Life Sciences 91 (2012) 959–967

Contents lists available at SciVerse ScienceDirect

Life Sciences
journal homepage: www.elsevier.com/locate/lifescie

Preventive effects of rutin on the development of experimental diabetic nephropathy

in rats
Hui-hui Hao a, Zhu-min Shao b, Dao-quan Tang a, c,⁎, Qian Lu a, Xu Chen a, c, Xiao-xing Yin a,⁎⁎,
Jing Wu a, c, Hui Chen a, c
a
Key Laboratory of New Drug and Clinical Application, Xuzhou Medical College, Xuzhou 221004, China
b
Department of Pharmacy, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China
c
Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical College, Xuzhou 221004, China

a r t i c l e i n f o a b s t r a c t

Article history: Aims: Diabetic nephropathy (DN) is an important microvascular complication and one of the main causes of
Received 29 March 2012 end-stage renal disease. In this study, the preventive effect and mechanism of rutin on the development of
Accepted 12 September 2012 DN in streptozotocin (STZ)-induced diabetic rats were investigated.
Main methods: After an early DN model was induced by STZ, rats were orally administered rutin at 3 doses for
Keywords: 10 weeks. Fasting blood glucose, creatinine (Cr), blood urea nitrogen (BUN), urine protein, kidney index,
Rutin
antioxidase, advanced glycosylation end products (AGEs), extracellular matrix (ECM) including collagen IV
Diabetic nephropathy
CTGF
and laminin, connective tissue growth factor (CTGF), phosphorylated Smad 2/3 (p-Smad 2/3) and Smad 7
Smads (p-Smad 7), and transforming growth factor-β1 (TGF-β1) were determined by different methods, respectively.
TGF-β1 The ultrastructural morphology was observed by a transmission electron microscope.
Oxidative stress Key findings: Compared with the DN group, rutin decreased the levels of fasting blood glucose, Cr, BUN, urine
protein, the intensity of oxidative stress and p-Smad 7 significantly. The expression of AGEs, collagen IV and
laminin, TGF-β1, p-Smad 2/3 and CTGF was inhibited by rutin significantly. Moreover, rutin was observed to
inhibit proliferation of mesangial cells and decrease thickness of glomerular basement membrane (GBM) by
electron microscopy.
Significance: The preventive effect of rutin on the development of DN is closely related to oxidative stress and
the TGF-β1/Smad/ECM and TGF-β1/CTGF/ECM signaling pathways. Those results suggest that rutin can prevent
the development of experimental DN in rats.
© 2012 Elsevier Inc. All rights reserved.

Introduction
The early stage of diabetic nephropathy (DN) is characterized by
renal hypertrophy, glomerular hypertrophy, glomerular hyperfiltration,
and microalbuminuria (Wolf and Ziyadeh, 1999; Makino et al., 1996;
Raptis and Viberti, 2001). These changes are related to the subsequent
development of glomerular morphological abnormalities and the prog-
nosis of DN.
One of the characteristic pathological changes in DN is the accumu-
lation of extracellular matrix (ECM) components including collagens,
fibronectin and laminin in the glomeruli and the interstitium of kidney
(Kanwar et al., 2008). Hyperglycemia is usually considered as the main
determinant factor of the initiation and progression of DN which
increases the formation of advanced glycosylation end products
(AGEs), oxidative stress and transforming growth factor-β1 (TGF-β1)
⁎ Correspondence to: D.Q. Tang, Key Laboratory of New Drug and Clinical Application,
Xuzhou Medical College, Xuzhou 221004, China. Tel./fax: +86 516 83262136.
⁎⁎ Corresponding author. Tel./fax: +86 516 83262009.
E-mail addresses: tdq993@126.com (D. Tang), tdq993@hotmail.com (X. Yin).
mRNA expressions. TGF-β1 is the most potent growth factor contribut-
ing to ECM accumulation, which it does by stimulating ECM production
and suppressing its degradation (Border et al., 1996). Smad proteins are
transcription factors that mediate TGF-β/activin signaling by forming
complexes with each other. Smads 2 and 3 are specific for TGF-β signal-
ing, and they are phosphorylated by TGF-β receptor type 1 after binding
with TGF-β (Wang et al., 2005; Gagliardini and Benigni, 2006). Either
Smad 2 or 3 forms a heterodimer with Smad 4, and the complex imme-
diately translocates into the nucleus and induces transcription of vari-
ous genes by combining specific binding sites of genomic DNA. Smad
7 can be an inhibitory Smad protein that blocks the phosphorylation
of Smad 2/3, counterbalancing the TGF-β signaling (Stopa et al.,
2000). Connective tissue growth factor (CTGF) has been described as a
growth factor that acts downstream of TGF-β1 and is a potent inducer
of ECM in the fibrotic process (Hishikawa et al., 2001). TGF-β1/Smad/
ECM or TGF-β1/CTGF/ECM seems to be a linked response in the patho-
genesis of DN.
Rutin is found in many foods and many traditional Chinese medi-
cines, whose treatment on diabetic mellitus has been proved by the
Stanely Mainzen Prince group. Their innovative achievements have

