methods for post-hoc analysis and two-tailed t-test when appropriate. A value of po0.05 was considered statistically
significant. For statistical analysis, GraphPad Prism 5 soft- ware San Diego, CA, USA was used.
3 Results
3.1 Effect of curcumin treatment on body weight,
kidney weightbody weight ratio, blood glucose, creatinine clearance Ccr, BUN and
protein urine
At the end of the 11th wk, diabetic rats exhibited significantly increased plasma glucose levels and decreased body weight
compared with normal rats. Chronic treatment with curcumin in diabetic rats from 3 to 11 wk altered plasma glucose levels
significantly compared with those of untreated rats. Treatment with curcumin also prevented body weight loss in diabetic rats,
but this effect was not significant compared with that of untreated diabetic rats. Diabetic rats had increased kidney
weightbody weight ratio, a marker for the development of DN, and this ratio was significantly decreased by treatment
with curcumin Table 1. Diabetic rats also exhibited increased plasma creatinine, increased BUN, increased urinary protein
excretion and decreased CCr and curcumin treatment signifi- cantly reduced plasma creatinine, reduced BUN, reduced
urinary protein excretion and increased CCr Table 1.
3.2 Effect of curcumin on the expression and
activity of PKC-a, PKC-b
1
and p-ERK12 in STZ- induced diabetic rat kidneys
Induction of diabetes for 8 wk was associated with increased PKC-a and PKC-b
1
expression in total cell lysate
Fig. 1A–C, which was significantly reduced by curcumin treatment. In Fig. 1D and E, evidence is shown that diabetic
animals demonstrated PKC activation, with a significant increase in the ratio of membrane to cytosolic PKC-a and-b
1
which was reduced by curcumin. Moreover, Western blot- ting analysis using anti-phospho ERK12 antibody, which
recognizes phosphorylated threonine 202 and tyrosine 204 of p44p42 ERK, detected enhanced phosphorylation of p44
p42 ERK in the diabetic group Fig. 2A and B. Curcumin treatment significantly inhibited the phosphorylation of
ERK in diabetes.
3.3 Curcumin reduced protein expression of
NADPH oxidase subunits in STZ-induced diabetic rat kidneys
Since activation of NADPH oxidase has been reported to be induced by diabetes and is associated with increased
oxidant production induced by hyperglycemia, we assessed changes in protein expression of NOX4 and p67phox
subunits of NADPH oxidase. As shown in Fig. 2D–F, there was increased protein expression of NOX4 and p67phox in
the diabetic group as compared with that in the normal group. These increases were significantly reduced by
curcumin treatment.
3.4 Curcumin prevents changes in lipid peroxidation
and GPx activity in diabetic rats The diabetic group exhibited a significant increase in the
levels of MDA compared with the normal group, and chronic treatment with curcumin significantly reduced the
levels of MDA in diabetic rat kidney. GPx activity in the kidneys was also significantly lower in the diabetic group
Table 1. Effect of curcumin on body weight, kidney weight, KWBW ratio, plasma glucose, creatinine clearance, BUN, protein urine,
plasma creatinine, MDA level, and GPx activity
a
Variables Normal n 5 5
Diabetic n 5 5
b
Curcumin n 5 5 Body weight BW g
541.6715.05 328.6712.63
353.2716.55 Kidney weight KW g
1.5970.10 1.9370.08
1.7870.15 KWBW ratio1000
2.9370.12 5.8870.11
5.0170.23
c
Plasma glucose mgdL 140.8720.3
699.8717.3 599717.0
c
CCr mLmin 3.970.7
1.070.2 3.070.7
c
BUN mgdL 22.570.7
37.171.7 3071.8
c
Protein urine mg24 H 13.270.6
33.675.2 18.872.7
c
Plasma creatinine mgdL 0.3070.01
1.4370.02 0.3670.02
c
MDA nmolmg kidney tissue 0.3170.01
0.6470.04 0.4070.02
c
GPx Umg protein kidney tissue 0.1770.03
0.0470.01 0.1370.01
c
BUN: blood urea nitrogen, CCr: creatinine clearance, GPx: glutathione peroxidase, KWBW: kidney weightbody weight, MDA: malondialdehyde.
a Values are expressed as mean7SEM b po0.05 versus. to normal group based on two-tailed t-test.
c po0.05 versus to diabetic group based on two-tailed t-test.
