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INTRODUCTION |
Diabetes is the most common cause of end stage renal disease in
many countries. Approximately 30% of type 1 diabetic patients suffer
from diabetic nephropathy (1, 2). Therefore, tremendous efforts have
been made to elucidate the molecular mechanism of diabetic nephropathy
to develop an effective treatment. The feature characteristic of
diabetic nephropathy is persistent albuminuria and mesangial expansion
followed by glomerulosclerosis and a decline in renal function. The
development of glomerulosclerosis in diabetes mellitus is always
preceded by the early hypertrophic processes in the glomerular
compartment (3). Because it is important to regulate the early stage of
the disease process, extensive efforts have been made to elucidate
growth factors and cytokines involved in mesangial expansion or
hypertrophy (4). Transforming growth factor-
1
(TGF-
1)1 and angiotensin
II are found to be implicated in the development of diabetic
nephropathy among them (5). Although angiotensin-converting enzyme
inhibitors and/or type I angiotensin receptor blockers are effective to
some extent, angiotensin-converting enzyme inhibitors can decrease the
risk of developing diabetic nephropathy by only 12.5% in type 2 diabetic patients (6). Therefore, additional pathogenetic mechanisms
are being urgently investigated to help design novel therapies for
patients with diabetic nephropathy.
Among other potential growth factors for glomerular cells, we have
investigated the role of Gas6 (growth arrest-specific gene 6) in the
pathogenesis of kidney disease. Gas6, cloned from serum-starved fibroblasts (7), is posttranslationally activated by
-carboxylation of glutamate residues at its N terminus in the presence of vitamin K
and inhibited by the anticoagulant warfarin (8, 9). Recently we showed
that Gas6 is an autocrine growth factor for mesangial cells and that
Gas6 and its receptor Axl play a critical role in the development of
glomerulonephritis by showing that warfarin and the extracellular
domain of Axl inhibit mesangial cell proliferation by specific blockade
of the Gas6-mediated pathway in a mesangial proliferative model of
glomerulonephritis (10, 11). However, the role of Gas6 and Axl in
diabetic nephropathy is not determined.
The present study is designed to examine whether Gas6 and Axl can
contribute to the pathogenesis of diabetic glomerular hypertrophy in vivo and in vitro. In this study, we
specifically asked whether Gas6 and Axl can play an important role in
mesangial cell hypertrophy, which is a feature seen in the early phase
of diabetic nephropathy and whether inhibition of the Gas6/Axl pathway
can affect the progression of diabetic nephropathy in streptozotocin
(STZ) rats and mice.
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EXPERIMENTAL PROCEDURES |
Materials--
STZ was obtained from Wako Pure Chemical Inc.
Ltd. (Osaka, Japan). Recombinant human TGF-
1 was purchased from R&D
Systems, Inc. (Minneapolis, MN).
Animals and Cell Culture--
Male Sprague-Dawley rats weighing
170-200 g were purchased from Shimizu Laboratory Animal center
(Hamamatsu, Japan). Gas6 knockout mice were generated with a targeted
disruption of the Gas6 gene as previously described (11).
Control inbred C57BL/6 mice were obtained from Clea Japan, Inc. (Osaka,
Japan). The animals were housed under specific pathogen-free conditions
at the Animal Facilities of Kyoto University, Faculty of Medicine. All
of the animal experiments were performed in accordance with
institutional guidelines, and the Review Board of Kyoto University
granted ethical permission to this study. The glomerular mesangial
primary culture was established from glomeruli isolated from normal
4-week-old mice (C57BL/6JxSJL/J) and was identified according to the
method previously described (12, 13). Phenotypically stable mesangial cells, 12th to 16th passages, were plated on 100-mm plastic dishes (Nalge Nunc International, Roskilde, Denmark) and maintained in growth
medium (3:1 mixture of Dulbecco's modified Eagle's medium/Ham's F-12
medium modified trace elements) (Nissui Pharmaceutical Co., Ltd.,
Tokyo, Japan) supplemented with 1 mM glutamine, penicillin at 100 units/ml, streptomycin at 100 µg/ml (Invitrogen) and 20% fetal bovine serum (Cansera International Inc., Rexdale, Canada).