0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.lfs.2012.09.003
960 H. Hao et al. / Life Sciences 91 (2012) 959–967
published that rutin can improve glucose homeostasis of diabetic rats
by decreasing fasting plasma glucose and increasing insulin levels,
increasing the content of glycogen in the liver and muscle whereas
decreasing that in the kidney, increasing the activity of hexokinase
and decreasing the activities of glucose-6-phosphatase and fructose-
1,6-bisphosphatase in the tissues (Stanley Mainzen Prince and
Kamalakkannan, 2006). This research group also proved that rutin can
decrease the levels of lipids, low density and very low density lipopro-
tein cholesterol in plasma, increase the levels of plasma high density
lipoprotein cholesterol and the activity of plasma lipoprotein lipase
and lecithin cholesterol acyltransferase and decrease the levels of lipids
and glycoproteins and the activity of 3-hydroxy 3-methylglutaryl
coenzyme A reductase in the liver and in the kidney in diabetic rats
basing on its antioxidant property (Stanely Mainzen Prince and
Kannan, 2006). Moreover, Stanely Mainzen Prince group also reported
that rutin can protect the diabetic rats kidney by decreasing the content
of hydroxyproline and collagen and the levels of tissue inhibitors of
metalloproteinases, increasing the activity of matrix metalloproteinases
in the kidney (Kamalakkannan and Stanely Mainzen Prince, 2006).
Other scholars also proved that rutin can inhibit the formation of
AGEs (Cervantes-Laurean et al., 2006) in STZ-induced rats. All of these
offer us a potential pharmacological foundation of rutin for DN therapy.
We previously reported that rutin can decrease the accumulation
of ECM mediated by TGF-β1/Smads in high glucose cultured-rat
mesangial cells (Tang et al., 2011). Although Kamalakkannan and
Prince have reported that rutin can decrease fasting plasma glucose,
glycosylated hemoglobin thiobarbituric acid reactive substances and
lipid hydroperoxides, increase insulin, C-peptide, hemoglobin, pro-
tein levels and non-enzymic antioxidants in diabetic rats induced by
streptozotocin (STZ), their work haven't focused on the creatinine
(Cr), blood urea nitrogen (BUN), and urine protein in DN rats
(Kamalakkannan and Prince, 2006). Moreover, the protective effects
and mechanism of rutin on early experiment DN in vivo have not
been studied by Stanley Mainzen Prince group and other scholars to
date, which may restrict the clinical application of rutin. Thus, the
present study was designed to investigate the effect of rutin on the
kidney of STZ-induced type 1 diabetic rats by observing the changes
of renal morphology and some signaling including TGF-β1, phosphor-
ylated Smad 2/3 (p-Smad 2/3), phosphorylated Smad 7 (p-Smad 7),
CTGF, collagen IV, and laminin. The blood glucose, Cr, BUN, urine pro-
tein, kidney index, antioxidases, and AGEs in DN rats were simulta-
neously observed. Captopril (CAP) was used as a positive control drug
in this study.
Materials and methods
Animals
Adult male Sprague–Dawley rats (Certificate No. SYXK 2010-0011),
weighing 180 g–220 g, procured from the Laboratory Animal Center of
Xuzhou Medical College were used in this study. The rats were
maintained in a controlled environment (12 h light/dark cycle) and
temperature (21 ±2 °C). All animals were fed on a standard pellet
diet and water was freely available. The animals were acclimatized to
the laboratory conditions before starting the experiment. All the exper-
iments were approved by Institutional Animal Ethics Committee and
followed the Guiding Principles for Care and Use of Laboratory Animals
of Xuzhou Medical College.
Chemicals
Rutin and STZ were purchased from Sigma Chemicals Company
(St. Louis, MO, USA). Captopril (CAP, Lot No. 050050), serving as a
positive control drug, was kindly provided by Changzhou Pharmaceuti-
cal Factory (Changzhou, China).
Induction and assessment of diabetes
STZ was freshly dissolved in citrate buffer (0.01 mol/L, pH 4.4) and
maintained on ice before use. The overnight fasted rats were made
diabetic with a single intraperitoneal injection of STZ (60 mg/kg).
Age-matched control normal standard rats (NS group, n = 10) received
an equivalent amount of citrate buffer. Diabetes was confirmed in the
STZ-injected rats by measuring the fasting plasma glucose levels 72 h
post injection. After an overnight fast, rats with blood glucose levels
above 13.89 mmol/L (250 mg/dL) were considered as diabetic and
were used in this study. Treatment with rutin was started on the third
day after STZ-injection.
Experimental design
Diabetic rats were randomly allotted into 5 groups as follows: diabet-
ic rats were treated with 1% sodium carboxymethyl cellulose (CMC) so-
lution (DN group, n =8); diabetic rats with 10, 30, and 90 mg/kg rutin
for the RL group (low dose, n=8), the RM group (moderate dose, n=
9), and the RH group (high dose, n =9), respectively; the diabetic rats
were treated with 10 mg/kg of CAP (CAP group, n=9). The low dose
of rutin was calculated according to its content in the moderate effective
dose of Ginkgo biloba extract for therapy of DN published by our labora-
tory (Ginkgo biloba extract employed in that experiment (Lu et al., 2007)
contains 11.4% rutin (Tang et al., 2010)). Drugs were suspended in 1%
CMC solution and orally administered to rats using an intragastric tube
for a period of 10 weeks. The same volume of CMC solution was admin-
istered to the NS group (n= 10).
Normal and diabetic rats were housed 5 per cage in the barrier
environment referred to as breed specific pathogen-free grade animals.
At the end of the experiments, after a 24 h urine sample collection, the
blood samples of the rats were withdrawn from the abdominal aorta.
Serum was collected to assess the renal function by measuring plasma
levels of Cr and urea nitrogen. Kidneys were removed, after being
weighed, parts of fresh kidney cortexes were stored in 10% for-
maldehyde solution for immunohistochemical assays, and 1 mm×
1 mm× 1 mm cubes of renal cortices were fixed in 2.5% glutaraldehyde
for electron microscopic measurement. The rest of the kidneys were
stored at −80 °C for the later analysis.
Measurement of blood glucose and renal function
Blood glucose was measured by blood glucose meter (OneTouch
UltraEasy, USA). The 24 h urinary albumin was measured by ELISA kit
purchased from R&D Systems Company (R&D, USA). BUN and Cr were
determined by the automatic biochemistry analyzer (Olympus2000,
Tokyo, Japan). The kidney index was expressed as 1000 × kidney
weight/body weight.
Assessment of renal oxidative stress and determination of AGEs, collagen
IV and laminin
The kidney tissue was minced and a homogenate was prepared with
10% (w/v) phosphate buffered saline (0.1 mol/L, pH 7.4) using a ho-
mogenizer. The kidney homogenate was centrifuged at 15,000 rpm
for 15 min at 4 °C. The supernatant was collected to determine the con-
tent of protein by bicinchoninic acid (BCA) protein assay kit purchased
