Pirfenidone inhibits TGF-β1-induced fibrosis via downregulation of Smad and ERK pathway in MDCK cells

Abstract

The prevalence of chronic kidney disease (CKD) in dogs increases with age, and renal fibrosis is an important pathophysiological mechanism in this process. However, only a few drugs that can effectively inhibit fibrosis in the kidneys of dogs are currently available. In this study, we aimed to determine whether pirfenidone, a drug that has shown antifibrotic effects in various clinical studies, also exerts antifibrotic effects on canine renal tubular epithelial cells, Madin-Darby canine kidney cells (MDCK). To this end, we treated MDCK cells with various concentrations of pirfenidone, followed by transforming growth factor-beta1 (TGF-β1) to stimulate fibrotic conditions. A cell viability assay was performed to determine the effect of pirfenidone on cell survival. Fibrosis-related markers and TGF-β1 fibrotic pathway-related markers were assessed using qPCR, Western blot analysis and immunocytochemistry. A one-way analysis of variance (ANOVA) was performed, followed by Tukey's post-hoc test for multiple comparisons. Pirfenidone treatment significantly reduced the expression of profibrotic markers such as α-smooth muscle actin, fibronectin, and collagen. Additionally, it upregulated the expression of E-cadherin, an epithelial marker. Furthermore, pirfenidone effectively inhibited the phosphorylation of key factors involved in the TGF-β1 signaling pathway, including Smad2/3 and ERK1/2. These results demonstrate that pirfenidone suppresses TGF-β1-induced fibrosis in MDCK cells by attenuating epithelial-mesenchymal transition and the relevant signaling pathways.

Keywords: Chronic kidney disease; Dog; Epithelial-mesenchymal transition; Pirfenidone; Renal fibrosis.

Fig. 1

Effects of pirfenidone (PFD) on cell viability and appearance in MDCK cells. Cells are treated with medium only, 0.1% DMSO (vehicle), or PFD (100, 200, 300, 400–500 µg/ml) for 48 h. A Cell viability is measured via a CCK-8 assay. Data are presented as mean ± SEM. The experiments are performed three times, with similar results. **P < 0.01. B Evaluation of cell morphology using phase contrast microscopy. Scale bar: 500 μm

Fig. 2

Effects of pirfenidone (PFD) on the TGF-β1 induced mRNA expression of EMT markers and ECM components tested using qPCR. Each graph represents relative mRNA expression of α-SMA (A), E-cadherin (B), Collagen1 (C), Fibronectin (D), and Vimentin (E). Cells are treated with medium only, TGF-β1 1 ng/mL, and TGF-β1 1 ng/mL with PFD 200–400 µg/mL. PFD is added 1 h before the TGF-β1 stimulation and the total incubation period was 24 h except for fibronectin (6 h). Data are presented as mean ± SEM. The experiments were performed three times, with similar results. ****P < 0.0001, ***P < 0.001, **P < 0.01 and *P < 0.05. ECM, extracellular matrix; EMT, epithelial-mesenchymal transition

Fig. 3

Effect of pirfenidone (PFD) on the TGF-β1 induced protein expression of EMT markers and ECM components tested using Western blot. Cells are treated with medium only, TGF-β1 1 ng/mL, and TGF-β1 1 ng/mL with PFD 200–400 µg/mL. PFD is added 1 h before the TGF-β1 stimulation, and the total incubation period was 48 h. A Protein levels of EMT markers and ECM. The experiments are performed three times, with similar results. Quantitative analysis of protein expression is performed, and the results of inter-group comparisons are shown. Each represents a relative ratio to GAPDH of fibronectin (B), Collagen3 (C), α-SMA (D), E-cadherin (E), and Vimentin (F). Data are presented as mean ± SEM. ****P < 0.0001, ***P < 0.001, **P < 0.01. ECM, extracellular matrix; EMT, epithelial-mesenchymal transition

Fig. 4

Effect of pirfenidone (PFD) on the TGF-β1 induced fibronectin expression on immunocytochemistry. Cells are treated with medium only, TGF-β1 1 ng/mL, and TGF-β1 1 ng/mL with PFD 200–400 µg/mL. PFD is added 1 h before the TGF-β1 stimulation and the total incubation period was 48 h. Then, cells are immunostained with an anti-fibronectin antibody and nuclei are counterstained with DAPI. The experiments are performed three times, with similar results. Scale bar: 400 μm

Fig. 5

Effect of pirfenidone (PFD) on the TGF-β1 induced protein expression of TGF-β1 signaling pathway components tested using Western blot. Cells are treated with medium only, TGF-β1 1 ng/mL, and TGF-β1 1 ng/mL with PFD 200–400 µg/mL. PFD is added 1 h before the TGF-β1 stimulation and the total incubation period is 48 h. A Protein levels of TGF-β1 signaling pathway markers. The experiments are performed three times, with similar results. Quantitative analysis of protein expression is performed, and the results of inter-group comparisons are shown. Each represents the relative ratio of p-Smad/t-Smad (B) and p-ERK/t-ERK (C). Data are presented as mean ± SEM. ****P < 0.0001, *P < 0.05