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From the Department of Pathology, Nippon Medical School, Tokyo, Japan
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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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Recent studies of experimental GN indicate that after glomerular capillary destruction, a complete capillary repair in damaged glomeruli leads to full recovery of the glomerular architecture.4-6 However, impairment of capillary repair process in glomerular damage could be associated with the development of glomerular sclerosis and renal dysfunction.7 In the recovery model of Thy-1 GN, repair of glomerular capillary network with endothelial cell proliferation is associated with up-regulated expression of vascular endothelial growth factor (VEGF) and its receptor, flk.4 In addition, systemic administration of VEGF can mediate glomerular endothelial cell proliferation in an experimental rat model of thrombotic microangiopathy.8 On the other hand, in the recovery model of Thy-1 GN, administration of VEGF165 antagonist significantly reduced glomerular endothelial cell proliferation and inhibited glomerular capillary repair, leading to glomerular sclerosis.9 These findings demonstrate that capillary repair is a crucial event in the recovery from glomerular damage accompanied by destruction of glomerular capillary network, and indicate that VEGF plays an important role in endothelial cell proliferation and capillary repair in damaged glomeruli.
However, the changes of VEGF production and alteration of glomerular capillaries in the development of glomerular sclerosis in GN have not been fully investigated. In the present study, we determine the serial changes in VEGF production in damaged glomeruli, alteration of glomerular capillaries, development of glomerular inflammation, and progression to glomerular sclerosis after severe glomerular destruction in experimental GN. In addition, we examined the potential beneficiary effects of VEGF165 in glomerular repair and resolution of GN. Acute GN with severe glomerular destruction was induced in rats by injection of both anti-Thy-1.1 antibody and Habu-snake venom, and is termed here as Thy-1/Habu-snake venom GN. Injection of anti-Thy-1.1 antibody induces diffuse mesangial cell lysis,10,11 and administration of Habu-snake venom, which has a strong proteolytic enzyme activity, mainly induces mesangial matrix lysis.2 Because mesangial cells and matrix provide a strong support for glomerular capillaries,12 injection of both anti-Thy-1.1 antibody and Habu-snake venom induces marked mesangiolysis with extremely severe capillary destruction of glomeruli, leading to the development of GN with irreversible glomerular damage and chronic renal failure.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal experiments described in the present study were approved by
the Ethics Review Committee for Animal Experimentation of Nippon
Medical School. Male Wistar rats (Sankyo Laboratory, Tokyo, Japan)
weighing 180 g were used for all experiments. Thy-1/Habu-snake
venom GN was induced by injection of both monoclonal anti-Thy 1.1
antibody (OX7; Cedarlane Laboratories, Ontario, Canada) at a dose of 60
µg IgG/100 g body wt on day 0 and Habu-snake venom
(Trimeresurus flavoviridis; Wako Pure Chemical
Industries, Osaka, Japan) at a dose of 75 µg/100 g body weight on day
1. Rats were then treated with VEGF165 (Gibco
BRL, Tokyo, Japan) dissolved in saline (VEGF-treated group) or saline
alone (control group) injections. We have demonstrated that, in the
recovery models of Thy-1 or Habu-snake venom GN, glomerular capillary
regeneration in the early phase after injury, before occurrence of
massive mesangial cell proliferation, is necessary to complete
glomerular repair.5,6
Rats, therefore, were administered
by VEGF165 or vehicle using intraperitoneal
micro-osmotic pumps (Alzet osmotic pump, model 2ML1; Alza, Mountain
View, CA) starting at day 2 and ending at day 9 (1 week). The pumps
were filled with VEGF165 (140 µg/2 ml saline)
and had a delivery time of 7 days, therefore,
VEGF165 delivery was calculated as 
10 µg/100
g body weight/day. In each group, five rats were biopsied or sacrificed
on days 0, 2, 5, 7, and 9, and 2, 3, 4, 6, and 8 weeks after the
disease induction. To estimate renal function, urine and blood samples
were collected for measurement of urinary protein, plasma creatinine,
and blood urea nitrogen using an autoanalyzer (SRL, Tokyo, Japan).
