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*
Department of Pathology, Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands
Department of Internal Medicine, Chubu Rousai Hospital, Nagoya,
Japan
Department of Pathology University of Utrecht, Utrecht, The
Netherlands.
§
Department of Nephrology, University of Utrecht, Utrecht, The
Netherlands.
Correspondence to Dr. Jan Aten, Department of Pathology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, L2-256, 1105 AZ Amsterdam, The Netherlands. Phone: 31-20-566-4935; Fax: 31-20-696-0389; E-mail: j.aten{at}amc.uva.nl
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle
actin—positive fibroblasts. In cultured mesangial cells, TGF-ß1,
-ß2, and -ß3 transiently increased the CTGF/glyceraldehyde phosphate
dehydrogenase mRNA ratio up to threefold versus control at 4 h. In
GVEC, upregulation of CTGF mRNA by these TGF-ß isoforms was more
sustained, being 8- to 16-fold versus control at 24 h. The kinetics
of CTGF expression strongly suggest a role in glomerular repair, possibly
downstream of TGF-ß, in this model of transient renal injury. |
Introduction |
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In models of renal injury, platelet-derived growth factor B (PDGF-B) and transforming growth factor ß (TGF-ß), among a variety of other cytokines and proinflammatory factors, have been identified as major determinants in the transition of an acute inflammatory response to either resolution or chronic fibrosis (2,3,4).
The TGF-ß family of proteins influence, among others, cell proliferation and apoptosis, matrix turnover, and immunity (2) The pleiotropism of TGF-ß family members is partly effectuated through the release and action of other cytokines, one of which is connective tissue growth factor (CTGF).
CTGF is a cysteine-rich secreted growth factor that was originally identified in human umbilical vein endothelial cells (5). Recently, CTGF was reported to be overexpressed in several forms of fibrosis, including atherosclerotic blood vessels (6), in which expression of CTGF mRNA was 50- to 100-fold higher than in normal arteries (6). CTGF was found to induce chemotaxis, proliferation, and matrix synthesis by normal rat kidney fibroblasts (5,7). In addition, effects of TGF-ß on fibroblasts are thought to be partially mediated by CTGF (8,9,10,11).
We recently reported that CTGF mRNA is mainly expressed by glomerular visceral and parietal epithelial cells in control human kidney tissue. CTGF mRNA expression was found to be similar to controls or slightly increased by podocytes in glomerular diseases characterized by noninflammatory lesions with proteinuria, such as minimal-change nephrotic syndrome and membranous nephropathy. In contrast, CTGF expression was observed to be markedly increased in inflammatory glomerular and tubulointerstitial (TI) lesions associated with cellular proliferation and matrix accumulation, including IgA nephropathy, chronic transplant rejection, crescentic glomerulonephritis, focal and segmental glomerulosclerosis, and lupus nephritis. Furthermore, expression of CTGF mRNA was highly correlated with the degree of TI fibrosis. These findings suggest that CTGF is a common factor involved in renal fibrosis (12). Indeed, CTGF was recently shown to be able to induce synthesis of fibronectin, type I collagen, and type IV collagen by mesangial cells (13,14).
We studied the kinetics of CTGF expression in a well-established model of proliferative glomerulonephritis, induced by anti—Thy-1.1 antibody in rats, as well as the regulation of CTGF expression in cultured mesangial cells and podocytes. Our data strongly suggest that CTGF is involved in the renal response to injury.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental Protocol
Experimental proliferative glomerulonephritis was induced in female Wistar
rats by intravenous injection of the monoclonal antibody (mAb) ER4 (anti-rat
Thy-1.1, mouse IgG2a, dosage 1 mg/kg body wt), as originally described by
Bagchus et al. (15).
Groups of three rats each were killed at days 1, 4, 7, and 14. As control, a
corresponding dose of OKT3 (anti-human CD3 
, mouse IgG2a; hybridoma
obtained from the American Type Culture Collection, Rockville, MD) was
injected into six rats. Groups of three rats each were killed at days 1 and
14. ER4 and OKT3 mAb were purified from hybridoma culture supernatants by
protein A chromatography. In addition, three normal rats that did not receive
antibody injection were examined.
