Oral administration of pyrophosphate inhibits connective tissue calcification

Abstract

Various disorders including pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI), which are caused by inactivating mutations in ABCC6 and ENPP1, respectively, present with extensive tissue calcification due to reduced plasma pyrophosphate (PPi). However, it has always been assumed that the bioavailability of orally administered PPi is negligible. Here, we demonstrate increased PPi concentration in the circulation of humans after oral PPi administration. Furthermore, in mouse models of PXE and GACI, oral PPi provided via drinking water attenuated their ectopic calcification phenotype. Noticeably, provision of drinking water with 0.3 mM PPi to mice heterozygous for inactivating mutations in Enpp1 during pregnancy robustly inhibited ectopic calcification in their Enpp1-/- offspring. Our work shows that orally administered PPi is readily absorbed in humans and mice and inhibits connective tissue calcification in mouse models of PXE and GACI PPi, which is recognized as safe by the FDA, therefore not only has great potential as an effective and extremely low-cost treatment for these currently intractable genetic disorders, but also in other conditions involving connective tissue calcification.

Keywords: generalized arterial calcification of infancy; oral pyrophosphate treatment; pseudoxanthoma elasticum; soft tissue calcification.

© 2017 Institute of Enzymology, RCNS, Hungarian Academy of Sciences Published under the terms of the CC BY 4.0 license.

Figures

Figure 1. Uptake of PPi from drinking water

1. Oral uptake of tetrasodium pyrophosphate in humans. Volunteers ingested tetrasodium pyrophosphate solutions of 43, 72, 110 mM, pH 8.0, resulting in a dose of 40 mg/kg (n = 10) or 67 mg/kg (n = 10) or 98 mg/kg (n = 9), respectively. Plasma PPi levels were determined before (0 min), and 30, 60, 120, 240, and 480 min after ingestion. The insert shows the differences between the basal plasma PPi level (0 min) and that 30, 60, and 120 min after ingestion.

2. Oral uptake of 98 mg/kg tetrasodium pyrophosphate (n = 9) in human indicating individual differences.

3. Uptake from the oral cavity (n = 5), from the stomach (n = 5), and from the intestine (n = 5) of C57/BI6 mice after ligating the esophagus and applied oral delivery of 100 μl 50 mM PPi; or the pylorus followed by stomach delivery of 200 μl 50 mM PPi and then ligation of the esophagus; or injecting 200 μl 50 mM PPi into the intestine after ligation of the pylorus. In each experiment, including control (n = 7), blood was collected after 15 min and PPi concentrations were determined.

4. Time‐course of PPi uptake from the stomach of C57/BI6 mice upon gavage delivery of 200 μl of 50 mM PPi, n = 5–11.

5. Dose‐dependent PPi uptake from the stomach of C57/BI6 mice upon gavage delivery of 200 μl of PPi of concentration 0 (n = 6), 1 (n = 6), 10 (n = 7), 25 (n = 7), 50 (n = 8), and 100 (n = 6) mM. Blood was collected for PPi assay after 15 min of delivery.

Data information: Data were analyzed by two‐tailed Mann–Whitney nonparametric test. Results are expressed as mean ± SEM.

Figure 2. Oral PPi attenuates induced cardiac and spontaneous calcification of vibrissae in Abcc6−/− mice

1. A

   Calcification of the heart of Abcc6−/− mice after 4 days of cryo‐injury (drinking water, upper image) or (drinking water with 10 mM PPi, lower image). Ca‐precipitations are indicated by arrows, and calcium deposits were visualized by Alizarin Red staining. Scale bar = 1 mm.

2. B

   PPi was provided in 0 (n = 8), 1 (n = 8) or in 10 (n = 8) mM concentrations via the drinking water to Abcc6−/− mice starting a day before cryo‐injury for a total of 4 days. The calcium content of heart tissue was determined by complexometry.

3. C, D

   Show typical Alizarin Red‐stained sections of an animal of the control group and that of the 10 mM PPi group, respectively. Abcc6−/− mice were kept on 10 mM PPi (in drinking water) starting at an age of 3 weeks (after weaning) until they were 22 weeks old. The control group was drinking tap water. Tissue blocks with the vibrissae were removed, paraffin‐embedded, sectioned, and stained with Alizarin Red for calcium deposits, scale bar = 1 mm.

4. E

   The extent of calcification, control (n = 9) and 10 mM PPi (n = 9), was quantified by morphometry as described in the Materials and Methods.

Data information: Data were analyzed by two‐tailed Mann–Whitney nonparametric test. Results are expressed as mean ± SEM.

Figure 3. Prenatal PPi treatment of the Enpp1−/− mice attenuates calcification of the tissue surrounding the vibrissae and the arteries of the hind limbs and kidneys

1. A

   Calcium content of the tissue blocks of the vibrissae. The heterozygous mothers and their offspring were kept on tap water until the pups were 30 days old (“control”, n = 11); Group 1: as the control group, but for 9 days on 10 mM PPi solution after weaning at day 21 (n = 8); Group 2: the mothers and the pups were kept on 10 mM PPi during pregnancy, breastfeeding and for 9 days after weaning (n = 7). Group 3: The mothers were kept on 10 mM PPi during pregnancy (n = 9). Group 4: The mothers and the pups were kept on 1 mM PPi during pregnancy, breastfeeding, and for 9 days after weaning (n = 13). Group 5: The mothers were kept on 1 mM PPi during pregnancy (n = 8).

2. B, C

   Typical Alizarin Red‐stained sections of tissue blocks with the vibrissae of animals of different groups. Scale bar: 1 mm

3. D

   Calcification of renal arteries. The Enpp1+/− mothers were kept on tap water (n = 7) or on 0.3 mM PPi (n = 6) during pregnancy. Offspring was kept on tap water for 55 days.

4. E

   Calcium content of the hind limb arteries. The heterozygous mothers were kept on tap water (n = 7) or 0.3 mM PPi (n = 6) during pregnancy. Offspring was kept on tap water for 55 days.

5. F, G

   Typical kidney sections of a 55‐day‐old animal of the control group and of a 55‐day‐old animal from the group of 0.3 mM treatment only during pregnancy. Sections were stained by the Yasue procedure. Scale bar: 200 μm.

6. H, I

   Typical Alizarin Red‐stained hind limb arteries of a 55‐day‐old animal of the control group and those of a 55‐day‐old animal from the group of 0.3 mM treatment only during pregnancy. Scale bar: 1 mm.

Data information: Data were analyzed by two‐tailed Mann–Whitney nonparametric test. Results are expressed as mean ± SEM.