Ectopic mineralization of connective tissue in Abcc6-/- mice: effects of dietary modifications and a phosphate binder--a preliminary study

Abstract

Pseudoxanthoma elasticum (PXE), a heritable multisystem disorder, is caused by mutations in the ABCC6 gene. We have developed a murine model for PXE by targeted inactivation of the corresponding mouse gene. A feature of this mouse model is ectopic mineralization of connective tissue capsule surrounding the bulb of vibrissae. This study was designed to investigate the effect of dietary sevelamer hydrochloride (Renagel), a phosphate binder, and specific mineral modifications on ectopic mineralization of connective tissue in Abcc6-/- mice. Three groups were fed a specific diet: (i) a standard rodent diet, (ii) a standard rodent diet supplemented with sevelamer hydrochloride, and (iii) a custom experimental diet with specific mineral modifications (high phosphorus, low calcium and low magnesium). The degree of mineralization was determined in hematoxylin-eosin-stained sections using computerized morphometric analysis and by chemical assays to measure the calcium and phosphorus content of the vibrissae. The results indicated increased mineralization in the Abcc6-/- mice fed a standard diet or a diet with mineral modifications as compared with control mice fed a standard diet. However, feeding Abcc6-/- mice with diet supplemented with sevelamer hydrochloride did not improve mineralization, in comparison to mice fed with normal diet. Collectively, these results suggest that the mineralization process in PXE may be exacerbated by changes in mineral intake. The role of dietary minerals, and phosphorus in particular, as well as that of phosphate binders, in ectopic mineralization of PXE, merits further investigation.

Figure 1

Computerized morphometric analysis of mineralization of the connective tissue capsule surrounding the bulb of vibrissae in Abcc6^+/+ (left panel) and Abcc6^−/− (right panels) mice fed either with normal diet, diet supplemented with sevelamer hydrochloride (Renagel®), or experimental diet, high in phosphorous and low in calcium and magnesium (for details see Materials and Methods). Muzzle skin was biopsied and histopathologic sections stained with Hematoxylin-Eosin. The arrows indicate foci of mineralization, and thin black lines outline either the total area of vibrissae or the areas of mineralization, allowing computerized quantitation of the mineralization process.

Figure 2

Quantitation of the mineralization either by using computerized morphometric analysis, as shown in Fig. 1 (A), or by chemical determination of calcium plus phosphate content in the muzzle tissue containing the vibrissae (B). The bars represent mean ± S.E., n = 4–5 in each group. Note the statistical significance, as indicated in an inset in B.