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*
Second Department of Pathology, Nagasaki University School of Medicine,
Nagasaki, Japan
Department of Histology and Cell Biology, Nagasaki University School of
Medicine, Nagasaki, Japan
Department of Medicine, Kidney Center, Tokyo Women's Medical College,
Tokyo, Japan
Correspondence to Dr. Takashi Taguchi, Second Department of Pathology, Nagasaki University School of Medicine, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan. Phone: 81-95-849-7055; Fax: 81-95-849-7056; E-mail: taguchi{at}net.nagasaki-u.ac.jp
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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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The normal glomerular ECM is primarily composed of various types of collagens, laminin, fibronectin, and sulfated proteoglycans. In vivo and in vitro studies have demonstrated that these components are secreted by mesangial and visceral epithelial cells (1,2). It has also been demonstrated that phenotypically altered renal cells are mainly responsible for the increased production of these components, as well as disease-specific components that are not expressed in normal kidneys. Several lines of evidence suggest that an imbalance between synthesis and degradation of these components is closely associated with the accumulation of ECM and the subsequent progression of renal diseases (3).
Matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinases (TIMP) play an important role in ECM remodeling in various glomerular diseases (4,5). MMP are zinc-dependent proteases that play a critical role in the turnover of ECM components, including collagens, elastin, laminin, proteoglycans, fibronectin, and other glycoproteins. MMP are generally classified into five categories on the basis of their properties, i.e., collagenases (MMP-1, -8, -13, and -18), gelatinases (MMP-2 and -9), stromelysins (MMP-3 and -10), membrane-type MMP (MMP-14, -15, -16, and -17), and others (MMP-7, -11, -12, and -19) (6). Previous studies showed that glomerular resident cells express and secrete several types of MMP, including gelatinase A (MMP-2), stromelysin-1 (MMP-3), and gelatinase B (MMP-9). A variety of MMP are expressed by cultured mesangial cells and/or glomerular epithelial cells (GEC) and are thought to be involved in glomerular injury in animal models (7,8,9,10). MMP-9, known as type I collagenase, is expressed in cultured mesangial cells as well as GEC. In studies of the passive Heymann nephritis model, McMillan et al. (10) demonstrated a marked increase in MMP-9 synthesis within GEC. MMP-3 is also present in both animal and human kidneys. Altered expression of MMP-3 mRNA has been detected in renal biopsy specimens from patients with IgA nephropathy and diabetic nephropathy (11,12).
The ets-1 proto-oncogene is a member of the transcription factor family that demonstrates homology to the v-ets oncogene (13,14). Ets-1 protein binds to DNA sequences containing a central GGA (A/T) core sequence (PEA3) via a DNA-binding domain and cooperates with the c-Fos/c-Jun complex at the AP-1 site to activate the expression of certain promoters (15). This motif has been observed in the promoter regions of numerous genes, including MMP-1, -3, and -9, urokinase-type plasminogen activator (u-PA), and TIMP-1 (16,17,18,19,20,21). Furthermore, Ets-1 protein enhances the promoter activity of MMP-1, MMP-3, and u-PA genes, indicating that the ets-1 proto-oncogene plays a pivotal role in the regulation of matrix proteinase expression. Therefore, it seems that regulation of the activity of several proteinases is one of the most important functions of Ets-1 protein, although it exhibits broad biologic functions. Although many studies have been performed to elucidate the altered expression of several proteinases in animal and human glomerular diseases, the expression of the ets-1 proto-oncogene in the kidney has not yet been determined.
In this study, we investigated whether the expression of the ets-1 proto-oncogene is involved in the progression of renal injury in a rat model of accelerated anti-GBM-induced glomerulonephritis, using immunohistochemical analysis, Western blotting, reverse transcription (RT)-PCR, and in situ hybridization. This model is characterized by crescentic severe proliferative glomerulonephritis, significant glomerular accumulation of macrophages and T lymphocytes, and severe interstitial inflammation (22,23,24,25). These changes ultimately lead to glomerular sclerosis and interstitial fibrosis. Therefore, the model seems to be useful for investigating the imbalance between ECM production and degradation in advanced glomerulonephritis. We also examined whether upregulation of Ets-1 protein in the nephritis model corresponded with renal MMP-3 expression, by correlating Ets-1-positive cells with MMP-3-positive cells in this model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Renal Function and Proteinuria
For assessment of renal function, each rat was kept in a single-animal
metabolism cage and 24-h urine samples were collected at the time of
euthanasia. Urinary protein excretion was measured by the sulfosalicylic acid
method (SRL, Nagasaki, Japan). Blood samples were obtained from the tail
artery at the end of each experimental period, before euthanasia, and plasma
urea levels were measured by the urease ultraviolet assay.