from Beyotim Institute of Biotechnology (Nantong, China). The total
antioxidative capability (T-AOC), catalase (CAT), total superoxidase
dismutase (T-SOD) and glutathione-per-oxidase (GSH-Px) in the renal
cortex and the level of malonaldehyde (MDA) were measured by
using the commercially available kit (Nanjing Jiancheng Bioengineering
Institute, Nanjing, China).
AGEs play a critical role in DN by stimulating type IV collagen and
laminin, the main component of ECM. The quantity of type IV collagen,
 H. Hao et al. / Life Sciences 91 (2012) 959–967
laminin and AGEs in the renal cortex was determined by ELISA kit pur-
chased from R&D Systems Company (R&D, USA).
Immunohistochemical measurements of CTGF
Kidney cortexes, fixed in 10% formaldehyde previously, were
embedded in paraffin and 4 μm thick sections were prepared for
immunohistochemistry analysis of CTGF. The renal tissue sections
were incubated with goat polyclonal anti-CTGF (Lot No. B1030,
Zhongshan Golden Bridge Biological Technology, Beijing, China) at a
dilution of 1:100 at 37 °C for 2 h. After washing in PBS, the sections
were incubated with rabbit anti-goat IgG horseradish peroxidase
(Lot K115214D, Beijing, China) for 30 min at room temperature. After
washing, to visualize CTGF, the renal tissue sections were stained with
3,3′-diaminobenzidine (DAB) for 10 min and then washed in tap
water for 10 min. After the nuclei were stained with hematoxylin for
20 s, the sections were evaluated by a light microscope (×400) and
were photographed. Photomicrographs of at least 20 fields in each rat
were quantified for the intensity of positive staining in the renal tissue
with IPLab software (Scanalytics, Fairfax, VA).
Western blot analysis for Smads and TGF-β1 in the renal cortex
For detection of p-Smad 2/3, p-Smad 7 and TGF-β1 in the renal
cortex, gel electrophoreses and subsequent western blotting was
performed. Firstly, renal cortex tissues were homogenized in ice-
cold RIPA buffer containing protease inhibitors. The homogenates
were centrifuged at 15,000 rpm for 15 min at 4 °C and an aliquot of
the supernatant was kept for protein determination. The content of
protein in the supernatant was determined by BCA protein assay kit.
All samples were boiled for 5 min in Laemmli sample buffer before
loading. A 60 μg sample of total protein was separated by 10% sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and
then transferred to nitrocellulose membranes (Pall Corporation,
USA). After being blocked with 5% nonfat dry milk in TBS-Tween
(0.1%) for 2 h at room temperature, the membranes were incubated
separately with the following antibodies: rabbit anti-phospho-Smad
2/3 (Santa Cruz Biotechnology, USA), rabbit anti-total-Smad 2/3
(Bioworld Technology, USA), rabbit anti-phospho-Smad 7 (Santa
Cruz Biotechnology, USA), rabbit anti-total-Smad 7 (Abcam, UK), rabbit
anti-TGF-β1 (Santa Cruz Biotechnology, USA) and rabbit anti-GAPDH
(Beyotim Institute of Biotechnology, China) antibodies. The membranes
were probed with the primary antibodies overnight at 4 °C and then
washed three times in TBS-Tween for 5 min, followed by incubation
with the appropriate secondary horseradish peroxidase conjugate anti-
body for 1 h at room temperature. After extensive washing, the signals
were visualized by the enhanced chemiluminescence system (ECL;
Santa Cruz Biotechnology, USA) and the density of each band was deter-
mined with Image J analysis software version 1.34s (Wayne Rasband
National Institutes of Health, USA).
Table 1
Effects of rutin on blood glucose, urine protein, Cr, BUN and kidney index of rats (mean ± SD).
Group n Fasting blood glucose
(mmol/L)
NS 10 6.16 ± 2.16
DN 8 20.91 ± 2.02#
RL 8 17.83 ± 3.14⁎
RM 9 13.92 ± 2.64⁎⁎
RH 9 12.54 ± 1.75⁎⁎
CAP 9 18.54 ± 4.47
Mean ± SD.
NS: normal standard group. DN: diabetic nephropathy. RL, RM, RH: rutin 10, 30, 90 mg/kg. CAP: captopril 10 mg/kg.
#
P b 0.01 vs NS group.
⁎ P b 0.05 vs DN group.
⁎⁎ P b 0.01 vs DN group.
961
RT-PCR for the determination of relative quantities of TGF-β1 mRNA
A reverse transcription polymerase chain reaction (RT-PCR) proce-
dure was performed to determine the relative quantities of TGF-β1
mRNA in the renal cortex, while β-actin mRNA, a housekeeping gene,
being used as an internal control. The total RNA was extracted from
the renal cortex with RNA simple Total RNA Kit (Lot No. J9117,
TIANGEN Biotechnology Company, Beijing, China). The upstream and
downstream primers for rat TGF-β1 mRNA were: 5′-CCCGCATC
CCAGGAC CTCTCT-3′ and 5′-CGGGGGACTGGCGAGCCTTAG-3′, yielding
a 519 bp product; whereas those for β-actin were: 5′-GCTGCGT
GTGGCCCCTGAG-3′ and 5′-ACGCA-GGATGGCATGAGGGA-3′, yielding a
252 bp product. Equal amounts (1 μg) of each total RNA sample were
added in a 50 μL reaction mixture exerting one-step amplification
with Quant One Step RT-PCR Kit (Lot No. J9104, TIANGEN Biotechnology
Company, Beijing, China). RT-PCR conditions were set to reverse tran-
scription for 30 min at 50 °C, initial denaturation for 2 min at 94 °C,
40 cycles at 94 °C for 30 s, 55 °C for 45 s, 72 °C for 1 min, and final elon-
gation at 72 °C for 10 min. The RT-PCR products were separated by 1.5%
agarose gel electrophoresis, and the band densities were analyzed using
Image J analysis software version 1.34s. The relative quantities of
TGF-β1 mRNA in the renal cortex were represented by the ratio of
band density of TGF-β1 versus that of β-actin.
Electronmicroscopy for morphological observation
Renal tissues, fixed in glutaraldehyde, were postfixed with 1% OsO4
for 2 h and dehydrated with graded ethanol. Samples were embedded
with epoxy resin and polymerized at 37 °C for 24 h, 45 °C for 24 h,
and 60 °C for 24 h. The renal samples were cut into ultrathin sections
and then stained with plumbum citrate for ultrastructural observation
under a transmission electron microscope (H600A-2, Hitachi, Japan).
The images were amplified 10,000 and the photos were scanned
into an image analysis system (Leica Qwin Standard V2.6, Leica
Microsystems, Germany).
Statistical analysis
All the grouped data were analyzed by ANOVA and Dunnett's
t-test (2-side) for different groups using SPSS 16.0 software. The
values are expressed as mean ± SD. P value b 0.05 was considered sig-
nificant and included in the study.
Results
Effect of rutin on blood glucose, kidney index and the renal function of
rats
In our experiment, the fasting blood glucose levels, urine protein,
Cr, BUN, and the kidney index in rats of the DN group were signifi-
cantly higher than those of the NS group (P b 0.01), which suggested
24 h urine protein Kidney index BUN Cr
(μm/24 h) ×1000 (mmol/L) (μmol/L)
3.42 ± 0.24 7.88 ± 0.69 7.53 ± 1.27 37.17 ± 5.17
38.71 ± 3.73# 14.96 ± 1.89# 26.69 ± 5.90# 67.44 ± 9.21#
27.15 ± 2.46⁎ 13.26 ± 0.87⁎ 22.72 ± 2.56⁎ 60.42 ± 5.91⁎
20.18 ± 1.96⁎⁎ 12.84 ± 1.10⁎⁎ 18.78 ± 1.97⁎⁎ 50.13 ± 7.12⁎⁎
15.14 ± 1.69⁎⁎ 11.57 ± 1.32⁎⁎ 16.74 ± 1.92⁎⁎ 46.14 ± 6.52⁎⁎
14.50 ± 2.63⁎⁎ 11.34 ± 1.32⁎⁎ 17.44 ± 3.48⁎⁎ 44.63 ± 4.50⁎⁎
962 H. Hao et al. / Life Sciences 91 (2012) 959–967

