Background: 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.
Methods: 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.
Results: 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.
Conclusion: 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.