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Introduction
In many countries diabetic nephropathy (DN) is a major complication
of diabetes. At present, it affects about 15%_25%
of type 1 and 30%_40% of type 2 diabetic patients, causing disabilities and a high mortality rate. It is characterized by the
thickening of the basement membranes, mesangial expansion and proliferation, and excessive accumulation of extracellular
matrix (ECM), and ultimately leads to nodular glomerulosclerosis and chronic renal failure.
However, the mechanisms underlying the pathogenesis of DN are not completely understood. Many scholars consider
the progression of DN a result of the interaction of multiple factors, such as high glucose, the polyol pathway, oxidative
stress, protein non-enzymatic glycation, and
cytokines[1]. Studies have found that high glucose is presumed to be an
initiating agent which increases the formation of advanced
glycosylation end products (AGE) and
induces oxidative stress. It also increases transforming
growth factor-β1 (TGF-β1) expressions. TGF-β1 is thought
to be the key cytokine involved in the progression of
DN[2]. It increases the synthesis of ECM components, including
collagens, fibronectin, and laminin, and results in
hypertrophy of the mesangial cell and glomerulosclerosis.
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[3,4]. Matrix metalloproteinases (MMP) are the major physiological
regulator of ECM degradation in the glomerulus. A balance
between ECM synthesis and degradation is a prerequisite
for maintaining the structural and functional integrity of the
glomerulus. MMP and tissue inhibitors of metalloproteinase
(TIMP) keep this balance together[5]. For the complexity of
mechanisms, there are no definitive drugs to delay the
development of DN. Therefore, it is necessary to develop
new drugs for DN that can deal with more than 1
pharmacological target in this intricate mechanism.
The tree Ginkgo biloba has long been believed to have
medicinal properties, and its extracts are among the most
widely-sold herbal supplements in the world. Ginkgo biloba
extract (GbE), extracted from Ginkgo
biloba leaves, is a defined, complex mixture containing 24%
Ginkgo flavone glycoside (quercetin, kaempferol, and isorhamne) and 6%
terpene lactones (ginkgolides and bilobalide). It has been
used as a therapeutic agent in some cardiovascular and
neurological disorders[6,7]. Although the exact mechanism is
unknown, evidence accumulated in vitro
and in vivo shows that GbE has a number of benefits, including ameliorating
hemodynamics, suppressing the platelet-activating factor,
scavenging reactive oxygen species (ROS), and relaxing
vascular smooth muscles[8]. All of these offer us a
pharmacological foundation of GbE for DN therapy. However, there
are still few published reports that focus on the protective
mechanisms of GbE on DN. Therefore, it is worthwhile for
us to explore its potential effects in preventing the
progression of DN.
To evaluate the effects of GbE on DN, in our present work
we used captopril (CAP) as an antifibrotic control
drug[9,10], and studied the possible influences of
GbE on the parameters that indicate protective effects against the progress
of DN, such as blood glucose, AGE, TGF-β1, MMP-2, TIMP-2,
CTGF, collagen IV and laminin in the kidney cortex, anti-oxidases in the serum, and the thickness of the
glomerular base membrane (GBM) on early experimental DN
rats, and observed the morphological changes on DN rats.
Materials and methods
Drugs GbE (Lot No 040029) was provided by the Pizhou
Fuwei Biochemical Company (Xuzhou, China), and was
dissolved in 1% carboxymethyl cellulose (CMC) solution.
Streptozotocin (STZ, Lot No P7993b) was purchased from
Biomol Research Lab (Plymouth Meeting, PA, USA). Captopril (Lot
No 050050), serving as a positive control drug,
was kindly provided by Changzhou Pharmaceutical Factory
(Changzhou, China) and was suspended in 1% CMC solution.
Animals Male Sprague-Dawley rats (Certificate
No SYXK 2001-0050), weighing 165.7±10.2 g (150_190 g) were obtained
from the Laboratory Animal Center of Xuzhou Medical
College (Xuzhou, China), following the Guiding Principles for
Care and Use of Laboratory Animals of Xuzhou Medical
College.
