Pyrophosphate Supplementation Prevents Chronic and Acute Calcification in ABCC6-Deficient Mice

Abstract

Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6-/- mice fully inhibited the acute dystrophic cardiac calcification phenotype, whereas alendronate had no significant effect. We also found that daily injection of PPi to Abcc6-/- mice over several months prevented the development of pseudoxanthoma elasticum-like spontaneous calcification, but failed to reverse already established lesions. Furthermore, we found that the expression of low amounts of the human ABCC6 in liver of transgenic Abcc6-/- mice, resulting in only a 27% increase in plasma PPi levels, led to a major reduction in acute and chronic calcification phenotypes. This proof-of-concept study shows that the development of both acute and chronic calcification associated with ABCC6 deficiency can be prevented by compensating PPi deficits, even partially. Our work indicates that PPi substitution represents a promising strategy to treat ABCC6-dependent calcification disorders.

Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Pyrophosphate (PPi) and bisphosphonates. A: The generation of ABCC6-dependent hepatic PPi was estimated in liver perfusate. The concentration of PPi in the perfusate buffer was compared between wild-type (WT; +/+) and Abcc6−/− mice (−/−), and to Abcc6−/− mice transiently expressing the human ABCC6 in liver. The relative level of expression of ENPP1 in the liver (B) and kidneys (C) of Abcc6−/− mice and wild-type mice was determined by real-time PCR using specific TaqMan probes. The data were normalized to the mouse GAPDH. D: PPi plasma concentration steadily decreases after a single i.p. injection of PPi at 112 μmol/kg (50 mg/kg) in Abcc6−/− mice with an approximate half-life of 42 minutes. Average plasma PPi levels of wild-type and Abcc6−/− mice are indicated. E: Plasma PPi concentration was significantly reduced in Abcc6−/− mice at 3-, 6-, and 12-month-old mice. At each time point, PPi levels were significantly lower in Abcc6−/− mice, though age does not influence PPi levels in either wild-type (+/+) or Abcc6−/− mice (−/−). F: Alkaline phosphatase activity (ALK) expressed as units (μmol of p-nitrophenyl/L per minute) was measured in plasma samples from the same 3-, 6-, and 12-month-old wild-type (+/+) and Abcc6−/− mice (−/−) as shown in E. The number of mice per group is shown. Data are expressed as means ± SEM. n = 3 per data point (D). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 2

The effect of pyrophosphate (PPi) and bisphosphonates on the acute dystrophic cardiac calcification (DCC) phenotype of Abcc6−/− mice. A: Various amounts of PPi were i.p. injected in Abcc6−/− mice daily for 7 days after inducing DCC. A single injection (inj.) of 28 μmol/kg (12.5 mg/kg) of PPi immediately after cryoinjury was sufficient to inhibit DCC. The level of calcification was measured as total ventricular calcium. B: Two bisphosphonates, alendronate (Aln) and etidronate (Etd), were tested against DCC in Abcc6−/− mice. Etidronate (8 μmol/kg per day or 2.0 mg/kg per day) significantly inhibits cardiac calcification, whereas alendronate (0.062 μmol/kg per day or 0.02 mg/kg per day) has no significant effect as compared to saline injections. The number of mice per group is shown. Data are expressed as means ± SEM (A and B). ∗∗P < 0.01, ∗∗∗P < 0.001 versus saline.

Figure 3

The effect of pyrophosphate (PPi) on the chronic (pseudoxanthoma elasticum–like) calcification phenotype of Abcc6−/− mice. Abcc6−/− mice were injected daily for 16 to 20 weeks with either saline (white bars) or a PPi solution at 224 μmol/kg per day (eg, 100 mg/kg per day) (black bars). Injections started either at weaning before the onset of ectopic calcification in vibrissae and continued daily for 16 weeks or were initiated at 6 months of age (after onset) when whiskers calcification is well developed, and continued for 20 weeks. For comparison purposes, vibrissae calcification of untreated Abcc6−/− mice is shown (gray bars). The number of mice per group is shown. Data are expressed as means ± SEM. ∗∗∗P < 0.001. WT, wild-type.

Figure 4

Histological evaluation of vibrissae calcification of Abcc6−/− mice after pyrophosphate (PPi) supplementation. The effect of daily PPi or saline injections on the calcification of vibrissae (chronic pseudoxanthoma elasticum–like phenotype) of Abcc6−/− mice was evaluated by Alizarin S Red staining (red to dark red color). A: Representative images of whiskers with approximately 15 vibrissae are shown for the preonset and postonset treatment groups with saline controls. A higher magnification image of a selected vibrissa (arrows) is shown. Arrowheads point to discrete calcification. B: The relative area occupied by calcification, as visualized by Alizarin red staining, was also quantified by morphometric analysis using the ImageJ software and show significant reduction between the saline and PPi treatments. C: For comparison purposes, representative images of whiskers from untreated Abcc6−/− mice are included. A higher magnification image of a selected vibrissa (arrows) is shown. The number of mice per group is shown. Data are expressed as means ± SEM. ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 versus saline. Scale bars = 500 μm (A and C). Original magnification, ×200 (higher magnification images).

