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From the Departments of Geriatric
Medicine*
and Artificial
Kidneys,¶
Kyoto University, Kyoto, Japan; the
Discovery Research Laboratory,
Shionogi
Company, Limited, Osaka, Japan; the Department of
Science,
Tohoku University, Sendai, Japan;
the Department of Laboratory Medicine,||
Tokushima
University, Tokushima, Japan; and Amgen,§
Thousand Oaks, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gas6 is a vitamin K-dependent
growth-potentiating factor for smooth muscle cells16-19
and its activities depend on 
-carboxylation of glutamate residues at
its N terminus.20,21
Recently we demonstrated that Gas6
works as an autocrine growth factor for mesangial cells through its
cell surface receptor Axl.22
We have also shown that
warfarin and the extracellular domain of Axl conjugated with the Fc
portion of human IgG1 (Axl-Fc) inhibit mesangial cell proliferation by
interfering the Gas6/Axl pathway.22
These two inhibitors
exert their activities in different ways; warfarin inhibits mesangial
cell proliferation possibly by inhibiting -carboxylation of Gas6,
whereas Axl-Fc blocks the binding of Gas6 to the cell surface Axl by
scavenging Gas6. We have also shown the possibility that effective
concentrations of warfarin as an anti-proliferative agent are much
below those used as an anticoagulant.23
Therefore, one of
the aims of the current study is to confirm the in vivo role
of Gas6 and its receptor Axl, and to examine the effect of warfarin and
Axl-Fc as anti-proliferative agents in experimental mesangial
proliferative glomerulonephritis caused by the injection of anti-Thy1.1
antibody, Thy1 glomerulonephritis (Thy1 GN).24,25
We also
examined whether the expression of growth factors that stimulate
mesangial cell proliferation in Thy1 GN can be modulated when the
Gas6/Axl pathway is interfered.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wistar rats (8 to 12 weeks old, 180 to 200 g) were purchased from Shimizu Laboratory Animal Center (Hamamatsu, Japan). Rats were housed under specific pathogen-free conditions at the Animal Facilities of Kyoto University, Faculty of Medicine. All animal experiments were performed in accordance with institutional guidelines, and the Review Board of Kyoto University granted an ethical permission to this study.
Induction of Experimental Mesangial Proliferative Glomerulonephritis (Thy1 GN)
Thy1 GN was induced by a single intravenous injection of mouse anti-Thy1.1 monoclonal antibody OX-7 (1 mg/kg body weight; Cedarlane, Hornby, Canada) as described elsewhere.24,25 These rats were sacrificed to obtain renal specimens, total glomerular RNA, and protein at days 3, 5, 8, and 15 (n = 6 per group). Six rats were injected with vehicle only and sacrificed as controls.
Isolation of Glomeruli
Glomeruli were isolated from the renal cortex of rats using the differential thieving method.26,27 The purity of the glomeruli was >90%.
Northern Blot Analysis
Total RNA was extracted from rat glomerular preparations by Trizol (GibcoBRL, Paisley, Scotland). Glomerular denatured RNA (20 µg) was electrophoresed through formaldehyde 1% agarose gel, and transferred to a nylon membrane (Schleiecher & Schuell, Keene, NH) as previously described. The membranes were hybridized with the SalI/EcoRI 0.5-kb fragment of rat Gas6 cDNA, and the PstI/EcoRI 1.3-kb fragment of human PDGF-B cDNA (kindly provided by T. Collins, Boston, MA) radiolabeled with [32P]-dCTP (10 mCi/ml; Amersham Pharmacia Biotechnology, Buckinghamshire, UK) by random primer extension.26,27 All Northern blots were repeated at least three times with RNA from different sets of animals.
Western Blot Analysis
Isolated glomeruli were suspended in lysis buffer (0.1 ml of 50 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 0.5% Triton X, 1 mmol/L ethylenediaminetetraacetic acid, 10 µg/ml aprotinin, 1 mmol/L phenylmethyl sulfonyl fluoride) for 1 hour. After centrifugation of the samples, the supernatants were used as total cell lysates. Protein concentrations were measured by DC protein assay (Bio-Rad Laboratories, Richmond, CA). Sixty µg of each sample was applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to nitrocellulose filters (Schleiecher & Schuell). The blots were subsequently incubated with rabbit anti-Gas6 or anti-Axl polyclonal antibody, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham Pharmacia Biotechnology). The final reaction was developed by the chemiluminescent system (Amersham Pharmacia Biotechnology). All Western blots were repeated at least three times from different sets of animals.
