Curcumin ameliorates macrophage infiltration by inhibiting NF-κB activation and proinflammatory cytokines in streptozotocin induced-diabetic nephropathy.

Abstract

Chronic inflammation plays an important role in the progression of diabetic nephropathy (DN) and that the infiltration of macrophages in glomerulus has been implicated in the development of glomerular injury. We hypothesized that the plant polyphenolic compound curcumin, which is known to exert potent anti-inflammatory effect, would ameliorate macrophage infiltration in streptozotocin (STZ)-induced diabetic rats.
Diabetes was induced with STZ (55 mg/kg) by intraperitoneal injection in rats. Three weeks after STZ injection, rats were divided into three groups, namely, control, diabetic, and diabetic treated with curcumin at 100 mg/kg/day, p.o., for 8 weeks. The rats were sacrificed 11 weeks after induction of diabetes. The excised kidney was used to assess macrophage infiltration and expression of various inflammatory markers.
At 11 weeks after STZ injection, diabetic rats exhibited renal dysfunction, as evidenced by reduced creatinine clearance, increased blood glucose, blood urea nitrogen and proteinuria, along with marked reduction in the body weight. All of these abnormalities were significantly reversed by curcumin. Hyperglycemia induced the degradation of IκBα and NF-κB activation and as a result increased infiltration of macrophages (52%) as well as increased proinflammatory cytokines: TNF-α and IL-1β. Curcumin treatment significantly reduced macrophage infiltration in the kidneys of diabetic rats, suppressed the expression of above proinflammatory cytokines and degradation of IκBα. In addition, curcumin treatment also markedly decreased ICAM-1, MCP-1 and TGF-β1 protein expression. Moreover, at nuclear level curcumin inhibited the NF-κB activity.
Our results suggested that curcumin treatment protect against the development of DN in rats by reducing macrophage infiltration through the inhibition of NF-κB activation in STZ-induced diabetic rats.

Full text

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RESEARCH Open Access
Curcumin ameliorates macrophage infiltration by
inhibiting NF-?B activation and proinflammatory
cytokines in streptozotocin induced-diabetic
nephropathy
Vivian Soetikno1,2, Flori R Sari1, Punniyakoti T Veeraveedu1, Rajarajan A Thandavarayan1, Meilei Harima1,
Vijayakumar Sukumaran1, Arun Prasath Lakshmanan1, Kenji Suzuki3, Hiroshi Kawachi4and Kenichi Watanabe1*
Abstract
Background: Chronic inflammation plays an important role in the progression of diabetic nephropathy (DN) and
that the infiltration of macrophages in glomerulus has been implicated in the development of glomerular injury.
We hypothesized that the plant polyphenolic compound curcumin, which is known to exert potent anti-
inflammatory effect, would ameliorate macrophage infiltration in streptozotocin (STZ)-induced diabetic rats.
Methods: Diabetes was induced with STZ (55 mg/kg) by intraperitoneal injection in rats. Three weeks after STZ
injection, rats were divided into three groups, namely, control, diabetic, and diabetic treated with curcumin at 100
mg/kg/day, p.o., for 8 weeks. The rats were sacrificed 11 weeks after induction of diabetes. The excised kidney was
used to assess macrophage infiltration and expression of various inflammatory markers.
Results: At 11 weeks after STZ injection, diabetic rats exhibited renal dysfunction, as evidenced by reduced
creatinine clearance, increased blood glucose, blood urea nitrogen and proteinuria, along with marked reduction in
the body weight. All of these abnormalities were significantly reversed by curcumin. Hyperglycemia induced the
degradation of I?Ba and NF-?B activation and as a result increased infiltration of macrophages (52%) as well as
increased proinflammatory cytokines: TNF-a and IL-1b. Curcumin treatment significantly reduced macrophage
infiltration in the kidneys of diabetic rats, suppressed the expression of above proinflammatory cytokines and
degradation of I?Ba. In addition, curcumin treatment also markedly decreased ICAM-1, MCP-1 and TGF-b1protein
expression. Moreover, at nuclear level curcumin inhibited the NF-?B activity.
Conclusion: Our results suggested that curcumin treatment protect against the development of DN in rats by
reducing macrophage infiltration through the inhibition of NF-?B activation in STZ-induced diabetic rats.
Background
Diabetic nephropathy (DN) is the largest single cause of
end-stage renal failure worldwide. In spite of the avail-
able modern therapies of glycaemic and blood pressure
control, many patients continue to show progressive
renal damage [1]. Therefore, it is extremely important to
identify novel interventions to halt the progression of
DN.
Recently, DN has been considered as an inflammatory
disease [2], in which macrophage infiltration into glomer-
uli is associated with the progression of glomerular injury
[3]. Monocytes/macrophages are the principle inflamma-
tory cells found in the diabetic kidney [3-5], these cells
extravasculated from the blood-stream and attracted to
the target tissue through a process mediated by various
chemokines secreted from resident glomerular cells such
as monocyte chemotactic protein (MCP)-1 and adhesion
molecules such as intercellular adhesion molecule
(ICAM)-1 [6-8]. MCP-1 is a potent chemokine that is
known to influence both macrophage accumulation and
* Correspondence: watanabe@nupals.ac.jp
1Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences,
Niigata University of Pharmacy and Applied Life Sciences, Niigata City, Japan
Full list of author information is available at the end of the article
Soetikno et al. Nutrition & Metabolism 2011, 8:35
http://www.nutritionandmetabolism.com/content/8/1/35
© 2011 Soetikno et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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macrophage function and its expression increases gradu-
ally in diabetic kidneys in animal models [9,10]. Renal
expression of ICAM-1, a 90-kD cell surface glycoprotein
that plays a major role in the regulation of interactions
with immune cells whose expression is upregulated at
the sites of inflammation, is known to be increased in
experimental type 1 diabetic animals. Furthermore,
macrophage infiltration was found to be blocked by anti-
ICAM-1 antibody, confirming that ICAM-1 mediates
macrophage infiltration into diabetic kidney [11]. These
findings suggest that ICAM-1 and MCP-1 may play an
important role in the pathogenesis of DN via the induc-
tion of inflammatory cell infiltration.
Transcription factors such as nuclear factor-?B (NF-?B)
regulate the gene expression of several cytokines, chemo-
tactic and matrix proteins involved in inflammation,
immunological responses, and cell proliferation [12]. Stu-
dies have demonstrated that high glucose-induced NF-?B
activation in mesangial cells may play a role in diabetic
renal injury through upregulation of ICAM-1 protein and
mRNA expression in rat mesangial cells [13,14]. In addi-
tion, activated NF-?B shows an important role in the
upregulation of MCP-1 in human DN [15]. NF-?B is also
known to activate its downstream inflammatory mediators
such as transforming growth factor (TGF)-b1, inducible
nitric oxide synthase (iNOS), fibronectin, tumor necrosis
factor (TNF)-a and interleukin (IL)-1b [14,16].