Figure 5

The human ABCC6 transgene is expressed at low but detectable levels in liver. A: The level of expression of normal human ABCC6 in the liver of Abcc6−/− transgenic mice (Tg ABCC6) was determined by real-time PCR using TaqMan probes specific to the human ABCC6 and the mouse GAPDH and compared to the transient expression of the same human ABCC6 obtained by hydrodynamic tail vein injections (HTVIs; ABCC6). B: Frozen sections of livers from Abcc6−/− transgenic mice carrying the human ABCC6 cDNA were used to perform immunofluorescence colabeling of the human ABCC6 (green, using the M6II-31 monoclonal antibody) and the plasma membrane marker cadherin (red). DAPI was used as counter staining (blue). Scale bar = 100 μm (B).

Figure 6

Effects of the human wild-type (WT) ABCC6 and R1314W variant transgenes on the phenotype of Abcc6−/− mice. A: The plasma pyrophosphate (PPi) concentration in transgenic mice carrying the wild-type ABCC6 (Tg ABCC6) is significantly higher (27%) over the nontransgenic Abcc6−/− control mice, whereas there is no statistically significant difference with R1314W transgenics (Tg R1314W). B: The effects of the wild-type ABCC6 transgene on dystrophic cardiac calcification (DCC) was tested in 3- to 5-month-old mice (Tg ABCC6). The level of calcification was measured as total ventricular calcium. For control purposes, the results are compared to calcification in wild-type, Abcc6−/− mice and knockout animals transiently expressing the human ABCC6 or the LacZ cDNA [hydrodynamic tail vein injection (HTVI)] after cryoinjury. Sham-operated Abcc6−/− mice were also used as controls. The DCC phenotype is significantly reduced in the Tg animals. The effects of the human wild-type ABCC6 and R1314W variant transgenes on chronic calcification in vibrissae (C) and kidneys (D) was determined over a period of 18 months. Calcification levels are shown for wild-type mice, Abcc6−/− mice, Abcc6−/− mice with the human ABCC6 transgene, or the R1314W variant transgene. Note that the y axis scale for the kidney calcification data are approximately 8% of the vibrissae. The number of mice in each group is shown. Data are expressed as means ± SEM (A–D). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001; ††P < 0.01, †††P < 0.001, and ††††P < 0.0001 versus Abcc6−/− mice at corresponding time points.

Figure 7

Histological evaluation of vibrissae calcification in Abcc6−/− transgenic mice. The positive effects of both transgenes (normal ABCC6 and R1314W variant) on the calcification of vibrissae (chronic pseudoxanthoma elasticum–like phenotype) was evaluated by Alizarin Red S staining (red to dark red color). A: Representative images of whiskers from 3- to 18-month-old Abcc6−/− transgenic (Tg) mice carrying the normal human ABCC6 gene are shown. B: Representative images are from whiskers of Abcc6−/− mice with the ABCC6 R1314W transgene are shown. A higher magnification image of a selected vibrissa (arrows) is shown below each larger panel (A and B). Arrows within the higher magnification images point to discrete calcification spots (A and B). Scale bars = 500 μm (A and B). Original magnification, ×200 (higher magnification images).

Figure 8

A possible model for ABCC6-dependent pyrophosphate generation and inhibition of ectopic calcification. ABCC6 facilitates the release of ATP from the liver (and other tissues), which is converted into the calcification inhibitor pyrophosphate (PPi) and adenosine monophosphate (AMP) by ectonucleotide pyrophosphatase-phosphodiesterase 1 (ENPP1). Ecto-5′-nucleotidase (NT5E; alias CD73) converts AMP into adenosine, which is an inhibitor of tissue-nonspecific alkaline phosphatase (TNAP) synthesis. In peripheral tissues, such as bone, PPi is released by progressive ankylosis protein (ANK) and hydrolyzed into inorganic phosphate (Pi) by TNAP. ABCC6 deficiency causes pseudoxanthoma elasticum (PXE) and some cases of generalized arterial calcification of infancy (GACI). Mutations in ENPP1 primarily lead to GACI and also some cases of PXE. Nonfunctional ecto-5′-nucleotidase (NT5E) results in arterial calcification because of deficiency of CD73 (ACDC).