Histological Examination
Kidney tissues from each animal were processed for evaluation by
light and immunofluorescence microscopy. For light microscopy, the
tissues were fixed in methyl Carnoy’s solution and were embedded in
paraffin. Sections (3 µm) were stained with periodic acid-Schiff. For
immunofluorescence microscopy, the tissues were snap-frozen in cold
acetone in OCT compound (Miles Inc., Elkhart, IN), and cryostat
sections (4 µm) were stained using indirect immunofluorescence
procedure with the following primary antibodies: rabbit polyclonal
antibodies against rat Gas6 (1:100 dilution),18,22,28
human Axl (1:100 dilution),29,30
proliferating cell
nuclear antigen (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz,
CA), rat PDGF-B (1:100 dilution) (Santa Cruz Biotec.), rat type I
collagen (1:100 dilution),31
rat type III collagen (1:100
dilution; Chemicon International, Inc., Temecula, CA), rat type IV
collagen (1:500 dilution),31
rat fibronectin (1:100
dilution; Calbiochem, La Jolla, CA), and rat laminin B2 (1:100
dilution)31
and goat polyclonal antibody against rat CD68
(1:100 dilution) (Santa Cruz Biotechnology). To detect mesangial cells
in glomeruli, sections were incubated with biotin-conjugated OX-7
(1:100 dilution) (Cedarlane), and treated with fluorescein
isothiocyanate-avidin (1:100 dilution; Cappel, Aurora, OH). Detection
of 
-smooth muscle actin (a marker of activated mesangial
cells)32
was done by a direct immunofluorescence procedure
with fluorescein isothiocyanate-conjugated monoclonal antibody
against -smooth muscle actin (1A4; Sigma Chemical Co., St. Louis,
MO). Specificity of the procedures was confirmed by substituting the
primary antibody with equivalent concentrations of irrelevant murine
monoclonal antibodies or normal rabbit/goat IgG.
Semiquantitative Histological Study
For quantitation of proliferating cells [proliferating cell nuclear antigen (PCNA)-positive cells], a blinded observer evaluated more than 15 consecutive cross sections of cortical glomeruli in each specimen and mean values per glomerulus were calculated. Glomerular expression of PDGF-B and type IV collagen was graded semiquantitatively at five levels: 0, very weak or absent staining; 1+, positive staining in <25% of the glomerular tuft; 2+, 25 to 49% of glomerular tuft; 3+, 50 to 75% of the glomerular tuft; and 4+, >75% of the glomerular tuft stained.33
Immunofluorescent Double Staining
To determine the localization of Gas6 and Axl, double
immunostaining for -smooth muscle actin and Gas6 or Axl was done.
Both primary antibodies (Gas6/Axl and 1A4) were incubated overnight at
4°C followed by the incubation with rhodamine-conjugated anti-rabbit
IgG (Chemicon International Inc.). Double immunostaining for CD68 and
PCNA was done in the same way. Specificity was confirmed by the
negative results when replacing either one of the primary antibodies
with irrelevant mouse monoclonal antibody or rabbit preimmune IgG.
Specimens were observed with a Zeiss microscope equipped with proper
filters.
Protocol of the Treatment with Warfarin in Thy1 GN
Dosage and time of administration of warfarin potassium (provided by Esai Co. Ltd., Tokyo, Japan) were determined based on the results of preliminary studies. When rats were administered with 0.25 and 0.5 mg/ml of warfarin in drinking water, the serum concentrations of warfarin gradually increased during the first 5 days, and reached a plateau value required to abrogate mesangial cell proliferation in vitro, previously described.22 In these concentrations in drinking water, no remarkable bleeding tendency or anemia was encountered. Based on these results, rats were treated with warfarin in drinking water (0, 0.25, or 0.5 mg/L) from 5 days before the initiation of Thy1 GN to the day of sacrifice. Rats were divided into three groups: a group without treatment, a group treated with 0.25 mg/L warfarin, and a group treated with 0.5 mg/L warfarin. In each group, rats were sacrificed at day 0, 3, 5, 8, and 15 (n = 6 for each group). Blood was collected at sacrifice and prothrombin times, hematocrits, and serum concentrations of warfarin were assessed as described.23 Before sacrifice, a 24-hour urine collection for creatinine and albumin measurement (Nephrat; Exocell Inc., Philadelphia, PA) was obtained from each rat as described previously.27
Treatment with Axl-Fc in Thy1 GN
A construct to fuse the extracellular domain of Axl and Fc portion of IgG1 was described previously.19 A control plasmid containing the Fc portion was made by ligating the 105-bp signal sequence of Axl and Fc portion directly. Expression vectors of Axl-Fc and Fc were transiently transfected into COS-7 cells and the culture supernatant was collected after 48 hours to purify recombinant Axl-Fc and Fc using Protein A agarose (Roche Diagnostics, Mannheim, Germany) as previously described.19 Purity of these recombinant proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two hundred µg of recombinant Axl-Fc or Fc was intravenously administered to rats (n = 6 for each group) once a day from 24 hours after the injection of anti-Thy 1.1 antibodies to day 7. In this experiment, rats were sacrificed at day 8, and a 24-hour urine collection was obtained before sacrifice as described.
