Introduction
Diabetic nephropathy (DN) is one of the most serious
micro-vascular complications of diabetes and the leading cause of
end-stage renal failure (1). The
early stage of DN is characterized by renal hypertrophy, glomerular
hypertrophy, glomerular hyperfiltration and microalbuminuria
(2). One of the characteristic
pathological changes in DN is the accumulation of extracellular
matrix (ECM) components, including collagens, fibronectin (FN) and
laminin in the glomeruli and the interstitium of the kidneys
(3). Hyperglycemia is usually
considered the main determinant factor of the initiation and
progression of DN, which increases the expression of transforming
growth factor-β1 (TGF-β1) (4).
The increase in TGF-β1 expression levels has been recognized as a
marker of DN, and TGF-β1 is the most potent growth factor
contributing to ECM accumulation (5). Nuclear factor-κB (NF-κB) is a
transcription factor which, under basal conditions, is sequestered
as an inactive form in the cytoplasm through its interaction with
the inhibitory protein, IκB. Under diabetic conditions, activated
NF-κB translocates to the nucleus and triggers the expression of
its target genes, including TGF-β1 and FN, further causing ECM
accumulation (6).
In recent years, interest in the biological
activities of marine organisms has intensified (7,8).
Fucoidan, an extract of the seaweed, Fucus vesiculosus,
whose chemical structure is presented in Fig. 1A, has been widely investigated as
an antioxidant, anticancer and anti-inflammatory agent, and has
been shown to play an important role in cancer and inflammation
(9,10). Thus, in the present study, we
sought to determine whether fucoidan prevents or impedes the
development of DN related to spontaneous diabetes.
The Goto-Kakizaki (GK) rat is a non-obese rat
substrain originally derived by the repeated inbreeding of
glucose-intolerant Wistar rats (11). It is a model of spontaneous and
moderate non-insulin-dependent diabetes. Between 3 and 4 weeks of
age, GK rats develop mild hyperglycemia and hyperinsulinemia. Thus,
GK rats were selected for use in the present study and Wistar rats
were used as the controls.
In the present study, to the best of our knowledge,
the protective effects of fucoidan against DN related to
spontaneous diabetes were examined in vitro and in
vivo for the first time. Our results revealed that fasting
blood glucose, blood urea nitrogen (BUN), serum creatinine (Cr),
urine protein and collagen Ⅳ levels, and the expression of TGF-β1
and FN, as well as the NF-κB p65 nuclear translocation all
significantly increased in the GK rats compared with the control
Wistar rats. These effects were all reversed in the GK rats which
were orally administered fucoidan. Our observations prove that
fucoidan prevents or impedes the development of DN related to
spontaneous diabetes by inhibiting the NF-κB signaling pathway,
suggesting that fucoidan may be a novel therapeutic agent for the
treatment of DN.
Materials and methods
Animals
The experiments were carried out at the Animal
Experimental Center of Harbin Medical University (Harbin, China).
Animal care and the protocols were in accordance with the Animal
Experiment Guidelines of Harbin Medical University and ethical
approval was obtained from the Harbin Medical University. Male GK
rats and Wistar rats were obtained from CLEA Japan, Inc. (Tokyo,
Japan). The rats were housed in a controlled environment at a
temperature of 24±1°C and under a 12-h light:dark lighting cycle
with the lights turned on at 7 a.m. At 6 weeks of age, the rats
were allowed free access to standard rat food and water with or
without the recommended concentrations of fucoidan (Sigma-Aldrich,
Shanghai, China; 50 and 75 mg/kg body weight), as previously
described (12) for 13 weeks.
Cell culture
Rats glomerular mesangial cells (GMCs) were
separated from the glomeruli of the GK rats and the control Wistar
rats and characterized as previously described (13). Briefly, primary cultures were
established from freshly isolated glomeruli, which were
mechanically sieved and harvested by iterative selection on
specific mesh sizes (200, 100 and final 80 μm). Those
retained on the sieve were collected and washed by centrifugation
at 1,000 rpm for 5 min, and incubated with 250 units/ml collagenase
(type I) for 30 min at 37°C. The GMCs were kept in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine
serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin
(Sigma-Aldrich) under standard conditions with 5% CO2 at
37°C (Sanyo, Tokyo, Japan) for experimental use.