STZ-induced Diabetic Rats and Mice--
Male rats weighing
170-200 g were made diabetic by a single intravenous injection of STZ
(55 mg/kg body weight) in 0.05 mol/liter citrate buffer (pH 4.5).
Weight-matched 8-week-old mice (17-20 g) were made diabetic by two
consecutive daily intraperitoneal injections of STZ (150 mg/kg)
dissolved in 0.01 mol/liter citrate buffer. Rats and mice receiving an
injection of citrate buffer were used as controls. The levels of blood
glucose were determined 2 days after injection of STZ or vehicle, and
rats and mice with blood glucose levels more than >16.7 mmol/liter
were used as diabetic (14, 15). Twelve weeks after STZ injection, the
rats and mice were weighed and sacrificed.
Warfarin Treatment--
The rats were divided into four groups:
control rats without treatment, control rats with warfarin treatment,
diabetic rats without treatment, and diabetic rats with warfarin
treatment. The rats with warfarin treatment were administered with 0.25 mg/liter warfarin potassium (provided by Eisai Co. Ltd., Tokyo, Japan) in drinking water from the day of STZ injection. The dosage of warfarin
was determined based on the previous report (10). Because diabetic rats
drink much more water, the dosage of warfarin was reduced to 0.06 mg/liter from 2 days after injection of STZ. Twelve weeks after
injection, the rats were weighed and sacrificed. Blood was collected at
sacrifice. Prothrombin times, hematocrits, serum creatinine, and plasma
concentrations of warfarin were measured as described (10). HbA1c was
measured using DCA2000 analyzer (Bayer Medical, Tokyo, Japan). Before
sacrifice, creatinine and albumin were measured from 24-h urine collection.
Immunohistochemistry--
Kidney tissues from each animal were
snap frozen in cold acetone in optimal cutting temperature
compound (Sakura Finetechnical Co. Ltd., Tokyo, Japan), and cryostat
sections (4 µm) were stained by indirect immunofluorescence procedure
with the following primary antibodies: rabbit polyclonal antibodies
against rat Gas6 (16) and human Axl (generous gift from Dr. Brian
Varnum, Amgen, Thousand Oaks, CA).
Isolation of Glomeruli--
Glomeruli were isolated from renal
cortices of rats using the differential sieving method (17, 18). The
purity of the glomeruli was >90%.
Western Blotting--
Isolated glomeruli were suspended in RIPA
buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1%
Nonidet P-40, 0.25% SDS, 1 mM
Na3VO4, 2 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml of aprotinine), and
incubated for 1 h at 4 °C. After centrifugation, the
supernatants were used as total cell lysates. 60 µg of each sample
was applied to SDS-PAGE. After electrophoresis, the proteins were
transferred to nitrocellulose filters (Schleicher & Schuell). The blots
were subsequently incubated with rabbit anti-rat Gas6, anti-human Axl, rabbit anti-phospho-p44/p42 mitogen-activated protein (MAP) kinase polyclonal antibody (Cell Signaling Technology, Beverly, MA), or rabbit
anti-MAP kinase polyclonal antibody (Oncogene, San Diego, CA), followed
by incubation with horseradish peroxidase-conjugated goat anti-rabbit
IgG (Amersham Biosciences). The immunoreactive bands were visualized
using horseradish peroxidase-conjugated secondary antibody and the
enhanced chemiluminescent system (Amersham Biosciences).
Histological Examination--
The mesangial cell area was
measured in a hematoxylin eosin staining section by Image-Pro Plus
(Media Cybernetics, Silver Spring, MD). For each animal, 50 mesangial
cell areas were analyzed. The glomerular surface area and the periodic
acid-methenamine-silver-positive area were determined using an image
analyzer (Image Processor for Analytical Pathology; Sumitomo Chemical
Co., Tokyo, Japan) (19, 20). For each animal, 50 glomeruli were analyzed.
