Chen Xiangmei, Wang Jianzhong, Zhou Feng, Wang Xiaodan, Feng Zhe
Department of Nephrology, Chinese General Hospital of PLA, Beijing, China.
Kidney Int. 2003 Aug;64(2):459-67. doi: 10.1046/j.1523-1755.2003.00133.x.
Angiotensin II and tissue type inhibitor metalloproteinase-1 (TIMP-1) have been implicated in renal tubulointerstitial fibrosis, but the exact mediating signaling pathway is still unknown. Angiotensin II has been reported to activate signal transducers and activators of transcription (STAT) and induce proliferation of myocyte and vascular smooth muscle cells (VSMC). We hypothesized that the STAT signal pathway is involved in the process of renal tubulointerstitial fibrosis. Therefore, we designed the present study to explore whether angiotensin II could induce TIMP-1 expression in human proximal tubular epithelial cells, and whether it was mediated through the STAT signaling pathway.
Electrophoretic mobility shift assay (EMSA) was employed to determine the DNA-STAT binding activity. Supershift assay was used to test the components of activated STAT proteins. Nuclear translocation of activated STATs was observed with laser scanning confocal microscopy. TIMP-1 expression was analyzed with Northern and Western blots. Valsartan and PD123319 were used to block the effects of angiotensin II type 1 (AT1) and angiotensin II type 2 (AT2) receptors of angiotensin II, respectively.
Cultured human proximal tubular epithelial cells constitutively expressed TIMP-1. Angiotensin II induced TIMP-1, mRNA, and protein expressions in time- and dose-dependent manners, which could be inhibited by the AT1 receptor antagonist valsartan, but not by the AT2 antagonist PD123319. Angiotensin II also activated STAT-DNA binding activity in both dose-dependent and biphasic time-dependent manners, and increased the phosphorylation and nuclear translocation of STAT proteins. To examine the role of STAT in angiotensin II-induced TIMP-1 expression, STAT1 and STAT3 antisense oligonucleotides were used. Northern and Western blots showed that STAT1 and STAT3 antisense oligonucleotides could inhibit angiotensin II-induced TIMP-1 expressions, and STAT1 and STAT3 proteins, respectively, could be supershifted by their polyclonal antibodies.
STAT1 and STAT3 may, at least in part, mediate angiotensin II-induced TIMP-1 mRNA expression in human renal proximal tubular epithelial cells, implicating a role of the STAT signaling pathway in pathogenesis of renal tubulointerstitial fibrosis.
血管紧张素II和组织型金属蛋白酶抑制因子-1（TIMP-1）与肾小管间质纤维化有关，但确切的介导信号通路仍不清楚。据报道，血管紧张素II可激活信号转导子和转录激活子（STAT），并诱导心肌细胞和血管平滑肌细胞（VSMC）增殖。我们推测STAT信号通路参与肾小管间质纤维化过程。因此，我们设计了本研究，以探讨血管紧张素II是否能诱导人近端肾小管上皮细胞中TIMP-1的表达，以及其是否通过STAT信号通路介导。
采用电泳迁移率变动分析（EMSA）确定DNA-STAT结合活性。用超迁移分析检测活化STAT蛋白的成分。用激光扫描共聚焦显微镜观察活化STAT的核转位。用Northern和Western印迹分析TIMP-1的表达。分别用缬沙坦和PD123319阻断血管紧张素II 1型（AT1）和2型（AT2）受体的作用。
培养的人近端肾小管上皮细胞组成性表达TIMP-1。血管紧张素II以时间和剂量依赖性方式诱导TIMP-1、mRNA和蛋白表达，AT1受体拮抗剂缬沙坦可抑制其表达，而AT2拮抗剂PD123319则不能。血管紧张素II还以剂量依赖性和双相时间依赖性方式激活STAT-DNA结合活性，并增加STAT蛋白的磷酸化和核转位。为了研究STAT在血管紧张素II诱导的TIMP-1表达中的作用，使用了STAT1和STAT3反义寡核苷酸。Northern和Western印迹显示，STAT1和STAT3反义寡核苷酸可分别抑制血管紧张素II诱导的TIMP-1表达以及STAT1和STAT3蛋白，它们的多克隆抗体可使其发生超迁移。
STAT1和STAT3可能至少部分介导血管紧张素II诱导的人肾近端肾小管上皮细胞中TIMP-1 mRNA的表达，提示STAT信号通路在肾小管间质纤维化发病机制中起作用。