Cytotoxicity assay
The CellTiter 96® AQueous One Solution
Cell Proliferation assay (Promega, Madison, WI, USA), which has
been reported to be an effective assay for determining cytotoxicity
and the number of viable cells (14), was used to determine the
cytotoxicity of fucoidan. GMCs from the Wistar rats were inoculated
at a concentration of 2×104 cells/well and incubated in
96-well plates with conditioned DMEM medium at 37°C under a 5%
CO2 atmosphere for 2 days. The GMCs were treated with
various concentrations of fucoidan (0, 10, 50, 100, 500 and 1,000
μg/ml) followed by a further 24 h of incubation.
Subsequently, 20 μl of the CellTiter 96 AQueous One Solution
Cell Proliferation assay solution was pipetted into all 96-wells
and the GMCs were further incubated for 1 h. The absorbance at 490
nm was measured using an MTP-800 microplate reader (Corona Electric
Co., Ltd., Ibaraki, Japan).
Measurement of fasting blood glucose,
BUN, serum Cr and urine protein levels
Fasting blood glucose was measured using a blood
glucose meter (OneTouch UltraEasy, Edina, MN, USA). The 24-h
urinary albumin levels were measured using an ELISA kit (R&D
Systems, Minneapolis, MN, USA). BUN and serum Cr levels were
determined using an automatic biochemistry analyzer (Olympus-2000;
Olympus, Tokyo, Japan).
Western blot analysis
Electrophoresis was performed using a vertical slab
gel with 12% polyacrylamide content according to a previously
described method (15). The
transfer of proteins from the SDS polyacrylamide gel to a membrane
was performed electrophoretically according to a previously
described method (16) with
certain modifications using a Semi-Dry Electroblotter (Sartorius
AG, Goettingen, Germany) for 90 min with an electric current of 15
V. The membrane was treated with Block Ace™ (4%) for 30 min at
22°C. The first reaction was performed using rabbit immunoglobulin
(IgG) antibodies against TGF-β1 (SAB4502954), FN (AV41490) and
NF-κB (SAB4502610; Sigma-Aldrich) in PBS containing 0.03% Tween-20
for 1 h at 22°C. Following washing in the same buffer, the second
reaction was performed using horseradish peroxidase
(HRP)-conjugated anti-rabbit goat IgG (20 ng/ml) for 30 min at
22°C. Following washing, the Enhanced chemiluminescence (ECL)
reaction was performed on the membrane using the ECL Plus Western
Blotting Detection System™ (GE Healthcare Life Sciences, Tokyo,
Japan).
Measurement of collagen IV levels
The kidney tissue was minced and a homogenate was
prepared with 10% phosphate-buffered saline (PBS; 0.1 mol/l, pH
7.4) using a homogenizer (UH-50; SMT Co., Ltd., Tokyo, Japan). The
kidney homogenate was centrifuged at 15,000 rpm for 15 min at 4°C.
The supernatant was collected and the quantity of type IV collagen
in the renal cortex was determined using an ELISA kit (R&D
Systems).
Histopathological analysis
For histopathological analysis, the kidneys from the
GK and Wistar rats were fixed in 10% neutral-buffered formalin and
subsequently embedded in paraffin. Sections (4-μm-thick) of
paraffin-embedded tissues were stained with hematoxylin and eosin
(H&E) solution for the determination of the histopathological
characteristics, as previously described (17). The cross-section yielding the
maximum diameter of the glomerulus was photographed and converted
into a digital image by an examiner blinded to the tissue source
using a light microscope equipped with a camera (Olympus BX-50;
Olympus Optical, Tokyo, Japan).
