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in
glomerular immune injury
Division of Nephrology, Department of Medicine, University of Hamburg, 20246 Hamburg, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Glomerular
upregulation of monocyte chemotactic protein-1 (MCP-1), followed by an
influx of monocytes resulting eventually in extracellular matrix
deposition is a common sequel of many types of glomerulonephritis.
However, it is not entirely clear how early expression of MCP-1 is
linked to the later development of glomerulosclerosis. Because
transforming growth factor-
(TGF-) is a key regulator of
extracellular matrix proteins, we hypothesized that there might be a
regulatory loop between early glomerular MCP-1 induction and subsequent
TGF- expression. To avoid interference with other cytokines that may
be released from infiltrating monocytes, isolated rat kidneys were
perfused with a polyclonal anti-thymocyte-1 antiserum (ATS) and rat
serum (RS) as a complement source to induce glomerular injury. Renal
TGF- protein and mRNA expressions were strongly stimulated after
perfusion with ATS-RS. This effect was attenuated by coperfusion with a
neutralizing anti-MCP-1 but was partly mimicked by perfusion with
recombinant MCP-1 protein. On the other hand, renal MCP-1 expression
and production were stimulated by administration of ATS-RS. Additional
perfusion with an anti-TGF- antibody further aggravated this
increase, whereas application of recombinant TGF- protein reduced
MCP-1 formation. Our data demonstrate an intrinsic regulatory loop in
which increased MCP-1 levels stimulate TGF- formation in resident
glomerular cells in the absence of infiltrating immune competent cells.
glomerulonephritis; chemokines; monocyte chemotactic protein-1; transforming growth factor-; glomerulosclerosis; isolated perfused
kidney
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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LEUKOCYTE INFILTRATION, resident cell growth, and extracellular matrix formation are characteristic histological features of various types of glomerulonephritis (10, 18, 31). These components participate in the damage of glomerular integrity. A large number of mediators such as cytokines and chemokines are involved in these processes (25). However, their potential interactions are only partially understood. Glomerular monocyte/ macrophage infiltration is a typical feature of the rat anti-thymocyte-1 model of mesangioproliferative glomerulonephritis (31). This glomerular infiltration is paralleled or followed by the formation of extracellular matrix (26). In the single-shot model, the accumulation of extracellular matrix resolves, but glomerulosclerosis eventually develops after repetitive applications of the anti-thymocyte-1 antibody. Glomerular monocyte recruitment in this model is largely mediated by chemokines, among which monocyte chemoattractant protein-1 (MCP-1) plays a predominant role (31). Neutralization of MCP-1 using specific antibodies not only reduces glomerular monocyte/macrophage infiltration in various experimental models of glomerulonephritis but also attenuates glomerular and tubulointerstitial matrix formation, at least to some extent (26, 33).
In the model of anti-thymocyte-1 glomerulonephritis, transforming
growth factor- (TGF-) is the major cytokine that regulates extracellular matrix formation (20). We recently found
that the neutralization of MCP-1 with a specific antibody in
anti-thymocyte-1 glomerulonephritis not only reduced
monocyte/macrophage infiltration but also decreased collagen type IV
and TGF- production (26). This suggested that the
effect of MCP-1 on matrix formation may be mediated through TGF-.
Because a reduction in glomerular infiltration of monocytes/macrophages
could account for the reduced TGF- formation in this in vivo
setting, it remains unclear whether MCP-1 stimulates TGF- formation
in resident renal cells such as mesangial cells. Therefore, we further
characterized a potential interaction between MCP-1 and TGF- in a
model of glomerular immune injury in the isolated perfused rat kidney
in the absence of infiltrating inflammatory cells (11).
Our results show that a feedback loop exists between MCP-1 and TGF-:
MCP-1 is an inducer mediator of TGF-, whereas increased TGF-
exerts a negative feedback on MCP-1 and reduces its expression. This
interaction between MCP-1 and TGF- may have an important effect in
reducing local inflammation in glomerulonephritis but may do so at the
expense of stimulating extracellular matrix formation.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Kidney perfusion. Rat kidney perfusion was performed exactly as previously described (11). In brief, left kidneys of male Wistar rats were perfused in situ with a catheter through the aorta. The kidney was removed and placed in a 37°C organ bath. The ureter was cannulated, and venous effluent was collected. Experiments were performed at constant pressure (100 mmHg) and temperature (37°C) as single-pass perfusions. The perfusate was a modified Krebs-Henseleit solution that contained (all data in mM if not stated otherwise): 145 sodium, 5.0 potassium, 110 chloride, 1.0 magnesium, 1.0 calcium, 27.4 hydrogen carbonate, 0.66 hydrogen phosphate, 0.3 dihydrogen phosphate, 7.6 glucose, 6.0 urea, 200 mg/l inulin (Merck, Darmstadt, Germany), 0.4 glutamine (Sigma, Munich, Germany), and 22 ml/l amino acids (5% aminoplasmal paed; Braun, Melsungen, Germany). A gelatin preparation was used as a colloidosmotic agent (Hemaccel; Behringwerke, Marburg, Germany) at a concentration of 35 g/l. The solution was filtered through a 0.45-mm cellulose acetate filter before the experiment (Sartorius, Göttingen, Germany). The perfusate solution was pregassed for 30 min with 95% O2-5% CO2 before perfusion.