Table 2
Effects of rutin on T-AOC, T-SOD, CAT, GSH-Px and MDA in the renal cortex (mean ± SD).

Group n CAT T-AOC T-SOD GSH-PX MDA

(U/mgprot) (U/mgprot) (U/mgprot) (U/mgprot) (nmol/ml)

NS 10 55.44 ± 16.15 7.30 ± 1.77 329.54 ± 78.00 2169.90 ± 170.71 4.45 ± 1.49
DN 8 18.08 ± 5.83# 1.10 ± 0.30# 220.78 ± 38.37# 1037.50 ± 153.14# 12.71 ± 2.93#
RL 8 26.59 ± 3.99⁎ 2.35 ± 0.76⁎ 263.22 ± 24.15⁎ 1323.10 ± 138.82⁎ 10.77 ± 1.48⁎
RM 9 42.09 ± 8.15⁎⁎ 3.92 ± 1.22⁎⁎ 267.40 ± 21.62⁎⁎ 1444.70 ± 306.52⁎⁎ 8.05 ± 2.20⁎⁎
RH 9 43.19 ± 7.56⁎⁎ 4.11 ± 0.44⁎⁎ 277.40 ± 27.08⁎⁎ 1785.30 ± 317.74⁎⁎ 6.49 ± 1.11⁎⁎
CAP 9 25.70 ± 4.23⁎ 2.76 ± 0.65⁎⁎ 253.90 ± 39.96 1265.40 ± 89.59⁎ 10.95 ± 1.22⁎

Mean ± SD.
NS: normal standard group. DN: diabetic nephropathy. RL, RM, RH: rutin 10, 30, 90 mg/kg. CAP: captopril 10 mg/kg.
#
P b 0.01 vs NS group.
⁎ P b 0.05 vs DN group.
⁎⁎ P b 0.01 vs DN group.

Table 3 Effects of rutin on levels of AGEs, collagen IV and laminin in the renal
Effects of rutin on AGE, collagen IV and laminin in the renal cortex (Mean ± SD). cortex
Group n AGE Collagen IV Laminin
(pg/mgprot) (ng/mgprot) (ng/mgprot) The levels of AGEs, collagen IV and laminin of the DN group were
NS 10 24.88 ± 4.13 0.98 ± 0.19 0.48 ± 0.10 increased significantly, when comparing with those of the NS group
DN 8 76.64 ± 13.70# 4.38 ± 1.68# 3.14 ± 0.88# (P b 0.01). Compared with the DN group, there was a significant de-
RL 8 64.80 ± 8.21⁎ 3.41 ± 0.44⁎ 2.75 ± 0.12 crease in the laminin levels of the RM, RH and CAP groups (P b 0.01),
RM 9 50.82 ± 9.85⁎⁎ 2.17 ± 0.72⁎⁎ 1.99 ± 0.45⁎⁎
whereas the RL group had no significant difference on the laminin
RH 9 36.09 ± 9.36⁎⁎ 1.65 ± 0.66⁎⁎ 0.94 ± 0.29⁎⁎
CAP 9 58.94 ± 9.79⁎⁎ 3.35 ± 0.77⁎ 2.03 ± 0.78⁎⁎ (P > 0.05). Collagen IV and AGE levels of the RL, RM, RH, and CAP
groups were strikingly lower than those of the DN group (P b 0.05 or
Mean ± SD.
NS: normal standard group. DN: diabetic nephropathy. RL, RM, RH: rutin 10, 30, 90 mg/kg.
P b 0.01) (Table 3).
CAP: captopril 10 mg/kg.
#
P b 0.01 vs NS group.
⁎ P b 0.05 vs DN group. Effect of rutin on the expression of CTGF in the renal cortex
⁎⁎ P b 0.01 vs DN group.

