Fig. 5. (OEG₂)₂-IP4 prevents calcification in a vitamin D-induced rat model of VC.

Calcification was induced by either s.c. administration of 300,000 IU/kg/day vitamin D₃ for 5 days combined with high-phosphate diet (a–c) or by oral administration of 100,000 IU/kg/day vitamin D₃ for 4 days combined with a warfarin-supplemented diet (d–j). Bulk calcium in the carotids (a) and kidneys (b), and plasma creatinine (c) of vehicle (n = 8 (b), n = 10 (a, c)), groups treated with twice daily s.c. injections of 30 mg/kg (OEG₂)₂-IP4 (n = 11) or IP6 (n = 5), and sham (n = 7). One rat in the vehicle group died several hours after first vitamin D₃ injection without explanation. Animals in the IP6 group had to be sacrificed 2 days ahead of schedule and no direct comparison can be made. d Kaplan–Meier survival plot. e, g Calcium quantification (e) and von Kossa-positive area quantitation (g) in the abdominal aorta. f Representative images of aortic calcifications stained with von Kossa (black) (scale bar = 500 µm). Calcium (h) and von Kossa-positive area (i) quantification in the thoracic aorta. Calcium quantification in the carotid artery (j). In d, n = 40 for vehicle, n = 12 for 1 × 12 and 1 × 25 mg/kg/day, n = 18 for 2 x 25 mg/kg/day, and n = 19 for 1 × 50 and 2 × 50 mg/kg/day dose groups. In e–j, n = 7 (e–h) and n = 6 (j) for 1 × 12.5 mg/kg/day, n = 8 for 1 × 25 mg/kg/day, n = 14 (i) and n = 15 (e–g, j) for 1 × 50 mg/kg/day, n = 18 for 2 × 25 mg/kg/day, n = 16 for 2 × 50 mg/kg/day dose groups, n = 24 (g) and n = 25 (e, h–j) for vehicle. All data are presented as mean ± s.d. from n animals. Statistical significance was derived from non-parametric Kruskal–Wallis and multiple comparison was performed by Mann–Whitney test with Bonferroni correction, with *p < 0.05, **p < 0.01 and ***p < 0.001 vs. vehicle. Comparison of survival was performed by Mantel–Cox test.