Twenty-four-h urinary protein loss was measured by the biuret method at days 0, 1, 4, 7, and 14. Urine was collected from rats in metabolic cages with free access to food and water.
Isolation of Glomerular RNA
Rats were anesthetized by ether and killed after excision and perfusion of
the kidneys as detailed below. To preserve the integrity and stability of the
glomerular RNA, all steps were performed at 4°C under sterile conditions.
The left renal artery and vein were ligated, and the left kidney was excised.
The right kidney was perfused in situ with cold phosphate-buffered
saline (PBS, pH 7.4) containing 0.1% NaN3, 0.5 mM
phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO), 2 mM Benzamidine-HCl
(Sigma), 1 mM Pepstatin A (Sigma), 1 mM Leupeptin (Sigma), and 50 mM
-amino-n-caproic acid (Sigma). After removal of the medulla, glomeruli
were retained from minced cortex by sieving and washed with perfusion buffer.
Microscopy confirmed that the resulting preparation consisted of isolated
glomerular tufts that included extracapillary glomerular lesions in
preparations from ER4-injected rats at day 7 or 14; contamination by tubular
fragments was minimal. The isolated glomeruli were dissolved in TRIzol (Life
Technologies, Gaithersburg, MD). The average time required for the whole
procedure was 20 min.
Renal Histology and Immunohistology
The left kidney was processed for routine histology, immunohistology, and
in situ hybridization (ISH). Kidney specimens were cut into
transversal fragments. One part was fixed during 16 h in 10% buffered formalin
and embedded in paraffin by conventional techniques. Sections were stained
with hematoxylin and eosin and periodic acid-Schiff. Formalin-fixed tissue was
also used for ISH as detailed below. A second part was fixed in methacarn and
embedded in paraffin. Methacarn-fixed tissue sections were deparaffinized with
xylene, rehydrated, and washed with PBS. Endogenous peroxidase activity was
inhibited with 0.3% hydrogen peroxide in methanol, and nonspecific protein
binding sites were blocked with normal goat serum. Subsequently, the sections
were incubated with mAb ED1 (antimonocytes and macrophages, mouse IgG1;
Serotec, Oxford, UK), 1A4 (anti—-smooth muscle actin
[SMA], mouse IgG2a; DAKO, Glostrup, Denmark), and 19A2
(antiproliferating cell nuclear antigen [PCNA], mouse IgM; Coulter
Corporation, Miami, FL) for 2 h at room temperature. Immobilized mouse
antibodies were detected using biotinylated goat anti-mouse Ig antibodies and
a streptavidinbiotin-immunoperoxidase technique (StreptABComplex/HRP kit,
DAKO). A third part of the excised left kidney was snap-frozen in liquid
nitrogen. Four-µm-thick sections were cut by cryostat, air dried, and fixed
in acetone at room temperature for 10 min. Endogenous peroxidase activity was
inhibited with 0.1% NaN 3 and 0.3% hydrogen peroxide in PBS, and
nonspecific protein binding sites were blocked with normal goat serum. The
sections were incubated with rabbit polyclonal IgG anti—TGF- ß1,
anti—TGF- ß2, or anti—TGF- ß3 (Santa Cruz Biotechnology,
Santa Cruz, CA), followed by a conjugate of polyclonal goat anti-rabbit IgG
antibodies, horseradish peroxidase, and dextran backbones (EnVision System,
DAKO) as secondary reagent. The polyclonal anti—TGF- ß1,
anti—TGF- ß2, and anti—TGF- ß3 antibodies were raised
against peptides that map at or near the carboxy terminal ends of the
respective TGF- ß isoforms. The antibodies are expected to bind both the
bioactive and the latent conformations of the TGF- ß isoforms
(16). Finally, enzyme activity
of horseradish peroxidase was detected using 3-amino-9-ethyl-carbazole.
Negative controls were performed by replacement of first step antibodies by
species- and isotype-matched Ig.
