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Chemico-Biological Interactions 190 (2011) 45–53

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Resveratrol ameliorates early diabetic nephropathy associated with suppression

of augmented TGF-␤/smad and ERK1/2 signaling in streptozotocin-induced
diabetic rats
Kuan-Hsing Chen a,b , Cheng-Chieh Hung b , Hsiang-Hao Hsu b , Yu-Hong Jing c ,
Chih-Wei Yang b , Jan-Kan Chen d,∗
Graduate Institute of Clinical Medicine, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan
Kidney Research Center, Department of Nephrology, Chang Gung Memorial Hospital, Lin-Kou Medical Center, Taiwan
Institute of Anatomy and Embryology, School of Basic Medical Sciences, LanZhou University, LanZhou, China
Department of Physiology, College of Medicine, Chang Gung University, Kwei-Shan 333, Tao-Yuan, Taiwan

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

Article history: Diabetic nephropathy (DN) is the major cause of end-stage renal disease. The early changes in DN are
Received 8 October 2010 characterized by an increased in kidney size, glomerular volume, and kidney function, followed by the
Received in revised form 26 January 2011 accumulation of glomerular extracellular matrix, increased urinary albumin excretion (UAE), glomeru-
Accepted 31 January 2011
lar sclerosis, and tubular fibrosis. Resveratrol (RSV) has been shown to ameliorate hyperglycemia and
hyperlipidemia in streptozotocin-induced diabetic rats. In the present study, we examined the beneficial
effects of RSV on DN and explored the possible mechanism of RSV action.
Male Sprague–Dawley rats were injected with streptozotocin at 65 mg/kg body weight. The induction
Diabetic nephropathy
of diabetes mellitus (DM) was confirmed by a fasting plasma glucose level ≥300 mg/dL and symptoms
TGF-␤/smad of polyphagia and polydipsia. The DM rats were treated with or without RSV at 0.75 mg/kg body weight
ERK1/2 3 times a day for 8 weeks. Animals were sacrificed and kidney histology was examined by microscopy.
Fibronectin Urinary albumin excretion, glomerular hypertrophy and expressions of fibronectin, collagen IV, and TGF-
Nephrin ␤ in the glomeruli were alleviated in RSV-treated DM rats, but not in untreated DM rats. In addition, RSV
treatment reduced the thickness of the glomerular basement membrane (GBM) to the original thickness
and increased nephrin expressions to normal levels in DM rats. Moreover, RSV inhibited phosphorylation
of smad2, smad3 and ERK1/2 in diabetic rat kidneys. This is the first report showing that RSV alleviates
early glomerulosclerosis in DN through TGF-␤/smad and ERK1/2 inhibition. In addition, podocyte injuries
of diabetic kidneys are lessened by RSV.
© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction mechanisms contribute to the development of DN. Glomerular

hyperfiltration is noted in the early stage of DN and causes glomeru-
Diabetic nephropathy (DN) is the major cause of end-stage lar injury in types 1 and 2 diabetes [8]. Two hypotheses have been
renal disease worldwide [1], and approximately 30% of type 1 proposed to explain the changes in early DN, the vascular hypoth-
diabetic patients suffer from DN [2,3]. Streptozotocin (STZ) effec- esis [9] and tubular hypothesis [10]. Both mechanisms show that
tively induces renal injury in a type 1 diabetic animal model. The hyperfiltration occurs in early stage of DN and causes increase in
early changes in DN are characterized by an increase in kidney glomerular pressure, which further contributes to sclerotic process
size, glomerular volume, and kidney function, followed by the of the glomeruli [11]. In addition, hyperglycemia accelerates the
accumulation of glomerular extracellular matrix, increased urinary accumulation of diacylglycerol and activates protein kinase C (PKC),
albumin excretion (UAE), glomerular sclerosis, and tubular fibro- resulting in overexpression of the genes encoding transforming
sis. Late stage overt DN is clinically characterized by proteinuria, growth factor beta (TGF-␤) and extracellular matrix components
hypertension, and progressive renal insufficiency [4–7]. Several in the mesangial cells and glomeruli [12]. Advanced glycation
end-products (AGEs) stimulate mesangial cells and increase the
production of extracellular matrix proteins such as type IV collagen
∗ Corresponding author. Tel.: +886 3 2118800x5077; fax: +886 3 2118700. and fibronectin [13,14]. In addition, activated TGF-␤/smad signal-
E-mail address: jkc508@mail.cgu.edu.tw (J.-K. Chen). ing is observed in the kidneys of diabetic patients and rats [15].

0009-2797/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
46 K.-H. Chen et al. / Chemico-Biological Interactions 190 (2011) 45–53

Table 1
Effects of resveratrol (RSV) on the plasma, urine biochemical parameters, body, and kidney weights of normal, DM, and RSV-treated DM rats.

Normal rats (N = 7) DM rats (N = 7) DM + RSV rats (N = 7)

Serum glucose (mg/dL) 126.7 ± 14.9 521.2 ± 61.1*

474.2 ± 58.4* , #
Urine amount (ml/day) 26.9 ± 7.0 143.7 ± 24.4* 110.8 ± 23.8* , #
Serum creatinine (mg/dL) 0.41 ± 0.03 0.34 ± 0.05 0.35 ± 0.05
Renal Ccr (ml/min kg) 5.8 ± 1.25 9.5 ± 4.4 10.1 ± 6.5
Serum triglyceride (mg/dL) 53.8 ± 16.7 229.7 ± 125.3* 79.3 ± 42.1* , #
Serum cholesterol (mg/dL) 37.4 ± 4.6 53.0 ± 18.6* 50.9 ± 7.9*
Body weight (g) 453.5 ± 21.2 188.2 ± 49.5* 227.4 ± 55.9* , #
Kidney weight (g) 2.74 ± 0.22 3.56 ± 0.41* 2.89 ± 0.17* , #
Kidney weight/body weight (mg/g) 6.04 ± 0.20 18.91 ± 2.98* 12.71 ± 2.51* , #

RSV, resveratrol; DM, diabetes mellitus.