Glomerular Filtration Rate (GFR) and the Measurement of Urinary
Albumin Excretion--
Urine volume (Vu) was
measured at 12 weeks by 24-h urine collection from rats housed in
individual metabolic cages. During the urine collection, the rats were
allowed free access to food and water. Serum and urine creatinine
concentrations (Cp and
Cu) were measured, and GFR was calculated by the
following equation: GFR = (Cu/Cp) × Vu/body weight (21). The albumin concentration in the urine was measured by Nephrat (Exocell Inc., Philadelphia, PA).
[3H]Leucine Incorporation and Determination of Cell
Number--
Mesangial cells were plated at 1.5 × 104
cells/well in 24-well dishes. After 48 h, the cells were
serum-starved in Dulbecco's modified Eagle's medium containing 0.5%
bovine serum albumin (Sigma-Aldrich) for 48 h. The medium was then
replaced with the fresh starving medium including various
concentrations of agonist or left untreated. After 18 h, the cells
were labeled with [3H]leucine (2 µCi/ml; Amersham
Biosciences) for 6 h, and the incorporation of
[3H]leucine into acid-precipitable materials was then
determined. For determination of cell number, the cells were
trypsinized, resuspended in phosphate-buffered saline, and counted with
a Coulter counter Z1 (Coulter Electronics Ltd., Hialeah, FL). The data
were normalized by dividing incorporation counts by the cell number and
showed as fold increases over control.
Flow Cytometry--
Mesangial cells were plated at 4.5 × 105 cells/well in 100-mm plates. The cells were treated as
the [3H]leucine incorporation procedure. After treatment,
mesangial cells were harvested by tripsinization, washed with
phosphate-buffered saline, centrifuged at 1,500 rpm for 10 min, and
then resuspended in ice-cold 70% ethanol added dropwise while
vortexing. Ethanol-fixed mesangial cells were then analyzed by forward
light scattering on a Becton Dickinson flow cytometer (BD Biosciences,
San Jose, CA).
Statistical Analyses--
The data are expressed as the
means ± S.D. Comparison among each group was performed by one-way
analysis of variance followed by Neuman-Keuls test to evaluate the
statistical significance between two groups. A p value of
<0.05 was considered to be significant.
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RESULTS |
Expression of Gas6 and Axl in STZ-induced Diabetic Rats--
Our
preliminary data showed that glomerular hypertrophy and an increase in
GFR and albuminuria were observed after 12 weeks of STZ injection in
rats. Therefore, to examine the role of Gas6/Axl in the early phase of
diabetic nephropathy in vivo, we used STZ-induced diabetic
rat kidney. We analyzed the glomerulus after 12 weeks of STZ injection
and found that expression of both Gas6 and Axl was significantly
increased in the STZ-treated group and that they were mostly localized
at endothelial and mesangial cells (Fig.
1).

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Fig. 1.
Expression of Gas6 and Axl in STZ-induced
diabetic rat kidney. Kidney tissues from each animal were snap
frozen in cold acetone in OCT compound. The cryostat sections (4 µm) were stained using indirect immunofluorescence procedure with
anti-Gas6 or anti-Axl antibody. The original magnification was
×400.
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Warfarin Treatment Inhibits Induced Expression of Axl and
Phosphorylation of p44/42 MAP Kinase--
Because
expression of Gas6 and Axl was induced in diabetic rats, the Gas6/Axl
pathway seems to play a role in the development of diabetic
nephropathy in the early phase of the disease process. Therefore,
we next examined whether inhibiting this pathway can be effective in
treating this experimental diabetic nephropathy. We treated rats with
warfarin in drinking water as shown in Fig. 2. Plasma concentrations of warfarin in
these rats were 0.71 ± 0.05 and 0.67 ± 0.05 µM, which were significantly lower than the ordinary
therapeutic concentrations as an anticoagulant. The body weight and
kidney weight/body weight values were not changed by warfarin
treatment. Significant prolongation of prothrombin times, anemia, or
bleeding tendency was not observed in all the rats during the whole
period of warfarin treatment as we already found in our previous study
(data not shown).

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Fig. 2.
