ERK通路抑制剂对白蛋白诱导人近端肾小管上皮细胞细胞外基质合成的影响

Effects of ERK Pathway Inhibitors on Albumin-induced Excessive Accumulation of Extracellular Matrix of Human Proximal Tubular Epithelial Cells

目的探讨细胞外信号调节激酶(ERK)通路抑制剂对人血清白蛋白诱导人近端肾小管上皮细胞细胞外基质(ECM)合成的影响。方法将体外培养的人近端肾小管上皮细胞株HK-2细胞按是否加入干预剂分为4组:A组(空白对照组)、B组、C组和D组。A组不加入任何干预剂。B组加入人血清白蛋白5g·L-1。C组加入ERK1/2信号通路的特异抑制剂PD98059 10μmol·L-1。D组加入ERK1/2信号通路的特异抑制剂PD9805910μmol·L-1预处理45min。预处理后,再加入人血清白蛋白5g·L-1。各组细胞均放置水套式CO2培养箱培养0、12、24和48h。培养0、12、24和48h后收取细胞,用于MMP-9、TIMP-1和col-ⅣmRNA及蛋白的检测。应用逆转录-聚合酶链反应(RT-PCR)法检测4组0、12、24和48h时MMP-9、TIMP-1和col-ⅣmRNA表达水平。ELISA法检测4组0、12、24和48h时MMP-9、TIMP-1和col-Ⅳ蛋白表达水平。结果 B组、D组12、24h时MMP-9、TIMP-1和col-ⅣmRNA及蛋白表达水平均显著高于A组(均P<0.01),48h时MMP-9、TIMP-1和col-ⅣmRNA及蛋白表达水平均显著低于A组(均P<0.01);D组12、24和48h时MMP-9、TIMP-1和col-ⅣmRNA及蛋白表达水平均显著低于B组(均P<0.01),MMP-9、TIMP-1和col-ⅣmRNA及蛋白表达水平均显著高于0h(均P<0.01)。结论人血清白蛋白可能通过ERK通路作用于HK-2细胞,促进ECM的合成和抑制ECM的降解,诱导ECM积聚,从而参与肾小管间质纤维化。

Objective To explore the effects of extracellular signal-regulated kinase （ERK） pathway inhibitors on albumin-induced excessive accumulation of extracellular matrix （ECM） of human proximal tubular epithelial cells in vitro. Methods The cultured HK-2 cells were divided into four groups. Group A only received the vehicle. Group B and C were treated with human serum albumin（5 g . L-1） and ERK1/2 inhibitor PD98059（10 umol. L-1）,respectively. Group D was given human serum albumin（5 g . L-1） after pretreatment with PD98059（10umol . L-1） for 45 minutes. Cells were cultured for 0,12,24 and 48 hours in a CO2 incubator, respectively. The mRNA and protein expression of matrix metalloproteinase-9 （MMP-9）, tissue inhibitor of metalloproteinase-1（TIMP-1） and type W collagen（col-iv） were detected by RT-PCR and ELISA, respectively. Results Compared with group A, the mRNA and protein expression of MMP-9, TIMP-1 and col-IV in group B and D increased at 12 and 24 hours after treatment,but decreased in group D at 48 hours after treatment（P〈0.01）. Compared with group B, the mRNA and protein expression of MMP-9,TIMP-1 and col-IV in group D increased at 12,24 and 48 hours after treat- ment（P〈0.01）. In group D,the mRNA and protein expression of MMP-9,TIMP-1 and col-IV at 12,24 and 48 hours after treatment was higher than that at the beginning of treatment （P〈 0.01）. Conclusion Human serum albumin may promote ECM synthesis,inhibit ECM degradation and induce ECM accumulation through the ERK pathway to participate in renal tubulointerstitial fibrosis.