Streptozotocin-induced diabetic rats were fed with or without RSV (0.75 mg/kg, t.i.d.) for 8 weeks.
All the data are mean ± SD.
p < 0.05 between the DM and the normal group.
p < 0.05 between the DM and the DM + RSV group.

Extracellular signal-regulated kinase (ERK), a mitogen activated with a murine microalbuminuria ELISA Kit (Exocell Inc., PA) accord-
protein kinase (MAPK), is an important kinase in the intracellular ing to the manufacturer’s instructions.
signal transduction system leading to cell proliferation and extra-
cellular matrix protein synthesis. Previous reports of research, both
2.2. Experimental protocols
in humans and in animals, have revealed that activation of the ERK
pathway in glomeruli may play an important role in progression
Diabetes was induced by a single injection of STZ (65 mg/kg
during the early stages of DN [16,17].
body weight) through the femoral vein to overnight-fasted rats as
Many natural polyphenolics have been reported to ameliorate
previously described [23]. Age-matched normal rats received an
hyperglycemia and hyperlipidemia in diabetic rats [18,19]. Among
equal volume of normal saline as the control group. One week after
them, resveratrol (3,5,4-trihydroxylstilbene, RSV) has attracted
STZ injection, blood samples were collected through the tail vein
the most interest. RSV has been shown to possess antioxidant,
and plasma glucose levels were measured by plasma glucose test
antiplatelet, anticancer, anti-inflammatory, and life-span extend-
films and enzymatic diagnostic kits. Rats with plasma glucose lev-
ing effects in various experimental models [20–22]. We previously
els ≥300 mg/dL and with symptoms of polyuria, polyphagia, and
showed that RSV exhibits insulin-like effects in streptozotocin
polydipsia were considered diabetic. STZ-DM rats were randomly
(STZ)-induced diabetic (STZ-DM) rats and that it ameliorated
divided into 2 groups (n = 7 in each group) and were treated with-
hyperglycemia, hyperlipidemia and other common diabetic symp-
out or with RSV. RSV was administered through gastric intubation
toms [23]. Further, we showed that RSV can alleviate diabetes
at 0.75 mg/kg body weight 3 times a day at 8 h intervals for 8 weeks.
mellitus (DM)-induced vasculopathy by attenuating the advanced
The age-matched normal rats were fed an equal volume of normal
glycation end products/receptor for AGE/nuclear factor kappa B
saline. At the end of 8 weeks, overnight-fasted rats were anes-
(AGE/RAGE/NF-␬B) signaling pathway [24]. RSV has been shown
thetized with intraperitoneal pentobarbital sodium (30 mg/kg body
to attenuate DN by an antioxidative mechanism and by reducing
weight), blood samples were harvested via an intracardiac needle
the expression of Sir-2 and p38 in diabetic kidneys [25]. Further,
using a chilled syringe containing 10 IU heparin, and their kidneys
Ding et al. demonstrated that RSV attenuates renal hypertrophy
were dissected. Blood samples were centrifuged at 13,000 rpm for
in STZ-DM rats by AMPK activation [26]. However, the effects and
10 min at 4 ◦ C and the plasma was stored at −80 ◦ C prior to further
underlying mechanisms of RSV-induced amelioration of extracellu-
use. The middle third of the right kidney from each rat was fixed
lar matrix accumulation in early DN requires further investigation.
in 4% paraformaldehyde and embedded in paraffin for morphol-
The aim of this study was to investigate whether RSV protects
ogy evaluation and immunohistochemistry. The renal cortex of the
against DN in a model of STZ-induced diabetic rats.
left kidney and residual right kidney from each rat was used for
glomeruli isolation.
2. Materials and methods
2.3. Plasma biochemistry
2.1. Experimental animals and reagents
Plasma glucose, triglyceride and cholesterol levels were mea-
Male Sprague–Dawley (SD) rats ranging from 8 to 10 weeks sured using the respective enzymatic diagnostic kits. Plasma and
old were obtained from the National Laboratory Animal Center, urine creatinine were measured using HPLC. All data were obtained
Taiwan. Animals were housed under optimal conditions with food from 2 independent measurements, each with triplicate incuba-
and water ad libitum. All experimental protocols were approved by tions.
the institutional Animal Ethics Committee, Chang Gung University.
All efforts were made to minimize animal suffering and to reduce
the number of animals used. STZ, RSV, and pentobarbital were pur- 2.4. Isolation of glomeruli
chased from Sigma (St. Louis MO). Enzymatic diagnostic kits were
purchased from Randox (Crumlin Co., Antrim, UK). Plasma glucose Renal cortices were cut into small pieces and glomeruli were
test films were purchased from Fuji (Fujitsu, Japan). Nuclear and isolated by the mechanical graded sieving technique. The sieved
cytoplasmic extraction reagents were purchased from Pierce (Rock- specimens were collected, centrifuged for 10 min at 6000 rpm,
ford, IL). Fibronectin, collagen IV, nephrin, and ␤-actin antibodies and the supernatant was discarded. Glomeruli in the pellet were
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). suspended using a nuclear extraction kit (Panomics) containing
Rabbit polyclonal anti-ERK1/2, phospho-ERK1/2, phospho-smad2 protease inhibitors. Glomeruli were homogenized by sonication
and phospho-smad3 antibodies were obtained from Cell Signaling and insoluble material was removed by centrifugation. The protein
(Boston, MA). The urinary albumin excretion rate was measured content in lysates was determined by the Bradford method.
K.-H. Chen et al. / Chemico-Biological Interactions 190 (2011) 45–53 47

