Ferulic Acid Attenuates TGF-β1-Induced Renal Cellular Fibrosis in NRK-52E Cells by Inhibiting Smad/ILK/Snail Pathway

Abstract

Renal fibrosis is a common cause of renal dysfunction with chronic kidney disease. Central to this process is epithelial-mesenchymal transformation (EMT) of proximal tubular epithelial cells driven by transforming growth factor-β1 (TGF-β1) signaling. The present study aimed to investigate the effect of Ferulic acid (FA) on EMT of renal proximal tubular epithelial cell line (NRK-52E) induced by TGF-β1 and to elucidate its underlying mechanism against EMT related to TGF-β1/Smads pathway. The NRK-52E cells were treated for 48 h with TGF-β1 (5 ng/mL) in different concentrations of FA (0 to 200 µM). Fibronectin, a mesenchymal marker, was assessed by western blotting. Western blotting was also used to examine the EMT markers (E-cadherin, and α-smooth muscle actin (α-SMA)), signal transducer (p-Smad2/3), and EMT initiator (Snail). ILK was also assayed by western blotting. The results showed that TGF-β1 induced spindle-like morphological transition in NRK-52E cells. Smad2/3 signaling pathway activation, increased fibronectin, α-SMA, ILK, and Snail expression, and decreased E-cadherin expression in TGF-β1-treated NRK-52E cells. FA efficiently blocked P-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. These findings suggest that FA may serve as a potential fibrosis antagonist for renal proximal tubule cells by inhibiting EMT process.

Figure 1

FA reversed TGF-β1-induced morphological changes in NRK-52E cells. NRK-52E cells were incubated with 5 ng/mL of TGF-β1 for 48 h with different concentrations of FA (0, 25, 50, 100, and 200 μM). (a) Untreated NRK-52E cells showed that a pebble-like shape is clearly observed. (b) TGF-β1-treated cells showed a decrease in cell-cell contacts and adopt a more elongated morphological shape. (c), (d), (e), and (f) showed reversed TGF-β1-induced morphological changes by different concentrations of FA (magnification of 200x).

Figure 2

Effects of FA on TGF-β1-induced expression of fibronectin in NRK-52E cells. NRK-52E cells were incubated with 5 ng/mL of TGF-β1 for 48 h with different concentrations of FA (0, 25, 50, 100, and 200 μM). (a) The intercellular fibronectin was measured by western blotting with β-actin used as an internal control. (b) The fibronectin was quantitatively analyzed with Image J software. The data showing mean ± SD. ^# P < 0.05, ^## P < 0.01, and ^### P < 0.005 versus control (0 ng/mL TGF-β1). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.005 versus control (0 μM FA in the presence of 5 ng/mL TGF-β1).

Figure 3

Effects of FA on TGF-β1-induced expression of E-cadherin and α-SMA in NRK-52E cells. NRK-52E cells were incubated with 5 ng/mL of TGF-β1 for 48 h with different concentrations of FA (0, 25, 50, 100, and 200 μM). (a) Expression of E-cadherin and α-SMA at the protein level were determined by Western blotting with β-actin used as an internal control. (b) and (c) The expression level was quantitatively analyzed with Image J software. The data showing mean ± SD. ^# P < 0.05, ^## P < 0.01, and ^### P < 0.005 versus control (0 ng/mL TGF-β1). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.005 versus control (0 μM FA in the presence of 5 ng/mL TGF-β1).

Figure 4

Effects of FA on TGF-β1-induced expression of pSmad2/3 in NRK-52E cells. NRK-52E cells were incubated with 5 ng/mL of TGF-β1 for 48 h with different concentrations of FA (0, 25, 50, 100, and 200 μM). (a) Expression of Smad2/3 and pSmad2/3 at the protein level was determined by western blotting with β-actin used as an internal control. (b) The pSmad2/3 was quantitatively analyzed with Image J software. The data showing mean ± SD. ^# P < 0.05, ^## P < 0.01, and ^### P < 0.005 versus control (0 ng/mL TGF-β1). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.005 versus control (0 μM FA in the presence of 5 ng/mL TGF-β1).

Figure 5

Effects of FA on TGF-β1-induced expression of ILK and Snail in NRK-52E cells. NRK-52E cells were incubated with 5 ng/mL of TGF-β1 for 48 h with different concentrations of FA (0, 25, 50, 100, and 200 μM). (a) Expression of ILK and Snail at the protein level were determined by western blotting with β-actin used as an internal control. (b) and (c) The expression level was quantitatively analyzed with Image J software. The data showing mean ± SD. ^# P < 0.05, ^## P < 0.01, and ^### P < 0.005 versus control (0 ng/mL TGF-β1). ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.005 versus control (0 μM FA in the presence of 5 ng/mL TGF-β1).