Pathological calcification and the mystery of Lot's wife

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Mineral content of the maternal diet influences ectopic mineralization in offspring of Abcc6−/− mice

Arterial calcification is a life-threatening complication of atherosclerosis and is a common feature of aging, diabetes mellitus and end-stage renal disease. Vascular calcification is generally believed to be an active process that is regulated by osteogenic factors. What is most remarkable, and perhaps unexpected, about the process of calcification, whether in the skeleton or vasculature, is that control mechanisms are required to prevent rather than to induce mineralization, as calcium and phosphate are present in all tissues and body fluids at concentrations that would readily form an insoluble precipitate. The most significant inhibitor of crystal formation appears to be pyrophosphate, and the equilibrium between phosphate and pyrophosphate within the extracellular matrix determines whether crystallization will occur.¹ Neuman termed this condition, wherein we all exist in a supersaturated solution of calcium and phosphate, “Lot's wife's problem.”² Accordingly, one might ask whether Lot's wife, in turning back to watch fire and brimstone consume everything she valued, experienced an acute imbalance between phosphate and pyrophosphate that led to her sudden and tragic transformation into a pillar of salt.

In the present work, Li and co-authors³ have used the Abcc6^-/- knockout mouse to test the hypothesis that the mineral content of the maternal diet can influence the extent of calcification in the offspring, who carry increased risk genetically of ectopic calcification. The Abcc6^-/- mouse lacks the ATP-binding cassette subfamily C member 6 protein, an ATP-dependent efflux transporter this is expressed primarily in the liver. The Abcc6^-/- mouse is a useful model for the human disorder pseudoxanthoma elasticum (PXE), in which there is progressive accumulation of mineralization in elastic fibers of the blood vessels, the skin, eyes, and other tissues, due to inactivation of ABCC6.^4,5 Abcc6^-/- mice have been instrumental in demonstrating that PXE is a metabolic disease caused by the absence of an unknown factor in the circulation, the presence of which depends on normal expression of ABCC6 in the liver. In some patients, loss of ABCC6 causes a second form of generalized arterial calcification of infancy (GACI). This condition was initially ascribed to loss-of-function mutations in ENPP1,⁶ the gene encoding the ectonucleotide pyrophosphatase/phosphodiesterase that generates pyrophosphate through hydrolysis of extracellular ATP. Low levels of pyrophosphate are considered the principal basis for arterial calcification in GACI patients, and loss of ENPP1 explains reduced production of pyrophosphate. However, how loss of ABCC6 leads to low levels of pyrophosphate remains a mystery equal to that of Lot's wife.⁷

Remarkably, Li and associates³ demonstrated that offspring of pregnant mice fed an “acceleration diet,” one that is low in magnesium and rich in phosphorus and which promotes pathological calcification, developed more extensive calcification than offspring of mothers that had received a standard chow during their pregnancy. Ectopic calcification was obvious as early as 4 weeks of age in the pups whose mothers had been fed the acceleration diet, and was even more extensive at 12 weeks of age, when calcification was first noted in the mice whose mothers had received the standard chow diet. Moreover, the calcification was present not only in the dermal sheath of vibrissae, a useful marker of overall mineralization, but was also observed in the kidney and heart and in those pups whose mothers had received the acceleration diet, also in the aorta. The patterns of calcification were confirmed in 2 different mouse strains carrying the Abcc6^-/- genotype, but 129S1/SvImJ mice showed greater pathological calcification than mice on the C57BL/6J background.

These data confirm that inactivation of Abcc6 is sufficient to cause pathological calcification, and also indicate that other loci, which differ between the 2 mouse strains used in these experiments, exert important epistatic interactions on Abcc6 that influence the process of vascular calcification. Although these data suggest that the mineral content, and specifically I think the amount of phosphate, of the mother's diet can influence the development of ectopic mineralization in susceptible offspring, there are intriguing questions raised by the experimental design of the study that will need to be addressed in future studies. First, after weaning, the mice were maintained on the same diets that their mothers had received, and they were not evaluated until 4 weeks of age. Is it possible that the acceleration diet exerted some (or all?) of its effect on ectopic calcification postnatally? And second, what effect might a low phosphate maternal diet have on ectopic calcification in mice (and humans) that lack functional Abcc6 alleles?

Although I suspect that the mystery of Lot's wife will not be soon solved, I am nevertheless reassured by the work of Li et al.³ that we are making good progress unravelling the mechanisms of ectopic calcification. Something I am sure that even Lot's wife would look “forward” to.