Morphologic Analysis
Renal tissues were fixed overnight in 4%
paraformaldehyde/phosphate-buffered saline (PBS) (pH 7.4), embedded in
paraffin, sectioned at 4 µm, and stained with periodic acid-Schiff (PAS)
stain and Masson trichrome reagent. For each animal, glomerular and
tubulo-interstitial damage was evaluated as follows: (1) percentage
of glomerular crescents, (2) percentage of global sclerosis,
(3) scoring of glomerular hypercellularity, and (4) scoring
of tubulointerstitial damage. A minimum of 50 glomeruli/animal were assessed
to determine the glomerular score (1 to 3) for each animal. Glomerular
hypercellularity was evaluated as total glomerular cell counts/glomerular
cross-section (gcs) in PAS-stained sections, as described previously
(27), as follows: 0, normal
(<50 cells/gcs); 1, mild (50 to 80 cells/gcs); 2, moderate (80 to 120
cells/gcs); 3, severe (>120 cells/gcs). Cortical tubulointerstitial injury
was characterized by tubular atrophy and dilation, cast formation,
interstitial fibrosis, and inflammatory cell infiltration into the
interstitium. It was semiquantitatively analyzed in Masson trichrome-stained
sections and divided into four grades, as follows: 0, no abnormal findings;
1+, mild (<30% of the cortex); 2+, moderate (30 to 60% of the cortex); 3+,
severe (>60% of the cortex).
Immunohistochemical Analysis
Immunohistochemical analysis was performed on paraffin-embedded sections,
as described previously (28).
Briefly, paraffin-embedded sections (4 µm) that had been fixed with 4%
paraformaldehyde in PBS (pH 7.4) were deparaffinized with xylene. Sections
were blocked for 1 h with either 10% goat serum or 10% rabbit serum, with 5%
nonfat dry milk, and were then incubated overnight at 4°C with primary
antibodies to the following: Ets-1 (C-20, rabbit polyclonal antibody against
the carboxyl-terminal domain of the Ets-1 protein; Santa Cruz Biotechnology,
Santa Cruz, CA) (29), MMP-3
(55-2A4, mouse monoclonal antibody against human MMP-3; Fuji Chemical
Industries, Japan) (30),
-smooth muscle actin (Dako, Denmark), vimentin (Dako), desmin (Dako),
ED1 (monocytes/macrophages and some dendritic cells; Serotec, UK), CD8 (T cell
cytotoxic/suppressor subset; Antigenix America), CD4 (T cell helper/inducer
subset; Antigenix America), and Ki-B1R (B cells and plasma cells; Biomedicals
AG). After washing with PBS, sections were processed further using the
Histostain streptavidin-alkaline phosphatase kit (Zymed Laboratories, San
Francisco, CA), and reaction products were developed with a mixture of
5-bromo-4-chloro-3-indolyl phosphate and nitrobule tetrazolium chloride.
Preabsorption of the primary antibody with excess recombinant Ets-1 peptide
(Santa Cruz Biotechnology) was performed for negative control samples. Spleen
tissue served as the internal positive control for Ets-1 protein
immunostaining, and Ets-1 expression was confirmed by Western blot analysis.
As negative controls for the other antibodies, sections were reacted with 10%
normal rabbit serum with 5% nonfat dry milk or PBS, instead of specific
antibodies. In addition, sections were also reacted with polyclonal antibodies
to the following: laminin (rabbit polyclonal antibody; Chemicon
International), collagen III (rabbit polyclonal antibody; Sanbio Co., The
Netherlands), and collagen IV (rabbit polyclonal antibody; LSL Co., Japan).
