original article http://www.kidney-international.

org
& 2011 International Society of Nephrology

see commentary on page 490

Treatment with pyrophosphate inhibits uremic

vascular calcification
W. Charles O’Neill1,4, Koba A. Lomashvili1, Hartmut H. Malluche2, Marie-Claude Faugere2
and Bruce L. Riser3,4
1
Renal Division, Emory University School of Medicine, Atlanta, Georgia, USA; 2Division of Nephrology, Bone and Mineral Metabolism,
University of Kentucky, Lexington, Kentucky, USA and 3Renal Division, Baxter Healthcare, McGaw Park, Illinois, USA

Pyrophosphate, which may be deficient in advanced renal Pyrophosphate (PPi) is a potent inhibitor of calcium
failure, is a potent inhibitor of vascular calcification. To crystallization that is present in body fluids at concentrations
explore its use as a potential therapeutic, we injected sufficient to prevent hydroxyapatite formation in vitro,1–3
exogenous pyrophosphate subcutaneously or suggesting that it may function as an endogenous inhibitor of
intraperitoneally in normal rats and found that their plasma calcification. In particular, production of PPi by smooth
pyrophosphate concentrations peaked within 15 min. There muscle may be an important defense against medial vascular
was a single exponential decay with a half-life of 33 min. calcification. Rat aortas do not calcify in vitro unless the PPi
The kinetics were indistinguishable between the two routes produced by the vessels is removed.4 Furthermore, humans
of administration or in anephric rats. The effect of daily lacking ectonucleotide pyrophosphorylase1 (Enpp1), the
intraperitoneal pyrophosphate injections on uremic vascular enzyme that synthesizes extracellular PPi, develop severe
calcification was then tested in rats fed a high-phosphate diet medial arterial calcification at an early age.5–7
containing adenine for 28 days to induce uremia. Although Recent data suggest that PPi deficiency may contribute to
the incidence of aortic calcification varied and was not the increased incidence of medial vascular calcification in
altered by pyrophosphate, the calcium content of calcified advanced kidney disease. Levels of PPi are reduced in
aortas was significantly reduced by 70%. Studies were hemodialysis patients,8 and correlate inversely with the level
repeated in uremic rats given calcitriol to produce more of vascular calcification in patients with advanced chronic
consistent aortic calcification and treated with sodium kidney disease.9 Hydrolysis of PPi is increased in aortas from
pyrophosphate delivered intraperitoneally in a larger volume uremic rats because of upregulation of alkaline phospha-
of glucose-containing solution to prolong plasma tase,10 providing a mechanism for vascular deficiency of PPi.
pyrophosphate levels. This maneuver significantly reduced Therefore, the data suggest that exogenous PPi may be useful
both the incidence and amount of calcification. Quantitative in treating or preventing uremic vascular calcification.
histomorphometry of bone samples after double-labeling Studies conducted more than four decades ago showed
with calcein indicated that there was no effect of that subcutaneously administered PPi inhibited medial
pyrophosphate on the rates of bone formation or arterial calcification in vitamin D-toxic rats.11 However, large
mineralization. Thus, exogenous pyrophosphate can inhibit doses were required and the treatment resulted in marked
uremic vascular calcification without producing adverse skin necrosis. Nonhydrolyzable analogs of PPi, such as
effects on bone. bisphosphonates, were subsequently shown to inhibit vas-
Kidney International (2011) 79, 512–517; doi:10.1038/ki.2010.461; cular calcification in the same animal model, but at much
published online 1 December 2010 lower doses.3 More recently, bisphosphonates have been
KEYWORDS: activated vitamin D; bone; chronic kidney disease; uremia shown to prevent aortic calcification in uremic rats.12,13 One
potential problem with this therapy, however, is inhibition of
bone formation. The high levels of tissue-nonspecific alkaline
phosphatase (TNAP) in bone serve to remove PPi and allow
bone formation to occur in the face of inhibitory circulating
PPi concentrations.14 This is the basis for the skeletal abnor-
malities in hypophosphatasia, where TNAP is deficient.15
Correspondence: W. Charles O’Neill, Emory University School of Medicine, However, TNAP would not protect bone from the non-
Renal Division, WMB 338, 1639 Pierce Drive, Atlanta, Georgia 30322, USA. hydrolyzable bisphosphonates and, accordingly, the doses of
E-mail: [email protected] bisphosphonates that inhibit vascular calcification in uremic
4
These authors are equally contributing senior authors. rats also inhibit bone formation.13
Received 23 April 2010; revised 9 September 2010; accepted 21 Based on these previous results, we reasoned that
September 2010; published online 1 December 2010 exogenous PPi might inhibit uremic vascular calcification