Fig. 1. Effect of resveratrol (RSV) on the microalbuminuria and glomerular tuft size of rats. Microalbuminuria was significantly increased in DM rats but the increase was
reversed by RSV treatments (A). Data are represented as means ± SEM of 7 independent experiments. Glomeruli sections of rat kidneys were stained with hematoxylin and
eosin 400×. Histopathological changes in kidney structure were assessed in 50 randomly selected tissue sections from each group studied at 400× magnification. Glomerular
volumes were elevated in DM rats as compared to those in normal rats and RSV treatment reversed these changes (B). Data are represented as mean ± SEM. Representative
images of glomeruli morphology from normal, DM and DM + RSV rats are shown. DM rats showed glomerular hypertrophy and RSV treatment resulted in a lesser extent of
glomerular hypertrophy (C). # p < 0.05 between the normal and the DM groups; *p < 0.05 between the DM and the DM + RSV groups.

2.5. Assessment of kidney morphology and estimation of 2.7. Western blot analysis
glomerular volume by light microscopy
Cytosolic proteins extracted from glomeruli were used
Renal tissues were fixed in 10% formalin and embedded in for further sodium dodecyl sulfate polyacrylamide gel elec-
paraffin in a routine fashion and thick sections (4 ␮m) were cut trophoresis (SDS–PAGE) and western blot analysis. Cytosolic
and stained with hematoxylin and eosin. The mean glomeru- proteins (40 ␮g) were fractionated on 10% SDS–PAGE and
lar tuft volume (GV) was determined from the mean glomerular transferred onto polyvinylidene fluoride (PVDF) membranes. Mem-
cross-sectional area (GA) by light microscopy. Photomicrographs branes containing fractionated cytosolic proteins were blotted
were scanned and profile areas were traced using Image J 4.5 with anti-fibronectin (1:20,000), anti-collagen IV (1:2000), anti-
software. GA was determined as the average area of a total of ERK1/2 (1:2000), anti-phospho-ERK1/2 (1:1000), anti-phospho-
50 cortical glomeruli from each animal. GV was calculated as smad2 (1:500), anti-phospho-smad3 (1:500) and anti-␤-actin
GV = ˇ/k × (GA)3/2 , where ˇ = 1.38, which is the shape coefficient (1:20,000) antibodies. Membranes were subsequently blotted
for spheres (the idealized shape of glomeruli), and  = 1.1, which is with horseradish peroxidase conjugated secondary antibodies
a size distribution coefficient. and the immunoreactive protein bands were visualized by
enhanced chemiluminescence (Immobilon Western, Millipore).
2.6. Immunohistochemical staining for TGF-ˇ1, fibronectin and Positive immunoreactive bands were quantified densitometrically
collagen IV in glomeruli and compared with the normal groups.

Paraffin sections (4 ␮m) of renal tissue were used for immuno- 2.8. Electron microscopy
histochemistry analysis. Monoclonal antibodies against TGF-␤1
(1:500), fibronectin (1:500), and collagen IV (1:500) were used Glutaraldehyde (3% in PBS) fixed renal tissues were postfixed
for immunohistochemistry. Immunoreactivity in the sections was with 1% OsO4 for 90 min and dehydrated with graded ethanol. Sam-
identified by reactions with biotinylated secondary antibody ples were embedded with resin (Epon 812) and polymerized at
(1:200, 1 h at 37 ◦ C) and streptavidin–biotin–peroxidase (1:200, 72 ◦ C for 48 h. Ultrasections (50–70 nm) were stained with lead
1 h at 37 ◦ C), followed by counterstaining with hematoxylin, dehy- acetate and uranyl, and observed using a JEM1230-microscope
dration, and mounting. Analysis of the percentage area occupied (Tokyo, Japan). The images were captured with a Gatan-Image-
by TGF-␤1 and fibronectin, collagen IV positive staining was per- System (NY, USA).
formed using computer-assisted image analysis software (Meta
Morph, version 4.6, Universal Imaging Corporation). For each kid- 2.9. Statistical analysis
ney, 10 glomerular profiles, cut in equatorial section planes and
successively appearing in the 400× visual field of the microscope, All data are expressed as mean ± SD. The statistical signifi-
were examined. cance between 2 groups was evaluated by the Student’s t-test. One
48 K.-H. Chen et al. / Chemico-Biological Interactions 190 (2011) 45–53

Fig. 2. (A) Immunohistochemical photograph of glomeruli in diabetic rats with or without RSV treatment. As compared with the glomeruli in the kidneys of the normal
group, the glomeruli in diabetic kidneys expressed stronger fibronectin, collagen IV and TGF-␤ immunostaining. Decreased fibronectin, collagen IV, and TGF-␤ were observed
in the diabetic rats after RSV treatment. Specimens were observed under original magnification (400×). Representative data showing increases in glomerular tuft area
immunostained for fibronectin (B), collagen IV (C), and TGF-␤ (D). Data are represented as mean ± SEM. # p < 0.05 between the normal and the DM groups; *p < 0.05 between
the DM and the DM + RSV groups.

