistry was used to observe expression of Klotho and Egr-1 protein.2 Cell cultureHuman renal tubular epithelial cell line(HK2)was purchased from the Chinese Academy of Sciences cell bank.Cells were grown in DMEM with 1.0 g/L glucose containing 10%FBS.Cells were cultured in a 5%C02 incubator at 37℃.Medium was replaced every 2 to 3 days.3 Cell transfectionOn the day before transfection,HK2 cells were seeded in 12-well plates.When the cell density reached 60%or 80%,the siRNA or plamid transfection was conducted.For this experiment,50 nM siRNA or lug/ml plamid was transfected into HK2 cells using Lipofectamine 3000.4 RNA extraction and RT-qPCRTotal RNA was extracted from mouse kidney cortices and HK2 cells and with Trizol.Reverse transcription of the total RNA was performed with an M-MLV kit.The gene expression levels were determined using Roche Light Cycler480 Real-Time PCR System.The relative values for each gene were calculated by the comparative 2-△△Ct method with β-actin as an internal control.5 Western BlotEqual amount of proteins(20ug),extracted from mouse kidney cortices and HK2 cells,were electrophoresed in 10%SDS-PAGE gels,transferred to PVDF and blocked and then incubated with primary antibodies at 4℃ overnight.A fluorescent secondary antibody was then added and incubated with the blots at room temperature for 1 h.The images were acquired using an Odyssey infrared imaging system.Gel-Pro analyzer software was used for the densitometry analysis.6 Statistical AnalysisAll results are presented as mean ± SD.One-way ANOVA was used to compare three or more independent groups.A two-tailed Student’s t-test was used for comparisons of two independent groups.P values less than 0.05 were considered significant.Results1 The expression of Klotho and Egr-1 in renal cortices of diabetic mice induced by high lipid + STZ.(1)Compared with the control mice,plasm Klotho of DM mice significantly decreased at 6w and further decreased at 12w,24h urinary microalbumin significantly increased at 6w and further increased at 12w.(2)Compared with the control mice,the expression of Klotho in renal cortice of DM mice significantly decreased at 6w and further decreased at 12w,while the expression of Egr-1 significantly increased only at 12w.2 The effect of Klotho on EMT in DKD(1)The expression of Klotho decreased significantly at 12h,and further decresed at 24h in HK2 cells after HG or TGF-β1 treatment.(2)Compared with pcDNA-Vector,pcDNA-Klotho transfection-mediated Klotho overexpression inhibited down-regulation of E-cadherin and up-regulation of FN and Col I in HG or TGF-β1 treated HK2 cells.(3)Compared with si-Negative,siRNA-mediated Klotho silencing aggravated down-regulation of E-cadherin and up-regulation of FN and Col I in HG or TGF-β1 treated HK2 cells.3 The effect of Egr-1 on EMT in DKD(1)The expression of Egr-1 increased significantly at 0.5h in HK2 cells after HG or TGF-β1 treatment,and then decreased to baseline level at 6h.(2)Compared with si-Negative,siRNA-mediated Egr-1 silencing ameliorated down-regulation of E-cadherin and up-regulation of FN and Col I in HG or TGF-β1 treated HK2 cells.(3)Compared with pENTER-Vector,pENTER-Egr-1 transfection-mediated Egr-1 overexpression aggravated down-regulation of E-cadherin and up-regulation of FN and Col I in HG or TGF-β1 treated HK2 cells.4 The mechanism of Klotho down-regulating Egr-1 in progression of DKD(1)Compared with pcDNA-Vector,pcDNA-Klotho transfection-mediated Klotho overexpression down-regulated Egr-1 expression,accompanied by the decreased p-Smad3/Smad3 ratio and(p-ERK1/2)/(ERK1/2)ratio in HG or TGF-β1 treated HK2 cells.(2)Compared with si-Negative,siRNA-mediated Klotho silencing up-regulated Egr-1,accompanied by the increased p-Smad3/Smad3 ratio and(p-ERK1/2)/(ERK1/2)ratio in HG or TGF-β1 treated HK2 cells.These effects of si-Klotho could be abolished by the ERK1/2 inhibitor PD98059.Conclusion1 Klotho expression decreases significantly and Egr-1 expression increased significantly in renal cortice of early DKD mice.The decrease of Klotho was earlier than the increase of Egr-1.2 High glucose and TGF-β1 time-dependently down-regulated Klotho expression in cultured HK2 cells.Klotho prevents the progression of DKD by inhibiting EMT.3 High glucose and TGF-β1 transiently induced Egr-1 expression in cultured HK2 cells.Egr-1 promotes the progression of DKD by inducing EMT.4 Klotho down-regulates Egr-1 in HG or TGF-β1 treated HK2 cells by inhibiting TGF-β1/Smad3-ERK1/2 signaling pathway,which may be one of the important anti-fibrotic mechanisms of Klotho in DKD.