Histopathological and Immunohistochemical Examination
After removal of the kidney, renal tissues were fixed in 20% buffered formalin and embedded in paraffin for light microscopic examination. Tissues were stained with hematoxylin and eosin, periodic acid-Schiff (PAS), and periodic acid-methenamine silver for histopathological examination. The naphthol AS-D chloroacetate esterase staining was performed to detect infiltrating neutrophils.
For immunohistochemistry, 20% buffered formalin-fixed
paraffin-embedded tissue sections were used, and the specimens were
stained by the standard avidin-biotin-peroxidase complex technique. The
following antibodies were used for immunohistochemistry. 1) Polyclonal
rabbit anti-rat thrombomodulin (TM) antibody13
(kindly
provided by Dr. David Stern, Columbia University, New York, NY), which
reacts with the surface of endothelial cells. This antibody has been
used as a marker for endothelial cells.5-7,13
Unfortunately, this antibody sometimes may detect free form of TM in
glomerular sclerosis or glomerular proliferative areas, we, therefore,
determined glomerular endothelial cells carefully, using not only
expression of TM but also its location and morphology. In addition,
endothelial TM expression alters in renal disorders. Confirmation of
the glomerular endothelial cells displayed by TM was performed by
staining tissue with the antibody against a surface antigen expressed
on all rat endothelial cells (RECA-1)4
or Griffonia
(Baneirare) simplicifolia lectin.14
The results
demonstrated similar endothelial cell patterns in glomeruli; neither
was comparable in quality or specificity to TM (data not shown). This
indicates that loss of TM activity in this model reflects a loss of
glomerular endothelial cells, confirming data of a previous
study.5-7
2) Monoclonal mouse anti-human 
-smooth
muscle actin (-SMA) antibody (1A4; DAKO, Glostrup, Denmark), which
is a marker for activated mesangial cells. 3) Monoclonal mouse
anti-proliferating cell nuclear antigen (PCNA) antibody (PC10, DAKO),
which is a marker for cellular proliferation. 4) Polyclonal goat
anti-type IV collagen antibody (Southern Biotechnology Associates,
Birmingham, AL), which is used for evaluation of mesangial matrix
accumulation and glomerular sclerosis. 5) Polyclonal rabbit anti-VEGF
antibody,15
which can detect VEGF-producing cells. 6)
Monoclonal mouse anti-flk-1 antibody (A-3; Santa Cruz Biotechnology,
Santa Cruz, CA), which can detect cells that express VEGF receptor. 7)
Polyclonal rabbit anti-human CD3 antibody (DAKO), which can detect
infiltrating rat T cells. 8) Monoclonal mouse anti-rat ED1 antibody
(BMA, Augst, Switzerland), which can detect infiltrating macrophages.
For TM, type IV collagen, VEGF, flk-1, CD3, and ED-1, tissue sections
were incubated with 0.1% pepsin for 60 minutes, 0.1% pepsin for 30
minutes, and 0.1% proteinase for 5 minutes, 0.4% pepsin for 20
minutes, 0.1% proteinase for 5 minutes, 0.1% pepsin for 45 minutes,
and 0.1% pepsin for 45 minutes, respectively, before incubation with
the primary antibody. To optimize the detection of PCNA, sections were
microwaved for 10 minutes in 0.01 mol/L sodium citrate (pH 6.0) after
dewaxing. Proliferating endothelial cells were identified after
double-immunohistochemistry staining with TM and PCNA, using the color
modification method of 3,3'-diaminobendizine (DAB) precipitation by
nickel chloride, which changes DAB color from brown to
black.6,7
Sections were incubated with anti-rat TM
antibody, a peroxidase conjugated goat anti-rabbit IgG (Jackson
Immunoresearch Laboratories, West Grove, PA) followed by DAB containing
hydrogen peroxide (H2O2).
Sections were then incubated with PCNA followed by a
peroxidase-conjugated goat anti-mouse IgG (BML, Nagoya, Japan), and
H2O2, nickel chloride containing DAB (DAB
substrate kit for peroxidase; Vector Laboratories, Burlingame, CA).
