Telmisartan counteracts TGF-β1 induced epithelial-to-mesenchymal transition via PPAR-γ in human proximal tubule epithelial cells

Abstract

Chronic renal failure (CRF) mainly results from kidney fibrosis. Epithelial-to-mesenchymal transition (EMT) occurs in stressed tubular epithelial cells and contributes to renal fibrosis. Transforming growth factor-β1 (TGF-β1) has been shown to initiate and complete the whole EMT process. Peroxisome proliferators-activated receptor-γ (PPAR-γ) exerts anti-inflammatory, anti-fibrotic and vaculo-protective effects on different renal diseases. Telmisartan is a member of angiotensin II (Ang II) receptor blocker (ARB) family. Recent studies show that Telmisartan has a partial agonistic effect on PPAR-γ. Therefore, we tested the hypothesis that Telmisartan reverses the progression of induced EMT by TGF-β1 in cultured human renal proximal tubular epithelial (HK-2) cells. Cultured HK-2 cells were treated with TGF-β1 (3 ng/ml), a combination of TGF-β1 and Telmisartan (10-200 umol/L) and a combination of TGF-β1, Telmisartan and GW9662, a PPAR-γ antagonist for 48 hours. EMT was determined by quantitative real-time PCR analysis of E-cadherin (E-cad), Connective Tissue Growth Factor (CTGF) and PPAR-γ transcript expression and immunocytochemical analysis of E-cad, α-Smooth Muscle Actin (α-SMA) and PPAR-γ protein expression. TGF-β1 induced phenotypic EMT in cultured HK-2 cell line via significantly reduced E-cad expression and significantly increased CTGF, α-SMA expression in association with the loss of epithelial morphology. Telmisartan reversed all EMT markers in a dose-dependent manner which was inhibited by PPAR antagonist GW9662. In the present study, it was suggested that Telmisartan attenuated TGF-β1 induced EMT by agonistic activation of PPAR-γ.

Keywords: GW9662; PPAR-γ; TGF-β1; epithelial-to-mesenchymal transition; proximal tubular epithelial cells; telmisartan.

Figure 1

Measurement of mRNA level for CTFG, E-cad and PPAR-γ in absence or presence of TGF-β1 stimulation. Shown quantification of mRNA level of CTFG, E-cad and PPARr by RT-PCR measurement in absence or presence of TGF-β1 (3ng/ml) stimulation. Data were represented in triplicate experiments ± S.D.

Figure 2

Measurement of mRNA level for CTFG by TGF-β1 plus Telmisartan stimulation in absent or present GW9662. Shown quantification of mRNA level of CTGF by RT-PCR measurement after stimulating with TGF-β1 (3ng/ml) plus Telmisartan at different concentrations (0, 3, 10, 30, 100 or 200μM) in absence or presence of GW9662 (0, 10, 30 or 100μM). Data were represented in triplicate experiments ± S.D.

Figure 3

Measurement of mRNA level for E-cad by TGF-β1 plus Telmisartan stimulation in absence or presence of GW9662. Shown quantification of mRNA level of E-cad by RT-PCR measurement after stimulating with TGF-β1 (3ng/ml) plus Telmisartan at different concentrations (0, 10, 30 or 100μM) in absence or presence of GW9662 (0, 10, 30 or 100μM). Data were represented in triplicate experiments ± S.D.

Figure 4

Measurement of mRNA level for PPAR-γ by TGF-β1 plus Telmisartan stimulation in absence or presence of GW9662. Shown quantification of mRNA level of PPAR-γ by RT-PCR measurement after stimulating with TGF-β1 (3ng/ml) plus Telmisartan at different concentrations (0, 10, 30 or 100μM) in absence or presence of GW9662 (0, 10, 30 or 100μM). Data were represented in triplicate experiments ± S.D.

Figure 5

Immunofluorescence labelling of HK-2 cells for detecting expression of α-smooth muscle actin, E-cad and PPAR-γ Labelling HK-2 cells with mouse anti-human PPAR-γ (A), mouse anti-human E-cad (B) or mouse anti-human α-SMA (C) followed with donkey anti-mouse Alexa Fluor 594 for α-SMA and PPAR-γ or goat anti-mouse Alexa Fluor 488 for E-cad in non-stimulation, stimulation with TGF-β1 (3ng/ml), TGF-β1 plus Telmisartan (100μM) or TGF-β1 plus Telmisartan (100μM) and GW9662 (100μM). Mouse IgG (mIgG) labeling controls are shown. Bar 20μm.