512 Kidney International (2011) 79, 512–517

WC O’Neill et al.: PPi treatment inhibits uremic vascular calcification original article

without adversely affecting bone. Therefore, we explored the additional study performed with both renal arteries and veins
therapeutic potential of PPi in uremic vascular calcification occluded to determine the renal contribution to PPi clear-
by examining both its pharmacokinetics and its effects on ance, the half-life was 35 min (not shown). Similar results
aortic calcification and bone formation in a model of medial were obtained after intraperitoneal injection (Figure 1b),
arterial calcification in uremic rats. with a half-life of 34.1±2.6 min. However, the peak concen-
tration as a fraction of the dose (0.336 and 0.340 mM/mmol/kg)
RESULTS was greater than after subcutaneous injection (0.196±0.041
Pharmacokinetics mM/mmol/kg), suggesting better delivery by the intraperitoneal
Initial studies were performed in three rats using a single route. With either route, the half-life was not affected by the
subcutaneous injection of 80 mmol/kg of PPi containing dose (shown in Figure 1b). Oral administration was not
tracer 32PPi. At 15 min, 51.9±3.5% of the radioactivity in the attempted because of rapid PPi hydrolysis in the intestinal
plasma was in the form of orthophosphate and this increased tract.11
to 91.4±0.8% at 120 min, indicating rapid hydrolysis of PPi.
Plasma 32PPi peaked at 15 min and decreased exponentially Effect on vascular calcification
thereafter (Figure 1a). The r2 for the linear regression of the To determine whether PPi administration reduces uremic
logarithmically transformed data ranged from 0.95 to 0.98, vascular calcification, rats fed a high-phosphate diet contain-
indicating a single-exponential decline and yielded a half-life ing adenine first underwent daily subcutaneous injection of
of 31.4±1.4 min. Extrapolation to the ordinate yielded an sodium PPi. However, this route resulted in subcutaneous
apparent volume of distribution of 4.1±0.5 l/kg. In an nodules and was therefore abandoned. In contrast, daily
intraperitoneal injection of sodium PPi in a volume of
5 ml/kg appeared to be well tolerated and was therefore used
a 25
to test our hypothesis. Body weights and plasma chemistries
of these rats at the time of killing are presented in Table 1.
Increase in plasma PPi (µM)

20 There was no mortality in the control group. One rat treated

with 160 mmol/kg/day died at 10 days (13% mortality).
15 Two rats treated with 80 mmol/kg/day died at 11 and 27 days
(25% mortality). The aorta from the latter rat was included
10
in the analysis and was not calcified. PPi treatment did not
alter animal weight or the plasma concentrations of calcium
and phosphorus. Plasma alkaline phosphatase activity was
5
slightly reduced in the rats treated with the lower dose of PPi
but not the higher dose. The urea concentration was slightly
0 higher in the rats treated with the higher dose of PPi but
0 20 40 60 80 100 120 140 160
this was not observed in subsequent studies at the same dose
Time (min)
(see below), and there were no other signs (weight loss,
b 0.4 physical appearance) that these rats were more uremic. There
was no correlation between any plasma parameter or the
Plasma PPi/dose (µM/µmol /kg)

adenine dose and aortic calcium content.