Laser scanning confocal microscopy
(LSCM)
GMCs from the kidneys of the GK and Wistar rats were
grown on glass coverslips. The GMCs were washed with PBS, fixed
with 4% paraformaldehyde in PBS for 20 min, and permeabilized with
0.1% Triton X-100 for 5 min at room temperature. After further
washing, the cells were blocked with 10% goat serum for 30 min at
room temperature. The cells were then incubated with anti-rat p65
(dilution 1:100) antibody (sc-8008; Santa Cruz Biotechnology, Inc.,
Dallas, TX, USA) at room temperature for 2 h followed by incubation
with the secondary antibody (Alexa Fluor® 488-conjuaged
goat anti-mouse IgG, dilution 1:1,000) (A-11029; Invitrogen,
Carlsbad, CA, USA) for 1 h. Nucleus were counterstained with
Hoechst 33342 solution (5 μg/ml in PBS) for 10 min. The
coverslips were mounted on glass lides with anti-fade mounting
medium (Invitrogen), and images were acquired using the Carl Zeiss
LSM 710 laser confocal fluorescence microscope (Carl Zeiss AG,
Jena, Germany).
Statistical analysis
Data are expressed as the means ± standard
deviation. Each experiment was repeated at least 3 times. The
Student’s t-test was used and a value of P<0.05 was considered
to indicate a statistically significant difference.
Results
Fucoidan is not cytotoxic to GMCs
GMCs separated from the Wistar rats were inoculated
at a concentration of 2×104 cells/well and incubated in
96-well plates with conditioned DMEM medium at 37°C under a 5%
CO2 atmosphere for 2 days. The GMCs were treated with
various concentrations of fucoidan (0, 10, 50, 100, 500 and 1,000
μg/ml) for 24 h, and then 20 μl of the CellTiter 96
AQueous One Solution Cell Proliferation assay solution was pipetted
into all 96-wells and the GMCs were further incubated for 1 h.
Fucoidan did not exert any obvious cytotoxic effects on the GMCs
(Fig. 1B).
Fucoidan decreases the high fasting blood
glucose, BUN, serum Cr and urine protein levels in the GK rats
Male GK and Wistar rats were housed in a controlled
environment at a temperature of 24±1°C and under a 12-h light:dark
lighting cycle with the lights turned on at 7 a.m. At 6 weeks of
age, the rats were allowed free access to standard rat food and
water with or without fucoidan (50 and 75 mg/kg body weight) for 13
weeks. Fasting blood glucose (Fig.
2A), BUN (Fig. 2B), serum Cr
(Fig. 2C) and urine protein
(Fig. 2D) levels were measured
using different methods, as described in the Materials and methods.
The fasting blood glucose, BUN, serum Cr and urine protein levels
were significantly increased in the GK rats (diabetes) compared
with the control Wistar rats (normal; P<0.01). The increased
fasting blood glucose, BUN, serum Cr and urine protein levels were
significantly decreased in the GK rats which were orally
administered fucoidan (50 mg/kg body weight, P<0.05; 75 mg/kg
body weight; P<0.01). There were no significant differences
observed between the Wistar rats treated with fucoidan and the
control (untreated) Wistar rats (data not shown).
 | Figre 2Fucoidan reduces the high fasting
blood glucose, blood urea nitrogen (BUN), serum creatinine (Cr) and
urine protein levels in Goto-Kakizaki (GK) rats. Male GK and Wistar
rats were allowed free access to standard rat food and water with
or without fucoidan (50 and 75 mg/kg body weight) from 6 weeks of
age for 13 weeks. Fasting blood glucose, BUN, serum Cr and urine
protein levels were measured using different methods as described
in the Materials and methods. (A) Fasting blood glucose, (B) BUN,
(C) serum Cr and (D) urine protein levels were significantly
increased in the GK rats compared with control Wistar rats. The
increased fasting blood glucose, BUN, serum Cr and urine protein
levels were significantly reduced in the GK rats which were orally
administered fucoidan. Data are expressed as the means ± standard
deviation (n=8). P<0.05 was considered to indicate a
statistically significant difference [**P<0.01, GK
rats (diabetes) vs. control Wistar rats (normal);
#P<0.05, GK rats administered fucoidan vs. untreated
GK rats; ##P<0.01, GK rats administered fucoidan vs.
untreated GK rats]. |
Fucoidan decreases the increased collagen
Ⅳ levels in the renal cortex of GK rats
Male GK and Wistar rats were housed in a controlled
environment at a temperature of 24±1°C and under a 12-h light:dark
lighting cycle and allowed free access to standard rat food and
water with or without fucoidan for 13 weeks from 6 weeks of age.