CTGF mainly expresses in tubular epithelial cells and some inter-

that the early DN model was successful. When comparing with the DN
group, low, moderate and high doses of rutin significantly reduced
blood glucose, urine protein, Cr, BUN, and kidney index levels in the
DN rats (P b 0.05 or P b 0.01). The administration of CAP did not alter
plasma glucose levels, but the urine protein, Cr, BUN, and kidney
index levels were significantly reduced compared with the DN group
(P b 0.01) (Table 1). These results suggest that rutin can effectively
decrease blood glucose and improve renal function of DN rats.
stitial cells. With the increase of interstitial lesions, the expression
of CTGF is increased. The results of immunohistochemistry stained
CTGF was brown yellow (Fig. 1A–F) and the intensity of positive
staining was used to quantify CTGF (Fig. 1G). Compared with the NS
group, brown yellow granules increased on the mesangial cells, vascular
endothelial cells and epithelial cells of renal tubules, which suggested
that the CTGF levels of the DN group was significantly increased
(P b 0.01). The levels of CTGF in the RL, RM, RH and CAP groups were
significantly lower than that of DN group (P b 0.05 or P b 0.01). These
results indicated that treatment of DN rats with rutin significantly
reduced the expression of CTGF in the renal cortex.

Effects of rutin on oxidative stress

As listed in Table 2, renal MDA levels, index of lipid peroxidation,
were higher in the DN group rats than those of the NS group (P b 0.01)
and markedly decreased by rutin. The T-AOC and the activities of the
CAT, GSH-Px, and T-SOD in the DN group were markedly decreased
(P b 0.01), which suggested that the oxidative stress was exhibited
in the DN group. When comparing with the DN group, low, moderate,
and high doses of rutin increased the T-AOC and the activities of
antioxidase (P b 0.05 or P b 0.01). These results indicated that rutin
could ameliorate the oxidative stress state of DN rats. CAP also signif-
icantly decreased MDA levels and increased T-AOC and the activities
of CAT and GSH-Px (P b 0.05 or P b 0.01), but it had no evident effect
on T-SOD (P > 0.05) (Table 2).
Effect of rutin on p-Smad 2/3 and p-Smad 7 in the renal cortex
The protein levels of p-Smad 2/3 and p-Smad 7 in rat renal cortex
were separated into distinct bands using Western blotting. As shown
in Fig. 2A and B, the level of p-Smad 2/3 was strikingly higher and
p-Smad 7 level was significantly lower in the DN group than those
of the NS group (P b 0.01). Compared with the DN group, the expres-
sion of p-Smad 2/3 in the RL, RM, RH and CAP groups was significantly
decreased (P b 0.05 or P b 0.01), whereas the level of p-Smad 7 in the
RL, RM, RH and CAP groups was significantly increased (P b 0.05 or
P b 0.01). These results strongly revealed that Smad 2/3 activation in
DN rat kidney was significantly inhibited by rutin whereas the activa-
tion of Smad 7 increased obviously.

Fig. 1. Immunohistochemical micrographs of CTGF in the renal cortex. CTGF was stained brown yellow (A–F, magnification ×400). Compared with the NS group (A), the expression of
CTGF in the kidney of the DN group (B) was increased. Rutin 90 mg/kg (D), 30 mg/kg (E), and 10 mg/kg (F) treatments decreased the overexpression of CTGF. Captopril 10 mg/kg
(C) also obviously decreased the CTGF level. The intensity of positive staining of CTGF (G) was decreased by rutin. n=6, mean±SD, #Pb 0.01 vs NS group; ⁎Pb 0.05, ⁎⁎Pb 0.01 vs DN group.
H. Hao et al. / Life Sciences 91 (2012) 959–967 963
964 H. Hao et al. / Life Sciences 91 (2012) 959–967

Fig. 2. Effects of rutin on the relative levels of p-Smad 2/3 (A) and p-Smad 7 (B). Protein
extracts from the renal cortex were probed via Western blotting using anti-(p-Smad 2/3),
anti-(total Smad 2/3), anti-(p-Smad 7) and anti-(total Smad 7) antibodies. The density of
each band was determined with Image J analysis software version 1.34s. The NS group and
DN group were treated with 1% CMC solution. RL, RM, and RH were treated with rutin 10,
30, and 90 mg/kg; CAP group was treated with 10 mg/kg of captopril. n=6, mean±SD.
#
Pb 0.01 vs NS group; ⁎Pb 0.05, ⁎⁎Pb 0.01 vs DN group.

Effect of rutin on the expression of TGF-β1 in the renal cortex
In this study, we examined whether rutin was capable of reducing
TGF-β1 expression in STZ-induced DN rats. As shown in Fig. 3A, the rel-
ative quantity of TGF-β1 mRNA in the kidney tissue of DN rats increased
significantly when comparing with that of the NS rats (P b 0.01). West-
ern blot analyses also showed that expression of TGF-β1 protein was
markedly upregulated in DN group (P b 0.01) (Fig. 3B).
In comparison with the DN group, the levels of TGF-β1 mRNA and
TGF-β1 protein expressions in the RL, RM, RH and CAP groups were
significantly decreased (P b 0.05 or P b 0.01). These findings suggest
that rutin can significantly inhibit the overexpression of TGF-β1 in
the renal cortex of DN rats.
Fig. 3. RT-PCR and Western blotting for the determination of the expression of TGF-β1 in
renal cortex. (A) Agarose electrophoresis of RT-PCR products amplified from the total RNA
extracts of the renal cortex, beta-actin was used as the internal standard in each sample.
The data for relative quantity of TGF-β1 mRNA was performed by densitometric analysis.
NS group and DN group were treated with 1% CMC solution. RL, RM, and RH were treated
with rutin 10, 30, and 90 mg/kg; CAP group was treated with 10 mg/kg of captopril. n=6,
mean±SD. #Pb 0.01 vs NS group; ⁎Pb 0.05, ⁎⁎Pb 0.01 vs DN group. (B) Western blotting
analysis TGF-β1 protein using anti-TGF-β1 and anti-GAPDH antibodies. The density of
each band was determined with Image J analysis software version 1.34s. NS group and
DN group were treated with 1% CMC solution. RL, RM, and RH treated with rutin 10, 30,
and 90 mg/kg; CAP group was treated with 10 mg/kg of captopril. n=6, mean±SD.
#
Pb 0.01 vs NS group; ⁎Pb 0.05, ⁎⁎Pb 0.01 vs DN group.