Statistical Analysis
Statistical analyses of serum concentrations of warfarin, prothrombin times, and urinary albumin/creatinine index were done by Student’s t-test. Numbers of PCNA-positive cells per glomeruli, and grading of expression of PDGF-B and type IV collagen were analyzed by two-way repeated analysis of variance followed by the Fisher’s post hoc test. P values <0.01 were considered significant. Data are expressed as means ± SD. Analysis was performed by simple regression using StatView program (Abacus Concepts Inc., Barkeley, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Thy1 GN, proliferation of mesangial cell begins at day 2, peaks
at day 8, and subsides in 15 days after injection of the antibody.
First, to examine whether expression of Gas6 and Axl is correlated with
mesangial proliferation, glomerular expression of Gas6 and Axl in Thy1
GN was determined. Signal intensity of Northern blot is determined by
NIH image, and is normalized to 28S ribosomal RNA. Glomerular
expression of Gas6 mRNA at day 0 was very scarce, however, the
expression increased, peaking at day 8 (2.3-fold), and returned to the
basal level at day 15, when mesangial-cell proliferation subsided
(Figure 1A)
. Expression of Gas6 protein
also increased by 3.8-fold (at day 5) and 6.6-fold (at day 8) at
maximum, and returned to the basal level at day 15 (Figure 1B)
. Next,
we examined the glomerular expression of Axl. Two major immunoreactive
proteins of approximately 140 kd (full length) and 120 kd (splice
variant) were detected, which are compatible with our previous studies
in mesangial cells. Expression of Axl increased by 3.2-fold (day 5),
and 2.9-fold (day 8), and resolved at day 15 (Figure 1C)
. Next we
studied the localization of Gas6 and Axl by immunostaining. At day 0,
Gas6 was hardly detected in glomeruli (Figure 1D, a)
, however in the
glomerulus of day 8, Gas6 was extensively expressed in a typically
expanded mesangial pattern (Figure 1D, b)
. No significant staining was
detected in any sections treated with irrelevant antibody or anti-Gas6
antibody preincubated with an excess amount of recombinant Gas6 (data
not shown). Simultaneously, double immunostaining for Gas6
and -smooth muscle actin was done to determine whether activated
mesangial cells express Gas6. Figure 1D, d
, demonstrates that the
majority of Gas6-positive cells at day 8 expressed -smooth
muscle actin, indicating that Gas6 appears to be produced
predominantly by mesangial cells in this experimental model. A minor
portion of glomerular cells was Gas6-positive and -smooth muscle
actin-negative (arrows), and Gas6-negative and -smooth muscle
actin-positive cells (asterisks) at the hylus of glomerulus
seemed to be smooth muscle cells in the arteriole. Similar to the
results of Gas6, Axl was hardly detected in the glomeruli at day 0
(Figure 1E, a)
, but at day 8, glomerular cells were highly positive for
Axl (Figure 1E, b)
. No significant staining was detected in any
sections treated with irrelevant antibody or anti-Axl antibody
preincubated with an excess amount of recombinant Axl-Fc (data not
shown). Finally, double immunostaining for Axl and -smooth muscle
actin was performed. Figure 2E, d
,
demonstrates that the majority of Axl-positive cells at day 8
expressed -smooth muscle actin. A minor portion of glomerular
cells was positive for Axl and negative for -smooth muscle (arrows).
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Because expression of Gas6 and Axl was induced dramatically in
parallel with disease severity of Thy1 GN, the Gas6/Axl pathway seems
to play an important role in the development of glomerulonephritis.
Therefore, we examined whether inhibiting this pathway might be
beneficial in treating this experimental glomerulonephritis. We
administered warfarin in drinking water at various concentrations (0,
0.25, or 0.5 mg/ml). Serum concentrations of warfarin in these rats
were 0.28 ± 0.05 µmol/L (0.25 mg/L) and 1.23 ± 0.4
µmol/L (0.5 mg/L) (Table 1)
, which were
within the serum concentrations that inhibit mesangial cell
proliferation in vitro. Significant prolongation of
prothrombin times, anemia (Table 1)
, or bleeding tendency was not
observed in rats during the whole period of warfarin treatment.