way analysis of variance (ANOVA) was conducted for comparisons (p < 0.01) in RSV-treated DM rats as compared with those of STZ-DM
among multiple groups and Tukey’s post hoc-test was used to eval- rats (Table 1). Daily urine output was reduced from 143.7 ± 24.4 ml
uate the significance among the means of the data groups. In all to 110.8 ± 23.8 ml (p < 0.01) in RSV-treated DM rats as compared to
analyses, p ≤ 0.05 was considered significant. that of STZ-DM rats. Kidney weight (2 kidneys) was significantly
greater in STZ-DM rats than in the normal rats, whereas the kidney
3. Results weight was significantly lower than that of STZ-DM rats after RSV
3.1. RSV attenuated body weight loss, hyperglycemia, and lipid
disorder in STZ-DM rats 3.2. Microalbuminuria and diabetic glomerular hypertrophy
were ameliorated by RSV
Plasma glucose, triglyceride, total cholesterol, creatinine, urine
creatinine, and urine output were measured as described in the At 8 weeks, UAE, one of the functional parameters in DN,
Materials and Methods. STZ-DM rats (8 weeks after STZ induction) was significantly higher in STZ-DM rats than in the normal rats.
exhibited increased levels of plasma glucose, triglyceride, creati- In addition, the severity of microalbuminuria was ameliorated
nine, urine MDA and urine output, and decreased body weight, by RSV in STZ-DM rats (Fig. 1A). Glomerular volume was used
as compared with the age-matched normal rats (p < 0.01). Plasma to assess glomerular hypertrophy. The mean glomerular volume
glucose was lowered by approximately 20% and plasma triglyc- in STZ-DM rats (2.34 × 106 ␮m3 ) was significantly greater than
eride was reduced from 229.7 ± 125.3 mg/dL to 79.5 ± 42.1 mg/dL that of the normal rats (1.77 × 106 ␮m3 ). However, the mean
K.-H. Chen et al. / Chemico-Biological Interactions 190 (2011) 45–53 49

Fig. 3. Effects of resveratrol (RSV) on the expression of fibronectin and collagen IV in the glomeruli of DM rats. The Western blotting was performed four times with glomeruli
protein extracts prepared from rats obtained from four independent experiments. A representative blot is shown for fibronectin (A) and collagen IV (B). ␤-Actin was used
as a loading control. The intensity of the immunoreactive band of the 4 blotting results was quantified by densitometry and the relative abundance of fibronectin (C) and
collagen IV (D) was expressed as mean ± SEM. # p < 0.05 between the normal and the DM groups; *p < 0.05 between the DM and the DM + RSV groups.

glomerular volume in RSV-treated DM rats was significantly lower (MAPK) family members, ERK1/2, are activated by phosphoryla-
(1.90 × 106 ␮m3 ) than that in STZ-DM rats (Fig. 1B). Histological tion. ERK1/2 plays a critical role in matrix expansion and fibrosis
examination revealed obvious glomerular hypertrophy in STZ-DM in DN. To assess the effects of RSV on ERK1/2 in DN, the levels of
rats and RSV treatment lessened the glomerular hypertrophy of the phosphorylation of ERK1/2 in the isolated glomeruli of normal, STZ-
DM rats (Fig. 1C). DM, and RSV-treated DM rats (n = 4 in each group) were analyzed.
ERK1/2 phosphorylation was markedly higher in STZ-DM rats than
3.3. Glomerular fibronection and collagen IV expression in in normal rats, and RSV treatment significantly inhibited the ERK1/2
diabetic nephropathy of STZ-DM rats was attenuated by RSV activation (p < 0.05) in RSV-treated DM rats as compared to the
untreated DM rats (Fig. 4E and F).
Fibronectin and collagen IV expression were evaluated by
immunohistochemistry to investigate the effect of RSV admin-
3.5. RSV attenuated glomerular basement membrane thickening
istration on the accumulation of extracellular matrix protein in
and increased nephrin expression of podocytes in STZ-DM rats
the glomeruli. Expressions of fibronectin and collagen IV were
increased in STZ-DM rats, and RSV treatment decreased the
Glomerular basement membrane (GBM) thickening is an early
increased expression of fibronectin and collagen IV in diabetic rats
characteristic of DN. The glomerular ultrastructural changes and
(Fig. 2A). Quantitative analysis revealed a significant inhibitory
thickness of basement membranes were evaluated by electron
effect of RSV on glomerular expression of fibronectin and collagen
microscopy. GBM thickening and foot process effacement of
IV in the diabetes group as compared with the normal group (Fig. 2B
podocytes were obvious in STZ-DM rats (Fig. 5A); however, these
and C). These observations were further confirmed by western blot
conditions began to revert to normal following RSV treatment.
analysis of fibronectin and collagen IV in protein lysates from iso-
Nephrin expression in the isolated glomeruli was also evaluated.
lated glomeruli. (Fig. 3A and B). The quantified densitometrical
Nephrin expression in the glomeruli of diabetic kidneys was lower
analysis of the western blots is shown in Fig. 3C and D.
than that in the glomeruli of normal kidneys 8 weeks after STZ
induction (Fig. 5B and C), and RSV increased the expression of
3.4. Activated TGF-ˇ/smad signaling and ERK1/2 phosphorylation nephrin in the diabetic rats.
in STZ-DM rats was suppressed by RSV

TGF-␤/smad signaling is upregulated in the progression of 4. Discussion

glomerulosclerosis in the diabetic rat kidney. To assess the effects of
RSV on TGF-␤/smad signaling, TGF-␤ immunohistochemistry and DN is the leading cause of end-stage renal disease and the STZ
the levels of phosphorylated smad2 in the isolated glomeruli of nor- diabetic rat model has been widely used to study early diabetic
mal, STZ-DM and RSV-treated DM rats (n = 4 in each group) were renal changes. In the present study, we treated STZ-DM rats with
analyzed. TGF-␤ expression was higher in STZ-DM rats than in nor- or without RSV for 2 months prior to experimentation. We showed
mal rats, and RSV treatment reduced TGF-␤ expression in diabetic that RSV treatment attenuated microalbuminuria and renal hyper-
rats (Fig. 2A and D). In addition, phosphorylation of smad2 and trophy of DM rats. In addition, RSV reduced the accumulation of
smad3 were markedly increased in STZ-DM rats as compared to extracellular matrix proteins such as fibronectin and collagen IV in
normal rats. Smad2 and smad3 activation were significantly inhib- the glomeruli of diabetic kidneys. Moreover, RSV normalized the
ited (p < 0.05) in RSV-treated DM rats as compared to untreated increased GBM thickness and increased nephrin expression in the
DM rats (Fig. 4A, C and B, D). The mitogen-activated protein kinase glomeruli of diabetic rats.
50 K.-H. Chen et al. / Chemico-Biological Interactions 190 (2011) 45–53