Effects of rutin on morphological change in kidneys NS group, significant thickening of the GBM, mild mesangial expansion,
and local foot process effacement were found in the DN group. Com-
An electronic microscopy was used to look into the ultrastructural pared with the DN group, these phenomena in the RL, RM, RH and
changes in the kidneys. As shown in Fig. 4, when comparing with the CAP groups were ameliorated.
 H. Hao et al. / Life Sciences 91 (2012) 959–967 965

Discussion
DN is the leading cause of end-stage renal disease and approximately
30% of type 1 diabetic patients suffer from DN (Krolewski et al., 1996).
STZ effectively induces renal injury in a type 1 diabetic animal model
and the STZ diabetic rat model has been widely used to study early DN.
Typical morphological changes in the diabetic kidney are represented
by diffuse GBM thickening, mesangial expansion, hyalinosis of the
mesangium and arteriolar walls, broadening and effacement of podocyte
foot processes, reduction in podocyte number, glomerulosclerosis and

Fig. 4. Effects of rutin on the ultrastructural changes in kidneys were shown by the transmission electron microscopy (×10,000). NS group: the thickness of the glomerulus basement
membrane was normal; there were no mesangial cell proliferation and extracellular matrix deposition. DN group: the glomerular basement membrane was thickened partly and foot
process effacement was obvious. RL group (rutin 10 mg/kg): a part of visceral epithelial cell foot showed microvillous transformation. RM and RH groups (rutin 30 and 90 mg/kg) and
CAP (captopril 10 mg/kg) group: there were no significant mesangial cell proliferation and mesangial matrix deposition; the glomerular basement membrane was normal. Among
them, the apoptosis of mesangial cell was shown obviously in the RH group.
966 H. Hao et al. / Life Sciences 91 (2012) 959–967
tubulointerstitial fibrosis (Fioretto and Mauer, 2007). Several decades of
extensive research has elucidated various pathways implicated in the
development of diabetic kidney disease. It has been reported that high
glucose can enhance the level of reactive oxygen species (ROS) and stim-
ulate the expression of TGF-β1 (Fukami et al., 2004). TGF-β1 can induce
the accumulation of ECM mediated by signals such as CTGF, Smads, pro-
tein kinase C (PKC), mitogen-activated protein kinase (MAPK), Ca2+,
etc., and these signals interact with each other in a complicated network
that aggravates DN.
The hyperglycemic condition and predisposition to oxidative
stress are well-documented conditions underlying the DN condition
(Tan et al., 2007), and formation of ROS is a direct consequence of
hyperglycemia. Several pathways have been related to an increased
production of oxidative stress in the context of DN. Moreover, the
intensity and durability of oxidative stress facilitate the formation of
AGEs. At the same time, the interactions between AGEs and RAGE
induce the activation of oxidative stress and stimulate the production
and release of cytokines, which amplify tissue damage (Suzuki et al.,
2006; Yuan et al., 2011). Thus, oxidative stress and AGEs interact
mutually and upregulate each other, which can lead to ECM accumu-
lation and mesangial cell hypertrophy. In our study, after the DN rats
were treated with rutin, it was found that the T-AOC and activities of
T-SOD, CAT, and GSH-Px all were increased significantly, and MDA
levels were decreased obviously in the renal cortex. The AGE levels
of rutin groups were also significantly reduced. These results strongly
suggest that rutin has the characteristics of antioxidant and anti-AGEs
in vivo, and this could be of benefit for the prevention of DN.
DN is characterized by increased urinary protein and loss of renal
functions (Ziyadeh and Wolf, 2008). In this study, the DN rats showed
the overexcretion of urinary protein, decrease of creatinine clearance
and glomerular enlargement. Albuminuria in diabetes is considered
to have both homodynamic (glomerular capillary hypertension and
hyperfiltration) and structural/cellular basis (changes in GBM,
mesangial cell matrix, and podocyte function) (Gruden et al., 2005;
Fowler, 2008). In our study, albuminuria of DN group was significant-
ly higher than that of the NS group, but rutin treatment groups were
found to attenuate microalbuminuria. Interestingly, we also found
that significant thickening of the GBM, mild mesangial expansion,
and local foot process effacement phenomena were alleviated when
DN rats were treated with rutin using electronic microscopy. There-
fore, we suggest that the amount of proteinuria may be related with
the degree of renal ultrastructural damage.
TGF-β1 plays a critical role in renal fibrosis and the accumulation
of mesangial ECM. The Smad protein following TGF-β1 is thought to
be one of the most important factors in the process of ECM accumula-
tion (Wolf, 2003). TGF-β1 signals through a heteromeric receptor com-
plex of the type I and type II receptors to activate the downstream
intracellular mediators Smad 2 and Smad 3 by phosphorylation. The
p-Smad 2/3 will then activate TGF-β1‐responsive genes in the process
of tissue fibrosis. Furthermore, increased p-Smad 2/3 is thought to be
some of the most important factors in the process of ECM accumulation
