Fig 1.
Urine PPi concentration and renal gene expression in Npt2a-/- mice.
Urine pyrophosphate concentration (U-PPi, A) following an overnight fast and renal gene expression as indicated on the y-axis for ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1, B), progressive ankylosis (Ank, C), ectonucleoside triphosphate diphosphohydrolase 5 (Entpd5, D), tissue nonspecific alkaline phosphatase (Tnsalp, E) in mice fed regular chow for 10 weeks. The data represent mean±SEM of 4–19 mice, p-values shown above the lines of comparisons were calculated by one-way ANOVA using Tukey’s adjustment for multiple comparisons (A) and Student’s t-test (B-E).
Fig 2.
The hypomorphic Enpp1asj allele worsens renal mineralization area seen in Npt2a-/- mice on regular chow.
Histomorphometric analysis of renal mineralization (%calcified area = 100*mineralization area/tissue area, A; calcification size = mineralization area/number of calcifications, um2, B) in 10 um sections of kidneys from mice fed regular chow for 10 weeks. The data represent individual animals (closed circles) with the means±SEM, p-values shown above the lines of comparisons were calculated by one-way ANOVA using Tukey’s adjustment for multiple comparisons, no significant differences were detected between groups in panel B.
Fig 3.
Urinary pyrophosphate concentration is inversely associated with renal mineralization size in a combined bivariate linear regression analysis of all mice.
All experimental WT and mutant mice from Fig 2 (n = 28) for which urine was available were evaluated using linear regression analysis to determine the association of renal mineralization with the urine pyrophosphate concentration (U-PPi) (% calcified area = 100*calcified area/total area A and calcification size = calcified area/number of mineralization B). Data points represent values of individual animals. Results of the linear regression analysis are shown as solid line with 95% confidence interval (stippled lines), R2 and p-values.
Fig 4.
Intraperitoneal injection of Na-pyrophosphate reduces cortical and medulary renal mineralization in Npt2a-/- mice.
Light micrographs of 10 um renal sections prepared from paraffin-embedded kidneys, obtained from mice with various genotypes fed regular chow for 10 weeks (A, upper panels: von Kossa, methylene green staining, 4X, and A, lower panels: von Kossa, hematoxylin and eosin staining, 40X); Transmission electron micrographs showing microspheres in double mutant mice on regular chow, inset with larger magnification shown to the right (B); Two weeks old Npt2a-/- pups treated with i.p. injections of vehicle or sodium pyrophosphate (160 micromole/Kg/day) for two weeks (C); Histomorphometric analysis of renal mineralization (%calcified area = 100*mineralization area/tissue area, (D); calcification size = mineralization area/number of calcifications, um2, (E), and plasma pyrophosphate levels (F) and urine pyrophosphate (U-PPi) (G) of two weeks old Npt2a-/- pups treated with i.p. injections of vehicle or sodium pyrophosphate (160 micromole/Kg/day) for two weeks, measured after overnight fast and 18–24 hrs. following the last treatment. The data represent individual animals (closed circles) with the means±SEM, p-values shown above the lines of comparisons were calculated by Student’s t-test.