蛋白激酶Cα激活参与肿瘤坏死因子α引起的肝肾综合征肾小球滤过率下降

Activated Protein Kinase C alpha Participates in the Decreased Glomerular Filtration Rate in Hepatorenal Syndrome Induced by Tumor Necrosis Factor Alpha

目的研究肿瘤坏死因子α(TNF-α)对肾小球系膜细胞蛋白激酶Cα(PKC-α)活性的影响,揭示TNF-α引起肝肾综合征肾小球滤过率(GFR)下降的机制。方法选择大鼠系膜细胞株进行体外培养。按TNF-α处理的不同时间点(0、4、8、24h)分4组。分别应用Western blot、免疫荧光、免疫电镜及PKC-α活性定量检测方法,观察TNF-α对PKC-α表达量及活性的影响。结果 TNF-α处理组PKC-α蛋白表达与对照组比较无统计学差异(4h:0.75±0.13;8h:0.76±0.12;24h:0.78±0.10,vs 0h:0.84±0.11,P>0.05)。免疫荧光及免疫电镜发现:对照组PKC-α在胞质中呈弥漫性分布,核内无表达。TNF-α处理8、24h组PKC-α存在明显核周聚集,胞核内也可见少量PKC-α表达,以TNF-α处理8时最明显。PKC活性定量检测TNF-α处理8、24h组PKC-α活性明显增强(4h:1.11±0.96,P=0.612;8h:1.87±0.25,P=0.000;24h:1.68±0.14,P=0.000 vs 0h:1.07±0.06),以TNF-α处理8h组最明显(P=0.017 vs 24h)。结论 TNF-α对GMCs中的PKC-α蛋白表达量无影响,却能明显增加其PKC-α活性,后者可能是TNF-α引起GFR下降的重要信号。

Objective To investigate the effect of tumor necrosis factor alpha（TNF - c~ ） on the expression and activity of protein ki-nase C alpha（ PKC -ct ）in glomerular mesangial cells （GMCs） in order to delineate the mechanisms of decreased glomerular filtration rate in hepatorenal syndrome caused by TNF -c~. Methods We choosed GMCs line from rats as our material. GMCs were divided into TNF - a - treated 0, 4, 8,24h groups. We identified the effect of TNF - ct on the expression and activity of PKC - ot by Western blot,im- munofluorescence staining, immune electron microscopy and PKC activity assays. Results TNF -ct could not affect the protein level of PKC-a（4h：O.75＋0.13; 8h：0.76 ~0.12; 24h：0.78 ~0.10,vs0h： 0.84 ＋0.11,P〉0.05）. Immunofluorescence staining and im- mune electron microscopy for PKC -a showed that in unstimulated cells, PKC -ct was detected only in the cytoplasm. PKC -c~ in GMCs treated by TNF - ct showed subcellular localization to perinucleus and into nucleus which was the sign of activated PKC - ct. TNF - ot in- duced an increase in PKC - c~ activity. PKC - ct activity was globally upregulated in TNF - ct - treated 8,24h groups（4h ： 1.11 ~ O. 96, P =0.612; 8h：1.87 ~0.25,P=0.000; 24h：1.68 ＋0.14,P=O. 000 vs 0h：l.07 ＋0.06）. The maximal effect was seen at TNF-α treated 8h group（P = 0. 017 vs 24h）. Conclusion The phenomenon that TNF - α can not affect the protein expression levels of PKC - α but increase the activity of PKC - ct in GMCs may be an important signal in the mechanisms of decreased GFR caused by TNF - or.