Rat CTGF Sequencing
A part of the cDNA sequence of rat CTGF was determined by dye terminator
cycle sequencing with an ABI-377XL sequencer (Perkin Elmer Corporation,
Norwalk, CT) using cDNA from rat mesangial cells. Synthetic oligonucleotides,
sense 5'-TCC CGA TCA TGC TCG CCT CCG TCG C-3' and antisense
5'-TTA CAG AAG AAA ATG AGA TGC AAC-3', were derived from the cDNA
sequence of mouse CTGF (GenBank accession number M70642) and used for specific
primers.
ISH for CTGF mRNA
Nonradioactive ISH was performed as detailed previously
(12). Briefly, the 1.5-kb
EcoRI/KpnI fragment of CL59 cDNA of human CTGF
(6) was used to produce
digoxigenin (DIG)-labeled antisense and sense riboprobes applying an RNA
labeling Kit (Roche Diagnostics, Mannheim, Germany). The DIG-labeled antisense
CTGF probe bound specifically to rat CTGF as was shown by Northern blot
analysis (12). After the ISH
reaction on 4- µm sections of formalinfixed, paraffin-embedded renal
tissue, the bound probes were detected using alkaline phosphatase-conjugated
Fab fragments of sheep anti-DIG antibody and then visualized with nitroblue
tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, toluidine salt
according to the DIG nucleic acid detection kit protocol (Roche Diagnostics).
Incubation with DIG-labeled sense CTGF probe was performed as negative
control. The sections were counterstained with hematoxylin or periodic
acid-Schiff without hematoxylin. Kidney sections from all 21 animals were
processed simultaneously.
Combined Detection of CTGF mRNA and SMA Protein
ISH in combination with immunohistology was performed on the same section
to detect simultaneously CTGF mRNA and SMA protein, as described
previously (12). Briefly,
sections first were hybridized with DIG-labeled RNA probe to detect CTGF
transcripts. After sections were washed with PBS, endogenous biotin was
blocked with streptavidin (Zymed, San Francisco, CA) and d-biotin (Sigma) in
two successive steps. Sections were subsequently stained for SMA
protein using the 1A4 mAb. The results of the double-labeling experiments
showed staining patterns and staining intensities similar to those obtained in
simultaneously performed single-staining experiments.
Morphometric Analysis of Immunohistology and ISH
For each kidney and for each type of staining, cross sections of 20
different glomeruli and 20 different TI areas of 0.01 mm2 were
examined. The fractions of the glomerular surface area that were positively
stained for ED1 protein, CTGF mRNA, and SMA protein were measured by
computer-aided planimetry using the NIH Image software (National Institutes of
Health, Bethesda, MD), as described previously
(17). For each kidney,
PCNA-positive cells were counted in 20 different glomerular cross sections and
the average number of PCNA-positive cells in 0.01 mm2 glomerular
surface area was calculated. The expression of SMA protein and CTGF
mRNA in periglomerular areas was assessed separately, using NIH Image software
to quantify the percentage of the interstitium immediately contiguous to
Bowman's capsule showing positive staining.
Culture of Glomerular Visceral Epithelial Cells and Mesangial
Cells
Established cell lines of rat glomerular visceral epithelial cells (GVEC)
and of rat mesangial cells were cultured and maintained as described
previously
(12,18).
GVEC and mesangium cells were cultured in medium that contained 5% NuSerum
(Becton Dickinson, Bedford, MA) or 20% fetal calf serum (Life Technologies,
Breda, The Netherlands), respectively, until they covered the bottom of the
culture flask for 70 to 80% and had become confluent. The cultures
subsequently were washed twice with serum-free medium and cultured in
serum-free medium for 24 h before incubation with recombinant human
TGF-ß1, TGF-ß2, or TGF-ß3 (all from R&D Systems, Abingdon,
UK) or human PDGF-BB (Roche Diagnostics), which were diluted in serum-free
medium. Dose-response studies were conducted using incubation durin g 4 h with
each growth factor at 0, 0.04, 0.2, 1, 5, and 25 ng/ml. The concentration
chosen for subsequent time-course studies was 5 ng/ml for each factor. At
harvesting, the cells were washed with PBS and lysed with TRIzol.