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Expression of OX-7 was also reduced in glomeruli of Thy1 GN
treated with warfarin (Figure 2B, c)
. To examine the effect of warfarin
on glomerular cell proliferation, the number of PCNA-positive cells
were counted. The number of PCNA-positive cells in the glomeruli of
rats treated with warfarin was significantly reduced in a
dose-dependent manner at each point studied (Figure 2C)
. To examine the
participation of infiltrating macrophages in the number of
PCNA-positive cells per glomerulus, double immunostaining of PCNA and
CD68 was performed. The number of PCNA/CD68-positive cells was 0.03 +
0.18 at day 0, 0.30 + 0.46 at day 3, 0.90 + 0.53 at day 5, 1.83 + 0.63
at day 8, and 0.37 + 0.63 when treated with warfarin (0.5 mg/L). We
also examined the expression of extracellular matrix protein, such as
collagen type I, type III, type IV, fibronectin, and laminin B2. By
semiquantitave analysis, Thy1 rats had a substantial increase in
immunostaining of these extracellular matrix proteins at day 8, whereas
in the rats treated with warfarin, expression of these extracellular
matrix proteins was markedly decreased (69% reduction in collagen type
I, 78% in collagen type III, 75% in collagen type IV, 41% in
fibronectin, and 68% in laminin B2 with 0.5 mg/L of warfarin
treatment) (Figure 2D)
. These data indicate that the reduction of
glomerular cell proliferation was associated with a decrease in matrix
expansion. Finally, we assessed the urinary protein excretion. Figure 2E
shows that warfarin treatment significantly inhibited urinary
albumin excretion at day 8 in a dose-dependent manner. Axl-Fc Inhibits Glomerular Cell Proliferation in Vivo
Next, we examined the effect of another inhibitor of the Gas6/Axl
pathway, Axl-Fc in Thy1 GN. Axl-Fc is supposed to capture Gas6 and
block its binding to endogenous cell surface Axl. Rats were daily
injected with vehicle, Axl-Fc, or Fc for 24 hours after the
administration of anti-Thy1.1 antibody. A significant reduction of
mesangial cells and matrix expansion in the glomeruli of Thy1 rats was
observed when treated with Axl-Fc but not with Fc (Figure 3A)
. Differences in the expansion of
mesangial cells were further confirmed by immunostaining for
OX-7 (Figure 3B)
. Expression of OX-7 in glomeruli
was significantly reduced when treated with Axl-Fc, but not with Fc.
The number of PCNA-positive cells in the glomeruli of rats treated with
Axl-Fc was also significantly reduced (89%) (Figure 3C)
, but not with
Fc. These data indicate that Axl-Fc could inhibit mesangial cell
proliferation. Finally, we examined urinary protein excretion.
Treatment with Axl-Fc significantly inhibited urinary albumin excretion
at day 8, but not with Fc (Figure 3D)
.
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Finally, we tried to determine whether the inhibition of the
Gas6/Axl pathway could affect the expression of other growth factors,
such as PDGF-B that are known to play critical roles in Thy1
GN.6-8
Expression of PDGF-B mRNA was induced in Thy1 GN,
whereas the induction was abolished when treated with warfarin or
Axl-Fc (Figure 4A)
. Expression of PDGF-B
protein in glomeruli was also abolished in Thy1 GN treated with
warfarin or Axl-Fc (Figure 4B)
. Semiquantitation for localization for
PDGF-B was shown in Figure 4C
(62% reduction in warfarin treatment,
79% reduction in Axl-Fc treatment, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of Gas6 and Axl was dramatically increased in Thy1 GN in parallel with the degree of mesangial cell proliferation. Double-staining immunohistochemistry indicated that most part of Gas6 and Axl was co-localized mainly in mesangial cells. Thus, it is conceivable that Gas6 is secreted from mesangial cells to stimulate the mesangial cell surface receptor, Axl, in an autocrine and paracrine manner in vivo.
We have already shown that warfarin and Axl-Fc block the mesangial proliferation induced by Gas6 in vitro.22 We extended these in vitro results to in vivo experiments. We administered these two inhibitors of the Gas6/Axl pathway to Thy1 rats to examine their anti-proliferative effects. Both agents significantly inhibited glomerular cell proliferation demonstrated histologically, and by the number of PCNA-positive cells. Warfarin treatment also significantly suppressed extracellular matrix accumulation and urinary protein excretion. Therefore, we concluded that warfarin and Axl-Fc inhibited mesangial cell proliferation and attenuated glomerulonephritis in Thy1 GN by blocking the Gas6/Axl pathway.
