The influence of fasudil on renal proximal tubular cell epithelial-mesenchymal transition induced by parathormone

Abstract

Background: Renal fibrosis is a common pathway through which a variety of chronic kidney diseases progress to end-stage renal disease. Epithelial-mesenchymal transition (EMT) of renal proximal tubular cells is one of the most important factors in renal fibrosis. This study investigates if fasudil could influence EMT of renal proximal tubular cells.

Methods: HK-2 cells in passage 3-4 were used for all experiments. The cells were divided into five groups and treated with different concentrations of PTH and then observe cellular morphological changes at 0, 24 and 48 h using an inverted microscope and investigate the expression of the epithelial cell marker E-cadherin and the renal fibroblast marker α-smooth muscle actin (α-SMA).

Results: PTH significantly induced EMT, fasudil-inhibited EMT induced by PTH to different degrees, and the inhibitory effect of fasudil was most pronounced at 20 μmol/L.

Conclusion: Monitoring PTH levels, early prevention and control of hyperparathyroidism and reducing the concentration of PTH are important means to improve prognosis and delay the progression of chronic kidney disease. Fasudil can restrain EMT induced by PTH; this conclusion provides experimental data for the application of fasudil in the clinical prevention and treatment of renal fibrosis.

Keywords: Renal proximal tubular epithelial cells (HK-2); epithelial–mesenchymal transition (EMT); fasudil; parathormone; renal fibrosis.

Figure 1.

The impact of fasudil on cell morphology. HK-2 cells were treated with 0, 10, 20 and 30 ng/mL Fasudil in the presence of 10⁻¹⁰ mol/L PTH for 48 h. Cells were observed with an inverted microscope at 100× magnification.

Figure 2.

Effect of fasudil on HK-2 cells. (A) HK-2 cells were cultured in each group as discussed in the Methods section. RT-PCR was performed and the expression of E-cadherin and α-SMA was normalized to actin. Results are from three independent experiments performed in triplicate and are displayed as the relative expression normalized to the control samples. Values are the means with S.E.M. shown by vertical bars. *p < .05. (B) Densitometric analysis for western blots. Values are the means from triplicate determinations with the S.E.M. shown by vertical bars. *p < .05. (C) Western blot for E-cadherin and α-SMA using HK-2 cells treated as aforementioned. (D) The number of positive-stained cells by immunofluorescence is shown by vertical bars. *, ** and #p < .05.

Figure 3.

Effect of fasudil on cell immunofluorescence. Immunofluorescence staining of HK-2 cells captured by inverted fluorescence microscope at 100× magnification. The number of cells from 10 random fields for each group was calculated. Data are presented as the mean ± S.E.M. *p < .05 versus control. Red: E-cadherin expression; Green: α-SMA expression; Blue: nuclei (DAPI). Cells coexpressing α-SMA and E-cadherin are yellow. Results are from three experiments performed in triplicate.