Figure 9.

Klotho attenuated endothelial injury and mesenchymal transition induced by high phosphate in cultured media. A–C: MS1 cells were incubated with Klotho (0.4 nM) in the presence of normal phosphate (N P_i; 1.0 mM) or high phosphate (H P_i; 2.0 mM). Culture media were collected for lactate dehydrogenase (LDH) release assay and cells for immunoblot analysis and immunohistochemistry at 24 h. A: LDH release in culture media. B: immunoblot analysis for CD31, vascular endothelial growth factor receptor type 2 (VEGFR2), and β-actin in total cell lysates extracted from MS1 cells. B, left: representative immunoblots. B, right: quantitative analysis of all immunoblots from six independent experiments. C: representative immunofluorescent images for endothelial marker (CD31; red) and VEGFR2 (green) in MS1 cells from six independent experiments. Scale bar = 25 µm. White arrowheads indicate CD31 costained with VEGFR2. D: MS1 cells were incubated in the conditions described in A–C but with 48-h incubation. Immunoblot analysis for α-smooth muscle actin (α-SMA) and β-actin in total cell lysates. D, left: representative immunoblots. D, right: quantitative analysis of all immunoblots from six independent experiments. A, B, and D: quantitative data are expressed with scatterplots of individual data points and means ± SD (bars and errors) of six independent experiments. Statistical significance was evaluated by two-way ANOVA followed by a Student–Newman–Keuls test, and significance was accepted when *P < 0.05 and **P < 0.01 between two groups. The sample number in each group is presented in parentheses underneath each corresponding bar.