The collagen Ⅳ content in the renal cortex was significantly
increased in the GK rats (diabetes) compared with the control
Wistar rats (Fig. 3C, normal;
P<0.01). The increased collagen Ⅳ content was significantly
decreased in the GK rats which were orally administered fucoidan
(P<0.01).
Oral administration of fucoidan decreases
the expression of TGF-β1 and FN in the renal cortex of rats and
GMCs
At 6 weeks of age, male GK and Wistar rats were
housed in a controlled environment at a temperature of 24±1°C and
under a 12-h light:dark lighting cycle with the lights turned on at
7 a.m. and allowed free access to standard rat food and water with
or without fucoidan for 13 weeks. The expression levels of TGF-β1
(Fig. 3A) and FN (Fig. 3B) were determined by western blot
analysis. The expression levels of TGF-β1 and FN were significantly
increased in the GK rats (diabetes) compared with the control
Wistar rats (normal). The oral administration of fucoidan
significantly reduced the increased expression levels of TGF-β1 and
FN in the GK rats (P<0.01).
Similar effects were observed in our in vitro
experiments with the GMCs. The GMCs obtained from the GK rats
(diabetes) showed increased levels of TGF-β1 and FN compared with
the GMCs obtained from the Wistar rats (normal). However, in the
GMCs treated with fucoidan, these levels were decreased (Fig. 3D and E).
Fucoidan attenautes the histopathological
changes in the kidneys of GK rats
The kidneys from the GK and Wistar rats were fixed
in 10% neutral-buffered formalin and subsequently embedded in
paraffin. Sections (4-μm-thick) of paraffin-embedded tissues
were stained with H&E solution for the determination of the
histopathological characteristics. Vacuolation and the regeneration
of renal tubular epithelial cells, and inflammatory cell
infiltration in the renal interstitium were evident in the kidneys
from the GK rats (diabetes) compared with those from the control
Wistar rats (normal). Fucoidan significantly attenuated these
histopathological changes in the kidneys of GK rats (Fig. 4A).
Oral administration of fucoidan
attenuates the nuclear translocation of NF-κB p65 in GMCs
At 6 weeks of age, male GK and Wistar rats were
allowed free access to standard rat food and water with or without
fucoidan for 13 weeks, and the GMCs were then separated from the
glomeruli of the GK rats and the control Wistar rats. The results
of LSCM (Fig. 4B) and western
blot analysis (Fig. 5) revealed
that the expression of NF-κB was significantly increased in the
GMCs from the GK rats (diabetes) compared with the GMCs from the
control Wistar rats (control). The nuclear translocation of NF-κB
p65 was significantly attenuated by the oral administration of
fucoidan (Fig. 5B;
P<0.01).
Discussion
To the best of our knowledge, the present study
demonstrates in vitro and in vivo, for the first
time, that fucoidan reduces hyperglycemia and prevents or impedes
the development of DN related to spontaneous diabetes. DN is one of
the most serious microvascular complications of diabetes and the
leading cause of end-stage renal failure (1). The early stage of DN is
characterized by renal hypertrophy, glomerular hypertrophy,
glomerular hyperfiltration and microalbuminuria (2). These changes are related to the
subsequent development of glomerular morphological abnormalities
and the prognosis of DN (18).