Because warfarin is an inhibitor of -carboxylation,34
there is a possibility that warfarin might prevent -carboxylation of
unknown vitamin K-dependent growth factors other than Gas6. However,
because Axl-Fc is supposed to be a specific scavenger of Gas6, it may
well be that the Gas6/Axl pathway plays an important role in mesangial
cell proliferation in vivo.
One of the important findings in this study is a new aspect of warfarin as an anti-proliferative agent. Warfarin has long been used as an anticoagulant to prevent thrombosis and embolism,35,36 and patients prescribed this agent are monitored by measuring prolongation of prothrombin times to achieve its anticoagulant effect. These patients have to be monitored for the risk of bleeding.37 However, the anti-proliferative effect of warfarin was achieved at serum concentrations of 0.28 to 1.23 µmol/L, which were significantly lower than the ordinary therapeutic concentrations as an anticoagulant (4 to 5 µmol/L).23 The prothrombin times of rats treated with warfarin in our experiments were not significantly prolonged, and no bleeding tendency or anemia was observed, whereas mesangial cell proliferation was significantly reduced.
The benefit of restricting the amount of warfarin is not only to
prevent bleeding tendency, but also to reduce the incidence of
osteoporosis,38
because long-term prescription of warfarin
is known to cause osteoporosis by inhibiting -carboxylation of
matrix Gla proteins in the bone. Lower doses of warfarin are also
beneficial in terms of drug interaction, because warfarin is known to
affect drug absorption from the intestine.39
Despite our findings about the efficacy of warfarin on reducing mesangial cell proliferation, clinical studies have not provided sufficient evidence about the beneficial action of warfarin on glomerulonephritis.40-43 One of the reasons for this discrepancy might be that all studies so far have investigated the combination of warfarin with dipyridamole or even with cyclophosphamide. Further, in these studies, anticoagulant doses of warfarin are used except the recent report of Woo and colleagues44 regarding the benefit of low-dose warfarin on mesangial proliferative glomerulonephritis. Therefore, the adverse effect of warfarin might overcome its beneficial effect. Moreover, these studies include cases with advanced forms of glomerulonephritis with severe matrix expansion and glomerulosclerosis without active proliferation. An anti-proliferative agent might not be able to exert its effect in such cases. We speculate that low-dose warfarin should be prescribed at the earlier phase of mesangial cell proliferation to exert its effectiveness.
Our data strongly support a notion that Axl-Fc could be a candidate for therapeutic agents for glomerulonephritis. This fusion protein inhibited mesangial cell proliferation, reduced urinary protein excretion, and restored renal function possibly by inhibiting the binding of Gas6 to the cell surface Axl.28 Axl-Fc might be better anti-proliferative agent than warfarin, because it specifically inhibits the Gas6/Axl pathway with fewer side effects.
Despite the existence of many potent growth factors known to stimulate mesangial cell proliferation in Thy1 GN,4-12 inhibition of the Gas6/Axl pathway results in a significant reduction of mesangial cell proliferation. Therefore, we examined the expression of other growth factors in the glomeruli treated with warfarin/Axl-Fc. Blocking the Gas6/Axl pathway abolished the induction of PDGF-B mRNA and protein in Thy1 GN. Therefore, it is conceivable that Gas6 stimulates mesangial cell proliferation not only directly, but also by modulating the expression of other potent growth factors. However, reduced PDGF-B expression in the glomeruli treated with warfarin or Axl-Fc might be because of reduced mesangial cell contents. Therefore, research is now in progress to identify the regulatory mechanism by which Gas6 modulates PDGF-B production in mesangial cells.
In summary, we have presented several lines of evidence that the Gas6/Axl pathway plays a critical role in Thy1 GN, and agents that block this pathway would be potent inhibitors of mesangial cell proliferation in vivo. We also demonstrated that low-dose warfarin is effective in blocking mesangial proliferation and restoring renal function, without altering hemostasis. Finally, identification the Gas6/Axl pathway as a critical mediator of mesangial cell proliferation provides another target for the treatment of human glomerular diseases.
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Acknowledgements |
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Footnotes |
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Supported by Grants-in Aid from the Ministry of Education, Science, Sports, and Culture of Japan (04263104, 054040439, 0557052, 04304051, 08407026); the International Scientific Research Program grants 05044163, 07044254, and 09044293 from the Japanese Ministry of Education, Science, Sports, and Culture; a research grant for health sciences from the Japanese Ministry of Health and Welfare; and grants 5A-2 and A8-1 for cardiovascular diseases from the Japanese Ministry of Health and Welfare.
Accepted for publication January 12, 2001.
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