Mol. Nutr. Food Res. 2013, 57, 1649–1659
1651 spectrophotometer Ultraspec 3100, Amersham Biosciences,
Cambridge, UK. The oxidation of nicotinamide adenine din- ucleotide phosphate reduced NADPH to nicotinamide ade-
nine dinucleotide phosphate was measured by the decrease in absorbance at 340 nm.
2.5 Longitudinal measurement of blood pressure by tail plethysmography
Blood pressure BP was noninvasively measured by a volume pressure-recording sensor and an occlusion tail-cuff CODA
System, Kent Scientific, Torrington, CT, USA. In brief, be- fore surgery, the conscious animal was placed in a restrainer
and permitted to rest for 10 to 15 min. The cuff was then placed on the tail and was inflated and released several times
to condition the animal to the procedure. After stabilization, BP was measured three times, and the average of the recorded
values was used. In this study, we report the mean systolic BP SBP of each group. On the third day before surgery, BP was
recorded week 0. Two weeks after the second surgery, the animals were retrained and BP was recorded week 3. Similar
BP recording was carried out in weeks 6 and 10 postsurgery.
2.6 Kidney homogenates and nuclear extracts preparation
Briefly, 100 mg of kidney cortex was homogenized in 0.5 mL of buffer A containing 10 mM HEPES, pH 7.8, 10 mM KCl,
2 mM MgCl
2
, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 1 M pepstatin, and 1 mM p-aminobenzamidine using a tis-
sue homogenizer for 20 s. Homogenates were kept on ice for 15 min, and then 125 L of a 10 Nonidet p40 solution were
added and mixed for 15 s and the mixture was centrifuged for 2 min at 12 000 rpm. The supernatant containing cytoso-
lic proteins was collected. The pelleted nuclei were washed once with 200 L of buffer A plus 25 L of 10 Nonidet p40,
centrifuged, then suspended in 50 L of buffer B containing 50 mM HEPES, pH 7.8, 50 mM KCl, 300 mM NaCl, 0.1 mM
EDTA, 1 mM DTT, 0.1 mM PMSF, and 10 vv glycerol, mixed for 20 min, and centrifuged for 5 min at 12 000 rpm.
The supernatant containing nuclear proteins was stored at –80⬚C [16]. The protein concentrations in kidney ho-
mogenates and nuclear extracts were determined by the bicin- choninic acid method.
2.7 Western blot analysis
Protein abundance of cycloxygenase-2 COX-2, NADPH oxi- dase subunits p22
phox
and p67
phox
, transforming growth fac- tor TGF-1, tumor necrosis factor TNF-␣, HO-1, Keap1,
Nrf2 nuclear translocation, and p65 NF-B nuclear transloca- tion were measured by the Western blot technique. Antibod-
ies against COX-2, p22
phox
, p67
phox
, TNF-␣, HO-1, Keap1, and Nrf2 were purchased from Santa Cruz Biotechnology Santa
Cruz, CA, USA. Antibodies against p65 NF-B were pur- chased from Cell Signaling Technology Denver, CO, USA.
Antibody against TGF-1 was purchased from Promega Madison, WI, USA. Antibodies to -actin Cell Signaling
Technology and Lamin A Santa Cruz, CA, USA were used for measurements of the housekeeping proteins for cytosolic
and nuclear target proteins, respectively. Aliquots containing 70 g of total protein cytosol and 50 g of total protein nu-
clear extracts were separated on a 7.5–12.5 SDS PAGE gel, and proteins were transferred to a nitrocellulose membrane.
After being briefly washed in Tris-buffered saline TBS; 10 mM Tris HCl, 0.85 NaCl, pH 7.2 containing 0.05
Tween-20 TBS-T and blocked for 1 h in 5 nonfat dry milk, membranes were incubated with appropriate antibodies in
5 nonfat dry milk overnight at 4⬚C. After being washed three times for 5 min in TBS-T, membranes were subse-
quently incubated with secondary antibody appropriately di- luted in TBS-T for 1 h at room temperature. The membrane
was washed four times and detection was performed using the enhanced chemiluminescence system Amersham Bio-
sciences, Buckinghamshire, UK and exposed to X-ray film.
2.8 Immunohistochemistry of fibronectin
Formalin-fixed, paraffin-embedded kidney tissue sections were used for immunohistochemical staining. After deparaf-
finization and hydration, the slides were washed in TBS. Endogenous peroxidase activity was quenched by incubat-
ing the slides in methanol and 0.3 H
2
O
2
in methanol. After overnight incubation with the primary antibody, that
is, fibronectin diluted 1:50 Santa Cruz, CA, at 4⬚C, the slides were washed in TBS and then horseradish peroxidase-
conjugated secondary antibody was added and the slides were further incubated at room temperature for 1 h. The
slides were washed in TBS and incubated with diaminoben- zidine tetrahydrochloride as the substrate and counterstained
with hematoxylin. A negative control without primary anti- body was included in the experiment to verify the antibody
specificity. For morphometry analysis, the percentage of pos- itive cells stained brown was determined with a computer-
assisted color image analyzer CIA-102; Olympus, Tokyo, Japan. Ten random fields per kidney were studied at ×100
magnification for comparison of the different groups [17].
2.9 Histopathological analysis
Light microscopy was performed in the formalin-fixed sec- tions 4 m stained with Azan Mallory to stain collagen as
an indicator of fibrosis and periodic acid-Schiff to analyze glomerulosclerosis and tubulointerstitial injury. Glomeru-
losclerosis was graded from 0 to 4 grade 0; normal, grade 1;
25 involvement of the glomerular tuft, grade 2; 25–50 involvement of the glomerular tuft, grade 3; 50–75 in-
volvement of the glomerular tuft, and grade 4; sclerosis in more than 75 of the glomerular tuft and the score was
calculated with the following formula: [1 × n glomeruli with
C
2012 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim www.mnf-journal.com
1652
V. Soetikno et al. Mol. Nutr. Food Res. 2013, 57, 1649–1659
1+ + 2 × n glomeruli with 2+ + 3 × n glomeruli with 3+ + 4 × n glomeruli with 4+] × 100total number of
glomeruli examined. Tubulointerstitial damage was graded according to the extension of tubular damage infiltra-
tion, fibrosis, and tubular dilatationatrophy. Interstitial fi- brosis was determined in ten random fields of the cortical
area at ×100 magnification with use of a computer-assisted color image analyzer CIA-102; Olympus, with the observer
blind to the study group. The results are presented as the ratio of the fibrotic area to the whole area of the kidney. The
same instrument was used to determine glomerulosclerosis and tubulointerstitial injury [17].
2.10 Determination of TNF-␣ expression by quantitative real-time RT-PCR