Double immunostaining with PCNA and -SMA was also performed to
detect proliferating and activated mesangial cells, using the same
technique. Sections were first stained with anti--SMA antibody, a
peroxidase-conjugated goat anti-mouse IgG (BML) followed by
H2O2 containing DAB.
Sections were then incubated with PCNA followed by a
peroxidase-conjugated goat anti-mouse IgG (BML), and
H2O2, nickel chloride
containing DAB. To detect VEGF-producing mesangial cells, double
immunostaining with VEGF and Thy-1 (rat mesangial cell marker) was also
performed. Four-µm frozen sections were stained with polyclonal
rabbit anti-VEGF antibody and followed by fluorescein
isothiocyanate-labeled goat anti-rabbit IgG antibody (Zymed, San
Francisco, CA). Sections were then incubated with anti-Thy-1 antibody
(OX7, Cedarlane Laboratories) and followed by Texas Red-conjugated goat
anti-mouse IgG antibody (Leinco Technologies, St. Louis, MO), and the
nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI;
Vector Laboratories). Specimens were examined under a confocal laser
scanning microscope (CLSM, TCS-SP; Leica Lasertechnik, Heidelberg,
Germany) based on an upright microscope (DMRB, Leica Lasertechnik)
equipped with a krypton/argon laser. For all biopsies, negative
controls were used in which the primary antibody was substituted with
equivalent concentrations of an irrelevant antibody or normal rabbit
IgG (DAKO). All control sections were negative.
For electron microscopic examination, the kidney tissue was fixed in 2.5% glutaraldehyde solution in phosphate buffer (pH 7.4) and postfixed with 1% osmium tetroxide, dehydrated, and embedded in Epok 812. Ultrathin sections were stained with uranyl acetate and lead citrate, and then examined with an electron microscope (model H7100; Hitachi Corp., Ibaragi, Japan).
Isolation of Glomeruli and Western Blot Analysis for VEGF165
To examine the production of VEGF in glomeruli before and after disease induction, Western blotting was performed using polyclonal rabbit anti-VEGF antibody (147; Santa Cruz Biotechnology). For this purpose, glomeruli were isolated as described previously,11 using a standard three-stage sieving method. Isolated glomeruli were homogenized in lysis buffer (150 mmol/L NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mmol/L NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, and 20 mmol/L Tris-HCl, pH 7.4). After centrifugation at 15,000 x g for 30 minutes at 4°C, the supernatant was collected and used for analysis. Samples containing 10 µg of protein per lane were separated on 10% acrylamide gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the separated protein was transferred to a Hybond-P nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK) and incubated with anti-VEGF antibody (1:2000). Bound antibody was detected with peroxidase-conjugated anti-rabbit IgG antibody (1:1000; Jackson ImmunoResearch) with the enhanced chemiluminescence detection system (ECL Western blotting detection regents, Amersham). Membranes were washed and then exposed to film. Densitometric analysis of the bands was performed with NIH Image software.
Quantification of Histopathological Findings
In each kidney sample, more than 20 cross-sections of glomeruli
were examined sequentially for the following parameters: 1) total
number endothelial cells, the mean number of nuclei of TM+ cells per
glomerular cross-section; 2) proliferating endothelial cells; the mean
number of both PCNA+ and TM+ cells per glomerular cross-section; 3)
capillary repair; the mean number of glomerular capillary lumina
surround by TM+ cells, per glomerular cross-section; 4) proliferating
and activated mesangial cells; the mean number of both PCNA+ and
-SMA+ cells per glomerular cross-section; 5) infiltrating
neutrophils; the mean number of naphthol AS-D chloroacetate esterase+
cells per glomerular cross-section; 6) infiltrating T cells; the mean
number of CD3+ cells per glomerular cross-section; and 7) infiltrating
macrophages; the mean number of ED-1+ cells per glomerular
cross-section. In addition, for evaluation of 8) activated mesangial
cell area and 9) sclerotic area in glomeruli, more than 20
cross-sections of glomeruli were graded semiquantitatively on specimens
stained for -SMA and type IV collagen, respectively, using a graded
system9,16
(0 = diffuse, very weak, or absent
glomerular staining and no localized increases of staining; 1+ = up to
25% of the glomerular tuft showed focal increase of staining; 2+ = 25
to 50% of the glomerular tuft showed focal increase of staining; 3+ =
50 to 75% of the glomerular tuft showed focal increase of staining; 4+
= >75% of the glomerular tuft showed focal increase of staining).