Induction of DN model and study protocol
Diabetes mellitus was induced in the male Sprague_Dawley rats, by ip
injection of 60 mg/kg of the beta-cell toxin STZ (dissolved in
pH 4.5 citrate buffer immediately before the injection), while
controlled normal standard rats (NS group,
n=13) received 6 mL/kg citrate buffer. The induction of the diabetic state was
confirmed by the blood glucose level measurement on the
third day after STZ administration. The rats with fasting
blood glucose concentrations=13.88 mmol/L were randomly
allotted into 5 groups: DN rats were treated with 1% CMC
solution (DN group, n=13); DN rats with 50, 100, and 200
mg/kg GbE for the GL group (low dose,
n=12), the GM group (moderate dose, n=14), and the GH group (high dose,
n=14), respectively; the DN rats were treated with 10 mg/kg of
captopril (CAP group, n=11). The same volume of CMC
solution was administered to the NS group
(n=13). The animals housed in the barrier environment refer to breed
specific pathogen-free grade animals, and they were allowed
food and water ad libitum. After 12 weeks, the urine and
blood samples were collected. After the animals were
sacrificed, fresh kidney cortices were stored in formaldehyde
solution for immunohistochemical measurements, and 1 mm×1
mm×1 mm cubes of kidney cortices were fixed in 2.5%
glutaraldehyde for electron microscopic measurement. The rest
of the kidneys were stored at _80 oC for the later analysis.
Measurement of renal function and biochemical
parameters Blood glucose was measured by the glucose oxidase
method with kits purchased from Dong-Ou Bioengineering
(No 2005110008, Wenzhou, China). The values of urine
protein, creatinine (Cr), and blood urea nitrogen (BUN) were
determined by the automatic biochemistry analyzer
(Olympus-2000, Tokyo, Japan). The kidney index was 1000×kidney
weight/body weight.
Collagen IV and laminin, the main component of ECM,
were measured by radioimmunoassay kits from Shanghai
High Biotech Center (Lot No 20060601, Shanghai, China).
Total antioxidative capability (T-AOC), catalase (CAT),
total superoxidase dismutase (T-SOD) and
glutathione-peroxidase (GSH-Px) activities in the serum were measured by
spectrophotometry, using kits from Jiancheng
Bioengineering Institute (Lot No 20050522, Nanjing, China).
AGE plays a critical role in diabetic nephropathy by
stimulating ECM synthesis. AGE either in the renal cortex or in the
serum was measured by fluorescence spectrophotometry
(fluorospectrophotometer F-4500, Hitachi, Tokyo, Japan), and
the concentration of AGE was represented by the
fluorescence optical density. The final value of AGE in the tissue
was modulated by the total protein in the tissue which was
measured by the Lowry method[11].
Immunohistochemical measurements of MMP-2,
TIMP-2, and CTGF The glass slides were sealed with 10%
polylysine. The 4 µm renal tissue sections were used to
perform immunohistochemical staining for MMP-2, TIMP-2,
and CTGF. The renal tissue sections were incubated with
rabbit polyclonal anti-MMP-2 (Lot No 200604, Boster
Biological Technology Company, Wuhan, China) at a dilution
of 1:50 at 37 oC for 2 h. After washing, goat anti-rabbit
IgG-horseradish peroxidase (Lot No 015090, Zhongshan Golden
Bridge Biotechnology Company, Beijing, China) was added.
To visualize MMP-2, the renal tissue sections were stained
with 3,3'-diaminobenzidine (DAB) for 10 min and then
examined by light microscopy (×400). All steps were performed at
room temperature. The TIMP-2 and CTGF measurements
were identical to MMP-2. The stained MMP-2, TIMP-2, and
CTGF were quantified by gray scale analysis (Leica Qwin
Standard V2.6, Leica Microsystems, Welzlar, Germany).
RT-PCR for the relative quantities of TGF-β1 mRNA
in the kidney cortex[12] A RT-PCR procedure was performed to
determine the relative quantities of TGF-β1 mRNA in the
kidney cortex, while β-actin mRNA, a housekeeping gene,
was used as an internal control. The total RNA was
extracted from the kidney cortex with the Promega Total RNA
Isolation System (Lot No 182207, Promega, Madison,
USA). The upstream and downstream primers for rat
TGF-β1 mRNA were: 5'-CCCGCATCCCAGGACCTCTCT-3' and 5'-CGGGGGACTGGCGAGCCTTAG-3', yielding a 519 bp
product; whereas those for β-actin were:
5'-GCTGCGTGTG-GCCCCTGAG-3' and 5'-ACGCAGGATGGCATGAGGGA-3',
yielding a 25 -bp product. Equal amounts (3 µL) of each total
RNA sample were added in a 50 µL reaction mixture exerting
one-step amplification with the Promega RT-PCR System (Lot
No 199676, Promega, USA). The reaction mixture was
incubated at 48 oC for 45 min to reverse
transcript, then went into cycles. The cycle conditions were set to initial denaturation
for 5 min at 94 oC, 40 cycles at 94
oC for 1 min, 57 oC for 50 s,
72 oC for 1 min, with a final elongation at 72
oC for 7 min. The RT-PCR products were separated by 1% agarose
electro-phoresis, and the band densities were analyzed using laser
densitometry. The relative quantities of TGF-β1 mRNA in
the kidney cortex were represented by the ratio of band
density of TGF-β1 versus that of b-actin.