1658
V. Soetikno et al. Mol. Nutr. Food Res. 2011, 55, 1655–1665
2011 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim www.mnf-journal.com
N D
Cur PKC α cytosol
PKC-α membrane Cur
D N
D A
PKC-β1 total PKC-α total
β -actin
-
2 2,5
3 0,5
1 1,5
B
Cur D
N
1 2
PKC-
β
1
β
-actin arb.unit
N D
Cur arb.unit
PKC-
α
membranecytosol
PKC-β1 cytosol PKC-β1 membrane
N D
Cur
2,5 3
4 5
C E
1 1,5
2 1
2 3
4
0,5 N
D Cur
arb.unit PKC-
β
1 membranecytosol 1
N D
Cur un
it
C-
α
β -actin arb.
u
PK
Figure 1. Expression of PKC-a and PKC-b
1
in the total cell lysate, cytosol and membrane
fraction of renal cortex. A Representative Western blots showing specific bands for
PKC-a, PKC-b
1
and b-actin as an internal control. These bands are representative of
five separate experiments. B–C Densito- metry measurements of protein analysis.
The mean density values of PKC-a and PKC- b
1
are expressed as ratios relative to that of b-actin. D and E Representative Western
blots of cytosolic and membranous frac- tions of PKC-a and PKC-b
1
are presented. PKC-a
and PKC-b
1
activity ratio
of membrane bound to cytosolic fraction of
each PKC was increased by diabetes and reduced with treatment by curcumin. Each
bar represents
mean7SEM. N,
age- matched normal rats; D, diabetic-treated
rats administered with vehicle; Cur, diabetic rats treated with curcumin 100 mgkgday.
po0.05 versus N;
]]
po0.05 versus D based on one-way ANOVA followed by
Tukey’s test.
N D
Cur NOX4
N D
Cur p-ERK12
ERK12 N
p67phox β-actin
A D
2 3
4 3
4 5
B E
1 2
N D
Cur p-ERK12
ERK12 arb. unit
1 2
NOX4
β
-actin arb.unit N
D Cur
1,5 4
C F
0,5 1
1 2
3
N D
Cur
m R
N A
p 3
G A
P D
H
N D
Cur p67phox
β
-actin arb.unit
Figure 2. A Representative Western blots showing specific bands for phosphorylated
ERK12 and ERK12 as an internal control. Equal amounts of protein sample obtained
from whole
kidney homogenates
were applied in each lane. These bands are
representative of five separate experiments. B Densitometric data of protein analysis.
The mean density values of p-ERK12 are expressed as ratios relative to that of ERK12.
C
Real-time RT-PCR
analysis showing
diabetes-induced upregulation
of p300
mRNA, which is downregulated by curcumin treatment. D Representative Western blots
showing specific bands for NOX4, p67phox and b-actin as an internal control. E and F
Densitometric data of protein analysis. The mean density values of NOX4 and p67phox
are expressed as ratios relative to that of b-actin. Each bar represents mean7SEM. N,
age-matched normal rats; D, diabetic-treated rats administered with vehicle; Cur, diabetic
rats treated with curcumin 100 mgkgday.
po0.05 versus N;
]]
po0.05 versus D based on one-way ANOVA followed by
Tukey’s test. Mol. Nutr. Food Res. 2011, 55, 1655–1665
1659
2011 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim www.mnf-journal.com
than in the normal group. However, chronic treatment with curcumin prevented this decrease in GPx activity in the
diabetic group Table 1.
3.5 Effect of curcumin on extracellular matrix