Glomerular cross-sections containing only a small portion of the
glomerular tuft were excluded from the analysis. All histopathological
evaluations were performed by investigators blinded to the treatment
modality (saline versus VEGF). These results were expressed
as the mean ± SD, and statistical analysis was performed using
the Student’s t-test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thy-1/Habu-snake venom GN is produced by injecting on day 0 an
antibody to the Thy-1 antigen, which is present on mesangial cells.
This results in an acute, complement-dependent mesangial
cell-lysis.10,11
Subsequently, Habu-snake venom is
administered on day 1 to induce mesangial matrix decomposition, because
of proteolytic enzyme activity.2
Although anti-thymocyte
serum or Habu-snake venom classically results in a focal
lesion,5,10
the rats developed diffuse and marked
mesangiolysis on day 2 after injection of both anti-Thy-1.1 antibody
and Habu-snake venom. In accordance with the failure of the capillary
support function of mesangial cells and matrix, the capillary network
was destroyed, and mesangiolytic lesions progressed to multiple and
large microaneurysmal ballooning. Multiple and large microaneurysmal
ballooning lesions were observed in >60% of glomeruli on day 2, when
the most severe glomerular damage was noted in this model (Figure 1A)
. Glomerular endothelial cells were
lost in the ballooning lesions with destruction of glomerular capillary
network as evident in both immunostaining for endothelial surface
protein, TM (Figure 1B)
, and ultrastructural studies (Figure 1C)
.
|
. In addition
to VEGF staining in podocytes in kidney tissue samples of
Thy-1/Habu-snake venom GN, proliferating mesangial cells and
infiltrating leukocytes also expressed VEGF on day 5 to week 2 (Figure 2, B and C)
. In parallel with the increase in the relative area of VEGF
expression in glomeruli, the protein level of
VEGF165 increased in isolated glomeruli between
days 5 and 7 (Figure 3)
. However,
VEGF165 protein level subsequently decreased at a
rapid rate until week 2. VEGF receptor flk-1 was expressed on
glomerular endothelial cells in normal, damaged, and regenerating
capillaries (Figure 2; E, F, and G
). Little or no expression of flk-1
was detected in proliferating mesangial cells.
|
|
. In contrast, many proliferating and
activated mesangial cells (PCNA+/-SMA+) were found in these lesions
(Figure 4B)
. In proliferative lesions, glomerular endothelial cells and
capillary structures were rarely detected on days 5 to 9 (Figure 4C)
.
Mesangial hypercellularity and mononuclear cell infiltration were
persistently seen without capillary regeneration in damaged glomeruli
on weeks 2 to 4 (Figure 5)
. Thereafter,
glomerular inflammation progressed without capillary repair, and
accumulation of mesangial matrix including type IV collagen advanced
with the development of glomerular sclerosis. In parallel with the
histopathological and immunohistochemical evidence of a progressive
glomerular damage, changes in blood and urine indicated a gradual
deterioration of renal function evident by proteinuria and worsening of
plasma creatinine and blood urea nitrogen (Figure 6)
.
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We also examined the effects of VEGF165-induced angiogenesis on the course of Thy-1/Habu-snake venom GN. In the treatment group, rats were infused intraperitoneally with recombinant human VEGF165 (10 µg/100g body weight/day) from days 2 to 9 when minimal endothelial cell proliferation and impaired capillary repair occurred after disease induction in the control group.