Morphological observation and measurement of the
thickness of GBM The kidney cortex samples stored in
formaldehyde solution were embedded with paraffin and stained
with periodic acid-Schiff (PAS). Every PAS-stained sample
in each group was observed under light microscope. Three
kidney samples from each experimental group were randomly
chosen for electron microscopic observation. The
specimens were embedded in epoxy resin and cut into ultra-thin
sections and then stained with plumbum citrate for
ultrastructural observation under a transmission electron
microscope (H600A-2, Hitachi, Japan). Five photos were taken at
different views for each kidney sample. The images were
amplified 6 K and the photos were scanned into a computer
so that the thickness of GBM was measured by an image
analysis system (Leica Qwin Standard V2.6, Leica Microsystems, Germany).
Statistical analysis Statistical analysis was performed
to compare the effects of GbE on early DN rats using ANOVA
and Dunnett's t-test (2-side) for different groups using SPSS
10.0 (Chicago, USA). Data were expressed as mean±SD.
Differences were considered to be significant at
P<0.05.
Results
Effects of GbE on physical behaviors, blood glucose,
urinary protein, Cr, BUN, and the kidney index
In our experiment, the rats in the DN group had hypopraxia,
cachexia, polyuria/polydipsia, yellowish and damp fur,
kyphosis, and tardy weight gain; the rats in the NS and GH
groups were vibrant and vigorous, had white and tidy fur,
and weight gain.
Table 1 shows that the fasting blood glucose level, urine
protein, Cr, BUN, and the kidney index of the DN group were
significantly higher than those of the NS group
(P<0.01), suggesting that our early DN model was successful. Low
doses of GbE markedly reduced blood glucose and Cr in the
DN rats (P<0.05), but was of no significant difference in
reducing BUN, urine protein, and the kidney index.
Moderate and high doses of GbE and captopril significantly
reduced blood glucose, urine protein, Cr, BUN, and kidney
index levels in the DN rats (P<0.01).
Effects of GbE on oxidative stress
The activities of the CAT, GSH-Px, T-AOC, and T-SOD of the DN group were all
lower than those of the NS group (P<0.01), suggesting that
the DN group exhibited oxidative stress. We also found that
low, moderate, and high doses of GbE increased these 4
anti-oxidase activities (P<0.05 or
P<0.01). These results
indicated that GbE could ameliorate the oxidative stress state
of DN rats. Captopril also significantly increased CAT and
GSH-Px activities (P<0.05), but it had no evident effect on
T-AOC and T-SOD (P>0.05; Figure 1).
Effects of GbE on collagen IV and laminin level in the
kidney cortex The levels of collagen IV and laminin in the
kidney cortex of early DN rats significantly increased when
compared with those of the normal rats (P<0.01). The
collagen IV levels of the GM, GH, and CAP groups were
strikingly lower than those of the DN group
(P<0.05 or P<0.01). The laminin levels of the GL, GM, GH, and all the CAP groups
decreased (P<0.01). These results suggested that
GbE could decrease the expressions of collagen IV and laminin of DN
rats, and that captopril's capability of decreasing
expressions of collagen IV was between that of
GbE's moderate and low dose (Figure 2).
Effects of GbE on AGE and the thickness of GBM
The AGE levels in the kidney cortex and in the serum of the DN
rats were greatly higher than those of normal rats
(P<0.01). In the kidney cortex and in the serum, there was significant
decrease in the AGE levels of the GL, GM, and GH groups
when compared with those of the DN group (P<0.05 or
P<0.01), whereas captopril significantly decreased the AGE
levels (P<0.01).
There was a significant difference in the thickness of
the GBM between the NS group and DN group
(P<0.01). The thickness of GBM decreased as the doses of
GbE increased. There was significant decrease in the thickness
of the GBM of the GL, GM, GH, and CAP groups, compared with that of the DN group
(P<0.05 or P<0.01; Table 2).