Curcumin is the active ingredient in the traditional her-
bal remedy and dietary spice turmeric (Curcuma longa)
and is the subject of clinical trials for various diseases
such as cancer, Alzheimer’s disease, and ulcerative colitis
[17]. The polyphenol curcumin (diferuloylmethane) com-
prises 2-8% of most turmeric preparations and is gener-
ally regarded as its most active component, having potent
antioxidant, anti-inflammatory and anticarcinogenic
properties. Modern science has revealed that curcumin
mediates its effects by modulation of several important
molecular targets, including transcription factors (e.g.,
NF-?B, activator protein (AP)-1, Early growth response
gene (Egr)-1, b-catenin and Peroxisome proliferator-acti-
vated receptor (PPAR)-g), enzymes (e.g., cyclooxigenase 2
(COX2), 5-lipoxygenase (LOX), iNOS and hemeoxygen-
ase-1 (HO)-1), cell cycle proteins (e.g., cyclin D1 and
p21), cytokines (e.g., TNF-a, IL-1, IL-6 and chemokines),
receptors (e.g., Epidermal Growth Factor Receptor
(EGFR) and Human Epidermal growth factor Receptor2
(HER2)) and cell surface adhesion molecules. Because it
can modulate the expression of these targets, curcumin is
now being used to treat cancer, arthritis, diabetes,
Crohn’s disease, cardiovascular diseases, osteoporosis,
Alzheimer’s disease, psoriasis and other pathologies [18].
Several in vitro and in vivo studies have demonstrated
that curcumin inhibits activation of NF-?B proinflam-
matory signaling pathways and reduces the influx of
macrophages [19-21]. However, to the best of our
knowledge, studies have not been revealed the effect of
curcumin on macrophages accumulation in DN. In the
present study, we investigated the effect of curcumin on
macrophages accumulation and the expression of
ICAM-1, MCP-1 and TGF-b1in experimental diabetic
kidney. In addition, the relationship between the extent
of transcription factor NF-?B and proinflammatory
cytokines, TNF-a and IL-1b, expression in renal tissue
was elucidated.
Methods
Animals and experimental protocol
All animal studies were treated in accordance with the
guidelines for animal experimentation of our institute
[22]. Male Sprague-Dawley rats (Charles River Japan Inc.,
Kanagawa, Japan), weighing approximately 250-300 g (8
weeks of age), were randomly divided into three groups
(n = 5): non-diabetic normal animals used as a controls
(C), diabetic animals treated with vehicle 1% gum Arabic
(D), and diabetic animals treated with curcumin 100 mg/
kg body weight [23] diluted in vehicle 1% gum Arabic
(Cur). Diabetes was induced by a single intraperitoneal
injection of streptozotocin (STZ 55 mg/kg body weight
in 20 mM citrate buffer, pH 4.5), while the control ani-
mals received 20 mM citrate buffer solution. The use of
STZ to induce diabetes provides an animal model of type
1 diabetes, as the drug causes pancreatic b-islet cell apop-
tosis. Diabetes was confirmed by tail-vein blood glucose
levels using Medi-safe chips (Terumo Inc., Tokyo, Japan),
and diabetes was defined if blood glucose ≥ 300 mg/dL.
Curcumin was started at three weeks after STZ injection
and was administered via oral gavage for 8 weeks. Rats
were housed in a temperature-controlled room and were
given free access to water and standard laboratory chow
during the study period. All rats were sacrificed at 11
weeks after the induction of diabetes for analysis of renal
tissue.
Biochemical analysis
Each week, rats were weighed and their blood glucose
levels were measured. Urine samples were collected over
a 24 h period in individual metabolic cages for the mea-
surement of protein in urine at 1, 3, 7, and 11 weeks and
were determined by the Bradford method. Urine creati-
nine was determined by the Jaffe method at 11 week. At
the end of experimentation, heparinized whole blood was
collected from anesthetized rats via heart puncture.
EDTA-blood was centrifuged at 3000 g, 15 min at 4°C for
the separation of plasma. The plasma was used for the
estimation of blood urea nitrogen (BUN) and creatinine.
Creatinine clearance was calculated in individual rats as
follows: Creatinine clearance = urine creatinine × urine
volume/plasma creatinine × time [24].
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Chemicals
Unless otherwise stated all reagents were of analytical
grade and were purchased from Sigma-Aldrich (Tokyo,
Japan).
Histopathological analysis
The kidney was decapsulated. Half of the kidney was
immediately snap-frozen in liquid nitrogen for subsequent
protein extraction and enzymatic assays. The remaining
excised kidneys were cut into about 2-mm thick transverse
slices and fixed in 10% formalin. After being embedded in
paraffin, several transverse sections were obtained from
the kidney and stained with Periodic acid-Schiff (PAS) for
histological evaluation. The frequency and severity of
lesions in kidney were assessed semi-quantitatively by light
microscopy. Forty glomeruli in each kidney were graded in
accordance with their severity of glomerular damage (0, no
lesion; 1, sclerosis of < 25% of the glomerulus; 2, sclerosis
of 26-50%; 3, sclerosis of 51-75%; and 4, sclerosis of > 75%
of the glomerulus). The glomerulosclerosis indexes were
calculated using the following formula, (glomerulosclerosis
index = (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4)/n0+ n1+
n2+ n3+ n4, where nxis number of glomeruli in each
grade of glomerulosclerosis) as reported previously [25].
Immunohistochemistry
Formalin-fixed, paraffin-embedded kidney tissue sections
were used for immunohistochemical staining. After
deparaffinization and hydration, the slides were washed
in Tris-buffered saline (TBS; 10 mM/L Tris HCl, 0.85%
NaCl, pH 7.2). Endogenous peroxidase activity was
quenched by incubating with the slides in methanol and
0.3% H2O2in methanol. After overnight incubation with
the primary antibody, that is, mouse monoclonal anti-
ED1 antibody (diluted 1:50) (sc-59103; Santa Cruz Bio-
technology Inc. CA, USA) at 4°C, the slides were washed
in TBS and horseradish peroxidase (HRP)-conjugated
goat antimouse secondary antibody was then added and
the slides were further incubated at room temperature
for 1 h. The slides were washed in TBS and incubated
with diaminobenzidine tetrahydrochloride as the sub-
strate, and counterstained with hematoxylin. A negative
control without primary antibody was included in the
experiment to verify the antibody specificity. Intraglo-
merular ED-1-positive cells were counted in 200 glomer-
uli/group under 400-fold magnification and expressed as
cells/glomerular cross section (gcs) [26].
Immunofluorescence staining for ICAM-1
For immunofluorescence, tissues were fixed in 10% buf-
fered formaldehyde solution and embedded in paraffin.
