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Phosphate overload accelerates vascular

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Nephrol Dial Transplant (2003) 18 [Suppl 3]: iii86iii89
DOI: 10.1093/ndt/gfg1022
Phosphate overload accelerates vascular calcium deposition in
end-stage renal disease patients
Takashi Shigematsu1, Takashi Kono2, Kenichi Satoh2, Keitaro Yokoyama1, Toyohiko Yoshida2,
Tatsuo Hosoya1 and Kohji Shirai3
1
Division of Nephrology and Hypertension, Jikei University School of Medicine, 2Division of Urology and Blood
Purification, Mihama Hospital, Chiba and 3Center of Diabetes, Endocrinology and Metabolism, Sakura Hospital, Toho
University School of Medicine, Japan
Abstract
Cardiovascular disease is a major problem in end-stage
renal disease (ESRD) patients, with calcification being
one of the conspicuous features of arteriosclerotic
vessels. In the present study, clinical analysis and
in vitro cell culture were used to investigate factors pro-
moting vascular calcification in ESRD patients. The
aortic arch calcification score (AACS) was the method
used to estimate vascular calcification by evaluation
of the simple posterioranterior view chest X-rays.
Factors that relate significantly to vascular calcifica-
tion and the AACS are the Ca 3 Pi, age, dialysis period,
blood pressure, smoking and diabetes mellitus, but not
total cholesterol or triglyceride. The Ca 3 Pi, which
depends on the serum phosphate concentration, is the
only specific factor with the possibility for correction
in ESRD patients, and so control of serum phosphate
concentration is an important factor for reducing
vascular calcification. The effects of phosphate over-
load on calcium deposition in human vascular smooth
muscle cells (hVSMCs) using a primary cell culture
system were also investigated. hVSMCs were harvested
from the radial artery in ESRD patients and it was
found that they could secrete extracellular matrix with
a high affinity for calcium in a high phosphate medium
(Pis5.4 mgudl). Therefore, phosphate overload might
stimulate the hVSMCs to accelerate the calcium
deposition in ESRD patients. These results suggest
that the control of phosphate excess is important for
prevention of calcium deposition on arteriole walls in
ESRD patients.
Keywords: cardiovascular disease; end-stage renal dis-
ease (ESRD); hyperphosphataemia; vascular calcifica-
tion; vascular smooth muscle cells (VSMCs)
Correspondence and offprint requests to: Takashi Shigematsu, MD,
PhD, Assistant Professor, Division of Nephrology and Dialysis Unit,
Department of Internal Medicine, Jikei University School of
Medicine, Aoto-General Hospital, 6-41-2 Aoto, Katsushika-Ku,
Tokyo 125-8506, Japan. Email: aotaki@jikei.ac.jp
# 2003 European Renal AssociationEuropean Dialysis and Transplant Association
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Introduction
The number of patients with end-stage renal disease
(ESRD) undergoing chronic renal replacement therapy
is increasing worldwide, which is a major problem not
only in the medical field but also socially and econo-
mically, so cost-effective care is important in this field.
One of the unfortunate medical problems that many
ESRD patients have is vascular disease, which is a
major cause of mortality from ischaemic heart disease,
cerebral vascular disease and arteriosclerosis obliterans
[1]. Vascular calcification is often observed in ESRD,
even in young patients [2], and is related to the
mortality risk in ESRD patients. Identification of the
factors that cause vascular calcification is therefore
important.
Subjects and methods
Clinical study of factors modulating vascular
calcification
We analysed 286 ESRD patients (FuMs194u92) aged
59.6"11.5 years (mean"SD) undergoing long-term haemo-
dialysis for a period of 9.7"7.6 years. We used the aortic
arch calcification score (AACS) method to estimate vascular
calcification by examination of simple, posterioranterior
view chest X-ray films. We scored the area of calcification
from grade 0 (no calcification) to 5 (severe calcification) by
detailed measurement of the calcification in the aortic arch
(Figure 1). We checked the clinical significance of age,
dialysis period, serum calciumphosphate product (Ca 3 Pi),
blood pressure, smoking, original kidney disease (e.g.
diabetes mellitus, hypercholesterolaemia, hyperuricaemia),
intact parathyroid hormone (i-PTH) concentration and
gender.
The PTH concentration was measured by i-PTH assay
using the Allegro i-PTH IRMA Kit (Sumitomo-Metaphysics
Pham, Osaka, Japan). Other assays were performed by
standard methods with an enzyme technique as the basic
Effect of phosphate overload on hVSMCs iii87

Fig. 1. Vascular calcification was estimated by the aortic arch calcification score (AACS) by posterioranterior view on simple chest X-ray
film. We scored the size of the area from grade 0 (no calcification) to 5 (severe calcification), by detailed measurement of the area of
calcification in the aortic arch.

Downloaded from http://ndt.oxfordjournals.org/ by guest on June 7, 2013

procedure. All data are shown as mean"SD. Statistical
analysis was performed by analysis of covariance (ANOVA)
with multiple comparisons, with a significance level set at 5%.
Multiple factorial regression analysis with the Cox-Hazard
model was also used.

