介导血管紧张素Ⅱ上调近端肾小管上皮细胞金属蛋白酶组织抑制剂1表达的信号分子研究

Signalling molecules mediate the induction of TIMP-1 expression by angiotensin Ⅱ in human proximal tubular epithelial cells

目的 研究信号转导子和转录激活子(STAT)在血管紧张素Ⅱ(AngⅡ)诱导人近端肾小管上皮细胞系HK-2细胞表达基质金属蛋白酶组织抑制剂1(TIMP-1)过程中的作用。方法 采用凝胶阻滞电泳(EMSA)测定DNA-STAT结合活性变化。Supershift和Western印迹分析STAT蛋白的组成。激光扫描共聚焦显微镜观察活化STAT蛋白的核转位。采用RT-PCR方法检测HK-2细胞AT1和AT2受体的表达。Northern印迹检测TIMP-1的mRNA表达。结果 AugⅡ能够以剂量和时间依赖方式激活STAT1和STAT3。AngⅡ刺激后TIMP-1 mRNA的表达显著增加。HK-2细胞可表达AT1和AT2两种受体。AugⅡ激活STAT蛋白和上调TIMP-1的作用能被AT1受体拮抗剂阻断,而不能被AT2受体拮抗剂阻断。结论 AngⅡ通过激活近端肾小管上皮细胞的AT1受体来活化STAT1和STAT3信号分子,并可以进而上调TIMP-1的mRNA表达。

Objective To explore whether angiotensin Ⅱ can activate signal transducer and activator of transcription(STAT1/STAT3) and up-regulate tissue inhibitor of metalloproteinase-1 (TIMP-1) expression of HK-2 human renal proximal tubular epithelial cells. Methods Electrophoretic mobility shift assay(EMSA) was employed to determine STAT-DNA binding activity. Supershift and Western blotting assay were used to test the components of activated STAT proteins. Nuclear transloction of activated STAT proteins was examined under laser scanning confocal microscope. TIMP-1 mRNA expression in HK-2 was measured by Northern blotting analysis. AT1 and AT2 mRNA expression in HK-2 was measured by RT-PCR. Valsartan and PD123319 were used to block AT1 and AT2 receptors of angiotensin Ⅱ respectively. Results Angiotensin Ⅱ could activate STAT1 and STAT3 in both concentration and time dependent manners. Angiotensin Ⅱ could also up-regulate TIMP-1 mRNA expression. RT-PCR results showed that both AT1 and AT2 receptors of angiotensin Ⅱ were constitutively expressed in HK-2 cells. All the above effects activated by angiotensin Ⅱ could be blocked by valsartan but not PD123319. Conclusion Angiotensin Ⅱ can activate STAT1 and STAT3 through AT1 receptors, and up-regulate TIMP-1 mRNA expression.