RNA Extraction and Northern Blot Analysis
Total RNA was prepared from isolated glomeruli and cultured cells using
TRIzol reagent and quantified by spectrophotometry. Twenty µg of total RNA
was size-separated by electrophoresis in a 1.2% agarose-0.34 M formaldehyde
gel, transferred to N-Hybond membrane (Amersham Pharmacia Biotech, Roosendaal,
The Netherlands), and UV cross-linked. Before transfer, gels were stained with
ethidium bromide and examined by ultraviolet illumination to determine the
position of 28S and 18S ribosomal RNA and to assess the integrity of the RNA.
The 0.6-kb PstI fragment of CL59 cDNA clone of human CTGF
(6) was labeled with
(32P)-dCTP (Amersham Pharmacia Biotech) using the Random Primers
DNA Labeling System (Life Technologies). RNA hybridization was performed at
65°C for 16 h, using 107 cpm probe per ml of 0.5 M sodium
phosphate (pH 7.2), 7% sodium dodecyl sulfate, and 1 mM
ethylenediaminetetraacetate. The membranes were washed for 10 min with 1
x SSC containing 0.1% sodium dodecyl sulfate at room temperature. Bound
radioactivity was documented by phosphor imaging (Molecular Dynamics,
Sunnyvale, CA). To control for equivalent loading of RNA, we performed
rehybridization with a cDNA probe for glyceraldehyde phosphate dehydrogenase
(GAPDH). Data were analyzed using ImageQuant Software (Molecular Dynamics) and
are expressed as CTGF/GAPDH ratios.
Semiquantitative Reverse Transcriptase-PCR for CTGF and TGF-ß1
mRNA
For first-strand cDNA synthesis, 10 µg of total RNA was incubated in a
reaction mixture of 50 µl with 5 nmol of pd(N)6 as primer (Pharmacia
Biotech, Roosendaal, The Netherlands). The reaction contained 400 units of
Moloney murine leukemia virus reverse transcriptase (RT; Life Technologies), 8
mM dithiothreitol, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2,
and 60 units of RNAse inhibitor (Roche Diagnostics). The reaction was
performed at 40°C for 1 h. Subsequently, the reverse transcriptase was
inactivated by heating the sample at 95°C for 10 min.
PCR was performed in 25 µl of 1 x PCR buffer, containing 0.25 µM of each primer (Table 1), 1 unit of Taq polymerase (Life Technologies), 200 µM of each dNTP, and 1.5 mM MgCl2, using a thermal cycler (PTC-100; MJ Research Inc., Watertown, MA). The PCR consisted of a 5-min denaturation step at 95°C, followed by 30 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C. The reaction was terminated for 7 min at 72°C.
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Semiquantitative RT-PCR was performed in two ways to evaluate glomerular CTGF or TGF-ß1 mRNA expression: (1) Co-amplification was performed by combining primers for either CTGF or TGF- ß1 mRNA with primers for GAPDH mRNA in the same sample, considering GAPDH as a housekeeping gene and internal standard. Dilution series of cDNA input were analyzed after 29, 31, and 33 cycles of PCR to establish optimal conditions in the exponential phase of amplification to calculate ratios of either CTGF/GAPDH or TGF- ß1/GAPDH mRNA. (2) For competitive PCR, a standard was constructed by deleting the internal 187-bp SmaI fragment from the 526-bp CTGF amplicon; the ligated 339-bp fragment was subcloned. Based on dilution series, a constant amount of the resulting plasmid was amplified together with the target template using the same CTGF primer pair. The products of the competitive PCR were separated as singlestrand DNA by agarose electrophoresis under denaturing conditions using 50 mM NaOH and 1 mM ethylenediaminetetraacetate to prevent formation of heteroduplex products. For both types of semiquantitative RT-PCR, ethidium bromide—stained gels were imaged (Eagle Eye II, Stratagene, La Jolla, CA) and the fluorescence intensities of the PCR products were analyzed with SigmaScan software (Jandel Scientific Software, Erkrath, Germany). To ensure the proper identity of the PCR products, we subcloned the amplicons in pGEM-T and analyzed it by dye terminator cycle sequencing.