Antibodies.
Anti-thymocyte serum (ATS) was prepared in New Zealand rabbits by
repeated immunization with thymocytes from Lewis rats as described
(31). A polyclonal antibody against rat MCP-1 was also
raised in New Zealand rabbits by repeated immunization with 40 µg of
recombinant rat MCP-1 (IC Chemikalien, Munich, Germany). This antibody
has been characterized previously in detail (33). A
monoclonal antibody against TGF-1-3 was purchased
from Genzyme. Antibodies against MCP-1 and TGF- were used for
neutralization in perfusion experiments and for detection of the
proteins in Western blot analysis.
Experimental groups.
The following groups were studied: group 1, controls
(n = 5), perfusate only; group 2, ATS-rat
serum (RS; n = 5), ATS (300 µl in 1 ml perfusate),
immediately followed by 1 ml of 1:2 diluted RS as a complement source
that was applied 20 min after start of perfusion within 90 s;
group 3, ATS-RS + polyclonal rabbit antiserum against
rat MCP-1 (n = 6), same as group 2, but the perfusate contained anti-rat MCP-1 antiserum (1 ml/l) throughout the
perfusion; group 4, kidneys were perfused only with
polyclonal rabbit anti-rat MCP-1 (1 ml/l; n = 4);
group 5, ATS-RS with preimmune rabbit IgG (1 ml/l) in the
perfusate (n = 3); group 6, infusion of a
mouse monoclonal antibody against TGF-1-3, three
bolus infusions of 100 µg anti-TGF-1-3 each in
0.5 ml perfusate at minutes 30, 50, and
70 (n = 4); group 7, infusion of
normal mouse IgG instead of anti-TGF-1-3 antibody
(n = 3); group 8, infusion of recombinant
rat MCP-1 (100 ng in 0.5 ml perfusate) as a bolus at minute
20 (n = 4); group 9, ATS-RS + monoclonal mouse anti-TGF-1-3 antibody (3 × 100 µg as bolus at minutes 30, 50, and
70; n = 3); group 10, ATS-RS + recombinant TGF-1, 3 × 200 ng as a bolus at
minutes 30, 50, and 70 (n = 3); group 11, recombinant
TGF-1 alone (dosage as in group 10;
n = 3).
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Isolation of total RNA, Northern blot analysis, and RT-PCR. Glomeruli were isolated from all perfused kidneys at the end of the experiment by fractional sieving according to a technique described earlier (30). The purity of the glomerular preparation reached 95%. After the last washing step, glomeruli were separated into two fractions for isolation of RNA and protein. Cellular RNA from whole glomeruli was isolated by the guanidinium isothiocyanate method (6).
Primary mesangial cell cultures from naive rats were established and characterized according to methods established in this laboratory (30). Furthermore, a rat mesangial cell line, rat renal fibroblasts (line NRK-49F obtained from ATCC), and a rat glomerular endothelial line (34) were used. RNA was isolated from cells growing in medium with 5% FCS. Total RNA (15 µg) was electrophoresed through a 1.2% agarose gel containing 2.2 M formaldehyde. Equal loading of lanes was evaluated by ethidium bromide staining of the 18S and 28S rRNA. The RNA was transferred to nylon membranes (Zetabind; Cuno, Meriden, CT) by vacuum blotting and was ultraviolet cross-linked. The blots were hybridized with a cDNA probe for TGF-1 after
[32P]dCTP labeling by random oligonucleotide priming of
the cDNA insert (520-bp EcoR I fragment; Ref.
26). The membranes were washed to a final high
stringency in 0.1× SSPE/1.0% SDS [1× SSPE = 0.18 M NaCl-10 mM
sodium phosphate (pH 7.4)-1 mM EDTA] for 30-60 min at 65°C.
Autoradiography was performed with intensifying screens at 
70°C for
appropriate time periods. The size of the respective RNA was identified
by comparison of its mobility with the ethidium bromide-stained RNA
standards. The membranes were rehybridized with a cDNA probe encoding
for 18S rRNA to account for small loading and transfer variations.
