Figure 10.

Klotho cytoprotection against phosphotoxicity in endothelial cells was attenuated by vascular endothelial growth factor receptor type 2 (VEGFR2) inhibitor. A: mouse endothelial cells (MS1) were incubated with 0–5 nM Ki8751, a VEGFR2 inhibitor, in the presence of normal phosphate (N P_i; 1.0 mmol/L) or high phosphate (H P_i; 2.0 mmol/L) and cotreated with Klotho (0.4 nM) or normal saline (NS) as vehicle for 24 h. Culture media were collected for lactate dehydrogenase (LDH) assay. Data are expressed as averages from six independent experiments under each condition, and statistical differences among four groups at 5 nmol/L Ki8751 were determined by two-way ANOVA followed by a Student–Newman–Keuls test. Statistical significance was accepted when *P < 0.05 and **P < 0.01 between two groups. B: comparison of LDH release in culture media between normal- or high-phosphate media with or without Ki8751 (2 nM) and cotreatment of Klotho after 24-h treatment. C: immunoblot analysis for CD31, VEGFR2, and β-actin in total cell lysates extracted from MS1 cells after 24-h treatment. C, left: representative blots. C, right: quantitative analysis of all immunoblots from independent experiments. D: representative immunofluorescent images for endothelial marker (CD31; red) and VEGFR2 (green) in MS1 cells from six independent experiments. Scale bar = 25 µm. White arrows indicate CD31 costained with VEGFR2. B and C: quantitative data are expressed with scatterplots of individual data points and means ± SD of six independent experiments. Statistical significance was evaluated by two-way ANOVA followed by a Student–Newman–Keuls test. Statistical significance was accepted when *P < 0.05 and **P < 0.01 between two groups and $P < 0.05 and $$P < 0.01 between vehicle and Klotho treatment within the same experimental condition. The sample number in each group is presented in parentheses underneath each corresponding bar.