Statistical Analyses
All values are expressed as mean ± SD. Classes of data to be
compared were first analyzed for homogeneity of variance with Levene's test
applying Satterthwaite's rule
(19) for adjustment to the
appropriate degrees of freedom, according to Snedecor and Cochran
(20). To obtain normality and
homoscedasticity of the distribution of proteinuria values and of CTGF/GAPDH
ratios in Northern blot experiments, we performed analyses after
transformation to the natural logarithm of these data
(21). Subsequently, repeated
measurements ANOVA was performed for proteinuria values to analyze possible
effects of treatment over time. For each other variable considered, one-way
ANOVA was applied to determine whether an overall difference exists between
groups. For subsequent multiple comparisons of specific treatment groups
against the single control group, two-tailed Dunnett's t tests were
performed (22). Differences
were considered to be statistically significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SMA (Figure
1). At day 1, SMA expression was confined to blood vessels
(Figure 1B), as in control rats
(Figure 1A). At day 4,
SMA was focally overexpressed in the periglomerular and mesangial area
(Figure 1C). SMA
expression was maximal at day 7 and had declined slightly at day 14 in both
areas (Figure 1, D and E).
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Partial Sequence of Rat CTGF
Analysis of a 1077-bp fragment of rat CTGF cDNA revealed nucleotide
sequence identities of 94% and 89% to the corresponding fragments of mouse and
human CTGF (Genbank accession numbers M70642 and M92934, respectively) and
complete identity to that of the recently released rat CTGF cDNA (Genbank
accession number AF120275.1, positions 242 to 1318). Oligonucleotides
5'-AGA ACT GTG CAC GGA GCG TG-3'(sense strand) and 5'-CCT
GAC CAT TCA GAG ACG AC-3' (antisense strand) were derived from the rat
CTGF sequence and used as specific primers for rat CTGF in PCR experiments,
yielding a 526-bp amplicon that corresponded with Genbank accession
AF120275.1, positions 413 to 938.
CTGF mRNA Expression during Anti—Thy-1.1 Nephritis
CTGF mRNA expression in renal sections from control rats was low and mainly
confined to visceral epithelial cells, as shown by ISH
(Figure 2A). In the TI area,
neither CTGF mRNA nor SMA protein could be detected (Figures
1A and
2A). In
anti—Thy-1.1—treated rats, CTGF mRNA expression had increased in
podocytes and parietal epithelial cells already at day 1
(Figure 2B), before an increase
of SMA protein expression was detected
(Figure 1B). At day 4, strong
CTGF mRNA expression was present in parietal epithelial cells
(Figure 2C). CTGF mRNA
expression was maximal at day 7 and was found mainly in extracapillary
proliferative lesions and in the periglomerular area and now was also
upregulated in mesangial areas (Figure
2D). Double-labeling experiments indicated that at day 7, parietal
epithelial cells and periglomerular SMA-positive fibroblasts
(myofibroblasts) expressed CTGF mRNA in the Bowman's capsular lesions
(Figure 3). In the glomeruli at
day 7, CTGF mRNA was expressed by visceral epithelial cells that did not stain
for SMA as well as by some SMA-positive cells located in the
mesangial area (Figure 3). At
sites of TI injury in anti—Thy-1.1 nephritis at day 7, CTGF mRNA was
also found to be expressed by interstitial cells but not by tubular epithelial
cells (Figure 4A). At day 14,
CTGF mRNA expression overall had declined
(Figure 2E), whereas SMA
protein expression remained high in the periglomerular area
(Figure 1E).
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Morphometric analysis demonstrated an early and strong upregulation of CTGF
mRNA expression in both the glomerular and periglomerular areas (Figures
5B and
6, respectively), clearly
preceding the strong increase of SMA protein expression as depicted for
the periglomerular area (Figure
6). Semiquantitative RT-PCR analysis of glomerular mRNA, applying
both co-amplification with endogenous GAPDH mRNA and PCR in the presence of an
added truncated CTGF competitor (representative experiments shown in
Figure 5A), confirmed the
kinetics of glomerular CTGF mRNA expression as observed with ISH
(Figure 5B). Together, these
different types of analysis indicate a 6- to 10-fold upregulation of
glomerular CTGF mRNA expression at day 7 when compared with control rats
(Figure 5B).