Briefly, paraffin-embedded or frozen tissue samples were cut into 4-µm
sections and treated for 30 min with 0.3% H2O2 in
methanol at room temperature, to inactivate endogenous peroxidase. Sections
were then incubated overnight at 4°C with the primary antibody and stained
using the Histofine streptavidin-horseradish peroxidase (HRP) kit (Nichirei
Laboratories, Japan). The reaction products were observed with
3,3'-diaminobenzidine-4HCl and H2O2. As negative
controls, normal goat or rabbit serum, with 5% nonfat dry milk, was used at
the same dilution. The percentages of Ets-1-or MMP-3-positive cells, CD4- or
CD8-positive T cells, B cells, and ED1-positive macrophages were determined in
at least 40 gcs, as well as in 40 high-power fields of cortical
tubulointerstitial area selected at random in 1 mm2, through the
eyepiece of the microscope.
Double-Immunostaining
Double-immunostaining was performed to localize Ets-1-expressing cells, as
described previously (31).
Briefly, sections treated with monoclonal antibodies to MMP-3, -smooth
muscle actin, vimentin, desmin, ED1, CD4, CD8, or Ki-B1R were initially
stained using the alkaline phosphatase method and then treated with
5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium chloride, which
produced dark purple staining. Sections were then pretreated with
H2O2 to quench endogenous peroxidase activity, further
stained for Ets-1 using the peroxidase method, and treated with
H2O2 and aminoethylcarbazole, which produced intense red
staining (Histostain double-staining kit; Zymed). In some cases, sections were
initially stained for Ets-1 and then counterstained with the other antibodies,
as described above.
Western Blot Analysis of Ets-1 Protein Expression
Frozen samples of renal cortex were lysed (lysis buffer was composed of 10
mM phosphate buffer, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin,
2 µg/ml chymostatin, and 0.2% Triton X-100), and the protein concentration
was measured with an ultraviolet/visible spectrophotometer (UV-1600; Shimadzu,
Japan). Each 100-µg protein sample was subjected to electrophoresis on a
10% sodium dodecyl sulfate-polyacrylamide gel and transferred to
polyvinylidene difluoride paper. Then, after blocking for 2 h at room
temperature with 20 mg/ml bovine serum albumin dissolved in Tris-buffered
saline solution, Ets-1 protein was detected using the polyclonal rabbit
antibody (C-20; Santa Cruz Biotechnology). Blots were washed and incubated
with HRP-conjugated goat antibody to rabbit IgG (Jackson ImmunoResearch
Laboratories, PA). Immunoreactive protein was detected by enhanced
chemiluminescence (Amersham, Buckinghamshire, UK).
In Situ Hybridization
In situ hybridization was performed to localize ets-1
mRNA-expressing cells by using a T-T-dimerized synthetic oligo-DNA probe, as
described previously
(32,33).
Briefly, rat ets-1 mRNA was detected using an oligo-DNA probe
complementary to a fragment of rat mRNA. The sequence of the antisense probe
was 5'-GCCCAGCTTCATCACAGAGTCCTATCAGAC-3'. Two TTA repeats were
added to both the 5' and 3' ends of that sequence during synthesis
(BEX Co., Tokyo), and the probes were later irradiated with ultraviolet light,
at a dose of 10 kJ/m2, to form T-T dimers. The paraffinembedded
sections were cut on silane-coated glass slides (4 or 6 µm thick),
deparaffinized, and rehydrated using standard procedures. The slides were
treated with 0.2 N HCl (at room temperature for 20 min) and 50 µg/ml
proteinase K (at 37°C for 15 min). After fixation for 5 min with 4%
paraformaldehyde in PBS, sections were immersed in PBS with 2 mg/ml glycine
and maintained in 40% deionized formamide in 4x SSC (1x SSC
contained 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) for 30 min until
hybridized. Hybridization was performed overnight at 37°C with 1 µg/ml
T-T-dimerized antisense oligo-DNA for ets-1, dissolved in
hybridization medium [10 mM Tris-HCl, pH 7.4, 0.6 M NaCl, 1 mM
ethylenediaminetetraacetate, 1x Denhardt's solution, 250 µg/ml yeast
tRNA, 125 µg/ml salmon testis DNA, 10% dextran sulfate, 40% deionized
formamide]. The slides were then washed for 5 h at 37°C with 50% formamide
in 2x SSC. Sections were reacted with HRP-conjugated mouse anti-T-T
dimer antibody (Kyowa Medex, Japan), and the sites of peroxidase were observed
with a chromogen solution containing 3,3'-diaminobenzidine-4HCl,
H2O2, and nickel and cobalt citation. Some sections were
counterstained with PAS stain, to identify individual cells for localization
of ets-1 mRNA, and mounted
(28). To verify the signal
specificity, the T-T-dimerized sense probe was hybridized with adjacent
sections as negative controls. Furthermore, a competitive study was performed
by adding a 100-fold excess amount of homologous or unrelated unlabeled
oligonucleotides to the hybridization buffer together with the antisense
probe. In addition, we evaluated the levels of hybridizable RNA in each
section by using in situ hybridization of 28S rRNA, as described
previously (32).
