Figure 6.
Vascular endothelial growth factor receptor type 2 (VEGFR2) haploinsufficiency promoted high dietary phosphate-induced kidney fibrosis in kl/+ mice. A: kidneys from four different genotyped mice [wild-type (WT), VEGFR2+/−, kl/+, and kl/+;VEGFR2+/−] that were fed normal-phosphate (N Pi) or high-phosphate (H Pi) diet for 12 wk, respectively. 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 locate double staining with α-SMA and GFP. B, bottom: quantitative analysis of PE staining 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, top: representative blots. C, bottom: quantitative analysis of all immunoblots from each group. A−C: quantitative data are expressed with scatterplots of individual data point (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. Significant differences were 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.