0.3 In rats fed a normal diet, aortic calcium content was
20±3 nmol/mg (n ¼ 22). A calcium content of 53 nmol/mg,
which is 3 s.d. above that in normal rats, was employed
0.2 a priori to define pathological calcification. As shown in

Table 1 | Plasma chemistries in adenine-fed rats receiving

0.1
daily intraperitoneal injections of NaPPi in 10 ml/kg
PPi dose, lmol/kg/day
0.0 Baseline 0 (n=15) 80 (n=6) 160 (n=7)
0 20 40 60 80 100 120 140 160 180
Time (min) Body weight (g) 324±1 223±6 220±10 230±5
Urea (mmol/l) 10.2±0.5 23±2 24±3 36±6*
Figure 1 | Change in plasma pyrophosphate (PPi) Calcium (mmol/l) 1.96±0.04 1.79±0.10 1.62±0.09 1.99±0.13
concentration after injection of PPi containing tracer [32P]PPi. Phosphorus (mmol/l) 1.4±0.1 6.9±0.8 6.7±0.4 6.7±0.6
(a) Single subcutaneous injection of 80 mmol/kg sodium PPi in Alkaline phosphatase (IU/l) NA 76.3±1.2 54.8±1.5* 80.0±3.9
three different rats. (b) Single intraperitoneal injections of Abbreviations: NA, not assayed; NaPPi, sodium PPi; PPi, pyrophosphate.
0.026 mmol/kg (solid symbols) or 2.6 mmol/kg (open symbols) *Po0.05.
sodium PPi in a volume of 5 ml/kg. The increment in plasma PPi Baseline chemistries were obtained from six rats of similar age fed normal rat chow
concentration was calculated from the specific activity of the (23% protein).
injected 32PPi. Results are means±s.e.m.

Kidney International (2011) 79, 512–517 513

original article WC O’Neill et al.: PPi treatment inhibits uremic vascular calcification

3500 20 * ** *
18
3000 16 *
#
14

Plasma PPi (µM)

Aortic calcium (nmol/mg)

2500
12
#
10
2000
8
6
1500
4

1000 2
0
0 60 120 180 240
500
Time (min)
Figure 3 | Plasma pyrophosphate (PPi) concentration after
0
single intraperitoneal injections of 160 lmol/kg sodium PPi in
0 80 160 a volume of 40 ml/kg. Results are means±s.e.m. of four animals.
PPi dose, µmol/kg/day *Po0.01, **Po0.001, #Po0.05 vs baseline.

Figure 2 | Effect of pyrophosphate (PPi) on aortic calcium

content in uremic rats. Rats were fed a diet consisting of 2.5%
protein, 1.06% phosphorus, 0.92% calcium, and 0.75% adenine for 10,000
4 weeks and received sodium PPi in Hank’s buffered saline or
saline alone by daily intraperitoneal injection of 5 ml/kg. Each
point represents a single animal.
8000

Figure 2, aortic calcification is variable in this model and only

Aortic calcium (nmol/mg)