Protocol of warfarin treatment
(A) and physiological characteristics of control and
diabetic rats with or without warfarin treatment after 12 weeks
(B). A single intravenous injection of 55 mg/kg
of STZ was performed on day 1 to make diabetic rats. The rats were
separated into control and diabetic groups with or without warfarin
treatment. Rats with warfarin treatment (Wa) were
administered with 0.25 mg/liter warfarin in drinking water. Because
diabetic rats drink much more water, the dosage of warfarin was reduced
to 0.06 mg/liter from 2 days after injection of STZ. B,
after 12 weeks of STZ injection, the rats were weighed and sacrificed.
Blood was taken to evaluate HbA1c and plasma concentrations of
warfarin. Kidney weight was also measured.
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After 12 weeks of STZ injection, we isolated glomeruli from the rats
and found increased glomerular expression of Gas6 and Axl by Western
blotting (Fig. 3) as shown in Fig. 1.
When we treated STZ rats with warfarin, we found that the expression of
Axl was markedly inhibited in warfarin-treated STZ rats than untreated STZ rats. Although warfarin treatment did not affect Gas6 expression, it might be due to the fact that the antibody used for Western blotting
cannot discriminate active or inactive Gas6.

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Fig. 3.
Effect of warfarin on the expression of Gas6
and Axl and expression and phosphorylation of p44/42 MAP kinase in STZ
rat glomeruli. After 12 weeks of STZ injection, the rats were
sacrificed, and glomeruli were isolated by a sieving method. The
isolated glomeruli were suspended in RIPA buffer. After centrifugation,
the supernatants were used as total cell lysates. 60 µg of each
sample was analyzed by Western blotting with the antibodies indicated.
Each lane represents a representative Western blot for the cell
lysate from each rat. Wa, warfarin treatment;
Cont, control.
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Because we have shown that Gas6 can activate p44/42 MAP kinase in
vitro (16), we examined whether p44/42 MAP kinase can be
phosphorylated in diabetic glomerular lysates and whether warfarin treatment can affect the phosphorylation. As shown in Fig. 3, p44/42
MAP kinase was phosphorylated in the glomerular lysates in STZ rats,
and warfarin treatment abolished their phosphorylation.
Warfarin Shows a Beneficial Effect on Mesangial and Glomerular
Hypertrophy--
Because glomerular hypertrophy is one of the earliest
structural alterations in diabetic nephropathy, we measured mesangial cell and glomerular surface areas in diabetic rat kidney and examined the effect of warfarin on glomerular hypertrophy. After 12 weeks of STZ
injection, both areas were significantly enlarged compared with control
rats, and administration of warfarin prevented the increase of
mesangial and glomerular areas (Fig. 4).
Because accumulation of mesangial extracellular matrix components is
also an early structural change in diabetic nephropathy, we also
measured the periodic acid-methenamine-silver-positive area in both
groups. However, there was no change in the periodic
acid-methenamine-silver-positive area between control and diabetic
groups, indicating that there is no glomerular sclerotic change after
12 weeks of STZ injection (data not shown).

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Fig. 4.
Effect of warfarin on mesangial
(A) and glomerular hypertrophy
(B). The mesangial cell area was measured in a
hematoxylin eosin staining section by Image-Pro Plus. For each animal,
50 mesangial cell areas were calculated. The glomerular surface area
was determined using an image analyzer. The data are expressed as the
means ± S.D. (n = 6 in the control group and
n = 10 in the diabetic group). *, p < 0.01; **, p < 0.05.
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Warfarin Treatment Improves Hyperfiltration and Excretion of
Urinary Albumin--
In the early phase of diabetic nephropathy, GFR
is increased in most of diabetic patients. Therefore, we examined
whether GFR is increased in STZ rats and whether warfarin treatment can affect GFR. After 12 weeks of STZ injection, GFR and urinary albumin excretion were significantly increased, and the increased GFR and
albuminuria were suppressed by warfarin treatment (Fig.
5).

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Fig. 5.
