ABSTRACT
Objective To observe the expression of NOD2 and epithelial-mesenchymal transition (EMT) related proteins in podocytes in high glucose environment,and explore the molecular mechanism of NOD2 involved in EMT.Methods The human glomerular podocytes were the subjects of study.α-SMA and Nephrin expressions were detected by immunofluorescence;the mRNA and protein expressions of NOD2,Snail and EMT related proteins (α-SMA,Desmin,E-cadherin,Nephrin) were detected by real-time fluorescence quantitative PCR and Western blotting.The podocytes were stimulated by high-glucose after shRNA interfering the of NOD2 expression,and the expressions of Snail and subsequent EMT-related proteins were detected by Western blotting.Prior to the activation of NOD2 by muramyl dipeptide (MDP),shRNA was used to interfere with the expression of Snail.E-cadherin,Nephrin,Desmin,and α-SMA were detected by Western blotting.Results After 24 hours of high glucose stimulation,PCR and Western blotting results showed that the expressions of NOD2 and Snail were significantly increased;the expressions of epithelial phenotype proteins E-cadherin and Nephrin were down-regulated;and the expressions of interstitial phenotype proteins Desmin and α-SMA were increased (all P ＜ 0.05);while there was no significant change in the hypertonic control group.After interference with NOD2,the abnormal expression of Snail and EMT related proteins were all recovered.After interference with Snail expression,Compared with the MDP group,the protein expressions of E-cadherin and Nephrin were significantly increased (all P ＜ 0.05);the expressions of Desmin and α-SMA were significantly decreased.Conclusions High glucose can induce NOD2 expression in podocytes,and promote podocyte epithelial-mesenchymal transition by upregulating Snail expression.Gene intervention targeting the NOD2/Snail/EMT pathway can reduce high-glucose-induced podocyte injury and may provide new ideas for the treatment of diabetic nephropathy.
ABSTRACT
Objective To observe the expression of ChemR23 induced by Angiotensin Ⅱ (Ang Ⅱ) in podocyte and its role in renal injury.Methods Conditionally immortalized mice podocytes were cultured in vitro.Immunofluorescence was used to observe the sub-cellular location of ChemR23.The expressions of ChemR23,Nephrin and Podocin stimulated by different concentrations of Ang Ⅱ were detected by qRT-PCR and Western blotting.Lentivirus targeting ChemR23 was used.The expressions of Nephrin and Podocin and the phosphorylation state of NF-κB P65 were detected by Western Blot.The inhibitor of NF-κB P65 was added to the cultural medium for 2 h before Ang Ⅱ stimulation.The effect of NF-κB P65 inhibitor on Ang Ⅱ-induced expression of Nephrin and Podocin was detected by Western Blot.Results It is showed that ChemR23 was located in cytosol and membrane.Compared with the normal control,the expression of ChemR23 was significantly increased by Ang Ⅱ in mRNA and protein level,while the expressions of Nephrin and Podocin were decreased (P ＜ 0.05).When using Lentivirus vector to interfere the expression of ChemR23,Ang Ⅱ-repressed expressions of Nephrin and Podocin were restored (P ＜ 0.05).Western Blot showed the level of phosphorylated NF-κB P65 was significantly increased by Ang Ⅱ stimulation (P ＜ 0.05),which could be inhibited by interfering the expression of ChemR23.When adding the NF-κB P65 inhibitor,the low expression of Nephrin and Podocin induced by Ang Ⅱ stimulation was restored (P＜0.05).Conclusions Ang Ⅱ can induce ChemR23 expression,which activates NF-κB P65 signaling pathway,and then inhibits the expressions of Nephrin and Podocin.Targeting ChemR23 is a potential way to alleviate podocyte injury caused by Ang Ⅱ.