Sections underwent microwave antigen retrieval, were
blocked with 10% rabbit serum in 1% BSA and were then
incubated with polyclonal goat anti-ICAM-1 antibody
(sc-1511; Santa Cruz). Binding sites of the primary anti-
body were revealed with fluorescein isothiocyanate-
conjugated secondary antibody. Samples were visualized
with a fluorescence microscope at 400-fold magnification
(CIA-102; Olympus) [27].
Cytosolic and nuclear extract homogenization
The kidney was cut and immediately frozen in liquid
nitrogen and kept at -80°C until use. The frozen kidney
was ground to a powder and then mixed in ice-cold
HEPES buffer (10 mM HEPES, 0.2% Triton X-100,
50 mM NaCl, 0.5 mM sucrose, 0.1 mM EDTA, protease,
and phosphatase inhibitors) and homogenized with an ice-
chilled Dounce homogenizer at 4°C. This was spun at
10,000 rpm for 10 min, and the supernatant was aliquoted
and stored at -80°C as the cytosolic extract. The pellet was
suspended in ice-cold buffer (10 mM HEPES, 500 mM
NaCl, 10% glycerol, 0.1 mM EDTA, 0.1 mM EGTA, 0.1%
IGEPAL, and protease and phosphatase inhibitors) and
vortexed at 4°C for 15 min and centrifuged for 10 min at
14,000 rpm. The resulting supernatant was aliquoted and
stored as the nuclear extract at -80°C. The absence of
cross-reactivity with b-actin in Western blots confirmed
the purity of nuclear extracts. A small aliquot of cytosolic
and nuclear extract was kept at 4°C for protein estimation
[28].
Western blotting
Cytosol (70 μg total protein) and nuclear extracts (50 μg
total protein) were separated on a 7.5-15% polyacrilamide
gel and electophoretically transferred to nitrocellulose
membrane. Membranes were blocked with 5% non-fat
dry milk in Tris-buffered saline Tween (20 mM Tris, pH
7.6, 137 mM NaCl, and 0.1% Tween 20) for 3 h at room
temperature, followed by an overnight incubation at 4°C
with polyclonal antibodies to rat ICAM-1, I?Ba (sc-371;
Santa Cruz), TNF-a (sc-1350; Santa Cruz), IL-1b (sc-
7884; Santa Cruz), NF-?B p65 (Cell Signaling #4764),
p-NF-?B (Ser276) (sc-101749; Santa Cruz), TGF-b1(Pro-
mega G-1221), MCP-1 (sc-1785; Santa Cruz) or b-actin
(Cell Signaling). All antibodies were used at a dilution of
1:1000. Three times after washing with Tris-buffered sal-
ine Tween 20 (TBS-T), incubation with appropriate
HRP-conjugated secondary antibodies were performed
for 1 h at room temperature. After three additional TBST
washes, the immunoreactive bands were visualized by
enhanced chemiluminescence (Amersham Biosciences,
Buckinghamshire, UK) according to the manufacturer’s
instructions. The levels of b-actin and lamin A (sc-20680;
Santa Cruz) were estimated in cytosol samples and in
nuclear extract samples, respectively, to check for equal
loading of samples. Films were scanned and band densi-
ties were quantified with densitometric analysis using
Scion Image program (Epson GT-X700, Tokyo, Japan).
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RNA extraction
Kidney tissues were preserved by immersion in RNAlater
(Ambion Inc., Austin, TX) immediately after sampling.
Total RNA was extracted after homogenization using
Ultra TurraxT8 (IKA Labortechinik, Staufen, Germany)
in TRIzol reagent (Invitrogen Corp., Carlsbad, CA)
according to the standard protocol. cDNA was synthe-
sized by reverse transcription using total RNA (2 μg) as a
template (Super Script II; Invitrogen Corp).
Gene expression analysis by real-time RT-PCR
Gene expression analysis was performed by real-time
reverse transcription polymerase chain reaction (RT-PCR)
(Smart cycler; Cepheid, Sunnyvale, CA) using cDNA
synthesized from the diabetic speciemen. Primer
sequences were as follows: IL-1b (forward), CTTCAATC
TCACAGCAGCACATCTCG, (reverse), TCCACGGG-
CAAGACATAGGTAGC; TNF-a (forward), CCCCAAA
GGGATGAGAAGTT, (reverse), CACTTGGTGGTT
TGCTACGA; GAPDH (forward), GCTCATTTCCTGG-
TATGACAACG, (reverse), AGGGGTCTACATGG-
CAACTG. Real-time RT-PCR, monitoring with a TaqMan
probe (TaqMan Gene expression assays; Applied Biosys-
tems, Foster City, CA) was performed according to the fol-
lowing protocol: 600 seconds at 95°C, followed by thermal
cycles of 15 seconds at 95°C, and 60 seconds at 60°C for
extension. Relative standard curves representing several
10-fold dilutions (1:10: 100: 1000: 10,000: 100, 000) of
cDNA from kidney tissue samples were used for linear
regression analysis of other samples. Results were normal-
ized to GAPDH mRNA as an internal control and are
thus shown as relative mRNA levels.
Statistical analysis
All values are expressed as means ± SEM and were ana-
lyzed using one-way analysis of variance (ANOVA) fol-
lowed by Tukey’s methods for post hoc analysis and
two-tailed t-test when appropriate. A value of p < 0.05
was considered statistically significant. For statistical
analysis, GraphPad Prism 5 software (San Diego, CA,
USA) was used.
Results
Animal data
At the end of the 11thweek, body weight and the ratio of
kidney weight to body weight, a marker for the develop-
ment of DN was significantly decreased and increased,
respectively in diabetic rats, and this ratio was signifi-
cantly decreased in diabetic rat-treated with curcumin.
Diabetic rats also exhibited increased plasma creatinine,
increased BUN, and decreased creatinine clearance
(CCr). All of these abnormalities were significantly
decreased by curcumin treatment (Table 1). Treatment
with curcumin also prevented body weight loss in
diabetic rats, but this effect was not significant compared
with that of untreated diabetic rats. During the study per-
iod, plasma glucose (Figure 1A) and 24 h urinary protein
excretion (Figure 1B) increased progressively in diabetic
rats which was significantly abrogated by the administra-
tion of curcumin (p < 0.05).
Table 1 Biochemical parameters in experimental animals
at 11 weeks
C (n = 5)D (n = 5)Cur (n = 5)
Body weight (BW) (g)
Kidney weight (KW) (g)
KW/BW ratio × 1000
Plasma creatinine (mg/
dL)
CCr (mL/min)
BUN (mg/dL)
539.5 ± 19.24 325.8 ± 15.88* 341.5 ± 15.11
1.5 ± 0.041.94 ± 0.11*
2.78 ± 0.055.94 ± 0.12*
0.55 ± 0.021.06 ± 0.13*
1.65 ± 0.11
4.83 ± 0.19#
0.69 ± 0.03#
3.8 ± 0.9
22.63 ± 0.95
0.81 ± 0.08*
38.05 ± 1.8*
3.61 ± 0.48#
30.58 ± 2.2#
BUN: blood urea nitrogen, CCr: creatinine clearance, KW/BW: ratio of kidney
weight to body weight.