Exposed films were scanned with a laser densitometer (Hoefer Scientific
Instruments, San Francisco, CA), and relative RNA levels were
calculated. RNA for Northern blots was not pooled, and each lane
represents RNA isolated from one kidney.
For RT-PCR, 5 µg of RNA in 7.5 µl H2O were incubated at
65°C for 3 min to untie the secondary RNA structure. To minimize
differences between the tubes, a master mix containing 5.5 µl
first-strand buffer (250 mM Tris · HCl, 375 mM KCl, and 15 mM
MgCl2), 3.4 µl dNTPs (10 mM each), 2.7 µl
dithiothreitol (0.1 M), 0.5 µg poly(dT) primer (Pharmacia, Freiburg,
Germany), 14 units RNasin (Promega, Madison, WI), and 200 units Moloney
murine leukemia virus reverse transcriptase (GIBCO-BRL, Eggenstein,
Germany) per sample were prepared and added to each specimen in equal
quantity. The reaction was carried out for 90 min at 37°C. For
amplification of cDNA, the following primers were used based on the rat
MCP-1 cDNA (23): sense primer, 5'-ACA GTT GCT GCC TGT
AT-3', and antisense primer, 5'-CAC ACT TCT CTG TCA TAC-3'. To 5 µl
of each sample, 18.2 µl H2O, 2.5 µl 10× PCR buffer
(500 mM KCl, 100 mM Tris · HCl, and 1% Triton X-100), 1.5 µl
25 mM MgCl2, 0.8 µl sense and antisense primer (50 ng/ml
each), 1.0 µl of an internal standard or H2O (PCR MIMIC;
Clontech, Palo Alto, CA), and 0.5 units Taq DNA polymerase (Promega) were added. The standard concentrations used ranged between
0.01 and 0.1 pg/ml. Rat MCP-1 standard was prepared using the sense
primer 5'-ACA GTT GCT GCC TGT AT CGC AAG TGA AAT CTC CTC CG-3' and the
antisense primer 5'-CAC ACT AGT TCT CTG TCA TAC TTG AGT CCA TGG GGA GCT
TT-3' (23). These primers were annealed to a
BamHI/EcoRI v-erbB fragment according to the
MIMIC kit protocol. The v-erbB fragment was used to avoid interference
with the rat MCP-1 cDNA. The resulting 580-bp fragment exhibited
identical annealing sites for primers used for the amplification of the native MCP-1 fragment derived from glomerular cDNA. To correct for
potential transfer variations and quality differences of RNA and
reverse transcription, an additional PCR for rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed using
5'-AAT GCA TCC TGC ACC ACC AA-3' as the sense primer and 5'-GTA GCC ATA
TTC CAT TGT CAT A-3' as the antisense primer. The predicted size of the
GAPDH fragment is 520 bp. PCR was performed for 28 cycles using the following temperature profile: denaturation at 95°C for 10 s, annealing for 20 s at 57°C, and extension for 40 s at
72°C. Finally, an extension step for 5 min at 72°C was performed. A
total of 10 µl of each reaction was run on a 1.5% agarose gel that
contained ethidium bromide and was photographed over ultraviolet light. Negative films were analyzed by densitometry with a densitometer (Hoefer Scientific), and absorption was calculated with Quick Integration Algorithm of the program GS 365W (Hoefer Scientific). With
this approach, a linear standard curve between the PCR product and the
number of cycles can be obtained (10).
Quantitative PCR for TGF-1 was performed using the same
method as described above. The primer sequences used for
TGF-1 were based on the following rat cDNA: sense,
5'-CTC ACT GCT CTT GTG ACA GC-3', and antisense, 3'-AGC TGC ACT TGC AGG
AGG GC-5' (22). The resulting amplification product was
520 bp. An internal standard was synthesized as described above.
Amplifications for TGF-1 were performed for 32 cycles.
The presence of CCR2 receptor RNA in isolated glomeruli and various
cell lines was tested with RT-PCR. After reverse transcription as
described above, PCR was run for 28 cycles using the following temperature profile: 95°C for 10 s, 57°C for 20 s, and
72°C for 30 s. The following primers were used:
5'-TCCATACAATATTGTTCT-3' and 5'-TCCCCAGTGGAAGGAGTGAA-3'
(14). The size of the amplification product was 288 bp,
and sequence analysis of subcloned cDNA revealed the identity of the
amplified product.
Western blot analysis.