Fig. 4. Effects of resveratrol (RSV) on smad2, smad3 and ERK1/2 phosphorylation in isolated glomeruli. The Western blotting was performed four times with glomeruli protein
extracts prepared from rats obtained from four independent experiments. A representative blot is shown for phospho-smad2 (A), phospho-smad3 (B) and phospho-ERK1/2
(E). ␤-Actin and total ERK1/2 served as loading controls for phospho-smad2, phospho-smad3, and phospho-ERK1/2, respectively. The intensity of the immunoreactive band
of the 4 blotting results was quantified by densitometry and the relative abundance of phospho-smad2 (C), phospho-smad3 (D) and phospho-ERK1/2 (F) is expressed as
mean ± SEM. # p < 0.05 between the normal and the DM groups; *p < 0.05 between the DM and the DM + RSV groups.

Microalbuminuria is a functional parameter in the early stages RSV did not affect glomerular hemodynamic changes. Therefore, we
of DN. Albuminuria in DM is considered to have both hemody- suggest that RSV reduces glomerular size by suppressing mesan-
namic (glomerular capillary hypertension and hyperfiltration) and gial cell hypertrophy and extracellular matrix expansion, which
structural/cellular basis (changes in GBM, mesangial cell matrix, is mainly composed of type IV collagens, rather than by modulat-
and podocyte function) [5,27,28]. In previous studies, RSV has been ing hemodynamics. In addition, we found in a previous study that
demonstrated to attenuate microalbuminuria [26,29]. Consistent RSV attenuated diabetes-induced increases in vascular leakage in
with the recent study by Ding et al. [26], RSV treatment prevented diabetic kidneys, as evaluated by Evans blue leakage [24].
glomerular hypertrophy in the diabetic rats in our study. However, In Table 1, although serum glucose was only mildly reduced
RSV treatment did not change elevated clearance of creatinine in in RSV-treated DM rats, the daily water consumption was signifi-
diabetic rats at 8 weeks after induction of diabetes, indicating that cantly reduced in RSV-treated DM rats compared to that of STZ-DM
K.-H. Chen et al. / Chemico-Biological Interactions 190 (2011) 45–53 51

Fig. 5. Effects of resveratrol (RSV) on the glomerular basement membrane (GBM) thickness and nephrin expression in the glomeruli of DM rats. (A) GBM thickness and
foot process effacement of podocytes were obvious in STZ-DM rats from electron microscopy. RSV treatment reversed these changes toward normal levels. (B) The cytosolic
fractions of protein extracts from isolated glomeruli were used for the blotting of nephrin, and ␤-actin served as a loading control. All experiments were performed
independently 4 times and the relative abundance of nephrin (C) is expressed as mean ± SEM of 4 experimental groups. Arrows indicate the positive nuclei. # p < 0.05 between
the normal and the DM groups; *p < 0.05 between the DM and the DM + RSV groups.

rats. Therefore, urine volume was also significantly reduced in and phospho-smad3 are associated with fibronectin and collagen IV
RSV-treated rats compared to that of STZ-DM rats. The result is expression in the diabetic kidney [39]. Interestingly, RSV treatment
compatible with the previous report by Su et al. [23]. In addi- reversed these changes in diabetic kidneys.
tion, amelioration of hypertriglyceridemia in STZ-DM rats by RSV The beneficial effects of RSV may be explained by the follow-
treatment has also been reported previously [23]. However, the ing observations. First, RSV was shown to possess a hypoglycemic
underlying mechanism has not been well understood. In our study, effect in STZ-DM rats, that is insulin-independent [23,40]. Good
insulin was not prescribed to the STZ-DM rats, therefore, STZ-DM glycemic control with insulin has been demonstrated to ameliorate
rats exhibited lower body weight compared to that of normal rat DN in STZ-DM rats [41–43]. However, in our results, oral admin-
and could be under ketoacidotic condition. Insulin has not been istration of RSV to STZ-DM rats resulted in only a mild decline
used in conjunction with RSV to STZ-DM rats in other previous stud- in plasma glucose, suggesting that RSV possesses other reno-
ies [23–26,29]. Since RSV exerted only mild hypoglycemic effect in protective mechanisms beyond the sugar lowering effect. Second,
DM rats, its beneficial effects could be due to other biological effects, RSV has been shown to inhibit extracellular signal-regulated kinase
thus, the use of insulin could easily normalize the glucose level and 1/2 (ERK1/2) associated inflammatory responses to NF-␬B or cellu-
mask the effects of RSV. lar proliferation to various stimuli [44]. Increased phospho-ERK1/2
TGF-␤ is an effector molecule that has been studied extensively (p-ERK1/2)-mediated hypertrophy and extracellular matrix accu-
as a major mediator of the hypertrophic and sclerotic changes in mulation in DM animal models and in cultured renal cells has been
DN [30]. TGF-␤ mRNA and protein levels are increased in both widely studied [45,46]. In addition, treatments to inhibit increased
the glomerular and the tubular compartments of various mod- p-ERK1/2 have been shown to ameliorate DN [17]. In our results,
els of experimental diabetes in rats and mice [31–33]. TGF-␤ RSV suppressed ERK1/2 phosphorylation and possibly contributed
stimulates the synthesis of key extracellular matrix molecules to lower fibronectin and collagen IV expression in the glomeruli of
in diabetic glomeruli, especially fibronectin and type IV colla- diabetic kidneys. Third, RSV is a well-known anti-oxidant. Chronic
gen [34]. Fibronectin and collagen IV are both present in normal hyperglycemia is reported to generate not only more reactive oxy-
mesangium, but are increased in DN and increased collagen IV gen species (ROS) but also to attenuate endogenous antioxidative
expression in the GBM contributes to its thickening in DN. Increased mechanisms through glycation of the scavenging enzymes. Treat-
TGF-␤ is attributed to the following: high glucose concentration ment with RSV in STZ-DM rats has been reported to attenuate renal
[35], advanced products of nonenzymatic protein glycation [14,36], dysfunction and oxidative stress in previous studies [22,24].
and oxidative stress [37,38]. Our immunohistochemical and west- Podocyte slit diaphragm-associated proteins play a central role
ern blot analysis showed that the expression of fibronectin and in maintaining the size-selective barrier, and podocyte injury or
collagen IV were increased in the extracellular matrix of dia- activation contributes to albuminuria and overt proteinuria in DN
betic glomeruli. In addition, TGF-␤ expression was also elevated [27]. Nephrin, an essential component of the slit diaphragms, is
in diabetic glomeruli. Phosphorylation of smad2 and smad3 are necessary to maintain selective glomerular filtration. Decreased
important down-stream signaling of TGF-␤ pathway that con- nephrin expression has been described in both experimental
tributes to tissue fibrosis. Furthermore, increased phospho-smad2 [47,48] and human diabetes, and nephrin mRNA inversely corre-
52 K.-H. Chen et al. / Chemico-Biological Interactions 190 (2011) 45–53