Results are presented as the mean ± SEM.
• *p < 0.05 vs. C.
• #p < 0.05 vs. D.
A
B
**
**
**
**
**
Figure 1 Time-course changes in blood glucose (A) and 24 h
urine protein (B). Blood glucose as well as 24 h urine protein
increased progressively in the untreated diabetic rats following
induction of diabetes. Curcumin treatment significantly reduced
blood glucose and 24 h urine protein in the beginning of treatment
and these were maintained throughout the study period until
sacrifice. Values are means ± SEM. **p < 0.01 vs Curcumin.
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Histology
Normal histology was seen in the control rats (Figure 2A).
On the other hand, histological examination of the kidney
of diabetic rats had marked histological changes in the glo-
merular and tubular structure (Figure 2B). In the
untreated diabetic rats, 64% of the glomeruli were segmen-
tally sclerosed (Figure 2J), whereas curcumin-treated rats
developed only 27.4% (p < 0.05 compared with untreated
diabetic rats) segmental sclerosis (Figure 2C,J).
Effect of curcumin on macrophage infiltration
Kidneys from control rats did not show any significant
macrophage infiltration (Figure 2D). On the other hand,
diabetic rats demonstrated prominent macrophage (ED-
1-positive cells) infiltration in the glomerulus (Figure 2E,
K), where as diabetic rats treated with curcumin showed
marked reduction of macrophage influx by 32% (p <
0.01) (Figure 2F,K).
Effect of curcumin on NF-?B and I?Ba
In this study (Figure 3A,B), we observed that cytosolic
I?Ba in the kidney of diabetic rats was significantly
lower than in the control (p < 0.05) and curcumin treat-
ment significantly reduced I?Ba degradation (p < 0.05).
In addition, NF-?B was activated, which is measured by
the extent of phosphorylation of its p65 regulatory
**
##
J
**
##
K
50 ?m
A
50 ?m
B
50 ?m
C
50 ?m
D
50 ?m
E
50 ?m
F
G
H
I
Figure 2 Histological staining with Periodic acid-Schiff’s (PAS) in glomeruli (A-C) shows the glomerular and tubulointerstitial structure
of a normal rat kidney (A), diabetic rat kidney (B), in which significant changes in glomerular and tubulointerstitial structure were
noted. Diabetic rat developed the pathological characteristics of early diabetic nephropathy, including glomerular hypertrophy, mesangial
expansion and sclerosis of glomerulus (B). (C) Diabetic rat treated with curcumin showed amelioration of sclerosis of glomerulus. D-F:
immunohistochemistry staining for macrophage (ED-1-positive cells). G-I: immunofluorescence staining for ICAM-1 in glomeruli. Kidney tissues
were harvested from normal rat as a control (A, D, G), diabetic rat (B, E, H) and curcumin-treated rat (C, F, I). Quantification results are shown for
glomerulosclerosis index in each group (J) and number of macrophages (ED-1-positive cells) (K). Values are means ± SEM. **p < 0.05 vs C;##p <
0.05 vs D (J). **p < 0.01 vs C;##p < 0.01 vs D (K).
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subunit (p-NF-?B) in diabetic rats compared with that
of controls. Curcumin treatment to the diabetic rats
resulted in a mitigatory effect and showed decreased
levels of activated NF-?B compared with that of diabetic
rats (Figure 3E,F), in which nuclear NF-?B was 3.73 fold
higher in diabetic rats than in the control (p < 0.01) and
curcumin treatment decreases the activated NF-?B by
2.08 fold than in untreated diabetic rats (p < 0.05).
Effect of curcumin on renal expression of TNF-a and IL-1b
Renal TNF-a and IL-1b protein expression assessed by
Western blotting and were significantly increased in
diabetic rats compared with those in control rats (p <
0.05), where as curcumin treatment significantly abro-
gated these increases in diabetic rats (p < 0.01) (Figure
3A,C,D). In line with protein analysis, renal TNF-a and
IL-1b mRNA expression assessed by real-time PCR were
also significantly higher in diabetic rats compared with
those in control rats (p < 0.05), and curcumin treatment
significantly attenuated the increased renal TNF-a and
IL-1b mRNA expression (p < 0.05). The TNF-a/
GAPDH mRNA and IL-1b/GAPDH mRNA ratios was
2.3 and 2.6 fold higher in diabetic rats compared with
control rats, respectively, and curcumin treatment
A
B
C
E
**
##
D
**
##
F
I?B?
IL-1?
TNF-?
?-actin
C
D
Cur
p-NF-?B
NF-?B
C
D
Cur
Lamin A
**
##
**
##
Figure 3 Renal expression of I?Ba, TNF-a, IL-1b, p-NF-?B and NF-?B. (A) Representative Western blots showing specific bands for I?Ba, TNF-a,
IL-1b and b-actin 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-D) Graphical representation of data from Western blots analyses. The mean density
values of I?Ba, TNF-a and IL-1b are expressed as ratios relative to that of b-actin. Values are means ± SEM. **p < 0.05 vs C;##p < 0.05 vs D for I?Ba,
**p < 0.01 vs C;##p < 0.01 vs D for TNF-a and IL-1b. (E) Representative Western blots showing specific bands for phosphorylated NF-?B, NF-?B as
an internal control and lamin A for equal loading of nuclear sample. (F) Densitometric data of protein analysis. The mean density values of p-NF-?B
are expressed as ratios relative to that of NF-?B. C, age-matched normal rats; D, diabetic-treated rats administered with vehicle; Cur, diabetic rats
treated with curcumin 100 mg/kg/day. ** p < 0.01 vs. C;##p < 0.05 vs. D based on one way ANOVA followed by Tukey’s test.
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ameliorated these increases by 1.7 and 2.5 fold, respec-
tively (Figure 4A,B).
Effect of curcumin on renal ICAM-1 and TGF-b1protein
expression
Western blot analysis showed that the renal ICAM-1 pro-
tein expression was increased by 2.2 fold in the diabetic
rats compared with that in the control rats (p < 0.05) and
treatment with curcumin significantly decreased ICAM-1
protein levels in the diabetic kidney (p < 0.05) (Figure 5A,
B). This finding was correlated with immunofluorescence
staining, in which, ICAM-1 expression in renal tissues
was significantly enhanced in diabetic rats compared with
non-diabetic rats (Figure 2H), where as curcumin sup-
pressed the increased intensity of ICAM-1 expression in
the diabetic rats (Figure 2I). Western blot analysis also
demonstrated a significant increase of renal TGF-b1
expression in the diabetic rats when compared with that
in the control rats, which was significantly attenuated by
treatment with curcumin (Figure 5A,B).