Effect of warfarin on hyperfiltration
(A) and urinary albumin excretion
(B). Urine volume was measured at 12 weeks from
24-h urine collection. Serum and urine creatinine concentrations were
measured, and GFR was calculated by an equation described under
"Experimental Procedures." Albumin concentrations in the
urine were measured by Nephrat. The data are expressed as the
means ± S.D. (n = 6 in the control group and
n = 10 in the diabetic group). *, p < 0.01; **, p < 0.05.
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STZ-treated Gas6 Knockout Mice Showed Less Glomerular
Hypertrophy--
To confirm the specificity of warfarin on the
Gas6/Axl pathway, we used STZ-treated Gas6 knockout mice. Our
preliminary data showed that glomerular hypertrophy was observed after
12 weeks of STZ injection in mice. Therefore, we analyzed mesangial and glomerular hypertrophy in Gas6 knockout and wild type mice after 12 weeks of STZ injection. As shown in Fig.
6A, Gas6 knockout mice were
smaller than wild type mice, and HbA1c was higher in Gas6 knockout mice
than in wild type mice. However, the blood glucose levels were almost
the same throughout the study period (data not shown). Although the
kidney weight/body weight was smaller in diabetic Gas6 knockout mice
compared with diabetic wild type mice, there was no statistically
significant difference. As shown in Fig. 6 (B and
C), mesangial cell and glomerular surface areas in
diabetic wild type mice were significantly larger than those in wild
type untreated mice. However, in diabetic Gas6 knockout mice, the
increase of both areas was significantly suppressed. These data also
indicate that Gas6 is involved in the development of the initial phase
of diabetic nephropathy and suggest that warfarin inhibits diabetic
nephropathy specifically through the Gas6-mediated pathway.

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Fig. 6.
Effect of Gas6 deficiency on mesangial and
glomerular hypertrophy in STZ-treated mice. Diabetes was induced
by two consecutive daily intraperitoneal injections of STZ (150 mg/kg).
Wild type (WT) and Gas6 knockout mice (KO) (12 each) were divided into two groups: untreated (Control) or
STZ-treated (Diabetes). Twelve weeks after STZ injection,
HbA1c, body weight, and kidney weight were measured (A).
Mesangial cell (B) and glomerular surface areas
(C) were also measured as described under "Experimental
Procedures." For each mouse, 50 mesangial cell and glomerular surface
areas were calculated. The data are expressed as the means ± S.D.
(n = 6 in each group). *, p < 0.01;
**, p < 0.05.
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Gas6 Induces Mesangial Cell Hypertrophy in Vitro--
To
investigate the mechanism by which Gas6 is involved in glomerular
hypertrophy in diabetic rats, we examined whether Gas6 can cause
mesangial cell hypertrophy in vitro. We measured
[3H]leucine incorporation in mouse mesangial cells as a
marker of cellular hypertrophy after incubation with various
concentrations of Gas6. Recombinant Gas6 increased incorporation of
[3H]leucine incorporation dose-dependently,
with a 1.5-fold increase at maximum (Fig.
7). The same dose of Gla-defective Gas6,
which is an inactive form of Gas6 without
-carboxylation, did not
affect [3H]leucine incorporation in mouse mesangial
cells. The Gas6-mediated increase in [3H]leucine
incorporation was almost the same as that of TGF-
1 (1 ng/ml). To
clarify whether mesangial cell hypertrophy is mediated specifically
through the Gas6-Axl pathway, we used the recombinant extracellular
domain of Axl (Axl-Fc), which is a recombinant fusion protein of the
extracellular domain of Axl and human Fc portion, as an inhibitor of
the Gas6-Axl pathway. After preincubation with 10 nM Axl-Fc
in starving medium for 1 h, Gas6 (100 ng/ml) was added to the
serum-starved mesangial cells, and [3H]leucine
incorporation was then measured. The addition of Axl-Fc inhibited the
increased [3H]leucine incorporation by Gas6, suggesting
that the effect of Gas6 on hypertrophy is specific for the Gas6-Axl
interaction. Further, we checked the cellular size of mesangial cells
by flow cytometry under the same protocol as [3H]leucine
incorporation (Fig. 8). We found that
treatment of the cells with Gas6 100 ng/ml or TGF-
1 increased the
cellular size by 1.1-fold but not Gla-defective Gas6.

