[Effects of leflunomide on high glucose-induced podocyte cytoskeleton and its mechanism]

To explore the effects of leflunomide active metabolite A771726 on high glucose-induced podocyte cytoskeleton and its possible signaling pathway.

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.

(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.

Active leflunomide metabolite A771726 may protect podocytes through blocking the high glucose-induced signaling pathway of NF-κBp65.