Reduced Cx43 expres sion increases fibrosis and pro arrhythmia in aged and pressure selleck chem overloaded mice due to enhanced fibroblast ac tivity. In our study, high glucose or silencing of Cx43 by Cx43 siRNA induced the upregulation of ICAM 1, TGF B1 and FN. Overexpression of Cx43 and Cx43CT at tenuated the increase in FN induced by high glucose in GMCs, confirming the importance of Cx43 in renal fibro sis. The c Src inhibitor PP2 also exhibited an inhibitory ef fect on the overexpression of ICAM 1, TGF B1 and FN induced by high glucose, thus confirming the role of c Src in the activation of NF B. Conclusions Our study describes a novel mechanism of NF B ac tivation in high glucose treated GMCs involving Cx43.
In summary, downregulation of Cx43 induced Inhibitors,Modulators,Libraries by high glucose Inhibitors,Modulators,Libraries activates c Src, which in turn promotes inter action between c Src and IB and contributes to NF B activation, leading to renal inflammation. The results presented in this study show that Cx43 induces NF B activation and fibrosis in GMCs, which is beneficial for the development of new therapies against DN. However, the mechanism by which regulation of Cx43 expression occurs requires further study. Methods Cell culture and transfection Rat GMCs were separated from the glomeruli of Sprague Dawley rats and identified via a specific assay as previously described. The cultured cells were used at confluence between the 5th and 8th passages. Con fluent cells were rendered quiescent by incubation for 24 h in serum free medium before treating with glucose or osmotic control for various times.
10 uM PP2 or 10 uM PP3 were added before high glucose for 30 min. Transfection of GFP Cx43, Flag Cx43CT and Cx43 siRNA plasmid were performed per the manufacturers instruction for Lipofectamine LTX Plus Reagent. Immunoprecipitation and immunoblotting The cell monolayers were lysed in a cell lysis buffer for im munoprecipitation. Immuno precipitation was performed by incubating Inhibitors,Modulators,Libraries 0. 5 mg cell lysate protein which was determined by bicinchoninic acid assay according to the manufactures instructions with 1ug of corre sponding antibody and protein GA agarose bead at 4 C overnight. Immunoblotting was performed as previously described. Kidney tissues were lysed, proteins were extracted as previously published. The nuclear and cytoplasmic proteins of GMCs were extracted using a commercially available assay kit and the total proteins were extracted as published.
The signals were visualized by a GE ImageQuant LAS4000mini, and analyzed using the Quan tity One Protein Analysis Inhibitors,Modulators,Libraries Software. The antibodies included mouse monoclo nal antibodies against connexin43, NF B p65, Inhibitor of B, p Tyr Inhibitors,Modulators,Libraries and FN, rabbit polyclonal antibody against c Src, goat polyclonal anti body against ICAM 1 and ZO 1, rabbit monoclonal Ruxolitinib structure antibodies against phospho c Src, connexin43, phospho IB and TGF B, rabbit monoclonal antibodies against Histone H1. 4 and tubulin, mouse monoclonal antibodies against Thy 1. 1 and RECA 1.