Effect of curcumin on renal expression of MCP-1
MCP-1 is a chemokine that play an important role in
the progression of DN. Using Western blot analysis we
found that renal MCP-1 protein expression was
increased by 1.5 fold in diabetic rats compared with that
in the control rats (p < 0.05). This increase in renal
MCP-1 protein expression was markedly suppressed by
curcumin treatment in the diabetic rats (p < 0.05) (Fig-
ure 5A,D).
Discussion
Macrophage accumulation and activation in the kidney
have been shown to correlate with the onset of DN [4].
Using accumulation of ED-1 as a marker of macrophage
activation, we have demonstrated increased activation of
macrophage in the glomeruli of kidney tissue from dia-
betic animals. In this study, we demonstrate for the first
time that macrophage infiltration in diabetic glomeruli is
ameliorated by the administration of curcumin. In addi-
tion, the results of this study suggest that the anti-inflam-
matory effect of curcumin in DN is partly mediated by
inhibiting ICAM-1 and MCP-1 expression under diabetic
conditions. We also found that curcumin significantly
suppressed NF-?B activity, a major transcriptional factor
of many proinflammatory genes as well as reduced the
degradation of cytosolic I?Ba: as a consequence, the level
of proinflammatory cytokines, TNF-a and IL-1b were
further significantly decreased.
Curcumin is a highly pleiotropic molecule capable of
interacting with numerous molecular targets involved in
inflammation. Curcumin has been reported to modulate
the inflammatory response by inhibiting the production of
the inflammatory cytokines IL-8, monocyte inflammatory
protein (MIP)-1a, MCP-1, IL-1b and TNF-a by monocyte
and macrophage [25]. The beneficial effects of curcumin
have also been demonstrated in several experimental kid-
ney disease models [20,28-32]. Ghosh et al. [28] showed
that curcumin treatment improved kidney function in ani-
mals with chronic renal failure by antagonizing the effect
of TNF-a elicited NF-?B activation and macrophage infil-
tration, indicating that the anti-inflammatory property of
curcumin may be responsible for alleviating chronic renal
failure in nephrectomized animals. A recent study also
demonstrated that curcumin treatment significantly
reduced obesity induced inflammatory response and
macrophage infiltration of white adipose tissue in murine
models of insulin-resistant obesity and decreased hepatic
NF-?B activity, an effect associated with decreased hepatic
expression of inflammatory molecules [33,34]. Chiu et al.
reported that curcumin prevents diabetes-associated
A
**
##
B
**
##
Figure 4 Effect of curcumin on renal messenger RNA
expression levels of TNF-a (A) and IL-1b (B) in rats with DN
were determined by quantitative RT-PCR. The expression level of
each sample is expressed relative to the expression level of GAPDH
gene. C, age-matched normal rats; D, diabetic-treated rats
administered with vehicle; Cur, diabetic rats treated with curcumin
100 mg/kg/day. ** p < 0.01 vs. C;##p < 0.01 vs. D based on one
way ANOVA followed by Tukey’s test.
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abnormalities in the kidneys through the inhibition of NF-
?B activation and p300 [20]. Curcumin is also known as
an established inhibitor of NF-?B activation [35] and has
recently been shown to specifically target inhibitory kappa
B kinase (I?B) [19]. However, it is still unknown whether
anti-inflammatory mechanism of curcumin through the
inhibition of macrophage infiltration, might offer the renal
protective effect in type I.
In recent years, several clinical and animal studies have
indicated that inflammatory cytokines play an important
roles in the development and progression of DN [36,37].
Schmid et al. have reported that upregulation of NF-?B
targets, a master transcriptional gene play a role in
inflammatory response in the kidney of the patients with
progressive DN [38]. NF-?B is present in the cytoplasma
complexed to its inhibitory protein known as I?B. After
activation by a number of physiological and nonphysiolo-
gical stimuli, such as IL-1b, TNF-a, or lipopolysacchar-
ide, I?B dissociates from NF-?B within minutes and
undergoes ubiquitination and degradation. Once NF-?B
is released from the inhibitory unit I?B, the NF-?B is
then translocated into the nucleus. Upon its nuclear
translocation, NF-?B undergoes phosphorylation on ser-
ine 276 in its p65 subunit and associates with surround-
ing chromatin components. It subsequently binds with
DNA and promotes the transcription of proinflammatory
cytokines, such as TNF-a and IL-1b. Previous study has
reported that phosphorylation on serine 276 is essential
for NF-?B p65-dependent cellular responses [39]. There-
fore, measurement of the phosphorylated p65 subunit of
NF-?B is an effective tool for determining NF-?B activa-
tion [12,40]. Our results showed that activation of NF-?B
and degradation of I?Ba, as well as proinflammatory
cytokines expression, TNF-a and IL-1b were increased in
diabetic rats compared with the levels in control rats
(Figures. 3 and 4). Curcumin treatment prevented all of
these alterations. We believe that the curcumin’s ability
to inactivate NF-?B [41] and thus inhibited the produc-
tion of proinflammatory cytokines [29] is its most likely
mechanisms of action.
Macrophages are known to cause early glomerular
injury in STZ-induced diabetes and also to be involved
in the mechanisms that cause progressive glomerular
and tubular damage [5,10,42,43]; in addition, ICAM-1
**
##
B
C
##
**
**
##
D
A
ICAM-1
?-actin
TGF-?1
C
D
Cur
MCP-1
Figure 5 Renal expressions of ICAM-1, MCP-1 and TGF-b1. (A) Representative Western blots showing specific bands for ICAM-1, MCP-1, TGF-
b1, and b-actin 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-D) Graphical representation of data from Western blots analyses. The mean
density values of ICAM-1, MCP-1 and TGF-b1are expressed as ratios relative to that of b-actin. Each bar represents mean ± SEM. C, age-matched
normal rats; D, diabetic-treated rats administered with vehicle; Cur, diabetic rats treated with curcumin 100 mg/kg/day. ** p < 0.05 vs. C;##p <
0.05 vs. D based on one way ANOVA followed by Tukey’s test.
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and MCP-1 are considered as a central molecule
involved in macrophage influx in DN [9,11,44-46]. Park
et al. have demonstrated that high glucose could upre-
gulate ICAM-1 protein and mRNA expression in rat
mesangial cells through the protein kinase C-NF-?B
pathways [11,14]. ICAM-1 is also induced by inflamma-
tory cytokines such as TNF-a, IL-1, and interferon-g
[47]. Recent studies provided evidences that both
ICAM-1 gene deficiency [10,45] and anti-ICAM-1
monoclonal antibody [11] obviously inhibited the infil-
tration of monocytes/macrophages into the glomerulus
and alleviated the extent of renal injury. Studies have
also demonstrated that NF-?B was involved in the
induction of MCP-1 in mesangial cell cultured under
high glucose condition and subsequently mediated
macrophage accumulation [9,48,49]. Consistent with
these previous reports, we have also observed that
ICAM-1 and MCP-1 expression were increased in rats
with experimental DN (Figure 5), which was associated
with marked macrophages infiltration (Figure 2), and
that these increases under diabetic conditions were ame-
liorated by curcumin treatment. This effect might be
mediated by curcumin’s inhibition of NF-?B activation.