RT-PCR
RT-PCR was used to detect ets-1 mRNA expression, as described
previously (34). Briefly,
total RNA was extracted from the renal cortex by using the acid guanidinium
phenol method. For this purpose, 3 µg of total RNA template was incubated
with 50 ng of random hexamers at 70°C for 10 min and then quickly chilled
on ice, followed by incubation for 10 min at room temperature. The sample was
then treated for 50 min at 42°C with 200 U of Superscript RT, 500 µM
dNTP mixture, 10 mM dithiothreitol, 20 mM Tris-HCl (pH 8.4), 5 mM KCl, and 2.5
mM MgCl2, in a final volume of 20 µl, using a single-stranded
cDNA synthesis kit (Superscript Preamplification System, Life
Technologies-BRL). The reaction was terminated by heating to 90°C for 5
min. The primers for rat ets-1 were
5'-GCCCAGCTTCATCACAGAGT-3' (upper) and
5'-TGTTGAAAGATGACTGGCTG-3' (lower), which were synthesized by BEX
Co. The PCR product from cDNA amplification using the aforementioned primers
was 296 bp. PCR was performed by using the GeneAmp PCR reagent kit (Perkin
Elmer Cetus, CT). Reactions were performed in a total volume of 25 µl,
containing 5 µl of the cDNA preparation, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM
MgCl2, 0.001% gelatin, 200 µM levels of each dNTP, 2.5 U of
Taq DNA polymerase, and 0.25 µM levels of each primer. Samples
were subjected to 29 cycles of PCR amplification using a thermocycler (PC701;
Astec Co., Japan). The PCR conditions for ets-1 were denaturation at
94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C
for 1.5 min. As a positive internal control for RT-PCR efficacy, primers were
derived for the constitutively expressed housekeeping gene ß-actin. The
primers for rat ß-actin were 5'-CTGATCCACATCTGCTGGAAGGTGG-3'
(upper) and 5'-ACCTTCAACACCCCAGCCATGTACG-3' (lower). The PCR
conditions were the same as described above, and the product from cDNA
amplification was 703 bp. The reaction products were subjected to
electrophoresis on a 2% agarose gel and stained with ethidium bromide.
Semiquantitative analysis was performed using an image analyzer (Chemi Imager;
Astec), and results were expressed as the Ets-1/ß-actin signal intensity
ratio. Sequential analysis of the PCR products was performed with an
autosequencer, to confirm specific amplification of ets-1 mRNA.
Briefly, ets-1 primers were labeled with fluorescein at the
5'-end by 5'-oligolabeling for fluorescein (Amersham). DNA
sequencing was performed using Thermo-Sequenase (Amersham) and the
fluorescein-labeled primer in a DNA sequencer (DSQ-1000L; Shimadzu, Japan).
The reactions were performed according to the instructions provided by the
manufacturer.
Statistical Analyses
Data were expressed as mean ± SEM. Differences between groups were
examined for statistical significance using the Mann-Whitney U test
or one-way ANOVA. Correlations were examined by Pearson's correlation
analysis. A P value of <0.05 indicated a statistically significant
difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histologic Findings
Injection of NTS resulted in the development of crescentic necrotizing
glomerulonephritis in all rats (Figure
2; Table 1). On day
21, treated rats exhibited moderate to severe hypercellularity, segmental
lesions, and marked cellular or fibrocellular crescents. Furthermore, marked
tubulointerstitial damage, such as tubular atrophy and degeneration,
interstitial fibrosis, and infiltration into the interstitium, was noted.
Although glomerular hypercellularity diminished on day 42, severe glomerular
sclerosis, including global sclerosis, was most pronounced. Furthermore,
extensive tubular damage and interstitial fibrosis were noted.