Inflammation has emerged as a key pathophysiological mechanism for
DN (19). One of the
characteristic pathological changes in DN is the accumulation of
ECM components, including collagens, FN and laminin in the
glomeruli and the interstitium of the kidneys (3). Hyperglycemia is usually considered
as the main determinant factor of the initiation and progression of
DN which increases the expression of TGF-β1 (4). The increase in the TGF-β1 expression
level is recognized as a marker of DN and TGF-β1 is the most potent
growth factor contributing to ECM accumulation (5). In accordance with these data, the
present study confirmed that the expression of TGF-β1, and the
levels of FN and collagen Ⅳ were significantly increased in the GK
rats compared with the control Wistar rats (Fig. 3). These factors lead to the
acceleration of the pathogenesis of glomerulosclerosis and
tubulointerstitial fibrosis (20)
and an increase in BUN, serum Cr and urine protein levels (Fig. 2).
In recent years, interest in the biological
activities of marine organisms has intensified (7,8).
Fucoidan, an extract of the seaweed, Fucus vesiculosus, has
been widely investigated as an antioxidant, anticancer and
anti-inflammatory agent, and has been shown to play an important
role in cancer and inflammation (9,10).
Fucoidan can suppress various inflammatory cytokines, such as
interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α),
interferon-γ (IFN-γ) and cyclo-oxygenase-2 (COX-2) (21). In the present study, the effects
of fucoidan against DN related to spontaneous diabetes were
investigated in vitro and in vivo. Fucoidan did not
exert any cytotoxic effects on GMCs separated from Wistar rats, and
there were no significant differences observed between the Wistar
rats administered fucoidan and the control (untreated) Wistar rats
(data not shown). Following the oral administration of fucoidan for
13 weeks, the increased fasting blood glucose, BUN, serum Cr, urine
protein and collagen Ⅳ levels in the renal cortex, and the
expression levels of TGF-β1 and FN in the renal cortex and GMCs
from GK rats were all significantly reduced (Figs. 2 and 3). Histopathological analysis also
revealed that histopathological changes, such as vacuolation and
the regeneration of renal tubular epithelial cells, and
inflammatory cell infiltration into the renal interstitium in the
kidneys of GK rats were attenuated by the oral administration of
fucoidan (Fig. 4).
NF-κB is a transcription factor (6). The nuclear NF-κB family of
transcription factors regulates the induction and resolution of
inflammation. NF-κB regulates the expression of numerous genes that
play a key role in the inflammatory response during human and
experimental kidney injury (22).
It is believed that the activation of NF-κB and chronic
inflammation play pivotal roles in the pathogenesis of DN (23). Under basal conditions, NF-κB is
sequestered as an inactive form in the cytoplasm through its
interaction with the inhibitory protein, IκB (6). Under diabetic conditions, following
the phosphorylation and degradation of IκB-α, NF-κB p65
translocates to the nucleus and binds to a specific DNA sequence to
activate the transcription of target genes, including TGF-β1 and
FN, further causing ECM accumulation (24,25). To better understand the mechanisms
of action of fucoidan in the prevention of the development of DN
related to spontaneous diabetes, we investigated the nuclear
translocation of NF-κB p65. Our results revealed that the increased
NF-κB nuclear translocation and NF-κB p65 expression in the GMCs
were significantly attenuated in the GK rats orally administered
fucoidan (Figs. 4 and 5), suggesting that the NF-κB signaling
pathway plays an important role in the development of DN. However,
fucoidan attenuated the NF-κB nuclear translocation in the GMCs
from the GK rats, but did not completely block it, suggesting that
other mechanisms are involved in this process. It has been reported
that the NF-κB nuclear translocation is also regulated by reactive
oxygen species (ROS) and protein kinase C in GMCs, and
phosphoinositide 3-kinase has also reported to be involved in this
event (26). The complex process
and mechanisms involved require further investigation.
In the present study, to the best of our knowledge,
the protective effects of fucoidan against DN related to
spontaneous diabetes were determined in vitro and in
vivo for the first time. Our observations prove that fucoidan
prevents or impedes the development of DN related to spontaneous
diabetes by inhibiting the activation of the NF-κB signaling
pathway; thus, fucoidan may be considered as a novel therapeutic
agent for the treatment of DN.
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