In the present study, diabetic glomerulosclerosis was
ameliorated along with the inhibition of macrophage
infiltration (Figure 2). Accumulating evidence suggests
that, in DN, glomerulosclerosis is associated with TGF-b1
expression [50,51], which is related to macrophage infil-
tration in glomeruli [52]. TGF-b1is assumed to mediate
inflammatory response and exaggerate the progression of
DN [53]. In vitro and in vivo studies have reported that
macrophages stimulates mesangial cells to produce ECM
proteins through TGF-b [44,54,55]. TGF-b can in turn
induce ECM overproduction from mesangial cells in
autocrine and paracrine fashions [45]. Previous study has
revealed that infiltrated monocytes/macrophages release
lysosomal enzymes, nitrous oxide, reactive oxygen inter-
mediates and TGF-b, which have been reported to play
an essential role in renal damage and the depletion of
macrophage by irradiation decreased the gene expression
of TGF-b and type IV collagen in the glomeruli of dia-
betic rats at 4 weeks after induction of diabetes, suggest-
ing the pathological role of macrophages in the increased
expression of ECM proteins [56,57]. From the above
results it is obvious that macrophage infiltration is an
important inducer of TGF-b1. The TGF-b1contains a
sequence located -715 to -707 bp where NF-?B binds to
target TGF-b1gene expression [58]. Thus, following NF-
?B activation, marked infiltration of macrophages subse-
quently upregulated the TGF-b1expression which further
promotes the increased ECM synthesis in diabetes. Our
results show that renal TGF-b1protein expression was
significantly increased in diabetic rats. Curcumin treat-
ment obviously decreased renal TGF-b1expression and
this improvement was achieved most probably via the
inhibition of NF-?B activation. Taken together, all the
above results suggest that beneficial effect of curcumin in
rats with DN is at least in part through inhibition of
macrophage infiltration via inhibiting NF-?B mediated
inflammatory response.
Conclusion
We have shown that the curcumin treatment amelio-
rated DN in rat model of type I diabetes through the
inhibition of macrophage infiltration in glomerulus due
to its anti-inflammatory effect. In addition, our results
support the findings that curcumin has a property to
inhibit the activity of NF-?B as well as the degradation
of I?Ba and as a result decreased the expression of
proinflammatory and profibrotic cytokines. Given these
promising preclinical findings, we believe that the curcu-
min might be considered as potential adjuvant entity for
preventing DN.
Acknowledgements
This research was supported by a Yujin Memorial Grant, Ministry of
Education, Culture, Sports and Technology of Japan and by a grant from the
Promotion and Mutual Aid Corporation for Private Schools, Japan. We thank
Wawaimuli Arozal, Sayaka Mito, Yoshiyasu Kobayashi and Somasundaram
Arumugam for their assistance in this research work.
Author details
1Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences,
Niigata University of Pharmacy and Applied Life Sciences, Niigata City, Japan.
2Department of Pharmacology, Faculty of Medicine, University of Indonesia,
Jakarta, Indonesia.3Department of Gastroenterology and Hepatology, Niigata
University Graduate School of Medical and Dental Sciences, Niigata, Japan.
4Department of Cell Biology, Institute of Nephrology, Niigata University
Graduate School of Medical and Dental Sciences, Niigata, Japan.
Authors’ contributions
VS and KW made substantial contributions to the conception and design of
the study, analysis and interpretations of data, as well as gave final approval
for the version to be published. FRS, PTV, and RAT participated in revising
the manuscript. MH, APL, and HK, helped in the interpretation of data. VKS
and KS participated in carrying out the RT-PCR. All authors contributed to
the drafting of the manuscript and agreed on the final version of the
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 18 March 2011 Accepted: 10 June 2011
Published: 10 June 2011
References
1.Svensson M, Sundkvist G, Arnqvist HJ, Björk E, Blohmé G, Bolinder J,
Henricsson M, Nyström L, Torffvit O, Waernbaum I, Ostman J, Eriksson JW:
Diabetes Incidence Study in Sweden (DISS). Signs of nephropathy may
occur early in young adults with diabetes despite modern diabetes
management: results from the nationwide population-based Diabetes
Incidence Study in Sweden (DISS). Diabetes Care 2003, 26:2903-2909.
2.Tuttle KR: Linking metabolism and immunology: diabetic nephropathy is
an inflammatory disease. J Am Soc Nephrol 2005, 16:1537-1538.
3. Furuta T, Saito T, Ootaka T, Soma J, Obara K, Abe K, Yoshinaga K: The role
of macrophages in diabetic glomerulosclerosis. Am J Kidney Dis 1993,
21:480-485.
Soetikno et al. Nutrition & Metabolism 2011, 8:35
http://www.nutritionandmetabolism.com/content/8/1/35
Page 9 of 11

Page 10

4.Chow F, Ozols E, Nikolic-Paterson DJ, Atkins RC, Tesch GH: Macrophages in
mouse type 2 diabetic nephropathy: correlation with diabetic state and
progressive renal injury. Kidney Int 2004, 65:116-128.
Sassy-Prigent C, Heudes D, Mandet C, Bélair MF, Michel O, Perdereau B,
Bariéty J, Bruneval P: Early glomerular macrophage recruitment in
streptozotocin-induced diabetic rats. Diabetes 2000, 49:466-475.
Amann B, Tinzmann R, Angelkort B: ACE inhibitors improve diabetic
nephropathy through suppression of renal MCP-1. Diabetes Care 2003,
26:2421-2425.
Wada T, Furuichi K, Sakai N, Iwata Y, Yoshimoto K, Shimizu M, Takeda SI,
Takasawa K, Yoshimura M, Kida H, Kobayashi KI, Mukaida N, Naito T,
Matsushima K, Yokoyama H: Up-regulation of monocyte chemoattractant
protein-1 in tubulointerstitial lesions of human diabetic nephropathy.
Kidney Int 2000, 58:1492-1499.
Banba N, Nakamura T, Matsumura M, Kuroda H, Hattori Y, Kasai K: Possible
relationship of monocyte chemoattractant protein-1 with diabetic
nephropathy. Kidney Int 2000, 58:684-690.
Chow FY, Nikolic-Paterson DJ, Ozols E, Atkins RC, Rollin BJ, Tesch GH:
Monocyte chemoattractant protein-1 promotes the development of
diabetic renal injury in streptozotocin-treated mice. Kidney Int 2006,
69:73-80.
Chow FY, Nikolic-Paterson DJ, Atkins RC, Tesch GH: Macrophages in
streptozotocin-induced diabetic nephropathy: potential role in renal
fibrosis. Nephrol Dial Transplant 2004, 19:2987-2996.