Administration of VEGF165 significantly enhanced
endothelial cell proliferation and glomerular capillary repair by day
9. Numerous proliferating endothelial (PCNA+/TM+) cells were found in
the mesangiolytic and ballooning lesions (Figure 7)
. Furthermore, the number of
proliferating endothelial cells was increased significantly on days 5
to 7 (Figure 8)
, which was followed by a
rapid recovery in the number of total TM+ glomerular endothelial cells
by week 2 (Figure 8)
. Glomerular capillary repair occurred through
capillary regeneration from the remaining endothelial cells as well as
new capillary growth from the glomerular vascular pole (Figure 7; A, B,
and C
). The regenerating capillaries with activated endothelial cells
were seen in the proliferative lesions (Figure 7, E and F)
. Thereafter,
new glomerular capillary network developed by week 8 (Figure 9)
. In parallel with capillary
regeneration, the number of glomerular capillary lumina per glomerular
cross-section also increased (Figure 8)
. On the other hand, numerous
PCNA+/-SMA+-proliferating and -activated mesangial cells were found
in proliferative lesions from the early phase of GN (Figure 7D)
.
Subsequently, mesangial hypercellularity was noted on day 7 with marked
mesangial proliferative GN (Figure 9)
. However, this process was
transient, and correlated with the development of glomerular capillary
network; mesangial cell hypercellularity and matrix expansion gradually
subsided in damaged glomeruli (Figure 9)
. By week 8, most of the
damaged glomeruli healed and only minimal segmental sclerotic lesions
were noted with -SMA+ or type IV collagen+ areas (Figure 10)
. During recovery, VEGF-treated rats
showed a rapid fall in mesangial cell proliferation and/or activation,
and leukocyte infiltration, together with a significant decrease in the
number of these cells (Figure 11)
.
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, in
parallel with the recovery from damaged glomeruli. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGF is as an endothelial cell-specific mitogen and could play a highly dynamic role in the regulation of both physiological and pathological angiogenesis.17-19 Through alternative splicing of RNA, VEGF could exist in one of four different isoforms: two isoforms are diffusible (VEGF121, VEGF165), and two (VEGF189, VEGF206) are mostly bound to the extracellular matrix. Its binding sites, shown recently to include the tyrosine kinase receptors flt-1 (VEGFR-1), flk-1/KDR (VEGFR-2), and flt-4 (VEGFR-3), are present in endothelial cells,17-21 in agreement with the hypothesis that VEGF is an endothelial cell-specific factor. Consistent with certain features of VEGF, systemic administration of VEGF165 in the present study significantly enhanced glomerular capillary repair with endothelial cell proliferation in Thy-1/Habu-snake venom GN. Importantly, even in rats treated with VEGF165, endothelial cell proliferation is precisely regulated and VEGF-mediated endothelial cell proliferation can be inhibited before occurrence of endothelial cell overproliferation. Indeed, in the present study, proliferating endothelial cells were rapidly reduced in VEGF-infused rats on day 9. Moreover, in this model, administered VEGF165 at a dose of 10 µg/100 g body weight/day did not mediate endothelial cell proliferation in normal organs, including liver, lungs, heart, and digestive tract (data not shown).
Recent studies have indicated that VEGF receptors are also expressed on mesangial cells,22,23 and VEGF165 induces mesangial cell proliferation,23 suggesting an autocrine mechanism of VEGF/VEGF receptors in mesangial cell proliferation in GN. In the present study, however, proliferation and/or activation of mesangial cells even in rats treated with VEGF165 significantly decreased compared with the control group, indicating that treatment with VEGF165 at a dose of 10 µg/100 g body weight/day does not enhance the proliferation and activation of mesangial cells. Because impaired capillary regeneration in severely damaged regions relates to the continuation of mesangial cell proliferation and activation, capillary regeneration can either directly or indirectly influence the suppression of mesangial cell proliferation and activation, may associate with a reciprocal interaction between endothelial cells and mesangial cells. VEGF increases the permeability of capillaries and is sometimes referred to as vascular permeability factor,17-19,24 and is also thought to be involved in the development of proteinuria.25,26 However, recent studies have shown that VEGF does not affect the development of proteinuria in renal diseases.27-29 Our results in the present study also demonstrated that administration of VEGF165 did not worsen proteinuria in GN. VEGF is also known as a chemoattractant for leukocytes.18,19 In our study, however, we could not detect a significant effect of VEGF165 on infiltration of naphthol AS-D chloroacetate esterase+ neutrophils, CD3+ T lymphocytes, and ED-1+ macrophages in the glomeruli during treatment. Our results, therefore, indicate that VEGF165 specifically mediates capillary repair through endothelial cell proliferation in GN.