developed in 40% of vehicle-treated rats. The lowest calcium

content that met the definition of pathological calcification as 6000
outlined above was 235 nmol/mg, indicating a clear distinc-
tion between calcified and noncalcified aortas. The incidence
of aortic calcification was decreased by PPi at 160 mmol/kg 4000
(29 vs 40%), but the difference was not significant. Although
there was a dose-dependent decrease in the calcium content
of the calcified aortas up to 74% (1898±466 vs 965±319 vs
2000
489±205 nmol/mg), the difference was not significant
because of the variability in calcification.
To better demonstrate the effect of PPi, changes were
made in order to prolong the effect of PPi and to reduce the 0
variability in calcification. To slow the absorption of PPi 0 32 160*
from the peritoneum, the volume of fluid was increased to
PPi dose, µmol/kg/day
40 ml/kg and glucose was added. As shown in Figure 3, this
resulted in a threefold increase in the plasma PPi level, and Figure 4 | Effect of peritoneal infusion of pyrophosphate (PPi)
levels remained elevated for B240 min. To reduce the on uremic vascular calcification. Rats were fed a diet consisting
of 2.5% protein, 1.06% phosphorus, 0.92% calcium, and 0.75%
variability in aortic calcification, uremic rats were given adenine for 4 weeks and given calcitriol 40 ng/kg by
40 ng/kg of calcitriol three times per week by subcutaneous subcutaneous injection 3 times per week. PPi was infused daily
injection. This resulted in extensive aortic calcification in all through a peritoneal catheter in a volume of 40 ml/kg. Each point
animals (Figure 4), but did not increase aortic calcium represents a single animal. *Po0.005 vs control.
content in nonuremic rats, even at the substantially higher
dose of 100 ng/kg (33.3±4.3 nmol/kg; n ¼ 6), indicating that
this effect is specific for uremia. The lower dose did not alter calcification (4744±418 nmol/
Two PPi solutions were tested in the studies shown in mg). Body weight (276 þ 10 vs 271 þ 14 g), plasma urea (25 þ 5
Figure 4, 0.8 and 4 mmol/l, both injected daily at 40 ml/kg, vs 25 þ 3 mmol/l), plasma calcium (1.50 þ 0.06 vs 1.58 þ 0.07
resulting in doses of 32 and 160 mmol/kg/day. The mmol/l), and plasma phosphorus (6.7 þ 0.3 vs 6.5 þ 0.3 mmol/l)
higher dose significantly reduced aortic calcium content did not differ between rats treated with 160 mmol/kg and vehicle-
(1040±762 nmol/mg vs 4773 þ 565 nmol/mg, Po0.005) and treated rats. Plasma PPi levels at the time of killing (24 h after the
the incidence of calcification (67 vs 100%, Po0.05). last peritoneal infusion of PPi) were the same in control rats

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WC O’Neill et al.: PPi treatment inhibits uremic vascular calcification original article

Table 2 | Parameters of bone formation in normal rats and in adenine-fed rats treated with or without 160 lmol PPi/kg/day
Uremic
No calcitriol Calcitriol
Normal (6) Control (7) PPi (7) Control (6) PPi (6)

Bone volume (% of total) 12.5±1.6 14.2±2.5 15.8±1.6 14.4±1.1 11.4±0.8

Osteoblasts (no. per 100 mm) 29±2 494±198 176±82 453±110 399±278
Osteoblast surface 0.39±0.04 6.2±2.2 2.5±1.1 5.6±1.3 5.3±3.3
Osteoid thickness (mm) 3.6±0.5 4.6±0.9 4.4±1.1 4.6±0.9 3.5±0.3
Mineral apposition (mm/day) 2.0±0.3 5.7±0.6 5.5±0.6 6.8±0.5 6.8±0.6
Bone formation (mm3/cm2/year) 9.9±1.9 24.9±6.3 21.9±5.2 43.2±8.5 45.3±6.2
Abbreviation: PPi, pyrophosphate.
None of the differences between control and PPi-treated rats were significant by Mann–Whitney U-test.
Parentheses indicate the number of animals in each group.

