Figure 8.
Klotho more significantly reduced high dietary phosphate-induced kidney fibrosis than vascular endothelial growth factor receptor type 2 (VEGFR2) haploinsufficiency-associated kidney fibrosis. A: kidneys from four different genotypes [wild-type (WT), VEGFR2+/−, Tg-kl, and Tg-kl;VEGFR2+/−] fed normal-phosphate (N Pi) or high-phosphate (H Pi) diet for 12 wk, respectively, as shown in Fig. 7A. Kidney sections were subjected to trichome (TC) stain to evaluate kidney fibrosis. A, top: representative microscopic images. Scale bar = 200 µm. A, bottom: quantitative analysis of fibrotic scores on TC-stained kidney sections with ImageJ software. B: immunofluorescent images for endothelial marker (PE; white), α-smooth muscle actin (α-SMA; red), and exogenous inserted gene [green fluorescent protein (GFP); green] in kidney sections. B, top: representative immunofluorescent images. Scale bar = 100 µm. White arrows indicate double staining of α-SMA with GFP. B, bottom: quantitative analysis of PE signal density on kidney sections with ImageJ software. C: immunoblot analysis for α-SMA, connective tissue growth factor (CTGF), and β-actin in total kidney lysates. C, left: representative blots. C, right: quantitative analysis of all immunoblots of all mice from each group. A–C: quantitative data are expressed with scatterplots of individual data points (open circles indicate male mice and pink circles indicate female mice) and means ± SD (bars and errors) of all mice from each group. 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 within the same phosphate diet treatment and $P < 0.05 and $$P < 0.01 between normal- and high-phosphate diet within the same genotype. The sample number in each group is presented in parentheses underneath each corresponding bar. PE, a cocktail of primary antibodies against platelet-endothelial cell adhesion molecule-1 and endomucin.