Protective effect of DMDD, isolated from the root of Averrhoa carambola L., on high glucose induced EMT in HK-2 cells by inhibiting the TLR4-BAMBI-Smad2/3 signaling pathway.

Hyperglycemia stimulated epithelial-mesenchymal transition (EMT) plays a critical role in initiating and progressing renal fibrosis in diabetic kidney disease (DKD). It is crucial to explore novel renal protective drugs for the treatment of DKD.The present study is to confirm our hypothesis and to accumulate the information for the application of DMDD (2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione) as a novel therapeutic agent to potentially inhibit renal fibrogenesis and EMT in the DKD.High glucose induced renal proximal tubular epithelial cell line (HK-2 cells) was cultured and treated with DMDD. The cell viability and DMDD cytotoxicity were assessed by CCK8. Immunofluorescence was used for detection of TLR4 and downstream protein in normal and high glucose induced HK-2 cells. HK-2 cells were transfected with lentivirus codifying for BAMBI (BMP and activin membrane bound inhibitor) and interfering RNA for determination of the effect of BAMBI over-expression and silencing, respectively. TLR4-BAMBI-Smad2/3 pathway was analyzed by means of RT-PCR and western blot.A high concentration (60mM) of glucose induced significant EMT process and TLR4 expression was increased obviously in this circumstance. DMDD inhibited high expressions of TLR4 and Smad2/3 in HG induced cells and decreased the expression of BAMBI. In addition, the effects of decreased BAMBI expression and increased Smad2/3 expression in HG cultured cells were reversed in the cells of TAK-242 (TLR4 signaling inhibitor) intervention. BAMBI gene silencing dramatically increased EMT process and the over-expression of BAMBI was opposite in HK-2 cells with HG condition. These observations of EMT were ameliorated when the HK-2 cells were pre-treated with DMDD.Our study demonstrates that DMDD treatment improves EMT in the HG induced HK-2 cells. In addition, DMDD significantly inhibits EMT by TLR4-BAMBI-Smad2/3 pathway, which hints that DMDD may be an alternative approach in diabetic renal injury.