| Background and objects: Chronic kidney disease is usually accompanied by inflammation and fibrosis,and renal fibrosis involves inflammation.Renal tubular epithelial cells(RTECs),epithelial mesenchymal transition(EMT)and macrophages play critical roles in kidney inflammation and fibrosis.In our previous study,we found that RTECs could promote kidney inflammation and fibrosis through expressing DC-SIGN in transdifferentiaton,and we further found that DC-SIGN interacting with TLR4 in macrophages may promote kidney inflammation and fibrosis.RUNX1 is a master regulator in hematopoietic stem cells.Recently,we found that RUNX1 may regulate TGF-β-induced EMT in our previous work and because inflammation is very closely related to fibrosis,we speculate that RUNX1 could play important roles in kidney inflammation and fibrosis.In this study,we explored the role of RUNX1 in regulating macrophages and RTECs in kidney inflammation and fibrosis based on the preliminary work,and we also explored the molecular mechanisms in this process.Methods:(1)Using RT-qPCR and Western blot analysis,RUNX1 expression at the mRNA and protein level were detected in human monocytic cell line(THP-1),mouse macrophage-like cell line(RAW 264.7),and primary mouse peritoneal macrophages(PEM)stimulated with 100 ng/mL or 1000 ng/mL LPS;Using specific siRNA to knock down RUNX1 in PEM,inflammatory cytokines(IL-1β,IL-6 and TNF-α)were detected by RT-qPCR and ELISA analysis after PEM were stimulated with LPS;Using a stable RAW 264.7 cell line that overexpressed RUNX1,inflammatory cytokines(IL-1β,IL-6 and TNF-α)were detected by RT-qPCR and ELISA analysis after this stable cell line was stimulated with LPS;Furthermore,inflammatory cytokines were also detected by RT-qPCR and ELISA analysis after the macrophages were intervened by RUNX1 inhibitor Ro 5-3335;(2)Using specific siRNA to knock down RUNX1 in PEM,JNK,ERK,p38 and p65 phosphorylation levels and IκBα degradation were detected by Western blot analysis;Moreover,the effect of RUNX1 and its different truncations(1-242 aa,50-178 aa and 243-453aa)on p50,p65 and p105 induced NF-κB luciferse activity was examined by a dual-luciferase reporter assay;The interaction between RUNX1 and NF-κB family members(p50,p65 and p105)were detected by co-immunoprecipitated experiments.(3)Inflammatory cytokines(IL-1β,IL-6 and TNF-α)were detected in different organs and serum of murine model of LPS-induced endotoxic shock intervened by RUNX1 inhibitor;(4)Using RT-qPCR and Western blot analysis,RUNX1 expression at the mRNA and protein level were detected in HK-2 cells stimulated with 5 ng/mL TGF-β;Using specific siRNA to knock down RUNX1 in HK-2 cells,the morphology of cells were observed by optical microscope after HK-2 cells were stimulated by TGF-β,and EMT maker(Snai2 and Vimentin)were examined by RT-qPCR and Western blot analysis;Using a stable NRK-52 E cell line that overexpressed RUNX1,EMT maker(Snail,Snai2,Vimentin and α-SMA)were detected by RT-qPCR and Western blot analysis;Moreover,RUNX1,TGF-β,Snail,Vimentin,α-SMA,Col1a1 and Fibronectin were detected by RT-qPCR in kidney of murine fibrosis model of unilateral ureteral occlusion(UUO).Results:(1)RUNX1 is constitutively expressed in macrophages,and LPS decreases RUNX1 levels in macrophages.Upregulation or downregulation of RUNX1 expression could promote or diminished production of inflammatory cytokines,including IL-1β and IL-6.(2)RUNX1 did not affect the downstream of TLR4 activation,including the ERK,JNK,p38,p65 phosphorylation levels and IκBα degradation in PEMs,but it significantly increased the p50,p65 and p105 induced NF-κB reporter activity,and the RUNX1 inhibitor,Ro 5-3335,partly reversed the synergized effect between RUNX1 and p50,p65 or p105.Besides,we further found that RUNX1 could interact with p50.(3)In vivo experiments indicates that A RUNX1 inhibitor protected against LPS-induced septic shock in vivo and inhibited the expression of IL-6 in kidney.(4)On the other hand,EMT inducer TGF-β could promote RUNX1 expression in RTECs;Upregulation or downregulation of RUNX1 expression could promote or block epithelial mesenchymal transition and expression of EMT maker,including Snai2 and Vimentin.(5)In vivo experiments further indicates that RUNX1 is markedly induced in murine fibrosis model of UUO,and EMT or fibrosis maker,including Snail,Snai2,Vimentin,fibronectin,Col1a1,CCL2 and TGF-β were also overexpressed in this fibrosis model.Conclusion: The interaction between RUNX1 and p50 may be the molecular mechanism that RUNX1 can regulate NF-κB signaling pathway of macrophages in kidney inflammation.Besides,RUNX1 may promote kidney fibrosis through epithelial mesenchymal transition,it indicates that RUNX1 may be the key regulator in kidney inflammation and fibrosis.The RUNX1 inhibitor can attenuate kidney inflammatory response and it suggests that this inhibitor may be the therapeutic target in renal inflammatory diseases or other inflammatory diseases. |