lates with the degree of proteinuria [28,49]. Nephrin expression [15] T. Yamamoto, T. Nakamura, N.A. Noble, E. Ruoslahti, W.A. Border, Expression
has been described as being transiently increased early in experi- of transforming growth factor beta is elevated in human and experimental
diabetic nephropathy, Proc. Natl. Acad. Sci. U.S.A. 90 (5) (1993) 1814–1818.
mental diabetes [47,48], perhaps due to activation of PKC [50], but [16] M. Toyoda, D. Suzuki, M. Honma, G. Uehara, T. Sakai, T. Umezono, H. Sakai, High
nephrin expression reportedly declines after a longer duration of expression of PKC-MAPK pathway mRNAs correlates with glomerular lesions
experimental diabetes and increasing glomerular injury [47,48], in human diabetic nephropathy, Kidney Int. 66 (3) (2004) 1107–1114.
[17] S. Hagiwara, Y. Makita, L. Gu, M. Tanimoto, M. Zhang, S. Nakamura, S. Kaneko,
as found in the present study. However, RSV treatment reversed T. Itoh, T. Gohda, S. Horikoshi, Y. Tomino, Eicosapentaenoic acid ameliorates
the decreased expression of nephrin in diabetic kidneys toward diabetic nephropathy of type 2 diabetic KKAy/Ta mice: involvement of MCP-
normal levels, and podocyte injuries were attenuated by RSV as 1 suppression and decreased ERK1/2 and p38 phosphorylation, Nephrol. Dial.
Transplant. 21 (3) (2006) 605–615.
was evidenced from electron microscopy data. This is the first [18] M. Pinent, M. Blay, M.C. Blade, M.J. Salvado, L. Arola, A. Ardevol, Grape
report to show that RSV attenuates podocyte injury in early diabetic seed-derived procyanidins have an antihyperglycemic effect in streptozotocin-
nephropathy, but the detailed mechanisms need to be elucidated induced diabetic rats and insulinomimetic activity in insulin-sensitive cell
lines, Endocrinology 145 (11) (2004) 4985–4990.
in the future.
[19] N. Yamabe, K.S. Kang, Y. Matsuo, T. Tanaka, T. Yokozawa, Identification of
In conclusion, RSV treatment decreased urinary albumin antidiabetic effect of iridoid glycosides and low molecular weight polyphenol
excretion and prevented glomerulosclerosis. Overproduction of fractions of Corni Fructus, a constituent of Hachimi-jio-gan, in streptozotocin-
TGF-␤/smad signaling, fibronectin and type IV collagen expres- induced diabetic rats, Biol. Pharm. Bull. 30 (7) (2007) 1289–1296.
[20] J.A Baur, D.A. Sinclair, Therapeutic potential of resveratrol: the in vivo evidence,
sion was significantly decreased by RSV treatment in this animal Nat. Rev. Drug Discov. 5 (6) (2006) 493–506.
model of type 1 DN. Activated ERK1/2 signaling in DN was also [21] L. Bordone, L. Guarente, Calorie restriction, SIRT1 and metabolism: understand-
suppressed by RSV. Furthermore, RSV treatment reversed the ing longevity, Nat. Rev. Mol. Cell Biol. 6 (4) (2005) 298–305.
[22] M. Jang, L. Cai, G.O. Udeani, K.V. Slowing, C.F. Thomas, C.W. Beecher, H.H. Fong,
decreased expression of nephrin in diabetic kidneys toward nor- N.R. Farnsworth, A.D. Kinghorn, R.G. Mehta, R.C. Moon, J.M. Pezzuto, Cancer
mal levels. Taken together, these results suggest that RSV could be chemopreventive activity of resveratrol, a natural product derived from grapes,
a new therapeutic agent for slowing the progression of early DN. Science 275 (5297) (1997) 218–220.
[23] H.C Su, L.M. Hung, J.K. Chen, Resveratrol, a red wine antioxidant, possesses
an insulin-like effect in streptozotocin-induced diabetic rats, Am. J. Physiol.
Conflict of interest Endocrinol. Metab. 290 (6) (2006) E1339–E1346.
[24] Y.H. Jing, K.H. Chen, S.H. Yang, P.C. Kuo, J.K. Chen, Resveratrol ameliorates vas-
culopathy in STZ-induced diabetic rats: role of AGE-RAGE signalling, Diabetes
None declared. Metab. Res. Rev. 26 (3) (2010) 212–222.
[25] K. Tikoo, K. Singh, D. Kabra, V. Sharma, A. Gaikwad, Change in histone H3
phosphorylation, MAP kinase p38, SIR 2 and p53 expression by resveratrol in
Acknowledgments preventing streptozotocin induced type I diabetic nephropathy, Free Radic. Res.
42 (4) (2008) 397–404.
[26] D.F. Ding, N. You, X.M. Wu, J.R. Xu, A.P. Hu, X.L. Ye, Q. Zhu, X.Q. Jiang, H. Miao,
This work was supported by Chang Gung Memorial Hospital C. Liu, Y.B. Lu, Resveratrol attenuates renal hypertrophy in early-stage diabetes
Grants CMRPG370391 and CMRPC380901 to Jan-Kan Chen. by activating AMPK, Am. J. Nephrol. 31 (4) (2010) 363–374.
[27] K. Tryggvason, J. Wartiovaara, Molecular basis of glomerular permselectivity,
Curr. Opin. Nephrol. Hypertens. 10 (4) (2001) 543–549.
References [28] R.G. Langham, D.J. Kelly, A.J. Cox, N.M. Thomson, H. Holthofer, P. Zaoui, N.
Pinel, D.J. Cordonnier, R.E. Gilbert, Proteinuria and the expression of the
[1] E. Ritz, I. Rychlik, F. Locatelli, S. Halimi, End-stage renal failure in type 2 diabetes: podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: effects
a medical catastrophe of worldwide dimensions, Am. J. Kidney Dis. 34 (5) (1999) of angiotensin converting enzyme inhibition, Diabetologia 45 (11) (2002)
795–808. 1572–1576.
[2] M. Bojestig, H.J. Arnqvist, G. Hermansson, B.E. Karlberg, J. Ludvigsson, Declining [29] S Sharma, M. Anjaneyulu, S.K. Kulkarni, K. Chopra, Resveratrol, a polypheno-