Immunocytochemical analysis of MMP-2,
TIMP-2, and CTGF MMP-2 and TIMP-2 are mainly expressed in the
cytoplasm of mesangial cells and renal tubular epithelial cells.
The color of the stained MMP-2 and TIMP-2 protein was
brown. The staining intensity of TIMP-2 of the DN group
highly increased. On the contrary, MMP-2 markedly
decreased when compared with those of the NS group. We
used gray scale analysis to quantify MMP-2 and TIMP-2
proteins and found that the levels of MMP-2/TIMP-2 of the
DN group was significantly different from that of the NS
group (P<0.01). With the increased concentration of
GbE, the expressions of MMP-2/TIMP-2 in the GM group and the
GH group significantly increased/decreased
(P<0.05 or P<
0.01), respectively. The expressions in the CAP group had
the same changes (P<0.01), and the levels of MMP-2 and
TIMP-2 in the CAP group was between those of the GH
group and GM group (Figures 3,4). Like TIMP-2, the levels
of CTGF in the GL, GM, GH, CAP groups significantly
decreased (P<0.01 or P<0.05; Figure 5). All of these results
suggested that GbE had a potent influence on the
expressions of MMP-2, TIMP-2, and CTGF (Figure 6).
Effect of GbE on the relative quantity of
TGF-β1 mRNA in the kidney cortex The RT-PCR products of
TGF-β1 were separated by 1% agarose electrophoresis, after which we
could see distinct bands (Figure 7A). The relative quantity
of TGF-β1 mRNA in the kidney cortex of the DN group
was greatly higher than that of the NS group
(P<0.01). The TGF-β1 mRNA level of the GM, GH, and CAP groups strikingly
decreased when compared with that of the DN group
(P<0.05 or P<0.01). The level of the GH group was similar to that
of the CAP group. The results suggested that a high dose of
GbE had the same effect as captopril in decreasing the
expression of TGF-β1 mRNA (Figure 7B).
Effects of GbE on morphological change in kidneys
The light microphotograph showed the existence of glomerular
mesangial hyperplasia (Figure 8). In the transmission
electron micrographs, the ultrastructure of glomerulus of the DN
rat was changed. The GBM was wrinkled and partly
thicken-ed, but in the GH group, the thickness of the GBM appeared
to be almost normal (Figure 9).
Discussion
DN is the leading cause of end-stage renal disease and
the characteristics of this diabetic complication include
macrovascular and microvascular damage, ECM
accumu-lation, and eventually chronic fibrosis. Several decades of
extensive research has elucidated various pathways
implicated in the development of diabetic kidney disease.
Oxidative stress has been known to play an important
role in the development and progression of DN, and the
formation of ROS is a direct consequence of hyperglycemia. It
has been shown that ROS activate the protein kinase C (PKC),
mitogen-activated protein kinase (MAPK), and JAK-STAT
pathways[13,14], which lead to the activation of
redox-sensitive transcription factors including
NF-κB, AP-1 (Fos and Jun proteins), STAT, and
Egr-1[15]. All of these enhance the transactivation of genes coding for cytokines such as
TGF-β1 and CTGF, which upregulate ECM protein
expression[16]. Therefore, antioxidant treatment is a potential antifibrotic
therapy for DN. Moreover, the intensity and durability of
oxidative stress facilitate the formation of AGE. AGE are the
biochemical end products of non-enzymatic glycosylation
that are formed irreversibly. AGE are elevated in serum and
in many tissues in patients with
diabetes[17,18]. They can covalently crosslink and biochemically modify protein
structure and affect protein functions, particularly collagen.
Additionally, in recent years, cell surface receptors
for AGE (RAGE) have been
identified[19], and post-receptor signaling
pathways are being
defined[20]. Through an AGE
receptor-dependent mechanism, AGE induction of cytokines and
growth factors has been implicated in contributing to
end-organ changes that occur in tissues of patients with
diabetes[21]. At the same time, the interactions between AGE and
RAGE induce the activation of oxidative stress and
stimulate the production and release of cytokines, which amplify
tissue damage. Thus, oxidative stress and AGE interact
mutually and upregulate each other, which can lead to ECM
accumulation and mesangial cell hypertrophy. In our study, after
the DN rats were treated with GbE, the activities of T-AOC,
T-SOD, CAT, and GSH-Px (common indicators of changes in
the anti-oxidation system) all increased significantly, strongly
suggesting that GbE has a potent antioxidative
capability in vivo. Furthermore, we also found that
GbE, even in low doses, significantly decreased AGE levels both in the
kidney cortex and in the serum. These results are consistent
with a former report in which GbE inhibited oxidized low
density lipoprotein (LDL)-stimulated fibronectin production
through an antioxidant action in rat mesangial
cells[22]. These data strongly suggest that
GbE has the characteristics of antioxidant and anti-AGE, and this could be of benefit for
the prevention of DN.
