CARDIOVASCULAR JOURNAL OF AFRICA • Vol 23, No 6, July 2012
e2
AFRICA
of vascular calcification.
13
To further investigate the effects
of p53 on the osteogenic differentiation of VSMCs, we used
wild-type (p53
+
/
+
) and p53-deficient (p53–/–) mice in this
study to construct animal models of CKD-associated vascular
calcification. Some of the mice were subjected to five-sixth (5/6)
nephrectomy (Nx) and a high-phosphate (HP) diet. At different
time points following 5/6 Nx, we studied histological changes
in the kidney, aortic calcification, the markers of osteogenic
differentiation (such as BMP-2, RUNX2, Osx and ALP), a
special marker of VSMCs [alpha smooth muscle actin (
α
-SMA
)], and the expression of p53 protein in the aortic tissue. The
effects of p53 on the osteogenic differentiation of VSMCs were
evaluated.
Methods
p53–/– mice (F2) in C57BL/6 background mice (F2)
were obtained from the NIH and housed in a pathogen-
free environment. p53
+
/– mice were crossed with p53
+
/–
mice to generate homozygous p53–/– and p53
+
/
+
littermates.
Genotyping of the mice was performed by polymerase chain
reaction analysis of DNA extracted from the tail tips, as reported
by Donehower.
10,14
Male p53–/– and p53
+
/
+
mice aged two
months were used in this study.
The experiments were carried out in accordance with the
Research Council and Animal Care and Use Committee of
the Research Institute of Surgery, Daping Hospital, Third
Military Medical University (Chongqing, China). Experiments
conformed to the guidelines of the ethical use of animals. The
animal study protocol was reviewed and approved by the Animal
Ethics Committee of Chongqing. Efforts were made to minimise
animal suffering and the number of mice used.
We used a well-established murine model of a CKD-associated
vascular calcification model. Briefly, male mice weighing 19 to
24 g were housed with a 12-hour light and dark cycle and allowed
free access to food and water. All animals were anesthetised
with sodium pentobarbital (50 mg/kg i.p.) and then placed on a
warming table to maintain a rectal temperature of 37°C.
5/6 Nx as a model of CKD was achieved in a two-step surgical
procedure.
3,15,16
The left kidney was exposed by a flank incision
and the upper and lower poles of the right kidney were resected.
Two weeks later the left kidney was removed. The mouse was
allowed to recover in a warmed cage, and food and water were
given
ad libitum
. The day after the 5/6 Nx, the animals received
standard HP diet (Altromin C1049, Germany, containing 1.65%
phosphate, 0.24% sodium, 0.95% calcium, 0.07% magnesium,
0.7% potassium and 17% protein).
Experimental design
Mice were randomly assigned to six experimental groups: (1)
p53
+
/
+
mice sham-operated on a normal diet (0.6% Ca and 0.6%
P) were used as the control (group 1,
n
=
10) with the p53
+
/
+
group, (2) p53–/– mice sham-operated on a normal diet were
used as the control (group 2,
n
=
10) with the p53–/– group, (3)
5/6 Nx of p53
+
/
+
mice on a normal diet (group 3,
n
=
10), (4)
5/6 Nx of p53–/– mice on a normal diet (group 4,
n
=
10), (5)
5/6 Nx of p53
+
/
+
mice
+
HP diet (group 5,
n
=
10), and (6) 5/6
Nx of p53–/– mice
+
HP diet (group 6,
n
=
10).
Food consumption in groups 1 to 4 was the same, and that
in groups 5 and 6 was the same. The high mortality in 5/6 Nx
mice precluded maintaining them for more than 12 weeks in this
study. At eight and 12 weeks following 5/6 Nx, the mice were
anaesthetised with intraperitoneal pentobarbital (five animals at
each time point in each group). Blood was collected by retro-
orbital bleeding. The mice were killed by cervical dislocation
and the kidneys and aortic tissue were collected. Each kidney or
aorta was cut into three parts medially for the following analyses.
Analyses
Paraffin-embedded sections of 4 µm were prepared and stained
with haematoxylin and eosin (HE) stain and then examined in a
blinded manner by two examiners, with each section evaluated
twice. Glomerular cell number was determined by counting
the nuclei within the glomerular tuft. Glomerular sclerosis was
graded as follows: 0
=
none;
+
1
=
sclerotic changes in
<
25% of
the glomerulus;
+
2
=
25– 50% sclerosis;
+
3
= >
50% sclerosis.
17
The mean score per glomerulus in each kidney was determined
as the sclerosis index.
Tubulo-interstitial fibrosis was defined as tubular atrophy,
dilation and intratubular casts, as well as cellular infiltration and
widening of the interstitium. It was scored semi-quantitatively
according to the method of Shih
et al
.
18
as follows: 0
=
normal;
0.5
=
small focal area injured; 1
=
less than 10% of the cortex
injured; 2
=
10–25% of the cortex injured; 3
=
25–75% of the
cortex injured; and 4
=
>
75% of the cortex injured.
Measurements of body weight and blood pressure, and the
levels of haemoglobin, blood urea nitrogen (BUN), phosphate
(Pi) and calcium (Ca) were performed as described by Bro
et
al
.
17
Parathyroid hormone (PTH) level was determined by a
commercial ELISA test (BioSource, Belgium).
Mineral deposition (calcification) was assessed under the
light microscopic using the von Kossa assay. Dewaxed and
rehydrated sections (4
µ
m) of artery tissue were placed in 5%
silver nitrate solution for 30 min in the dark, then into revelator
solution (Kodak) for 5 min. They were fixed in 5% sodium-
thiosulfate solution for another 5 min. Finally the sections were
counterstained with 2% eosin.
Calcium deposits appeared as black areas.
19,20
For the vascular
calcification score, sections were graded from 0 to 4
+
for von
Kossa staining,
21
where 0
=
no calcification; 1
=
spots; 2
=
single segments of black staining; 3
=
multiple segments; and 4
=
diffuse, circumferential staining. The calcification score was
obtained by averaging all the scores from all sections.
The determination of BMP-2, RUNX2 and Osx in the aortic
tissue was performed on cryostat sections (4
µ
m) using indirect
immunofluorescence staining. Briefly, the sections were fixed
with 4% formaldehyde/PBS (pH 7.4) and treated with 3% H
2
O
2
in methanol for 10 min to inactivate endogenous peroxidase.
After washing in PBS, the sections were microwaved in 10
mM citrate buffer (pH 6.0) for 10 min to retrieve the antigen.
Sections were then incubated with 1.5% normal goat serum for
15 min, followed by incubation with primary antibodies, BMP-2
antibody (1:200, code: sc-6895, Santa Cruz, USA), RUNX2
antibody (1:200, code: sc-12488, Santa Cruz, USA), or Osx
antibody (1:200, code: sc-22536-R, Santa Cruz, USA) at 37°C
for three hours.
After removal of the unbound primary antibody and rinsing
with PBS, the sections were incubated with fluorescein
isothiocyanate-conjugated (FITC) antibody (code: AP186F,
