Nicotinamide treatment in a murine model of familial tumoral calcinosis reduces serum Fgf23 and raises heart calcium

Abstract

Mutations in the GALNT3 gene result in familial tumoral calcinosis, characterized by persistent hyperphosphatemia and ectopic calcific masses in soft tissues. Since calcific masses often recur after surgical removal, a more permanent solution to the problem is required. Nicotinamide is reported to lower serum phosphate by decreasing sodium-dependent phosphate co-transporters in the gut and kidney. However, its effectiveness in tumoral calcinosis remains unknown. In this study, we investigated nicotinamide as a potential therapy for tumoral calcinosis, using a murine model of the disease-Galnt3 knockout mice. Initially, five different doses of nicotinamide were given to normal heterozygous mice intraperitoneally or orally. Treatment had no effect on serum phosphate levels, but serum levels of a phosphaturic hormone, fibroblast growth factor 23 (Fgf23), decreased in a dose-dependent manner. Subsequently, high-dose nicotinamide (40mM) was tested in Galnt3 knockout mice fed a high phosphate diet. The radiographic data pre- and post-treatment showed that nicotinamide did not reverse the calcification. However, the treatment retarded calcification growth after 4weeks, while in the untreated animals, calcifications increased in size. The therapy did not affect serum phosphate levels, but intact Fgf23 decreased in the treated mice. The treated mice also had increased calcium in the heart. In summary, nicotinamide did not alter serum phosphate levels, likely due to compensatory decrease in Fgf23 to counteract the phosphate lowering effect of nicotinamide. Although increased calcium accumulation in the heart is a concern, the therapy appears to slow down the progression of ectopic calcifications.

Keywords: Calcification; Fgf23; Nicotinamide; Phosphate; Tumoral calcinosis.

Figure 1

Administration of nicotinamide in drinking water. Nicotinamide was infused in drinking water to treat heterozygous females. While nicotinamide did not change serum phosphorus, it had a significant effect on serum Fgf23 levels. Each column represents the mean ± SEM. N = 8–12 for serum measurements; N = 3–6 for urine measurements. ANOVA p-values between the five groups are shown above each graph. N.S., not significant; Ca, calcium; Pi, phosphorus; ALP, alkaline phosphatase; BUN, blood urine nitrogen; Ur, urine; Cr, creatinine.

Figure 2

Nicotinamide treatment of Galnt3 knockout mice. Galnt3 knockout mice were fed a high phosphate diet for 10 weeks, followed by 4 weeks on nicotinamide infused drinking water. Intact Fgf23 decreased significantly in treated males and females; however, total Fgf23 decreased only in males. Each column represents the mean ± SEM. N=8–12 for serum measurements, N=5–8 for urine measurements. T-test P < 0.05* and P < 0.01***, compared to sex-matched controls. Ca, calcium; Pi, phosphorus; ALP, alkaline phosphatase; BUN, blood urine nitrogen; Ur, urine; Cr, creatinine.

Figure 3

Effect of nicotinamide on ectopic calcifications in Galnt3 knockout mice. After ten weeks of the high phosphate diet, four Galnt3 knockout mice developed well-defined ectopic calcifications (white arrows). Two were treated with approximately 40 mM of nicotinamide, and the other two were untreated. While calcifications in the untreated mice (Untreated 1 and 2) became larger, more radiodense, or interconnected, there was no clear increase in the calcification sizes in the treated mice (Treated 1 and 2). Furthermore, an additional untreated mouse (Untreated 3) developed calcification in the tongue, which was not apparent in the pre-treatment radiograph.

Figure 4

Effects of nicotinamide treatment on kidney and heart tissues in Galnt3 knockout mice. Calcium and phosphorus in the kidney were not affected; however, calcium in the heart was significantly higher in the treated females. Each column represents the mean ± SEM. N=7–12 for kidney measures and N=4–9 in heart measures. T-test P< 0.01***, compared to same-sex control group.