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TGF-ß1, -ß2, and -ß3 Protein Expression during
Anti—Thy-1.1 Nephritis
Because TGF-ß1 is known to play an important role in the pathogenesis
of anti—Thy-1.1 nephritis
(3) and was described to be a
potent inductor of CTGF expression previously
(23), expression of
TGF-ß1 and of its family members TGF-ß2 and -ß3 was compared
with that of CTGF in this model. In control rats
(Figure 7A) and in rats at days
1 (Figure 7B) and 4
(Figure 7C), renal TGF-ß1
protein expression could not be detected by immunohistology. At day 7,
TGF-ß1 was found to be expressed in glomeruli
(Figure 7D) and to a larger
extent by interstitial cells and weakly by tubular epithelial cells in areas
of TI injury (Figure 4B). At
day 14, both glomerular and TI TGF-ß1 expression had decreased
(Figure 7E).
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In contrast, protein expression of TGF-ß2 was strongly upregulated as early as day 1 (Figure 7G) when compared with the TGF-ß2 expression in control animals, which was limited to a few glomerular and scattered interstitial cells (Figure 7F). The upregulation of TGF-ß2 expression at day 1 predominantly occurred in glomeruli but was also observed for tubular epithelium and interstitium (Figure 7G). From day 1 onward, TGF-ß2 expression gradually decreased (Figure 7, H through J).
In control rats, TGF-ß3 expression was detected in a distinct glomerular epithelial distribution pattern and was observed in a subset of tubular epithelial cells as well (Figure 7K). TGF-ß3 expression had somewhat increased at day 1 (Figure 7L). Glomerular expression of TGF-ß3 at days 4 (Figure 7M) and 7 (Figure 7N) had decreased and was detected in a more patchy pattern, in association with the loss of normal glomerular architecture during anti—Thy-1.1 nephritis. At day 14, glomerular TGF-ß3 expression had recovered to control levels (Figure 7O).
TGF-ß1 mRNA Expression during Anti—Thy-1.1 Expression
Because TGF-ß1 protein expression could not be detected at days 1 and
4, whereas glomerular CTGF mRNA expression was already clearly upregulated by
visceral and parietal epithelial cells in this early phase of
anti—Thy-1.1 nephritis (Figure 2, B
and C), glomerular TGF-ß1 was also analyzed at the mRNA level
by using semiquantitative RT-PCR and co-amplification with primers for GAPDH
mRNA. Similar kinetics as observed for CTGF mRNA were detected for TGF-ß1
mRNA; however, differences with control levels reached significance only at
days 7 and 14 of anti—Thy-1.1 nephritis when a sixfold increase of the
TGF-ß1/GAPDH ratio was observed
(Figure 8).
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CTGF mRNA Expression Induced by TGF-ß1, -ß2, and -ß3
In Vitro
In rat mesangial cells and in rat GVEC, TGF-ß1, -ß2, and -ß3
significantly induced early upregulation of CTGF mRNA expression
(representative Northern blot analyses shown in
Figure 9A). In mesangial cells,
a maximum was observed at 4 h of incubation when CTGF/GAPDH mRNA ratios had
increased with factors 3.1 ± 1.1 for TGF-ß1, 3.2 ± 1.7 for
TGF-ß2, and 2.6 ± 0.8 for TGF-ß3 when compared with the
ratios in simultaneous control cultures
(Figure 9B). At 24 h of
incubation, CTGF/GAPDH mRNA ratios had decreased to background levels
(Figure 9B). In rat GVEC, the
response was more pronounced and sustained than in the mesangial cells.
Maximal CTGF/GAPDH mRNA ratios were detected at 24 h of incubation and
amounted to 16.2 ± 8.8 for TGF-ß1, 8.2 ± 3.6 for
TGF-ß2, and 8.8 ± 6.1 for TGF-ß3 when compared with the
ratios obtained for simultaneous control cultures
(Figure 9C). PDGF-BB did not
significantly affect CTGF mRNA expression in these cell lines (not shown).