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Fig. 7.
[3H]Leucine incorporation in
mouse mesangial cells by Gas6. Mesangial cells were plated at
1.5 × 104 cells/well in 24-well dishes. After 48 h, the cells were serum-starved in Dulbecco's modified Eagle's medium
containing 0.5% bovine serum albumin for 48 h. Then the medium
was replaced with the fresh starving medium including various
concentrations of agonist or left untreated. After 18 h, the cells
were labeled with [3H]leucine (2 µCi/ml) for 6 h,
and the incorporation of [3H]leucine into
acid-precipitable materials was determined. The data were normalized by
dividing incorporated counts with cell number and showed as fold
increases over control. The values expressed are the means ± S.D.
of six independent experiments. *, p < 0.01.
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Fig. 8.
Flow cytometric analysis of mesangial cell
size. The cells were treated as described in Fig. 7. After
treatment, the mesangial cells were harvested by tripsinization, washed
with phosphate-buffered saline, centrifuged at 1,500 rpm for 10 min,
and then resuspend in ice-cold 70% ethanol. Ethanol-fixed mesangial
cells were then analyzed by forward light scattering on a Becton
Dickinson flow cytometer. The data are representative of six
independent experiments with qualitatively similar changes. Thin
line, control (Cont); bold line, agonist.
A, 100 ng/ml of Gas6; B, 100 ng/ml of
Gla-defective Gas6; C, 1 ng/ml of TGF- 1. D,
means of forward scatter of mesangial cells after treatment. The data
are shown as fold increases over control. The values are the
means ± S.D. of six independent experiments. **,
p < 0.05. FSC, forward scatter.
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DISCUSSION |
In this study, we have shown a novel mechanism of mesangial
hypertrophy in diabetic nephropathy mediated by Gas6. This is the first
demonstration that Gas6 can induce mesangial cell hypertrophy characteristic of the early stage of diabetic nephropathy and that
warfarin is effective to prevent the progression of diabetic nephropathy. Our study implies that Gas6 can be a novel growth factor
that plays a crucial role in the development of the initial phase of
diabetic nephropathy.
Here we have found a novel aspect of warfarin as an anti-hypertrophic
agent. Our data also show that warfarin treatment ameliorated hyperfiltration and urinary albumin excretion in STZ rats. Thus hypertrophy and hyperfiltration might be an interactive mechanism and
presumed to be causally linked (22). It is conceivable, therefore, that
blocking the Gas6/Axl pathway can improve the vicious cycle in diabetic
nephropathy. Therefore, treating diabetic patients with warfarin to
prevent the nephropathy would be one of the options for treatment.
However, the side effect of warfarin should be noted if we treat
diabetic patients with warfarin. Warfarin has long been used as an
anticoagulant to prevent thrombosis and embolism (23, 24), and patients
prescribed with this agent are monitored by measuring prolongation of
prothrombin times to achieve its anticoagulant effect. These patients
have to be treated for the risk of bleeding (25). However, the
anti-hypertrophic effect of warfarin was achieved at serum
concentrations of 0.7 µM, which is significantly lower
than the ordinary therapeutic concentrations as an anticoagulant (4-5
µM) (26). The prothrombin times of rats treated with
warfarin in our experiments were not significantly prolonged, and no
bleeding tendency or anemia was observed (data not shown), whereas
mesangial cell hypertrophy was significantly inhibited. Although we
have shown the clear effect of warfarin on the development of diabetic
nephropathy, the question remains about the specificity of the effect
of warfarin. We have already shown that in mesangial cells warfarin
specifically inhibits the Gas6/Axl pathway in vitro (16). To
further confirm the specificity, we have treated Gas6-deficient mice
(11) with STZ (15) and found that both mesangial and glomerular areas were significantly decreased in Gas6-deficient mice compared with wild
type mice. Therefore, we can conclude that this effect of warfarin
would be mediated specifically through the inhibition of Gas6.