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Immunohistochemical Assessment of Infiltrating Cells
Infiltrating ED1-positive macrophages, CD4-positive T cells, CD8-positive T
cells, and Ki-B1R-positive B cells were identified by immunohistochemical
analysis at different times during the progression of glomerulonephritis
(Table 2). A few macrophages
were detected in the glomeruli and interstitium in normal rat kidney. In
contrast, numerous ED1-positive macrophages were observed in both the
glomeruli and interstitium on day 7, and numbers further increased between day
14 and day 42. The maximal numbers of macrophages were observed on day 21 in
the glomeruli and on day 28 in the interstitium. The densities of CD4-positive
and CD8-positive T cells were also significantly increased in glomeruli on day
7; however, these cells were more frequently observed in the interstitium and
reached peak densities on day 28. Furthermore, there were significant
increases in B cell counts in both the glomeruli and interstitium, although
their infiltration was less than that of other cell types.
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Expression of Ets-1 and MMP-3 in Anti-GBM Disease
We also investigated the expression of Ets-1 protein in the renal cortex by
immunohistochemical analysis and Western blotting (Figures
3,
4, and
5). Ets-1-positive cells were
only occasionally detected in the glomeruli and tubulointerstitium in control
rat kidneys. In contrast, numerous Ets-1-positive glomerular intrinsic cells
and interstitial cells and some tubular epithelial cells were noted in
NTS-treated rat kidneys. Staining in immunopositive cells was limited to the
nuclei, as reported previously
(29). The number of
Ets-1-positive cells increased approximately 8.5- to 8.8-fold on days 14 to 21
in glomeruli in this model, compared with control kidneys, and increased
approximately 5.7- to 6.0-fold on days 21 to 28 in the interstitium. These
results indicated that the peak expression of Ets-1 occurred earlier in the
glomeruli (days 14 to 21) than in the interstitium (days 21 to 28)
(Figure 4). Moreover, we
demonstrated, by Western blotting, an Ets-1 band (54 kD) in the renal cortex
of diseased rats (days 14 and 21) (Figure
5).
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MMP-3 expression was also significantly upregulated in the glomeruli and interstitium during the progression of glomerulonephritis, compared with control kidneys (Figures 6 and 7). In particular, MMP-3-immunopositive cells were similar to Ets-1-expressing cells in the interstitium of diseased rats. Furthermore, the time course of MMP-3 expression closely resembled that of Ets-1 expression (Figure 7).
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Expression of ECM in Anti-GBM Disease
To investigate the expression of ECM known to be degraded by MMP-3, we
performed additional immunostaining, including staining for type III and IV
collagen and laminin. Positive immunostaining for type IV collagen and laminin
was present in the mesangium and GBM in the glomeruli of control rats, but no
intraglomerular immunostaining for type III collagen was observed. In the
tubulointerstitium of control rats, focal deposition of type III collagen was
observed in the interstitium, whereas type IV collagen and laminin were
observed mainly in the tubular basement membrane. As we previously described
for other types of human and/or animal nephritis
(28,31),
type III collagen was strongly present in interstitial fibrosis, fibrous
crescentic lesions, and the sclerotic matrix of glomeruli in NTS-treated rats.
The level of expression was proportional to the progression of glomerular
sclerosis and interstitial fibrosis (Figure
8). Type IV collagen was also identified in the sclerotic lesions
of glomeruli and in the thickened tubular basement membrane, compared with the
lesser accumulation of laminin (data not shown).
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Characterization of Ets-1-Positive Cells by
Double-Immunostaining
To identify cells expressing Ets-1 protein, we performed double-staining
for Ets-1 and various marker molecules, such as -smooth muscle actin,
desmin, and vimentin, as noted above (Figures
9 and
10). -Smooth muscle
actin and desmin were used as references for phenotypic changes of mesangial
cells and visceral epithelial cells in glomeruli, whereas vimentin and
-smooth muscle actin were used for identification of injured tubular
epithelial cells and myofibroblasts in the tubulointerstitium, respectively
(35,36,37).