(5.5±0.4 mM) and rats treated with the higher dose of PPi extracellular PPi production in the vessel wall is unknown,
(5.3±0.7 mM), indicating that there was no accumulation of PPi these data suggest a reduction in extracellular PPi levels in
in the plasma over the course of the experiment. uremic vessels. Owing to the short half-life of PPi in plasma,
only transient increases in the concentration of plasma PPi
Effect on bone occurred with treatment, and it is possible that the increases
Quantitative histomorphometric analysis was performed in tissue PPi were similarly transient. However, the data show
on femurs from uremic rats (receiving or not receiving that a 4 h increase in PPi levels with a peak tripling was
calcitriol) treated with or without PPi (high dose only), and sufficient to substantially inhibit calcification. This suggests
are shown in Table 2. For comparison, data from pair-fed that only intermittent correction of PPi deficiency can
nonuremic rats obtained in a previous study are also shown. prevent calcification.
All parameters of bone formation were greatly increased in As PPi could potentially inhibit any hydroxyapatite
uremic rats, but were unaffected by PPi treatment. Osteo- formation, effects on normally calcified tissues such as bone
clastic parameters were also unchanged by PPi treatment need to be considered. Despite the robust inhibition of
(data not shown). vascular calcification in uremic rats, there was no effect on
any parameters of bone formation. This selective action of
Other effects PPi is probably explained by the high levels of TNAP in bone.
There was no mortality in the second set of studies and there This ectoenzyme hydrolyzes PPi and enables mineralization
was no difference in the appearance or behavior of the PPi- to occur in the face of circulating PPi levels that can inhibit
treated rats. Examination of the knee joint revealed no crystal hydroxyapatite formation.14 The inhibition of bone formation
deposition in rats treated with PPi (not shown). Histology of that we previously observed with doses of bisphosphonates
the abdominal organs revealed a chronic inflammation of the sufficient to inhibit uremic vascular calcification13 can prob-
peritoneum covering the diaphragm in low- and high-dose ably be explained by the fact that bisphosphonates are not
groups (Figure 5) and fibrosis of the splenic capsule and hydrolyzed by TNAP and can thus directly inhibit bone
chronic inflammation involving the mesentery attached to mineralization. Another explanation for the selective effect
the spleen in the high-dose group. In general, the severity of of PPi is the immediate proximity of the vascular wall to
the peritoneal reaction on the diaphragm was marked in circulating PPi compared with bone tissue.
rats given 4 mmol/l PPi and only mild in rats receiving There was a significantly greater plasma urea concentra-
0.8 mmol/l. A mild inflammatory response was also observed tion with the highest PPi dose in the initial study. The fact
in the placebo group, presumably a response to the surgery, that this group did not have a reduction in adenine dose
the presence of the peritoneal catheter, or exposure to the compared with the other groups (see Materials and Methods)
vehicle. No animal showed overt signs of peritonitis. could have contributed. However, the animals did not show
other signs of increased uremia, and plasma urea was not
DISCUSSION increased in the second set of studies despite the same dose of
This study demonstrates that exogenous PPi can substantially adenine. There was no articular crystal deposition indicative
inhibit vascular calcification in uremic rats. This action is of calcium PPi deposition disease, suggesting that arthritis
presumably because of an increase in the concentration of will not be a side effect of PPi treatment. An inflammatory
PPi in the vessel wall, which directly inhibits hydroxyapatite reaction was present in the peritoneum, indicating that this
formation. Tissue levels are controlled by local production dose and frequency of PPi is not feasible for long-term
and hydrolysis as well as diffusion from the blood. We have intraperitoneal delivery.
previously shown that plasma levels of PPi are reduced in Our initial studies were hampered by the variability of
hemodialysis patients8 and that PPi hydrolysis is increased in vascular calcification in the adenine, high-phosphate diet
vessels from uremic rats.10 Although the effect of uremia on model, often with o50% of rats developing any calcification.

Kidney International (2011) 79, 512–517 515

original article WC O’Neill et al.: PPi treatment inhibits uremic vascular calcification

a tion has been observed in other studies and with other

models,17–19 but usually with higher doses We also found
that treatment of normal rats on a high-phosphate diet
with a 2.5-fold higher dose of calcitriol did not increase
aortic calcium content, indicating that calcitriol was acting
synergistically with the renal failure.
This study indicates that PPi may be a useful therapy for
uremic vascular calcification. Inhibition of calcification was
achieved with only small, transient daily increases in plasma
PPi that did not produce any adverse effects on bone.
Additional studies will be required to determine the optimal
dosing and route of administration and whether PPi can
reverse existing vascular calcification.