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Discussion |
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We have previously shown that CTGF mRNA is highly expressed in human renal disorders associated with proliferative and fibrotic lesions, such as crescentic glomerulonephritis, IgA nephropathy, focal and segmental glomerulosclerosis, diabetic nephropathy, and chronic transplant rejection (12). In these conditions, CTGF was found to be expressed by GVEC and parietal epithelial cells, fibroblasts, vascular smooth muscle cells, and endothelial cells (12). In the present study, ISH revealed a remarkable increase of CTGF mRNA expression, first by GVEC and parietal epithelial cells in the induction phase of the model and more extensively by these cell types at days 4 and 7, accompanied by expression of CTGF mRNA by mesangial cells at days 4 and 7 and by periglomerular myofibroblasts at day 7. The CTGF mRNA expression had decreased considerably at day 14. These observations corresponded with semiquantitative RT-PCR analyses of glomerular RNA for CTGF.
CTGF is a member of the CCN family of structurally related proteins, which also includes Cyr61, Nov, WISP-1, WISP-2, and WISP-3. The CCN family members consist of three or four structural modules that are homologous to regions of various extracellular matrix proteins and are encoded by separate exons (10,31,32). The mosaic nature of the CCN proteins infers their effects on diverse aspects of cell function and behavior, including adhesion, migration, mitogenesis, differentiation, and survival (reviewed in references 32 and 10).
The early upregulation of CTGF mRNA in anti—Thy-1.1 nephritis indicates that enhanced expression of CTGF is not restricted to advanced stages of sclerotic disorders and can be involved in the physiologic response to tissue injury as well. Indeed, CTGF also has been implicated in wound repair in skin (11,23) and may be of biologic significance in various stages of this complex process.
The upregulation of glomerular CTGF mRNA expression at day 1 preceded the
increased glomerular expression of SMA, which is regarded as a marker
for mesangial cell activation in this model
(33). Although mesangial cells
are considered to be the prime target cells in the anti—Thy-1.1 model,
expression of CTGF mRNA was confined to the glomerular epithelial cells at
this early stage. CTGF produced by glomerular epithelial cells may affect in a
paracrine way the behavior of other cell types known to be involved in the
pathogenesis of anti—Thy-1.1 glomerulonephritis, such as platelets,
endothelial cells, mesangial cells, or fibroblasts. In addition, as has been
described for TGF-ß1
(34), CTGF may be an autocrine
factor for mesangial cells
(14) in later stages of
anti—Thy-1.1 glomerulonephritis.
TGF-ß1 is a potent inducer of CTGF expression in fibroblasts (5) and vascular smooth muscle cells (6). Recently, culture in high glucose was also found to induce CTGF expression in mesangial cells (13,14) via TGF-ß- and protein kinase C-dependent pathways (13). Indeed, the 5' flanking promoter region of the human CTGF gene does contain a unique TGF-ß response element (35). Therefore, we compared the expression patterns of CTGF first with those of TGF-ß1. By immunohistochemistry, we could detect TGF-ß1 at day 7 after disease induction, but to our surprise not at day 1 when CTGF mRNA was already clearly upregulated. Semiquantitative RT-PCR analysis confirmed the significantly increased presence of TGF-ß1 message in glomerular RNA at day 7 and also at day 14. It should be noted that the mean TGF-ß1/GAPDH ratio had increased at days 1 and 4 but not significantly when compared with untreated control rats. Overall, however, the levels of the glomerular mRNA ratios of CTGF/GAPDH and of TGF-ß1/GAPDH followed similar kinetics during anti—Thy-1.1 glomerulonephritis. Also, at sites of TI injury at day 7, ISH for CTGF mRNA was associated with immunostaining for TGF-ß1 in interstitial cells.
In addition to TGF-ß1, the isoforms TGF-ß2 and TGF-ß3 may be important in both regulation of wound repair and development of fibrosis. By immunohistochemistry, we detected a remarkable, strong increase of glomerular expression of TGF-ß2 already at the first day after induction of anti—Thy-1.1 glomerulonephritis. Glomerular TGF-ß3 protein expression, which was abundant in control animals, had somewhat increased at day 1 but seemed to decrease at days 4 and 7 of anti—Thy-1.1 glomerulonephritis. The diminished staining for TGF-ß3 may rather reflect the temporary loss of glomerular cells than decreased TGF-ß3 expression on a per cell basis.