incidence of nephropathy in insulin-dependent diabetes mellitus, N. Engl. J. lic phytoalexin, attenuates diabetic nephropathy in rats, Pharmacology 76 (2)
Med. 330 (1) (1994) 15–18. (2006) 69–75.
[3] M. Krolewski, P.W. Eggers, J.H. Warram, Magnitude of end-stage renal disease [30] W.B. Reeves, T.E. Andreoli, Transforming growth factor beta contributes to
in IDDM: a 35 year follow-up study, Kidney Int. 50 (6) (1996) 2041–2046. progressive diabetic nephropathy, Proc. Natl. Acad. Sci. U.S.A. 97 (14) (2000)
[4] T. Deckert, J.E. Poulsen, M. Larsen, Prognosis of diabetics with diabetes onset 7667–7669.
before the age of thirty-one. I. Survival, causes of death, and complications, [31] T. Nakamura, M. Fukui, I. Ebihara, S. Osada, I. Nagaoka, Y. Tomino, H. Koide,
Diabetologia 14 (6) (1978) 363–370. mRNA expression of growth factors in glomeruli from diabetic rats, Diabetes
[5] G.C. Viberti, R.D. Hill, R.J. Jarrett, A. Argyropoulos, U. Mahmud, H. Keen, Microal- 42 (3) (1993) 450–456.
buminuria as a predictor of clinical nephropathy in insulin-dependent diabetes [32] K. Sharma, F.N. Ziyadeh, Renal hypertrophy is associated with upregulation of
mellitus, Lancet 1 (8287) (1982) 1430–1432. TGF-beta 1 gene expression in diabetic BB rat and NOD mouse, Am. J. Physiol.
[6] C.E. Mogensen, C.K. Christensen, Predicting diabetic nephropathy in insulin- 267 (6 Pt 2) (1994) F1094–F1101.
dependent patients, N. Engl. J. Med. 311 (2) (1984) 89–93. [33] C.W. Yang, M. Hattori, H. Vlassara, C.J. He, M.A. Carome, E. Yamato, S. Elliot,
[7] K. Borch-Johnsen, S. Kreiner, Proteinuria: value as predictor of cardiovascular G.E. Striker, L.J. Striker, Overexpression of transforming growth factor-beta 1
mortality in insulin dependent diabetes mellitus, Br. Med. J. (Clin. Res. Ed.) 294 mRNA is associated with up-regulation of glomerular tenascin and laminin
(6588) (1987) 1651–1654. gene expression in nonobese diabetic mice, J. Am. Soc. Nephrol. 5 (8) (1995)
[8] K. Hiragushi, H. Sugimoto, K. Shikata, T. Yamashita, N. Miyatake, Y. Shikata, J. 1610–1617.
Wada, I. Kumagai, M. Fukushima, H. Makino, Nitric oxide system is involved in [34] K. Sharma, F.N. Ziyadeh, Biochemical events and cytokine interactions link-
glomerular hyperfiltration in Japanese normo- and micro-albuminuric patients ing glucose metabolism to the development of diabetic nephropathy, Semin.
with type 2 diabetes, Diabetes Res. Clin. Pract. 53 (3) (2001) 149–159. Nephrol. 17 (2) (1997) 80–92.
[9] G.T. O’Bryan, T.H. Hostetter, The renal hemodynamic basis of diabetic [35] F.N. Ziyadeh, K. Sharma, M. Ericksen, G. Wolf, Stimulation of collagen gene
nephropathy, Semin. Nephrol. 17 (2) (1997) 93–100. expression and protein synthesis in murine mesangial cells by high glucose is
[10] S.C. Thomson, A. Deng, D. Bao, J. Satriano, R.C. Blantz, V. Vallon, Ornithine decar- mediated by autocrine activation of transforming growth factor-beta, J. Clin.
boxylase, kidney size, and the tubular hypothesis of glomerular hyperfiltration Invest. 93 (2) (1994) 536–542.
in experimental diabetes, J. Clin. Invest. 107 (2) (2001) 217–224. [36] F.N. Ziyadeh, D.C. Han, J.A. Cohen, J. Guo, M.P. Cohen, Glycated albumin stimu-
[11] T.H Hostetter, Hyperfiltration and glomerulosclerosis, Semin. Nephrol. 23 (2) lates fibronectin gene expression in glomerular mesangial cells: involvement of
(2003) 194–199. the transforming growth factor-beta system, Kidney Int. 53 (3) (1998) 631–638.
[12] D. Koya, M.R. Jirousek, Y.W. Lin, H. Ishii, K. Kuboki, G.L. King, Characterization of [37] M. Brownlee, Biochemistry and molecular cell biology of diabetic complica-
protein kinase C beta isoform activation on the gene expression of transforming tions, Nature 414 (6865) (2001) 813–820.
growth factor-beta, extracellular matrix components, and prostanoids in the [38] M.C. Iglesias-De La Cruz, P. Ruiz-Torres, J. Alcami, L. Diez-Marques, R. Ortega-
glomeruli of diabetic rats, J. Clin. Invest. 100 (1) (1997) 115–126. Velazquez, S. Chen, M. Rodriguez-Puyol, F.N. Ziyadeh, D. Rodriguez-Puyol,
[13] G Pugliese, F. Pricci, G. Romeo, F. Pugliese, P. Mene, S. Giannini, B. Cresci, Hydrogen peroxide increases extracellular matrix mRNA through TGF-beta in
G. Galli, C.M. Rotella, H. Vlassara, U. Di Mario, Upregulation of mesan- human mesangial cells, Kidney Int. 59 (1) (2001) 87–95.
gial growth factor and extracellular matrix synthesis by advanced glycation [39] S.R. Mulay, A.B. Gaikwad, K. Tikoo, Combination of aspirin with telmisartan
end products via a receptor-mediated mechanism, Diabetes 46 (11) (1997) suppresses the augmented TGFbeta/smad signaling during the development of
1881–1887. streptozotocin-induced type I diabetic nephropathy, Chem. Biol. Interact. 185
[14] C.W. Yang, H. Vlassara, E.P. Peten, C.J. He, G.E. Striker, L.J. Striker, Advanced gly- (2) (2010) 137–142.
cation end products up-regulate gene expression found in diabetic glomerular [40] J.Y. Deng, P.S. Hsieh, J.P. Huang, L.S. Lu, L.M. Hung, Activation of estrogen
disease, Proc. Natl. Acad. Sci. U.S.A. 91 (20) (1994) 9436–9440. receptor is crucial for resveratrol-stimulating muscular glucose uptake via
K.-H. Chen et al. / Chemico-Biological Interactions 190 (2011) 45–53 53

