Cardiovascular Journal of Africa: Vol 23 No 6 (July 2012) - page 65

CARDIOVASCULAR JOURNAL OF AFRICA • Vol 23, No 6, July 2012 AFRICA e3
After the proteins were transferred, the blots and gels were
stained with Coomassie blue to check for complete protein
transference and equal loading. Membranes were blocked in
5% skimmed milk and hybridised to the following primary
antibodies against p53, BMP-2, RUNX2, Osx, ALP and
α-SMA as mentioned above (1:2 000) in anti-glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) mAb (1:2 000; code:
sc-47724, Santa Cruz, USA). The membranes were washed in
20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween-20,
and hybridised to the corresponding horseradish peroxidase-
conjugated secondary antibodies, goat anti-rabbit IgG-HRP
(code: SC-2004, Serologicals Corporation) 1:3 000 or goat anti-
mouse IgG-HRP (code: 12-349, Serologicals Corporation) 1:3
000 at room temperature for one hour.
Chemiluminescence detection was performed using
Supersignal (code: 34080, Pierce, IL, USA) according to the
manufacturer’s instructions and images were acquired on X-ray
film. Quantitative densitometry was performed on the identified
bands using a computer-based measurement system.
Statistical analysis
Results are expressed as mean ± SEM. Data analysis was
performed using SPSS (SPSS Inc, Chicago, IL, Version
11.0). Means of total cell number per glomerulus, glomerular
sclerosis index score and tubulo-interstitial fibrosis index score
(histological index score), as well as body weight, blood
pressure and serum chemistry parameters in non-size matched
experimental groups were compared using Kruskal-Wallis
non-parametric tests.
To determine the differences in vascular calcification and the
expression levels of the proteins in p53+ /+ and p53–/– mice at
different time points after 5/6 Nx, the Fisher exact test was used.
For calculating the correlation between the expression of p53
protein and vascular calcification or osteogenic differentiation
proteins, linear correlation analysis was done on the Western blot
data. Statistical significance was defined as p < 0.05.
Results
The morphology of the kidney was normal in groups 1 and
2 eight and 12 weeks after 5/6 Nx (data not shown). The
appearance of abnormalities, including glomerular hypertrophy,
cellular proliferation, glomerular sclerosis, intratubular casts,
tubular dilation and atrophy, and intertubular cell infiltration and
matrix accumulation occurred in groups 3 and 4, as well as in
groups 5 and 6 at eight weeks, and became progressively worse
Chemicon, Germany) diluted 1:100 in PBS at 37°C for 30 min.
The slides were washed three times with PBS and stained with
propidium iodide (Sigma) and Hoechst 33342 (Sigma). Negative
controls consisted of substituting the primary antibody with PBS.
Immunofluorescent images were visualised under a confocal
microscope (LSM 510 META, Carl Zeiss, Jena, Germany).
Positive results were identified by a green fluorescence.
Immunostaining for p53, ALP and α-SMA in the aorta tissue
was performed on cryostat sections (4 µm) using the standard
avidin–biotin complex method. Sections were processed as
previously described.10 Anti-mouse p53 polyclonal antibody
(1:200, code: SC-6243, Santa Cruz, USA), anti-ALP (1:200,
GmbH, Germany) and monoclonal mouse anti-α-SMA (1:200,
Sigma-Aldrich Co, St. Louis, MO, USA) were used as primary
antibodies. Sections were incubated with primary antibody at
37°C for three hours.
After removal of unbound primary antibody and rinsing
with PBS, the sections were incubated with avidin-biotinylated
horseradish peroxidase (code: pk-7200, Vectastain Elite ABC kit;
Vector Laboratories) for 60 min. The slides were washed again in
PBS,thenvisualisationwasperformedusingthediaminobenzidine
(DAB) substrate–chromogen system(code: 3468, Dako, Glostrup,
Denmark) and counterstained with 1% methylene green.
Finally, sections were washed with tap water, dehydrated and
mounted. Negative controls consisted of substituting the primary
antibody with PBS. A brown colour indicated a positive result.
The Western blot analysis was carried out for determination of
p53, BMP-2, RUNX2, Osx, ALP and α-SMA in the aortic tissue.
Pieces of dissected aortic tissue were immersed in lysis buffer
(50 mM Tris-HCl, pH 7.4, 0.1% SDS, 1% NP-40, 0.5% sodium
deoxycholate, 100 mM NaCl, 0.1 mM sodium orthovanadate,
1 mM sodium fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin,
10 μg/ml pepstatin, and 10 μg/ml PMSF), homogenised with
a dounce homogeniser, and then incubated on ice for 30 min.
After centrifuging at 20 000 × g and 4°C for 30 min, the protein
concentration of the collected supernatant was determined by
BCA kit (code: 23250, Pierce, Rockford, IL).
Samples of 40-μg protein were mixed with two × loading
buffer containing 125 mM Tris-HCl (pH 6.8), 5% glycerol,
2% sodium dodecyl sulfate (SDS), 2% β-mercaptoethanol and
0.001% bromophenol blue and were electrophoresed on 13%
sodium dodecyl sulfate-polyacrylamide gels for BMP-2 and
ALP, or on 10% gels for p53, RUNX2, Osx and α-SMA. The
proteins were transferred overnight to polyvinylidine difluoride
(PVDF) membranes (Millipore, Bedford, MA) using a Bio-Rad
Western blot apparatus.
TABLE 1. EFFECTS OF 5/6 NXAND HP TREATMENT ON BODYWEIGHT, BLOOD PRESSURE
AND SERUM CHEMISTRY IN P53+/+ AND P53–/– MICEAT 12WEEKS
p53+/+ mice p53–/– mice
Sh 5/6 Nx 5/6 Nx + HP Sh 5/6 Nx 5/6 Nx + HP
Body weight (g) 12.1 ± 1.4 17.3 ± 1.7a 16.8 ± 2.2a 22.6 ± 1.3 18.5 ± 2.1a 18.8 ± 2.5a
Blood haemoglobin (mmol/l) 10.6 ± 0.6 8.4 ± 0.4a 8.2 ± 0.3a 10.8 ± 0.5 8.7 ± 0.4a 8.6 ± 0.2a
Plasma urea (mmol/l) 12.5 ± 1.3 27.7 ± 4.5b 29.1 ± 3.8b 13.8 ± 2.1 30.2 ± 5.7b 32.8 ± 4.2b
Plasma calcium (mmol/l) 2.1 ± 0.2 2.8 ± 0.1a 3.7 ± 0.2cd 2.3 ± 0.2 3.1 ± 0.3a 4.1 ± 0.3cd
Plasma phosphate (mmol/l) 2.7 ± 0.3 3.6 ± 0.2c 4.8 ± 0.4cd 3.0 ± 0.4 3.8 ± 0.4c 5.0 ± 0.3cd
PTH (ng/ml) 67 ± 22 685 ± 221b 843 ± 301be 72 ± 27 702 ± 237b 862 ± 325be
Results are expressed as group mean ± SEM at 12 weeks after 5/6 nephrectomy (5/6 Nx) or sham-operation (Sh), n = 5 per group.
ap < 0.05, bp < 0.001, cp < 0.01 vs untreated Sh. dp < 0.01, ep < 0.001 vs 5/6 Nx mice. PTH = parathyroid hormone.