Although the three TGF-ß isoforms do not seem to differ at the level of membrane receptor binding and signal transduction (36), a differential role for the TGF-ß variants is suggested by the nonoverlapping phenotypes of the respective knockout mice (36) and by diverse temporal and spatial expression patterns in organogenesis (37,38,39) and skin wound healing (40) and in response to glomerular injury (41,42,43). Interestingly, in a model of membranous nephropathy, GVEC were found to have increased expression of TGF-ß2 and TGF-ß3 but not of TGF-ß1 (42).
Because immunohistochemistry revealed the early upregulation of TGF-ß2 and TGF-ß3, followed by that of TGF-ß1, in association with the appearance of increased CTGF mRNA expression during anti—Thy-1.1 glomerulonephritis, we investigated a possible causal relation in vitro. All three isoforms of TGF-ß were found to be able to induce an early and transient upregulation of CTGF mRNA in mesangial cells. In GVEC, the upregulation of CTGF mRNA expression was more sustained and also reached higher levels compared with those in mesangial cells. Again, all three TGF-ß isoforms were potent inducers of increased CTGF mRNA expression, indicating that in addition to TGF-ß1, TGF-ß2 and TGF-ß3 are candidate stimulators of glomerular CTGF upregulation in anti—Thy-1.1 glomerulonephritis. The glomerular protein expression of TGF-ß3 in control animals was confined to the visceral epithelial cells and may be involved in regulation of the basal expression of CTGF that was observed to be restricted at the mRNA level to glomerular epithelial cells as well. It should be stressed that an unknown but probably large fraction of the total amount of each of the TGF-ß isoforms may be noncovalently bound to latency-associated peptide and is not present in its bioactive conformation (16,44).
In the induction phase of anti—Thy-1.1 nephritis, podocytes are exposed to increased stretching forces as a result of the loss of mesangial cells (45). Stress damage may be the cause of the increased expression of TGF-ß2 and TGF-ß3, which may lead to rapid upregulation of CTGF in podocytes. Our findings on early upregulation of TGF-ß2 and TGF-ß3, possibly in response to podocyte stress, correspond to those obtained in experimental membranous nephropathy (42). However, TGF-ß independent induction of CTGF expression cannot be excluded.
The pathogenesis of periglomerular fibrosis is still unresolved but
involves the participation of SMA-positive fibroblasts. The clinical
prognosis for patients with IgA nephropathy correlated well to the number of
periglomerular myofibroblasts
(46). Interestingly,
SMA-positive cells were found to surround Bowman's capsule also of
nonsclerotic glomeruli, independent of the presence of TI fibrosis and
glomerulosclerosis
(46,47).
These findings suggest an interaction between periglomerular
SMA-positive cells and glomerular cells. Previously, we observed strong
expression of CTGF mRNA by periglomerular SMA-positive cells in
association with the presence of CTGF mRNA expressing visceral and parietal
epithelial cells in human biopsy specimens
(12). For anti—Thy-1.1
glomerulonephritis, we observed that increased CTGF mRNA expression preceded
that of SMA in the periglomerular area. Together, these findings
suggest that CTGF, expressed by parietal epithelial cells, may promote in a
paracrine way the appearance of SMA-positive cells around the
glomeruli. CTGF produced by periglomerular myofibroblasts may increase the
synthesis of extracellular matrix leading to periglomerular fibrosis.
Previous studies demonstrated the involvement of several growth regulators in various stages of the pathogenesis of anti—Thy-1.1 glomerulonephritis (3,4). On the basis of the transient expression of CTGF mRNA observed in this model and of the various functions of CTGF as discussed above, we propose that CTGF may be an important factor in the renal response to injury as well, possibly downstream of TGF-ß. Evidently, in vivo intervention studies are mandatory to provide further clues on the significance of CTGF and on its relation to the action of the many other factors in these processes.
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Acknowledgments |
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This work was presented in part at the 30th and 32nd annual meetings of the American Society of Nephrology, November 2-5, 1997, San Antonio, Texas, and November 5-8, 1999, Miami Beach, Florida, and has been published in abstract form (J Am Soc Nephrol 8: 517A, 1997 and J Am Soc Nephrol 10: 542A, 1999).
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Smooth muscle actin is a marker of
mesangial cell proliferation. J Clin Invest87
: 847-858,1991
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