Sugimoto H, Shikata K, Hirata K, Akiyama K, Matsuda M, Kushiro M,
Shikata Y, Miyatake N, Miyasaka M, Makino H: Increased expression of
intercellular adhesion molecule-1 (ICAM-1) in diabetic rat glomeruli:
glomerular hyperfiltration is a potential mechanism of ICAM-1
upregulation. Diabetes 1997, 46:2075-2081.
Guijarro C, Egido J: Transcription factor-kappa B (NF-kappa B) and renal
disease. Kidney Int 2001, 59:415-424.
Park CW, Kim JH, Lee JH, Kim YS, Ahn HJ, Shin YS, Kim SY, Choi EJ,
Chang YS, Bang BK: High glucose-induced intercellular adhesion
molecule-1 (ICAM-1) expression through an osmotic effect in rat
mesangial cells is PKC-NF-kappaB-dependent. Diabetologia 2000,
43:1544-1553.
Jiang Q, Liu P, Wu X, Liu W, Shen X, Lan T, Xu S, Peng J, Xie X, Huang H:
Berberine attenuates lipopolysaccharide-induced extracelluar matrix
accumulation and inflammation in rat mesangial cells: involvement of
NF-κB signaling pathway. Mol Cell Endocrinol 2011, 331:34-40.
Mezzano S, Aros C, Droguett A, Burgos ME, Ardiles L, Flores C, Schneider H,
Ruiz-Ortega M, Egido J: NF-kappaB activation and overexpression of
regulated genes in human diabetic nephropathy. Nephrol Dial Transplant
2004, 19:2505-2512.
Riad A, Du J, Stiehl S, Westermann D, Mohr Z, Sobirey M, Doehner W,
Adams V, Pauschinger M, Schultheiss HP, Tschöpe C: Low-dose treatment
with atorvastatin leads to anti-oxidative and anti-inflammatory effects in
diabetes mellitus. Eur J Pharmacol 2007, 569:204-211.
Hatcher H, Planalp R, Cho J, Torti FM, Torti SV: Curcumin: from ancient
medicine to current clinical trials. Cell Mol Life Sci 2008, 65:1631-1652.
Shishodia S, Sethi G, Aggarwal BB: Curcumin: getting back to the roots.
Ann N Y Acad Sci 2005, 1056:206-217.
Aggarwal S, Ichikawa H, Takada Y, Sandur SK, Shishodia S, Aggarwal BB:
Curcumin (diferuloylmethane) down-regulates expression of cell
proliferation and antiapoptotic and metastatic gene products through
suppression of IkappaBalpha kinase and Akt activation. Mol Pharmacol
2006, 69:195-206.
Chiu J, Khan ZA, Farhangkhoee H, Chakrabarti S: Curcumin prevents
diabetes-associated abnormalities in the kidneys by inhibiting p300 and
nuclear factor-kappaB. Nutrition 2009, 25:964-972.
Kuwabara N, Tamada S, Iwai T, Teramoto K, Kaneda N, Yukimura T,
Nakatani T, Miura K: Attenuation of renal fibrosis by curcumin in rat
obstructive nephropathy. Urology 2006, 67:440-446.
Watanabe K, Ohta Y, Nakazawa M, Higuchi H, Hasegawa G, Naito M, Fuse K,
Ito M, Hirono S, Tanabe N, Hanawa H, Kato K, Kodama M, Aizawa Y: Low
dose carvedilol inhibits progression of heart failure in rats with dilated
cardiomyopathy. Br J Pharmacol 2000, 130:1489-1495.
Jain SK, Rains J, Croad J, Larson B, Jones K: Curcumin supplementation
lowers TNF-alpha, IL-6, IL-8, and MCP-1 secretion in high glucose-treated
cultured monocytes and blood levels of TNF-alpha, IL-6, MCP-1, glucose,
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
and glycosylated hemoglobin in diabetic rats. Antioxid Redox Signal 2009,
11:241-249.
Bagheri F, Gol A, Dabiri S, Javadi A: Preventive effect of garlic juice on
renal reperfusion injury. Iran J Kidney Dis 2011, 5:194-200.
Thallas-Bonke V, Thorpe SR, Coughlan MT, Fukami K, Yap FY, Sourris KC,
Penfold SA, Bach LA, Cooper ME, Forbes JM: Inhibition of NADPH oxidase
prevents advanced glycation end product-mediated damage in diabetic
nephropathy through a protein kinase C-alpha-dependent pathway.
Diabetes 2008, 57:460-469.
Ohga S, Shikata K, Yozai K, Okada S, Ogawa D, Usui H, Wada J, Shikata Y,
Makino H: Thiazolidinedione ameliorates renal injury in experimental
diabetic rats through anti-inflammatory effects mediated by inhibition
of NF-kappaB activation. Am J Physiol Renal Physiol 2007, 292:F1141-F1150.
Thandavarayan RA, Watanabe K, Ma M, Veeraveedu PT, Gurusamy N,
Palaniyandi SS, Zhang S, Muslin AJ, Kodama M, Aizawa Y: 14-3-3 protein
regulates Ask1 signaling and protects against diabetic cardiomyopathy.
Biochem Pharmacol 2008, 75:1797-1806.
Ghosh SS, Massey HD, Krieg R, Fazelbhoy ZA, Ghosh S, Sica DA, Fakhry I,
Gehr TW: Curcumin ameliorates renal failure in 5/6 nephrectomized rats:
role of inflammation. Am J Physiol Renal Physiol 2009, 296:F1146-F1157.
Abe Y, Hashimoto S, Horie T: Curcumin inhibition of inflammatory
cytokine production by human peripheral blood monocytes and
alveolar macrophages. Pharmacol Res 1999, 39:41-47.
Kuhad A, Pilkhwal S, Sharma S, Tirkey N, Chopra K: Effect of curcumin on
inflammation and oxidative stress in cisplatin-induced experimental
nephrotoxicity. J Agric Food Chem 2007, 55:10150-10155.
Ghosh SS, Salloum FN, Abbate A, Krieg R, Sica DA, Gehr TW, Kukreja RC:
Curcumin prevents cardiac remodeling secondary to chronic renal
failure through deactivation of hypertrophic signaling in rats. Am J
Physiol Heart Circ Physiol 2010, 299:H975-H984.
Tirkey N, Kaur G, Vij G, Chopra K: Curcumin, a diferuloylmethane,
attenuates cyclosporine-induced renal dysfunction and oxidative stress
in rat kidneys. BMC Pharmacol 2005, 5:15.
Weisberg SP, Leibel R, Tortoriello DV: Dietary curcumin significantly
improves obesity-associated inflammation and diabetes in mouse
models of diabesity. Endocrinology 2008, 149:3549-3558.
Woo HM, Kang JH, Kawada T, Yoo H, Sung MK, Yu R: Active spice-derived
components can inhibit inflammatory responses of adipose tissue in
obesity by suppressing inflammatory actions of macrophages and
release of monocyte chemoattractant protein-1 from adipocytes. Life Sci
2007, 13:926-931.