both insulin-dependent and -independent pathways, Diabetes 57 (7) (2008) [46] H. Fujita, S. Omori, K. Ishikura, M. Hida, M. Awazu, ERK and p38 mediate high-
1814–1823. glucose-induced hypertrophy and TGF-beta expression in renal tubular cells,
[41] H. Gronbaek, I. Vogel, R. Osterby, I. Lancranjan, A. Flyvbjerg, H. Orskov, Effect of Am. J. Physiol. Renal Physiol. 286 (1) (2004) F120–F126.
octreotide, captopril or insulin on renal changes and UAE in long-term experi- [47] P. Aaltonen, P. Luimula, E. Astrom, T. Palmen, T. Gronholm, E. Palojoki, I.
mental diabetes, Kidney Int. 53 (1) (1998) 173–180. Jaakkola, H. Ahola, I. Tikkanen, H. Holthofer, Changes in the expression of
[42] J.W Scholey, T.W. Meyer, Control of glomerular hypertension by insulin admin- nephrin gene and protein in experimental diabetic nephropathy, Lab. Invest.
istration in diabetic rats, J. Clin. Invest. 83 (4) (1989) 1384–1389. 81 (9) (2001) 1185–1190.
[43] J. Petersen, J. Ross, R. Rabkin, Effect of insulin therapy on established diabetic [48] J.M. Forbes, F. Bonnet, L.M. Russo, W.C. Burns, Z. Cao, R. Candido, H. Kawachi, T.J.
nephropathy in rats, Diabetes 37 (10) (1988) 1346–1350. Allen, M.E. Cooper, G. Jerums, T.M. Osicka, Modulation of nephrin in the diabetic
[44] O.H. Kang, H.J. Jang, H.S. Chae, Y.C. Oh, J.G. Choi, Y.S. Lee, J.H. Kim, Y.C. Kim, kidney: association with systemic hypertension and increasing albuminuria, J.
D.H. Sohn, H. Park, D.Y. Kwon, Anti-inflammatory mechanisms of resveratrol Hypertens. 20 (5) (2002) 985–992.
in activated HMC-1 cells: pivotal roles of NF-kappaB and MAPK, Pharmacol. [49] M. Toyoda, D. Suzuki, T. Umezono, G. Uehara, M. Maruyama, M. Honma, T.
Res. 59 (5) (2009) 330–337. Sakai, H. Sakai, Expression of human nephrin mRNA in diabetic nephropathy,
[45] M. Mage, C. Pecher, E. Neau, E. Cellier, M.L. Dos Reiss, J.P. Schanstra, R. Couture, Nephrol. Dial. Transplant. 19 (2) (2004) 380–385.
J.L. Bascands, J.P. Girolami, Induction of B1 receptors in streptozotocin diabetic [50] S.X. Wang, P. Mene, H. Holthofer, Nephrin mRNA regulation by protein kinase
rats: possible involvement in the control of hyperglycemia-induced glomerular C, J. Nephrol. 14 (2) (2001) 98–103.
Erk 1 and 2 phosphorylation, Can. J. Physiol. Pharmacol. 80 (4) (2002) 328–333.