Hypercoagulability in glomeruli has also been reported in diabetic
nephropathy (27), which might worsen the renal function. Recently,
Angelillo-Scherrer et al. (28) reported that a deficiency of
Gas6 protects mice from thrombosis. In this study, we used a low dose
of warfarin, and at these concentrations we found no prolongation of
prothrombin time (data not shown). However, we already reported that
even under these concentrations, warfarin can inhibit the activation of
Gas6 in vitro and in vivo (10, 16, 29). It is
still possible that warfarin could affect the coagulation cascades and
prevent thrombotic events even at low concentrations. Therefore, Gas6
might affect the development of diabetic renal disease by improving the
coagulation state.
The increase of extracellular matrix followed by mesangial cell
hypertrophy is one of the major characteristics in diabetic nephropathy
(30). Mauer et al. (31) investigated the
structural-functional relationship in a cross-section of patients with
type 1 diabetes. They found a close correlation between mesangial
expansion and clinical manifestations of diabetic nephropathy. Ziyadeh
(3) also showed that the development of irreversible renal changes in
diabetes mellitus, such as glomerulosclerosis, is always preceded by
the early hypertrophic processes in the glomerular compartment. In our
experiment, however, we could not find an excessive accumulation of
extracellular matrix in STZ rats at 12 weeks, although other investigators have reported increased deposition of extracellular matrix in a later phase of nephropathy in STZ rats (32). Because our
purpose in this study is to determine the role of Gas6 in the initial
phase of diabetic nephropathy, we analyzed up to 12 weeks after STZ
injection. The correlation between Gas6 and diabetic glomerulosclerosis
should be evaluated by treating the rats with warfarin for a longer
time or using another animal model. Treating STZ rats with a high
protein diet would be another way to accelerate the glomerulosclerosis
(33) and should be tested in the future experiments.
We have already shown that Gas6 is a growth factor for mesangial cells
in vitro and that Gas6 plays a key role in acute and chronic
forms of glomerulonephritis in vivo (10, 16, 29). In those
studies, we have shown that Gas6 can induce mesangial proliferation
through a tyrosine kinase, Axl (11, 16) and a transcription factor
signal transducer and activator of transcription 3 (29). However, in
this study we have clearly shown that Gas6 can induce mesangial
hypertrophy in vivo and in vitro. Therefore, the
obvious question is why Gas6 only induces mesangial hypertrophy without
affecting the mesangial proliferation in this diabetic rat model.
Although we determined the mesangial cell area in the kidney of
diabetic rats, we did not notice that the glomerular cell number was
increased in diabetic rats (data not shown). Although Young et
al. (34) reported that diabetic nephropathy may be associated with
some glomerular cell proliferation, hypertrophy is the major finding of
diabetic nephropathy. It remains uncertain why Gas6 can be induced in
both glomerulonephritis and diabetic nephropathy and why Gas6 does not
induce cell proliferation in diabetic nephropathy. It is conceivable
that some other growth factor or cytokine is playing an additional role
in determining the fate of mesangial cell in the disease process.
We have clearly shown that p42/44 MAP kinase was phosphorylated in the
glomeruli after 12 weeks of STZ injection and that warfarin treatment
abolished the phosphorylation. In the case of insulin-like growth
factor 1, Akt seems to be responsible for its hypertrophic effect in
skeletal myotube (35), and endothelin-induced hypertrophy requires
activation of p42/44 MAP kinase, c-Jun N-terminal kinase/stress-activated protein kinase, and phosphatidylinositol 3-kinase pathways (36). Although we have no definite evidence to
indicate the role of p42/44 MAP kinase in mesangial cell hypertrophy so
far, phosphorylation of p42/44 MAP kinase might be used as a marker for
the hypertrophy in diabetic nephropathy. The molecular mechanism of
mesangial cell hypertrophy in diabetic nephropathy should be further
clarified in future studies.
In summary, this is the first demonstration that Gas6 and Axl are
involved in the development of the initial phase of diabetic nephropathy by inducing mesangial cell hypertrophy. This is a completely novel mechanism explaining the development of diabetic nephropathy. Blocking this pathway would be beneficial to prevent the
progression of nephropathy in diabetic patients.