Singh S, Khar A: Activation of NFkappaB and Ub-proteasome pathway
during apoptosis induced by a serum factor is mediated through the
upregulation of the 26S proteasome subunits. Apoptosis 2006, 11:845-859.
Navarro JF, Mora C: Role of inflammation in diabetic complications.
Nephrol Dial Transplant 2005, 20:2601-2604.
Wolkow PP, Niewczas MA, Perkins B, Ficociello LH, Lipinski B, Warram JH,
Krolewski AS: Association of urinary inflammatory markers and renal
decline in microalbuminuric type 1 diabetics. J Am Soc Nephrol 2008,
19:789-797.
Schmid H, Boucherot A, Yasuda Y, Henger A, Brunner B, Eichinger F,
Nitsche A, Kiss E, Bleich M, Gröne HJ, Nelson PJ, Schlöndorff D, Cohen CD,
Kretzler M: Modular activation of nuclear factor-kappaB transcriptional
programs in human diabetic nephropathy. Diabetes 2006, 55:2993-3003.
Okazaki T, Sakon S, Sasazuki T, Sakurai H, Doi T, Yagita H, Okumura K,
Nakano H: Phosphorylation of serine 276 is essential for p65 NF-kappaB
subunit-dependent cellular responses. Biochem Biophys Res Commun 2003,
300:807-812.
Jain SK, Velusamy T, Croad JL, Rains JL, Bull R: L-cysteine supplementation
lowers blood glucose, glycated hemoglobin, CRP, MCP-1, and oxidative
stress and inhibits NF-kappaB activation in the livers of Zucker diabetic
rats. Free Radic Biol Med 2009, 46:1633-1638.
Singh S, Aggarwal BB: Activation of transcription factor NF-kappa B is
suppressed by curcumin (diferuloylmethane). J Biol Chem 1995,
270:24995-5000.
Ikezumi Y, Hurst LA, Masaki T, Atkins RC, Nikolic-Paterson DJ: Adoptive
transfer studies demonstrate that macrophages can induce proteinuria
and mesangial cell proliferation. Kidney Int 2003, 63:83-95.
Nikolic-Paterson DJ, Atkins RC: The role of macrophages in
glomerulonephritis. Nephrol Dial Transplant 2001, 16:3-7.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
Soetikno et al. Nutrition & Metabolism 2011, 8:35
http://www.nutritionandmetabolism.com/content/8/1/35
Page 10 of 11

Page 11

44.Chow FY, Nikolic-Paterson DJ, Ozols E, Atkins RC, Tesch GH: Intercellular
adhesion molecule-1 deficiency is protective against nephropathy in
type 2 diabetic db/db mice. J Am Soc Nephrol 2005, 16:1711-1722.
Okada S, Shikata K, Matsuda M, Ogawa D, Usui H, Kido Y, Nagase R, Wada J,
Shikata Y, Makino H: Intercellular adhesion molecule-1-deficient mice are
resistant against renal injury after induction of diabetes. Diabetes 2003,
52:2586-2593.
Chow FY, Nikolic-Paterson DJ, Ma FY, Ozols E, Rollins BJ, Tesch GH:
Monocyte chemoattractant protein-1-induced tissue inflammation is
critical for the development of renal injury but not type 2 diabetes in
obese db/db mice. Diabetologia 2007, 50:471-480.
Wertheimer SJ, Myers CL, Wallace RW, Parks TP: Intercellular adhesion
molecule-1 gene expression in human endothelial cells. Differential
regulation by tumor necrosis factor-alpha and phorbol myristate
acetate. J Biol Chem 1992, 267:12030-12035.
Marumo T, Schini-Kerth VB, Busse R: Vascular endothelial growth factor
activates nuclear factor-kappaB and induces monocyte chemoattractant
protein-1 in bovine retinal endothelial cells. Diabetes 1999, 48:1131-1137.
Ha H, Yu MR, Choi YJ, Kitamura M, Lee HB: Role of high glucose-induced
nuclear factor-kappaB activation in monocyte chemoattractant protein-1
expression by mesangial cells. J Am Soc Nephrol 2002, 13:894-902.
Chen S, Jim B, Ziyadeh FN: Diabetic nephropathy and transforming
growth factor-beta: transforming our view of glomerulosclerosis and
fibrosis build-up. Semin Nephrol 2003, 23:532-543.
Ziyadeh FN, Hoffman BB, Han DC, Iglesias-De La Cruz MC, Hong SW,
Isono M, Chen S, McGowan TA, Sharma K: Long-term prevention of renal
insufficiency, excess matrix gene expression, and glomerular mesangial
matrix expansion by treatment with monoclonal antitransforming
growth factor-beta antibody in db/db diabetic mice. Proc Natl Acad Sci
USA 2000, 97:8015-8020.
Cohen MP, Shea E, Chen S, Shearman CW: Glycated albumin increases
oxidative stress, activates NF-kappa B and extracellular signal-regulated
kinase (ERK), and stimulates ERK-dependent transforming growth factor-
beta 1 production in macrophage RAW cells. J Lab Clin Med 2003,
141:242-249.
Murphy M, Crean J, Brazil DP, Sadlier D, Martin F, Godson C: Regulation
and consequences of differential gene expression in diabetic kidney
disease. Biochem Soc Trans 2008, 36:941-945.
Pawluczyk IZ, Harris KP: Macrophages promote prosclerotic responses in
cultured rat mesangial cells: a mechanism for the initiation of
glomerulosclerosis. J Am Soc Nephrol 1997, 8:1525-1536.
Leonarduzzi G, Scavazza A, Biasi F, Chiarpotto E, Camandola S, Vogel S,
Dargel R, Poli G: The lipid peroxidation end product 4-hydroxy-2,3-
nonenal up-regulates transforming growth factor beta1 expression in
the macrophage lineage: a link between oxidative injury and
fibrosclerosis. FASEB J 1997, 11:851-857.
Young BA, Johnson RJ, Alpers CE, Eng E, Gordon K, Floege J, Couser WG,
Seidel K: Cellular events in the evolution of experimental diabetic
nephropathy. Kidney Int 1995, 47:935-944.
Sassy-Prigent C, Heudes D, Mandet C, Bélair MF, Michel O, Perdereau B,
Bariéty J, Bruneval P: Early glomerular macrophage recruitment in
streptozotocin-induced diabetic rats. Diabetes 2000, 49:466-475.
Lan Y, Zhou Q, Wu ZL: NF-kappa B involved in transcription
enhancement of TGF-beta 1 induced by Ox-LDL in rat mesangial cells.
Chin Med J (Engl) 2004, 117:225-230.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
doi:10.1186/1743-7075-8-35
Cite this article as: Soetikno et al.: Curcumin ameliorates macrophage
infiltration by inhibiting NF-?B activation and proinflammatory
cytokines in streptozotocin induced-diabetic nephropathy. Nutrition &
Metabolism 2011 8:35.
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