To examine the relationship between Ets-1 and infiltrating cells,
double-immunostaining was performed for CD4, CD8, ED1, and Ki-B1R. In
addition, we also double-stained the tissues for Ets-1 and MMP-3. In glomeruli
of NTS-treated rats, Ets-1-positive cells were identified as predominantly
desmin-positive GEC (including crescentic cells) and glomerular endothelial
cells, whereas only a few ED1-positive macrophages and CD4-positive T cells
were positive for Ets-1 protein. Furthermore, only a few cells were positive
for both Ets-1 and MMP-3 in glomeruli
(Figure 9). In the
tubulointerstitium, Ets-1-positive cells were predominantly observed in
-smooth muscle actin-positive myofibroblasts and vascular smooth muscle
cells and in a small number of vascular endothelial cells. In addition,
ED1-positive infiltrative macrophages and CD4- and/or CD8-positive T cells
demonstrated modest Ets-1 expression. In contrast, the double-staining
procedure demonstrated that MMP-3-positive interstitial cells were more often
colocalized with Ets-1 expression than were those in the glomeruli
(Figure 10).
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Expression of ets-1 mRNA in Anti-GBM Disease
The expression of ets-1 mRNA in the renal cortex was determined by
RT-PCR analysis of RNA isolated from rats with anti-GBM-induced nephritis
(Figure 11). Expression of
ets-1 mRNA was present in all samples, including those from control
rats, and sequential analysis confirmed that the amplification product of 296
bp exhibited 100% homology with the published rat ets-1 cDNA sequence
(data not shown). Semi-quantitative analysis demonstrated that ets-1
mRNA levels, relative to ß-actin levels, were 3.7 to 6.5 times higher in
rats with anti-GBM-induced nephritis, compared with control rats. We also
examined the distribution of ets-1 mRNA expression in control and
nephritic rats on days 14 and 21 by in situ hybridization
(Figure 12). A few glomerular
and interstitial cells that were weakly positive for ets-1 mRNA
expression were observed in control kidneys. In contrast, in situ
hybridization revealed a significant increase in the number of ets-1
mRNA-positive glomerular and interstitial cells in the renal tissue of
NTS-treated rats. Glomerular cells expressing ets-1 mRNA were
identified as GEC or endothelial cells. A small number of infiltrating cells
also expressed ets-1 mRNA. However, there was little or no expression
in glomerular mesangial cells. In the tubulointerstitium, signals for
ets-1 mRNA were observed in some interstitial cells, as well as a few
vascular endothelial cells and infiltrating cells. These results for the
glomeruli and interstitium were similar to those for Ets-1 protein expression,
as detected by immunohistochemical analysis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ets-1 proto-oncogene is a transcription factor that was originally identified by sequence homology with the viral v-ets oncogene of the E26 avian leukemia retrovirus (13). Expression of the ets-1 proto-oncogene has been detected in various cells, and the role of the ets-1 gene expressed in mesodermal lineage cells, such as fibroblasts and endothelial cells, has drawn wide attention in the fields of embryogenesis and angiogenesis (29,38,39,40). Recent studies have also demonstrated that Ets-1 protein exhibits multiple activities in the transcriptional regulation of numerous genes. Among these activities, one of the most important functions seems to be activation of the transcription of matrix proteinases such as MMP-3 and u-PA (16,17,18,19,21). This study demonstrated that not only serial changes in expression but also the cellular distribution of MMP-3 was similar to that of Ets-1 protein. Moreover, MMP-3-positive interstitial cells in diseased kidneys often expressed Ets-1 protein, as demonstrated by double-immunostaining, thus confirming the close correlation between Ets-1 and MMP-3 expression. The upregulated expression of MMP-3 induced by Ets-1 might participate in matrix remodeling in injured renal tissues. However, only a few cells in the glomeruli exhibited coexpression by double-staining. Although the reason for this discrepancy is not fully understood, it is possible that the turnover of Ets-1 protein is too rapid for colocalization with MMP-3 in glomerular resident cells. Alternatively, it is possible that other transcriptional factors, such as c-Jun and c-Fos, are engaged in the induction of MMP-3 expression, in combination with Ets-1 protein (8, 41).
MMP-3 is important for the degradation of ECM components, such as type III, IV, and VI collagen, laminin, aggrecan, and fibronectin (6). As previously demonstrated in advanced glomerulonephritis (3), the expression of major matrix substrates for MMP-3, such as type III or IV collagen, increases in advanced glomerulonephritis and further increases with the progression of renal injury, as confirmed by immunohistochemical analysis. Furthermore, overexpression of these matrices in the kidney seems to coincide with the time course of enhanced MMP-3 expression. These results suggest that MMP-3 plays a pivotal role in ECM remodeling in our system.