Binding of 45Ca to the extracellular matrix produced

by hVSMCs
We harvested human vascular smooth muscle cells
(hVSMCs) from the radial artery of nine ESRD patients by
an explant method. The cells were cultured for 7 days in
2.7 mgudl phosphate Dulbeccos modified Eagles medium
(DMEM) with 10% fetal calf serum (FCS) after confluent
growth. In the tests using excess phosphate, the phosphate
concentration was increased 2-fold to 5.4 mgudl using
glycerophosphate. After 7 days, hVSMCs were removed
using KOH (0.01 molul). 45Ca (1.0 mCi) used as a tracer was
Fig. 2. Relationship between aortic arch calcification score (AACS)
added to the remaining extracellular matrix. After 3 h and serum Ca 3 Pi. The Ca 3 Pi )65 mgudl2, was significantly
incubation with 45Ca, the conditioned medium was washed, (P-0.01) related to the AACS. This effect on the abdominal aorta
and the radioactivity of the extracellular matrix was counted with ECG disorder (ischaemic change shown as STT change) was
in the calcium precipitate. also closely related to the serum Ca 3 Pi (data not shown).

We found that the powerful determinant for Ca 3 Pi

Results is the serum phosphate concentration (Figure 3).

Clinical study of the modulating factors of vascular

Binding of 45Ca to the extracellular matrix produced
calcification
by hVSMCs
The multiple factorial analysis was adjusted for age
The results of the in vitro study are shown in Figure 4.
and also for other aspects of the procedure. Factors
The extracellular matrix secreted from the conditioned
that can affect the AACS are age, dialysis period,
hVSMCs in the high phosphate medium had a high
Ca 3 Pi, blood pressure, smoking and diabetes mellitus.
affinity for 45Ca. The effect of phosphate excess was
Age, dialysis period and diabetes are impossible to
almost 2-fold.
modify, but hypertension and smoking are now
controllable risk factors, even in the normal popula-
tion. The control of Ca 3 Pi is specific for the
minimization of vascular calcification in ESRD Discussion
patients (Figure 2). High serum cholesterol, hyper-
uricaemia, i-PTH and gender are not significant Cardiovascular diseases have a great deal of influence
factors in the estimation of the AACS. on the long-term survival of ESRD patients [1]. A
iii88
Fig. 3. Correlation between the Ca 3 Pi and serum calcium or phosphate concentration. The Ca 3 Pi is determined almost only by the Pi
concentration and is closely correlated with serum phosphate (Ys10.4765.155, r2s0.914), but not serum calcium (r2s0.229).
Fig. 4. Impact of phosphorus on calcium precipitation in primary
cultured human vascular smooth muscle cells (hVSMCs) from
dialysis patients, which can secrete extracellular matrix with a high
affinity for calcium in a high phosphate medium.
major problem in ESRD patients is vascular calcifica-
tion, which is known to occur more in the intima
(where the VSMCs are located) than in the endothe-
lium [3]. In the present study, we investigated the
factors affecting vascular calcification by clinical
studies, and then evaluated the impact of elevated
phosphate on calcification by in vitro cell culture.
Typical vascular calcification seems to be similar to
that of bone in ESRD patients, which strongly suggests
that vascular calcification is related to bone mineral
metabolism. We analysed vascular calcification accord-
ing to the classical method of AACS (Figure 1). An
advantage of this method is that it can be carried out in
any hospital or haemodialysis centre, as opposed to
some more recent procedures such as electron beam
CT analysis. The AACS is related positively to serum
Ca 3 Pi (Figure 2), which is recognized as a signifi-
cant factor controlling vascular calcification in
ESRD patients [4]. It has been reported that the
Ca 3 Pi is ;65 mg2udl2, and our results corresponded
to that figure. We also demonstrated that phosphate
was the significant factor in determining the Ca 3 Pi
T. Shigematsu et al.
(Figure 3); so maintaining it at -65 mg2udl2 controls
hyperphosphataemia in much the same way as keeping
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the serum phosphate concentration at -6.06.5 mgudl.
Block et al. have reported that control of serum
phosphorus and Ca 3 Pi has a major impact in reducing
the mortality risk in chronic haemodialysis patients [5].
They concluded that patients with a serum phosphorus
concentration )6.5 mgudl have an increased mortality
risk, and our results are in agreement with their
conclusion.
In addition, we evaluated the effect of raised
phosphate on vascular calcification in vitro. hVSMCs
harvested by an explant method from the radial artery
of ESRD patients and cultured in a 2-fold more
concentrated phosphate medium (5.4 mgudl) than
controls can secrete extracellular matrix that has a
high affinity for calcium. This effect of phosphate
excess was almost 2-fold greater than in the normal
phosphate controls. The progression of calcium
deposition in vessels appears to be dependent on
phosphate overload. The composition of the extra-
cellular matrix is still obscure, but some factors, such
as Gla protein, osteocalcin and osteopontin, that can
cause calcium deposition, may be present in the matrix
[6,7]. The mechanism whereby hVSMCs recognize the
high phosphate signal is unknown, but there is some
information available about the relationship between
VSMCs and osteoblasts [8]. It has been reported that
osteoblasts can act as human Glvr-1 phosphate trans-
porting agents [9], and hVSMCs may possess the
phosphate transporting system that responds to the
high phosphate signal.
In conclusion, phosphate overload may accelerate
vascular calcium deposition, and control of phosphate
excess is important in the treatment of this condition in
ESRD patients.
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