(Mulay et al., 2010), whereas, Smad 7, as one of the inhibitory Smads,
blocks TGF-β signaling pathway by inhibiting Smad 2/3 phosphoryla-
tion and thereby exerting its anti-fibrotic effect (Henique and
Tharaux, 2012). In our experiments, the expression of type IV collagen
and laminin of the DN group strikingly increased. We also observed
an increase in the expression of p-Smad 2/3 and the relative TGF-β1
levels, whereas a decrease in the inhibitory signal, p-Smad 7. These
findings indicate that it is the high glucose that initially induces the
increased expression in TGF-β1, ultimately resulting in the accumula-
tion of type IV collagen and laminin. In other words, it is the activated
TGF-β1/Smad signaling pathway that induces the accumulation of
ECM. Therefore, TGF-β1/Smad/ECM is obviously a linked response. Our
results are consistent with the conclusions of other researchers (Li
et al., 2003; Yu et al., 2004; Gagliardini and Benigni, 2006). In our
study, after diabetic rats induced by STZ were treated with rutin, the
expressions of TGF-β1 and p-Smad 2/3 were lowered significantly, and
p-Smad 7 was raised greatly, suggesting that rutin can suppress the
TGF-β1/Smad/ECM response and prevent the development of DN.
Those findings are consistent with our previous report in vitro about
the protective effects of rutin on rat glomerular mesangial cells cultured
by high glucose (Tang et al., 2011).
CTGF, one of the most recent identified growth factors with a role in
DN, has been considered as downstream mediator of TGF-β1 and a
potent inducer of ECM in the fibrotic process (Nguyen et al., 2008).
Some scholars believe that the TGF-β1/Smad signaling should be greatly
responsible for CTGF expression. However, there is also evidence of a
TGF-β-independent regulation of CTGF, which is related to a direct acti-
vation of its synthesis by hyperglycemia, AGEs and static pressure
(Wahab et al., 2001). It is becoming clear that the coordinated expres-
sion of TGF-β1 and CTGF is crucial for the induction of ECM proteins
and thus, for the development of DN. Numerous studies indicate that
hyperglycemia induces an increase in TGF-β1 expression at both the
mRNA and protein levels in experimental and human diabetes, as well
as in cultured mesangial cells, and that increased signaling by TGF-β1
is also markedly influenced by CTGF. However, the expression of some
ECM proteins, such as fibronectin, is CTGF-dependent, and its promoter
region does not contain any Smad-binding elements. Thus, CTGF may
mediate the induction of the ECM protein expression both directly
and indirectly by potentiating the TGF-β1/Smad signaling pathway. In
other words, CTGF is a crucial mediator for the TGF-β1-stimulated
matrix protein expression. In our experiments, the expression of CTGF
in the DN group increased clearly and the accumulation of type IV colla-
gen and laminin had the same trend. After DN rats were treated with
rutin, those parameters were ameliorated significantly. In this study,
CTGF and TGF-β1 had the same trend, this finding may indicate that
there might be a TGF-β1/CTGF/ECM linked response.
The interaction of ROS, AGEs, CTGF and TGF-β1/Smads has become a
focus in research of DN. It has been found that AGEs-RAGE-mediated
ROS generation activates TGF-β1-Smad signals and subsequently
induces mesangial cell hypertrophy and ECM accumulation (Fukami
et al., 2004). It was concluded that ROS and TGF-β1-dependent signals
mutually interact and upregulate each other in the pathogenesis of
DN. In our present study, rutin, an anti-oxidant bioflavonoid, can atten-
uate the symptom of DN and downregulate TGF-β1/Smad and CTGF
expressions in rats. This means that rutin may exert a protective effect
on the early development of DN in STZ-induced diabetic rats.
Conclusion
Rutin can significantly decrease the levels of fasting blood glucose,
Cr, BUN, urine protein and the thickness of GBM, and also can amelio-
rate oxidative stress, inhibit the accumulation of type IV collagen and
laminin, decrease AGEs, TGF-β1, p-Smad 2/3 and CTGF, and increase
p-Smad 7 expression in the renal cortex of DN rats. All these results
indicated that rutin may postpone renal damage and have protective
effects on STZ-induced DN rats. Rutin may be a potential drug for the
prevention of early DN.
Conflict of interest statement
None.
Acknowledgments
This work was supported by the Project of the National Natural
Science Foundation of China (No. 81173104), the Natural Science
Research Funds of Jiangsu Province (No. BK2011211), a Project Funded
by the Priority Academic Program Development of Jiangsu Higher Edu-
cation Institutions, President Special Grant of Xuzhou Medical College
(09KJZ22 and 2010KJZ25) and the Natural Science Foundation of
Xuzhou City (No. XF10C074).
 H. Hao et al. / Life Sciences 91 (2012) 959–967
References
Border WA, Yamamoto T, Noble NA. Transforming growth factor beta in diabetic
nephropathy. Diabetes Metab Rev 1996;12:309–39.
Cervantes-Laurean D, Schramm DD, Jacobson EL, Halaweish I, Bruckner GG,
Boissonneault GA. Inhibition of advanced glycation end product formation on
collagen by rutin and its metabolites. J Nutr Biochem 2006;17:531–40.
Fioretto P, Mauer M. Histopathology of diabetic nephropathy. Semin Nephrol 2007;27:
