肿瘤坏死因子α增强肾小球系膜细胞胞内钙释放参与肝肾综合征发病机制

Enhanced calcium release induced by tumor necrosis factor alpha in glomerular mesangial cells par- ticipated in hepatorenal syndrome

目的研究肿瘤坏死因子α（TNF-α）对肾小球系膜细胞（GMCs）胞内钙离子浓度（[Ca2＋]i）及细胞收缩的影响，证明TNF-α通过1，4，5-三磷酸肌醇受体（IP3Rs）增强GMCs对缩血管物质的易感性是肝肾综合征的重要机制。方法应用大鼠GMCs株，测量时均选无钙缓冲液及内皮素（ET）刺激。分正常对照ET刺激组（0h组）、TNF-α处理4hET刺激组（4h组）、TNF-α处理24hET刺激组（24h组）；另设上述3组于ET前10min加入IP3Rs阻断剂2-氨基乙氧基联苯硼酸盐（2-APB）。应用Fluo-3／AM标记及激光共聚焦显微镜，观察TNF-α预处理后GMCs对ET刺激后[ca2＋]i的变化及2-APB阻断后的变化。同时测量各组GMCs收缩前后表面积的变化以明确收缩幅度。结果ET刺激可使GMCs在短时间内[Ca22＋]i迅速、显著增加，TNF-α4h、24h组[Ca2＋]i上升幅度尤为明显[4h：（648．08±267．11）nmol／L；24h：（879．30±260．29）nmol／L；0h：（619．93±258．94）nmol／L，F：5．486，4hvs0h：P〈0．05；24hvs0h：P〈0．05；24hvs4h：P〉0．05]。2-APB阻断后3组细胞对ET刺激均失去反应，[Ca2＋]i无上升。ET刺激可使各组细胞面积缩小；TNF-α 4h、24h组上述效应更加明显[4h：（2198±340）μm2；24h：（2260±553）μm2；0h：（2436±474）μm2，F=4．001，4hvs0h：P〈0．05；24hvs0h：P〈0．05；24hvs4h：P〉0．05）。结论TNF-α可增加ET刺激引起的[Ca2＋]i上升进而引起细胞收缩，并证明是通过IP3Rs通路产生效应的，这可能是肝肾综合征时肾脏对缩血管物质产生高敏性进而引起。肾小球滤过率下降的重要机制。

Objective To investigate the effect of tumor necrosis factor-α （TNF-α） on intracellular calcium concentration （ [ Ca2 ＋] i ） and the contraction of glomerular mesangial cells （ GMCs）, and prove that hypercontractility of GMCs induced by TNF-α in hepatorenal syndrome（HRS） was connected with inositol 1,4,5-trisphophate receptors （IP3Rs）. Methods GMCs were divided into TNF-α-treated 0 h, 4 h, and 24 h groups. Another 3 groups were blocked by 2-APB. The effect of TNF-αon [ Ca2 ＋ ] i was identified and observed whether it could be blocked by 2-APB. Contraction of GMCs was determined by accessing the surface area of cells before and after contraction. Results TNF-α significantly increased ET-induced calcium release, in that we found higher [ Ca2 ＋ ] i after stimulated by ET in TNF-α -treated 4 h group and 24 h group[ 4 h： （648.08 ± 267.11 ）nmol/L; 24 h： （879. 30 ±260. 29） nmol/L; 0 h： （619. 93 ± 258. 94） nmol/L,F =5.486,4hvs0h：P 〈0.05;24hvs0h：P 〈0.05;24hvs4h：P 〉0.05]. This phenomenon can be totally blocked by 2-APB in all groups. The change in planar surface area in response to ET was slightly in control cells but significantly enhanced in TNF-α -treated cells [ 4 h：（2198 ± 340）μm2; 24 h： （2260 ~553） μm2 ; 0 h： （2436 ±474） μm2, F =4. 001,4 h vs 0 h： P 〈0. 05 ; 24 h vs 0 h： P 〈0. 05 ;24 h vs 4 h ： P 〉 0. 05 ]. Conclusion TNF-α can enhance ET-induced sarcoptasmic reticulum Ca2 ＋ re-lease and increase the contractile responses of GMCs to ET, which is associated with IP3Rs. TNF-α is re-sponsible for hyperconstractility of glomeruli in HRS.