| Background and objects: Tubulointerstitial injury and interstitial fibrosis are the main causes and common pathological processes of end-stage renal failure due to different etiologies of chronic kidney disease.Inflammatory and immune responses are important physiological processes of tubulointerstitial lesions,and their inflammatory regulatory mechanisms are still not clear.Many evidences indicate that epithelial mesenchymal transition(EMT)plays an important role in renal interstitial fibrosis.Renal interstitial inflammation not only induces changes in local immune microenvironment but also induces EMT.Therefore,current researches focus on revealing the mechanism of tubulointerstitial lesions and fibrosis mechanism and the progress of renal disease,is the current research focus.Our previous study found that tubular epithelial cells in the early stage of inflammation can express dendritic cell(DC)phenotype DC-SIGN(DC-specific ICAM-3-grabbing non integrin)through transdifferentiation.DC-SIGN can induce T cell pro-inflammatory responses,and participate in the interrenal inflammation and fibrosis.As an innate immune molecule DC-SIGN,it can interact with TLR4 to mediate the DC-like function of renal tubular epithelial cells and participate in the regulation of renal local inflammation.However,how the renal tubular epithelial cells transdifferentiate into DC-like cells and its relationship with EMT are not yet clear.We further found that RUNX1(Runt-related transcription factor 1)was expressed in renal tubular epithelial cells with DC-like transdifferentiation and the process of EMT,suggesting that the transcription factor may also be involved in the DC-like transdifferentiation of renal tubular epithelial cells and EMT regulation,and there may be a correlation between the two.Therefore,this study from the perspective of inflammation and transdifferentiation,combined with DC-like transdifferentiation of renal tubular epithelial cells and its relationship with EMT,this study intends to further explore the transdifferentiation of renal tubular epithelial cells and their molecules by observing the molecular regulation of RUNX1 transdifferentiation and mechanism.Methods:(1)Human renal tubular epithelial cells(HK-2)were treated with TNF-α(20 ng/m L),and the expression of RUNX1 was detected by real-time quantitative PCR and Western blot.The expression of RUNX1 and DC-SIGN was detected by real-time quantitative PCR.Then the mouse model of nephritis injury was established and used to detect the expression of RUNX1 in renal tubular epithelial cells by real-time quantitative PCR.(2)The RUNX1-overexpressing HK-2 cells were stably transfected with TGF-β(20ng/m L),and real-time quantitative PCR was used to detect the expressions of RUNX1,DC-SIGN,E-cadherin(CDH1)and Snail2,and subsequently transfected with HK-2 cells overexpressing RUNX1.The cells were stimulated with TGF-β and the expression of PU.1 and DC-SIGN was detected by real-time quantitative PCR.Overexpression of HK-2cell stably transfected PU.1 was stimulated by TGF-β.The expression levels of PU-1 and DC-SIGN were detected by real-time PCR and Western blot.(3)The renal tissue samples of renal injury were collected and the expression of DC-SIGN and TLR4 in renal tissue were detected by immunohistochemical staining.The HK-2 cells were treated with LPS(100ng/m L)Western blot was used to detect the expression of DC-SIGN.HK-2 cells were used to stimulate LPS.The interaction and localization of DC-SIGN and TLR4 were detected by protein immunoprecipitation and immunofluorescence.In addition,DC-SIGN si RNA was used to stimulate LPS.The phosphorylation levels of p65,IKKε,TBK1,p38 and JNK were detected by real-time quantitative PCR and Western blot.Immunofluorescence staining was used to detect the nuclear translocation of p65 in LPS.(4)HK-2 cells were treated with TGF-β(20 ng/m L),dual-luciferase gene reporter system was used to detect the expression of RUNX1 On TGF-β-induced Smad3 activity status;followed by protein immunoprecipitation detection of RUNX1 and Snail,Slug and Twist1 and other interactions;then use HK-2 cells transfected RUNX1 si RNA,the effect of RUNX1 on the expression of ATP1B1 and HIF-1α induced by TGF-β was detected by Western blot.The cells were stably transfected with HK-2 cells transfected with RUNX1 si RNA and HK-2 cells overexpressing RUNX1 The effect of RUNX1 on TGF-β-induced phosphorylation of JNK,ERK,p38 and Akt was detected by Western blot.Finally,renal fibrosis model was constructed by knocking out Runx1 mouse renal tubular epithelial cells and RUNX1 in vivo validation of these effects.Results:(1)The results showed that TNF-α can significantly promote RUNX1 expression in HK-2 cells,and the stimulation effect was significantly higher than the control group with IL-4 and CSF1 induction effect.Compared with the control group mice,the tubular epithelial cells of nephritis injury model mice in vivo were confirmed low expression of RUNX1.Model group mice with impaired renal function significantly expressed RUNX1 in the isolated renal tubular epithelial cells.Then HK-2 cells with RUNX1 overexpression were used to find that compared with the HK-2 control group,the steady-state cells cells significantly expressed RUNX1 and DC-SIGN whether with the TNF-α stimulation or not.The expression of DC-SIGN was also further up-regulated in HK-2 cells treated with higher stimulation of TNF-α.(2)Using HK-2 cells with overexpression of RUNX1,stimulated with TGF-β,it was found that overexpression of RUNX1 can significantly downregulate E-cadherin expression and upregulate Snail,Slug,Vimentin and DC-SIGN expression.Overexpression of RUNX1 in HK-2 also promote the PU.1 expression.Then,the HK-2 cells stably transfected with the above PU.1-overexpressing PU.1 gene were stimulated by EMT-inducing factor TGF-β and found that overexpression of PU.1 could significantly promote DC-SIGN expression.(3)Using renal tissue samples from patients with renal injury,we found that renal tubular epithelial cells in renal injury all highly expressed DC-SIGN and TLR4,and both of them were located in renal tubular epithelial cells;then HK-2 cells were used and stimulated with LPS The results showed that DC-SIGN and TLR4 were co-localized on the surface of HK-2 cells with the complex of DC-SIGN.DC-SIGN si RNA could inhibit the activation of TBK1 and nuclear translocation of p65 in TLR4-Myd88-independent pathway induced by LPS,and also inhibited the secretion of the inflammatory cytokines IL-6 and TNF-α in HK-2 cells.(4)The RUNX1 si RNA intervention and overexpression of RUNX1 HK-2 cell stable transfected cells were found to inhibit or upregulate the EMT markers HK-2 cells SNAI1,SLUG,N-cadherin expression;subsequently found that RUNX1 regulation of EMT are It has nothing to do with the TGF-β/Smad3 pathway and its interaction with EMT-related regulatory molecules(Snail,Slug,Twist1),ATP,and hypoxia inducible factor(HIF-1α)Subunit p110δ is expressed and induces Akt activation,which in turn regulates the PI3K/Akt pathway.Further verification was performed using the renal fibrosis model of the renal tubular epithelial cells of Runx1 knockout mice.Conclusion: RUNX1 is involved in the regulation of DC-like transformation of renal tubular epithelial cells in inflammation and EMT.In the above process,RUNX1 can cooperate with PU.1 to regulate renal tubular epithelial cells to DC-like transdifferentiation,involved in the regulation of kidney local inflammation or immune damage.In addition,RUNX1 also participates in the occurrence of EMT and the formation of renal fibrosis by regulating the PI3K/Akt signaling pathway,suggesting that RUNX1 may be a key regulator of renal tubular epithelial cell transdifferentiation. |
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