195–207.
Fowler MJ. Microvascular and macrovascular complications of diabetes. Clin Diabetes
2008;26:77–82.
Fukami K, Ueda S, Yamagishi S, Kato S, Inagaki Y, Takeuchi M. AGEs activate mesangial
TGF-beta-Smad signaling via an angiotensin II type 1 receptor interaction. Kidney
Int 2004;66:2137–47.
Gagliardini E, Benigni A. Role of anti-TGF-beta antibodies in the treatment of renal
injury. Cytokine Growth Factor Rev 2006;17:89–96.
Gruden G, Perin PC, Camussi G. Insight on the pathogenesis of diabetic nephropathy
from the study of podocyte and mesangial cell biology. Curr Diabetes Rev
2005;1:27–40.
Henique C, Tharaux PL. Targeting signaling pathways in glomerular diseases. Curr Opin
Nephrol Hypertens 2012;21:417–27.
Hishikawa K, Oemar BS, Nakaki T. Static pressure regulates connective tissue growth
factor expression in human mesangial cells. J Biol Chem 2001;276:16797–803.
Kamalakkannan N, Prince PS. Antihyperglycaemic and antioxidant effect of rutin, a
polyphenolic flavonoid, in streptozotocin-induced diabetic Wistar rats. Basic Clin
Pharmacol Toxicol 2006;98:97-103.
Kamalakkannan N, Stanely Mainzen Prince P. The influence of rutin on the extracellular
matrix in streptozotocin-induced diabetic rat kidney. J Pharm Pharmacol 2006;58:
1091–8.
Kanwar YS, Wada J, Sun L, Xie P, Wallner EI, Chen S, et al. Diabetic nephropathy: mech-
anisms of renal disease progression. Exp Biol Med 2008;233:4-11.
Krolewski M, Eggers PW, Warram JH. Magnitude of end-stage renal disease in IDDM: a
35 year follow-up study. Kidney Int 1996;50:2041–6.
Li JH, Huang XR, Zhu HJ, Johnson R, Lan HY. Role of TGF-beta signaling in extracellular
matrix production under high glucose conditions. Kidney Int 2003;63:2010–9.
Lu Q, Yin XX, Wang JY, Gao YY, Pan YM. Effects of Ginkgo biloba on prevention of devel-
opment of experimental diabetic nephropathy in rats. Acta Pharmacol Sin
2007;28:818–28.
Makino H, Kashihara N, Sugiyama H, Kanao K, Sekikawa T, Okamoto K, et al. Phenotypic
modulation of the mesangium reflected by contractile proteins in diabetes. Diabetes
1996;45:488–95.
Mulay SR, Gaikwad AB, Tikoo K. Combination of aspirin with telmisartan suppresses the
augmented TGF-beta/smad signaling during the development of streptozotocin-
induced type I diabetic nephropathy. Chem Biol Interact 2010;185:137–42.
967
Nguyen TQ, Tarnow L, Jorsal A, Oliver N, Roestenberg P, Ito Y, et al. Plasma connective
tissue growth factor is an independent predictor of end-stage renal disease and
mortality in type 1 diabetic nephropathy. Diabetes Care 2008;31:1177–82.
Raptis AE, Viberti G. Pathogenesis of diabetic nephropathy. Exp Clin Endocrinol Diabetes
2001;109(Suppl. 2):S424–37.
Stanely Mainzen Prince P, Kannan NK. Protective effect of rutin on lipids, lipoproteins,
lipid metabolizing enzymes and glycoproteins in streptozotocin-induced diabetic
rats. J Pharm Pharmacol 2006;58:1373–83.
Stanley Mainzen Prince P, Kamalakkannan N. Rutin improves glucose homeostasis in
streptozotocin diabetic tissues by altering glycolytic and gluconeogenic enzymes.
J Biochem Mol Toxicol 2006;20:96-102.
Stopa M, Anhuf D, Terstegen L, Gatsios P, Gressner AM, Dooley S. Participation of
Smad2, Smad3, and Smad4 in transforming growth factor beta (TGF-beta)-induced
activation of Smad7. THE TGF-beta response element of the promoter requires
functional Smad binding element and E-box sequences for transcriptional regula-
tion. J Biol Chem 2000;275:29308–17.
Suzuki D, Toyoda M, Yamamoto N, Miyauchi M, Katoh M, Kimura M, et al. Relationship
between the expression of advanced glycation end-products (AGE) and the recep-
tor for AGE (RAGE) mRNA in diabetic nephropathy. Intern Med 2006;45:435–41.
Tan AL, Forbes JM, Cooper ME. AGE, RAGE, and ROS in diabetic nephropathy. Semin
Nephrol 2007;27:130–43.
Tang D, Yang D, Tang A, Gao Y, Jiang X, Mou J, et al. Simultaneous chemical fingerprint
and quantitative analysis of Ginkgo biloba extract by HPLC-DAD. Anal Bioanal Chem
2010;396:3087–95.
Tang DQ, Wei YQ, Gao YY, Yin XX, Yang DZ, Mou J, et al. Protective effects of rutin on rat
glomerular mesangial cells cultured in high glucose conditions. Phytother Res
2011;25:1640–7.
Wahab NA, Yevdokimova N, Weston BS, Roberts T, Li XJ, Brinkman H, et al. Role of con-
nective tissue growth factor in the pathogenesis of diabetic nephropathy. Biochem
J 2001;359:77–87.
Wang W, Koka V, Lan HY. Transforming growth factor-beta and Smad signalling in kid-
ney diseases. Nephrology (Carlton) 2005;10:48–56.
Wolf G. Growth factors and the development of diabetic nephropathy. Curr Diab Rep
2003;3:485–90.
Wolf G, Ziyadeh FN. Molecular mechanisms of diabetic renal hypertrophy. Kidney Int
1999;56:393–405.
Yu L, Border WA, Anderson I, McCourt M, Huang Y, Noble NA. Combining TGF-beta in-
hibition and angiotensin II blockade results in enhanced antifibrotic effect. Kidney
Int 2004;66:1774–84.
Yuan Y, Zhao L, Chen Y, Moorhead JF, Varqhese Z, Powis SH, et al. Advanced glycation
end products (AGEs) increase human mesangial foam cell formation by increasing
Golgi SCAP glycosylation in vitro. Am J Physiol Renal Physiol 2011;301:F236–43.
Ziyadeh FN, Wolf G. Pathogenesis of the podocytopathy and proteinuria in diabetic
glomerulopathy. Curr Diabetes Rev 2008;4:39–45.