b
MATERIALS AND METHODS
Plasma PPi kinetics
Male rats (230–400 g) were anesthetized with isoflurane for the
duration of these studies. Polyethylene tubing was placed in the
carotid artery and kept patent with heparinized saline. After
injection of 32PPi, 200 ml samples of blood were drawn into
heparized tubes and centrifuged to collect plasma. Plasma was
combined with an equal volume of 10% trichloroacetic acid and
centrifuged again. Orthophosphate was extracted as previously
described10 using 1 volume of supernatant, 10 volumes of 30 mmol/l
ammonium molybdate in 0.75 M sulfuric acid, and 20 volumes of
isobutanol/petroleum ether (4:1). Radioactivity in the organic phase
was counted and radioactivity remaining in the aqueous phase was
assumed to be PPi. The change in plasma PPi concentration was
c calculated from the specific activity of the injected PPi. In some
experiments, unlabeled PPi was injected and PPi was assayed
directly in the plasma.

Aortic calcification in uremic rats

Chronic renal failure was produced by feeding adenine to rats.20,21
Male Sprague-Dawley rats were obtained from Charles River
Laboratories (Wilmington, MA) and fed a standard rodent diet
until body weight exceeded 300 g. Rats were then fed a 2.5% protein
diet (Harlan Teklad, Madison, WI), which enhances vascular
calcification,12 supplemented with neutral sodium phosphate. This
diet contained 1.06% phosphorus and 0.92% calcium. The adenine
content was 0.75% and was reduced to 0.5% for rats that had a rapid
rate of weight loss. This occurred after 10 days in half the rats in the
control group and all the rats in the 80 mmol/kg group in the studies
shown in Figure 2, and after 21 days in 3 rats each in the control
group and the 32 mmol/kg group in the studies in Figure 4. For some
Figure 5 | Histology of the diaphragm and parietal peritoneum experiments, calcitriol (Calcitriol Injection, American Regent
in uremic rats given sodium pyrophosphate (PPi) by Shirley, NY) was given subcutaneously at a dose of 40 ng/kg every
intraperitoneal injection. (a) Vehicle; (b) 0.8 mmol/l PPi;
Monday, Wednesday, and Friday, concomitant with the adenine diet.
(c) and 4 mmol/l PPi. Hematoxylin and eosin staining.
Calcein (2 mg in 2 ml Hank’s solution) was injected intraperitoneally
on days 20 and 25 to label bone. Rats were killed after 28 days and
aortas were perfused with saline, harvested, and cleaned. All
protocols were approved by the Institutional Animal Care and Use
This has also been observed by other investigators, and the Committee.
mechanism responsible for this variability is unknown.12,16
Lowering the protein content of the diet, which eliminated Intraperitoneal PPi therapy
this variability in a previous study,12 was not effective in this In initial studies, sodium PPi was injected daily in 5 ml/kg of a
study, but treatment with calcitriol resulted in aortic buffered saline solution and was started simultaneously with the
calcification in all animals and increased the overall degree adenine diet. Larger volumes were used in subsequent studies and
of calcification. This effect of calcitriol on uremic calcifica- required peritoneal catheters. Three days before beginning adenine

516 Kidney International (2011) 79, 512–517

WC O’Neill et al.: PPi treatment inhibits uremic vascular calcification original article

treatment, peritoneal catheters were placed through a midline ACKNOWLEDGMENTS

abdominal incision and tunneled subcutaneously to a subcutaneous The authors thank Faten Hassounah for her expert technical
port implanted behind the neck (Clearport; Access Technologies, assistance.
Skokie, IL). Sodium PPi in a sterile, glucose-containing, buffered REFERENCES
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DISCLOSURE
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emplyee of Baxter Healthcare. All the other authors declared no histologic structure and remodeling dynamics of bone. Comput Programs
competing interests. Biomed 1981; 13: 191–202.

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