骨桥蛋白和核转录因子在慢性环孢素A肾毒性中的作用

Roles of osteopontin and nuclear transcription factor in chronic cyclosporine nephrotoxicity

目的探讨炎性介质骨桥蛋白(OPN)的表达和核转录因子在慢性环孢素A(CsA)肾毒性中的作用。方法雄性Sprague-Dawley大鼠喂食低盐(0.05%钠盐)饲料下分为两组,正常对照组给予皮下注射橄榄油(1 mL·kg-1·d-1);毒性组给予皮下注射CsA(15 mg·kg-1·d-1)4周。通过观察炎性细胞浸润(ED-1)和肾小管间质纤维化,比较两组大鼠的肾小管间质损伤程度;采用RNA印迹、免疫组织化学染色方法检测OPN在mRNA和蛋白水平的表达;用凝胶电泳迁移率实验(EMSA)分析法和免疫印迹法测定核转录因子(NF-κB和AP-1)的结合活性和IκB蛋白的表达。结果毒性组大鼠表现为肾小管间质带状纤维化[(38.9±3.3)%/5 mm2vs(0±0)%/5 mm2,P<0.01]和大量ED-1阳性细胞浸润[(89±9)vs(7±2),P<0.01]。与对照组相比,毒性组大鼠OPN mRNA和蛋白的表达增加[(729±37)%vs(103±4)%,P<0.01],分布于肾小管上皮细胞,其主要位于肾小管间质损伤部位;毒性组核转录因子NF-κB[(218±19)%vs(116±15)%,P<0.01]和AP-1[(735±225)%vs(101±4)%,P<0.01]结合活性增加,而IκB蛋白的表达减少[(9±7)%vs(105±7)%,P<0.01]。直线相关分析示OPN mRNA的表达与肾小管间质纤维化程度(r=0.959,P<0.001)和核转录因子的结合活性呈正相相关(NF-κB:r=0.773,P<0.01;AP-1:r=0.619,P=0.01)。结论炎性介质OPN和核转录因子参与了慢性CsA肾毒性肾小管间质的损伤。

Objective To investigate the roles of osteopontin（OPN） and nuclear transcription factor in a rat model of chronic cyclosporine A （CsA） nephrotoxicity. Methods Male Sprague-Dawley rats maintained on a low salt diet were treated daily with vehicle （olive oil, 1 mL · kg^-1 · d ^-1, s. c. ） and CsA （15 mL · kg^-1 · d^-1 , s. c. ） for 4 weeks. Renal histopathology was estimated by trichrome staining （tubulointerstitial fibrosis） and immunohistochemistry （ED-I） to assess the degrees of renal tubulointerstitial lesions. In addition, OPN mRNA and protein expression and nuclear transcription factor （NF-κB and AP-1） were studied by northern blotting analysis, immunohistochemistry, electrophoretic mobility shift assay, and immunoblotting analysis. Results CsA-treated rats displayed significantly striped tubuIointerstitial fibrosis （[38.9 ± 3.3]%5 mm^2 vs [0 ± 0]%5 mm^2 , P〈0.01） and increased ED-l-positive cells （89±9 vs 7±2, P〈0.01）. Compared with VH-treated rats, CsAtreated rats showed significantly upregulated OPN mRNA and protein expression in the proximal tubular cells, mainly localizing at areas of severe injured tissues. CsA-treated group also had significantly increased activities of NF-κB （[218 ± 19] % vs [116 ± 15]%, P〈0.01） and AP-1 （[735±225]% vs [101±4]%, P〈0.01）, and significantly decreased expression of （[9±7]% vs [105±71%, P〈0.01）. Correlation analysis revealed that upregulated OPN mRNA was positively correlated with tubulointerstitial fibrosis （r= 0. 959, P〈0. 001） and activities of NF-κB and AP-1 （NF-κB： r= 0. 773, P〈0. 01, AP-1 ： r= 0. 619, P=0.01, respectively）. Conclusion Our findings suggest that OPN, nuclear transcription factor NF-κB and AP-1 are involved in renal tubulointerstitial injury in chronic CsA nephrotoxicity.