Glomerular VEGF can be released by both resident glomerular cells and infiltrating leukocytes in GN.4,21,30-33 Of glomerular cells, epithelial,4,30 endothelial,31 and mesangial4,21,32,33 cells can produce VEGF in vitro. Several workers have reported that podocytes express VEGF in normal glomeruli.21,33 In addition, activated mesangial cells also produce VEGF in proliferative GN.4,32 Our results showed that glomerular epithelial cells, proliferating mesangial cells, and some infiltrating leukocytes expressed VEGF, and the protein levels of VEGF165 increased in damaged glomeruli in the early phase of experimentally induced GN. VEGF receptor flk-1 was expressed on normal, damaged, and regenerating glomerular endothelial cells. A variety of cytokines and growth factors are involved in the pathogenesis of GN,34 and several of these, such as transforming growth factor-ß, platelet-derived growth factor, interleukin-1ß, epidermal growth factor, and insulin-like growth factor stimulate VEGF production.17-19 In addition, VEGF is stimulated by hemodynamic changes, including hypoxia stress, mechanical stretch, and hypertension.17-19,35 Similarly, known inducers of VEGF receptors include platelet-derived growth factor and hypoxic stress.19,20 Thus, a series of stimuli involved in GN can induce VEGF/VEGF receptor.
In Thy-1/Habu-snake venom GN, however, proliferation of endothelial cells and capillary repair was rarely seen in severely damaged glomeruli. Rather, mesangial cell proliferation and/or activation continued, and the sequence of events showed progression from damaged glomeruli to global sclerosis associated with chronic renal failure. These results suggest that impaired angiogenic capillary repair is associated with progression of GN and irreversible glomerular damage. Angiogenesis, vascular maturation, and vascular remodeling are controlled by local actions of chemical mediators, extracellular matrix, metabolic gradients, as well as physical angiogenic, angiogenesis-inhibitory, and vascular remodeling-induced factors.36-39 It is probable that these parameters are not precisely regulated in this model. In addition, severe capillary destruction with severe injury and marked loss of endothelial cells may be associated with impaired angiogenesis. Because glomerular epithelial cells express desmin, a marker of glomerular epithelial injury in rats,40 from the early phase of Thy-1/Habu-snake venom GN (Dr. Y Masuda, personal communication), glomerular epithelial cell injury may also influence impaired angiogenesis. Although the mechanisms of impaired glomerular capillary repair in this model are not fully understood at present, acceleration of angiogenic capillary repair is necessary in the treatment of GN accompanied by severe endothelial injury and capillary destruction.
Recently, the therapeutic effects of angiogenic growth factors have been investigated clinically and in animal models, and administration of VEGF, basic fibroblast growth factor (FGF-2), or vectors including their encoding DNA results in improvement of hemodynamics and increased capillary density in ischemic tissues.19,41,42 In renal disease, two angiogenic growth factors, FGF-2 and VEGF, are known to mediate endothelial cell proliferation in damaged glomeruli.4,8 However, intravenous injection of FGF-2 results in glomerular podocyte injury and promotes glomerular sclerosis.43-45 FGF-2 is also known as a strong mitogen for kidney fibroblasts and may promote interstitial fibrosis.46 Based on this background, we examined the beneficial effects of angiogenesis in GN, using VEGF165 and demonstrated that stimulation of angiogenic capillary repair by VEGF165 enhanced glomerular repair and accelerated resolution of GN. Our results in the present study suggest that systemic administration of VEGF165 could be therapeutically effective in GN accompanied by extensive endothelial damage.
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Acknowledgements |
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Footnotes |
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Y. M. and A. S. contributed equally to this work.
Accepted for publication April 26, 2001.
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) express VEGF. C: In double
stain with VEGF (green)
and Thy-1 (red), the
expression of VEGF in proliferative mesangial cells is indicated by
yellow color. D–F: VEGF receptor flk-1 is expressed on
endothelial cells in normal, damaged, and regenerating glomerular
capillaries.
