The Effect Of TLR2 On Epithelial-mesenchymal Transition Of Diabetic Renal Tubular Epithelial Cells

Objective:Diabetic nephropathy?DN?is one of the most important complications of diabetes,and is also a main cause of Chronic Kidney Disease?CKD?.In China,as the incidence of diabetes increases by years,the incidence of diabetic nephropathy also shows an increasing trend.CKD continuously aggravates renal lesions and later develops into chronic renal failure,which seriously affects the quality of life and survival rate of every patient.Therefore,it is particularly important to explore the pathogenesis of CKD.Tubulointerstistisis fibrosis?TIF?is a marker of CKD and the best predictor of kidney damage.Tubular epithelial-mesenchymal transition?EMT?plays a central role in the development of TIF.Toll-like receptors 2?TLR2?is an important member of toll-like receptors?TLRs?,which is pasrticipate in a variety of kidney diseases development.Is TLR2 involved in DN renal tubular EMT?So,in this study,the purpose was to investigate the role of TLR2 in EMT of DN by using renal biopsy specimens,cultured HK2 cell and TLR2 knockout mice diabetes model.Methods:1.Renal biopsies obtained from DN patients and distant portions of renal tumors pathologically diagnosized as control were used.And the expressions of TLR2,?-SMA and E-cadherin in renal tissues were detected by immunohistochemistry?IHC?,and the correlation between TLR2 and?-SMA,TLR2 and E-cadherin was analyzed.2.After treated with high glucose?30 mM?for 0 h,24 h,48 h and 72 h,HK2 cells were collected and the expression of TLR2,?-SMA and E-cadherin protein was detected by Western Blot.3.HK2 cells were randomly divided into control group,High Glucose group,HG+shNC group and HG+shTLR2 group.The expression of?-SMA and E-cadherin was detected by Western Blot.ELISA was used to detect the content of TGF-?1 in the supernatant.4.The diabetic model was induced by streptozotocin in C57BL/6J wild type and TLR2^-/-mice with the same background.The mice were divided into control group,WT+STZ group and TLR2^-/-+STZ group.The mice tail DNA was extracted to identify the mice genotype,Western Blot was taken to detect the expression of?-SMA and E-cadherin protein.5.Some renal cortex of mice was taken to make frozen sections,the expression of fibronectin?FN?was detected by immunofluorescence?IF?.Some renal tissues were made into paraffin sections,the expression of Collagen?was detected by IHC.Renal tubulointerstitial fibrosis was detected by Masson staining.6.Paraffin sections of mice kidney tissue were taken and the expression of TGF-?1 was detected by IHC.Results:1.The upregulation of TLR2 might mediate the EMT in diabetic nephropathy.IHC showed that the expression of TLR2 and?-SMA protein was significantly up-regulated and the expression of E-cadherin protein was significantly down-regulated in renal tubular epithelial cells of diabetic nephropathy patients.And there was a significantly positive correlation between TLR2 and?-SMA expression?P<0.05,r=0.5188?.However,there was a significantly negative correlation between TLR2 and E-cadherin expression?P<0.05,r=?0.6625?2.Overexpression of TLR2 was contributed to the EMT induced by high glucose.Western Blot showed that the expression of TLR2 and?-SMA in HK2 cells exposed to high glucose increased and the expression of E-cadherin gradually decreased in time-depedent manner.3.Under the stimulation of high glucose,knockdown of TLR2 decreased the expression of?-SMA and increased the expression of E-cadherin induced by high glucose,meanwhile,TGF-?1 was involved in the EMT.Western Blot showed that the expression of?-SMA in HG+shTLR2 group was lower than that in HG+shNC group,but the expression of E-cadherin increased.The results of ELISA showed that the secretion of TGF-?1 in HG group was significantly higher than that in control group,but compared with HG+shNC group,knockdown of TLR2 expression could decrease the secretion of TGF-?1 induced by high glucose.4.Knockout of TLR2 gene could inhibit the EMT.PCR showed that the tail tissue of TLR2^-/-mice was negative for TLR2 wild-DNA and positive for TLR2 mutant-DNA.Western Blot showed that the expression of?-SMA increased and the expression of E-cadherin decreased in WT+STZ group compared with control group,indicating that EMT occurred in renal tubular epithelial cells of mice,but the expression of?-SMA decreased and the expression of E-cadherin increased in TLR2^-/-+STZ group compared with WT+STZ group.5.Knockout of TLR2 gene could inhibit extracellular matrix in renal tubules of diabetic mice.The results of IF,IHC and Masson showed that the expression of FN and Collagen?was up-regulated and the number of blue-stained collagen fibers in WT+STZ group was higher than that in control group.But in TLR2^-/-+STZ group,the expression of FN and Collagen?decreased and the blue-stained collagen fibers decreased.6.Knockout of TLR2 gene could reduce the expression of TGF-?1 in renal tubular cells of diabetic.The expression of TGF-?1 in WT+STZ group increased,after knockout of TLR2 expression,the expression of TGF-?1decreased in TLR2^-/-+STZ group.Conclusions:The overexpression of TLR2 played an important role in tubular EMT of diabetic nephropathy partly by upregulating TGF-?1 expression.