Previous studies demonstrated signals for MMP-3 mRNA in glomerular resident cells, including glomerular mesangial, epithelial, and endothelial cells, and cells of Bowman's capsule in human diseased kidneys, by in situ hybridization analysis (11, 12). However, these findings for NTS-treated rats demonstrated minimal expression of MMP-3 in glomerular mesangial cells. Considering that the major changes in the NTS model occur in the phenotype and mitotic rates of GEC, rather than mesangial cells (as is more common in other types of glomerulonephritis), the discrepancy in the expression of MMP-3 could be explained by the different features of glomerular damage. Further studies using other forms of experimental and human glomerulonephritis are necessary to clarify the relationship between the expression of MMP-3 and glomerulonephritis, including the involvement of MMP inhibitors such as TIMP-1.
High levels of ets-1 gene expression have been demonstrated in T lymphocytes, and there is evidence that such expression is essential for the maintenance of the normal pool of resting T cells (42, 43). Moreover, the Ets-1 binding site is present in the T cell receptor gene enhancer and CD4 gene core promoter (44, 45), and it was reported that the ets-1 proto-oncogene might play an important role in T cell activation (46). Because the model of nephrotoxic nephritis is known to be closely associated with the accumulation of T cells in the kidneys, as described above, we expected to observe the ets-1 gene predominantly expressed in T cells in nephritic kidneys. Unexpectedly, however, only a small number of T cells expressed ets-1 mRNA and protein. In this context, it should be noted that Ets-1 products are involved in maintaining T cells in a quiescent state and ets-1 gene levels are decreased after the activation of T cells (46, 47), indicating that most infiltrating T cells might already be activated in the kidneys of NTS-treated rats.
Finally, recent studies demonstrated that the ets-1 gene is essential for the normal development of mammalian kidneys and the maintenance of glomerular integrity and that the Ets-1 protein may act as an upstream regulator of the expression of FREAC-4, a winged helix transcriptional factor that is detected during kidney development (48, 49). In fact, kidneys from ets-1-knockout mice exhibited various glomerular abnormalities, including sclerosis, atrophy, and markedly fewer and immature glomeruli (48). These results might indicate a protective reaction against destruction of the normal structure of the kidney, in the presence of Ets-1 protein overexpression. Further studies are needed to determine whether the ets-1 proto-oncogene acts as a progression factor or a protection factor in various forms of glomerulonephritis.
In conclusion, this study demonstrated that the expression of the ets-1 proto-oncogene was upregulated in the renal tissue of a rat model of crescentic glomerulonephritis. Expression of ets-1 mRNA was observed primarily in glomerular resident cells, interstitial myofibroblasts, and some vascular endothelial cells and infiltrating cells. Overexpression of the ets-1 proto-oncogene might be associated with the pathogenesis of glomerulonephritis through the expression of MMP.
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 H. Tanaka, Y. Terada, T. Kobayashi, T. Okado, S. Inoshita, M. Kuwahara, A. Seth, Y. Sato, and S. Sasaki Expression and Function of Ets-1 during Experimental Acute Renal Failure in Rats J. Am. Soc. Nephrol., December 1, 2004; 15(12): 3083 - 3092. [Abstract] [Full Text] [PDF]
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 M. S. Razzaque, C. S. Foster, and A. R. Ahmed Role of Collagen-Binding Heat Shock Protein 47 and Transforming Growth Factor-{beta}1 in Conjunctival Scarring in Ocular Cicatricial Pemphigoid Invest. Ophthalmol. Vis. Sci., April 1, 2003; 44(4): 1616 - 1621. [Abstract] [Full Text] [PDF]
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 D. Baillat, A. Begue, D. Stehelin, and M. Aumercier ETS-1 Transcription Factor Binds Cooperatively to the Palindromic Head to Head ETS-binding Sites of the Stromelysin-1 Promoter by Counteracting Autoinhibition J. Biol. Chem., August 9, 2002; 277(33): 29386 - 29398. [Abstract] [Full Text] [PDF]
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J. Reisdorff, A. En-Nia, I. Stefanidis, J. Floege, D. H. Lovett, and P. R. Mertens Transcription Factor Ets-1 Regulates Gelatinase A Gene Expression in Mesangial Cells J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1568 - 1578. [Abstract] [Full Text] [PDF]
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