[Effects of leflunomide on high glucose-induced podocyte cytoskeleton and its mechanism].
摘要
OBJECTIVE: To explore the effects of leflunomide active metabolite A771726 on high glucose-induced podocyte cytoskeleton and its possible signaling pathway. METHODS: The conditionally immortal human glomerular podocytes were divided into normal glucose (NG), mannitol (MA), high glucose (HG), high glucose with PDTC (pyrrolidine dithiocarbamate, a NF-κBp65 inhibitor) and high glucose with active leflunomide metabolite A771726 groups. Western blot was used to measure the ratio of p-NF-κBp65 to total NF-κBp65. And the protein and mRNA expressions of NF-κBp65, TRPC6 and nephrin were detected by Western blot and reverse transcription polymerase chain reaction (PCR). Immunofluorescence staining was used to detect the changes in the skeleton of podocyte. RESULTS: (1) Podocytes with high glucose could activate the NF-κBp65 signaling pathway. There was a significant increase of p-NF-κBp65 protein at 60 min versus 0 min (1.20 ± 0.04 vs 0.79 ± 0.02, P < 0.01). Little activation of the pathways was observed in groups NG and MA. The up-regulated protein expression of p-NF-κBp65 induced with high glucose was significantly inhibited by PDTC and A771726 (both P < 0.05). The difference of NF-κBp65 mRNA expression was not statistically significant between the groups (all P > 0.05). (2) High glucose-induced podocyte activated the NF-κBp65 signaling path. Its downstream TRPC6 mRNA and protein expression significantly increased than NG while nephrin became down-regulated more than NG. PDTC and A771726 inhibited the high expression of TRPC6 while the expression of nephrin was elevated (all P < 0.05). (3) Immunofluorescent assay of high glucose-induced podocyte cytoskeleton showed disorderly F-actin and a disappearance of tensile fiber after 72 h. CONCLUSION: Active leflunomide metabolite A771726 may protect podocytes through blocking the high glucose-induced signaling pathway of NF-κBp65.