Of the complicated mechanisms, the pathway of
hyperglycemia-oxidative stress TGF-β1-ECM is assuredly very
important. ROS are considered the activators of overall
signaling pathways. TGF-β1 is the key cytokine mediating
the production of ECM proteins. Hyperglycemic conditions
generate ROS. ROS and AGE interact with and upregulate
each other and activate the TGF-β1/Smad signaling
path-way[23]. Furthermore, ROS generated intracellularly from the
glucose metabolism, and the AGE-RAGE interaction also
activate PKC (together with DAG) and MAPK pathways. These,
together with activated Smads, coordinate the transcription
of a wave of genes, including angiotensinogen,
thrombo-spondin-1 (TSP-1), and CTGF. Angiotensin II (Ang (II)
stimulates further generation of ROS and the expression of
TGF-β1. More recently, Ang II blockade is rapidly becoming a
standard antifibrotic therapy in renal diseases because ACE
inhibitors block TGF-β1 induced by Ang
II[24]. In our study, we also found that an ACE inhibitor (captopril) decreased
the relative quantity of TGF-β1 mRNA and the level of CTGF.
CTGF is another prosclerotic cytokine and has also been
shown to be involved in both the early and later stages of
DN[25]. Secreted CTGF works in concert with
TGF-β1 activated by TSP-1 and ROS to transactivate subsequent waves
of genes, including those encoding structural proteins whose
accumulation leads to glomerulosclerosis in DN. It is
becoming clear that the coordinated expression 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 experiment, after the DN rats were treated
with GbE, the relative quantity TGF-β1 mRNA in the kidney
cortex of the DN rats decreased significantly, strongly
suggesting that GbE has an inhibitive effect on
TGF-β1 mRNA in the kidney cortex. This is consistent with a previous
report about the effect of GbE on
TGF-β1[26]. We also found that the expression of CTGF on
GbE-treated DN rats markedly decreased. Therefore, in this aspect,
GbE can be a prime candidate for the prevention and treatment of DN.
The ECM is a complex structure that influences the
behavior of its resident cells, and its accumulation correlates
closely with renal impairment in diabetes and ultimately leads
to glomerular scarring. Therefore, a balance between ECM
synthesis and degradation is a prerequisite for maintaining
the structural and functional integrity of the glomerulus.
MMP are a group of zinc dependent endopeptidases with
similar biochemical natures that are capable of degrading all
components of ECM and basement membranes. Thus, changes in the MMP expression or activity will directly
translate into altered ECM turnover. MMP-2 is the main MMP
responsible for the degradation of collagen IV.
It is secreted in an inactive form that becomes activated on the cell
surface by a membrane type 1 MMP. The activity of MMP-2 is
also regulated by specific tissue inhibitors of MMP
(TIMP-1 and TIMP-2), that were modulated directly and indirectly
by TGF-β1. So the balance between MMP and TIMP is
important in the fibrotic process. MMP is one of the main
proteinases that participates in the degradation of ECM. In
our study, the levels of MMP-2 and TIMP-2 in the DN group
conspicuously changed. The expression of MMP-2 greatly
decreased and TIMP-2 increased in the DN group. After
treatment with GbE, the level of MMP-2 increased and
TIMP-2 decreased. This result strongly suggests that
GbE has a potent inhibitory effect on the accumulation of
ECM.
For the complexity of the mechanisms of DN, it is
necessary to develop new drugs to deal with more than 1
pharmacological target. In our STZ_induced, early DN rat model,
we found that GbE reduced the blood glucose level,
decreased the level of AGE, the intensity of oxidative stress,
the level of TGF-β1 mRNA, TIMP-2, CTGF, increased
MMP-2, further lowered the levels of collagen IV and laminin in the
kidney cortex, and deceased the thickness of GBM,
and therefore ameliorated the morbidity in physical behavior and
morphology. Therefore, GbE has protective effects on
several pharmacological targets in the complicated pathology
mechanism of DN. This means that GbE may exert a
protective effect on the early development of DN in STZ-induced
diabetic rats.
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