Isolated glomeruli were dissolved in 100 µl of Laemmli buffer and
boiled for 10 min (15). After centrifugation, 200 µg
protein were loaded per lane and were separated on an 8%
SDS-polyacrylamide gel. The separated proteins were electrophoretically
transferred to polyvinyl membranes. Blocking was performed in 5%
nonfat dry milk prepared in PBS with 0.1% Tween 20. MCP-1 was
identified by incubating the membrane with a polyclonal rabbit anti-rat
MCP-1 antiserum (1:1,000 dilution; for preparation of the antibody see Antibodies), followed by horseradish peroxidase-conjugated
goat anti-rabbit IgG (1:2,000 dilution). For the detection of
TGF- protein, separate gels were run, and the membranes were
incubated with a monoclonal mouse anti-human
TGF-1-3 antibody (1:1,000 dilution; Genzyme). This
antibody cross-reacts with rat TGF-. Selected blots were washed and
reprobed with a monoclonal antibody against -actin (Sigma) to
control for small variations in protein loading and transfer. Signals
were detected with a luminescence immunodetection assay (ECL Western
blotting system; Amersham). Bands were scanned by densitometry as
described above, and a ration between specific signal and -actin was
calculated. Western blot analysis was performed two times.
ELISA for measurement of urinary MCP-1 and TGF- protein.
Urinary excretion of MCP-1 and TGF- was measured using commercially
available ELISA kits [rat MCP-1 (Cytoscreen Elisa; BioSource International, Camarillo, CA); rat TGF- (Predicta Elisa; Genzyme, Cambridge, MA)]. Both ELISA are sandwich assays in which a specific antibody has been coated on microtiter plates to capture MCP-1 or
TGF-1. A second specific biotinylated antibody then
binds to a different epitope. We have tested whether addition of
anti-TGF-1-3 antibodies directly to standard curves
of the rat MCP-1 ELISA and direct administration of anti-MCP-1 antibody
to the TGF- ELISA do not interfere with measurements (data not shown).
Statistical analysis. Data are given as means ± SD. Multiple groups were analyzed with ANOVA. Individual comparisons between two groups were made with Student's t-test. A value of P < 0.05 was considered as significant.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Expression of TGF- RNA and protein in the isolated perfused
kidney.
Northern blot analysis showed that glomerular TGF-1 mRNA
expression doubled after perfusion with ATS-RS (Fig.
3). When the perfusate contained an
antibody against rat MCP-1, this increase was attenuated. Perfusion
with antibody alone decreased the TGF-1 signal below
control levels [Fig. 3; controls: 1.0, ATS-RS: 2.0 ± 0.2 (P < 0.05 vs. controls), ATS-RS + anti-MCP-1:
1.41 ± 0.1 (P < 0.05 vs. ATS-RS only),
controls + anti-MCP: 0.57 ± 0.08 (P < 0.05 vs. controls) relative changes in mRNA expression normalized to 18S,
n = 2]. TGF-1 mRNA expression was
evaluated additionally with quantitative RT-PCR. In kidneys that were
treated with ATS-RS mRNA, expression for TGF-1 was
increased significantly compared with controls (Fig.
4). Coperfusion with an anti-rat MCP-1
antibody attenuated this increase, but TGF-1 expression
was still above control levels (Fig. 2). In kidneys that received only
anti-MCP-1 with the perfusate, TGF-1 transcripts were
reduced below controls. A bolus infusion of recombinant rat MCP-1
protein significantly increased TGF-1 mRNA compared with
control perfusions (Fig. 4).
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protein expression, mainly as a monomer form of TGF- (Fig.
5). This increase was reduced when the
anti-MCP-1 antibody was added to the perfusate (Fig. 5). The antibody
alone reduced TGF- protein expression below control levels.
Perfusion with recombinant rat MCP-1 slightly increased TGF-
synthesis [Fig. 5; controls: 1.0, ATS-RS: 5.0 ± 0.4 (P < 0.01 vs. controls), ATS-RS + anti-MCP-1:
2.1 ± 0.3 (P < 0.05 vs. ATS-RS only),
controls + anti-MCP: 0.9 ± 0.1, controls + rec-MCP-1:
1.7 ± 0.1 (P < 0.05 vs. controls) relative
changes in TGF- expression normalized to -actin,
n = 2].
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excretion was near the detection limit of the assay in
perfusion of normal kidneys (Fig. 6).
However, treatment with ATS-RS led to a significant increase in urinary
TGF- concentration already after 10 min. Coperfusion with anti-MCP-1
antibody almost completely prevented this increase in urinary TGF-
ecxretion (Fig. 6). However, the ATS- and RS-induced urinary excretion
of TGF- was not reduced by coperfusion with rat nonspecific IgG (data not shown). When isolated kidneys were perfused with recombinant rat MCP-1, a clear increase in TGF- protein was found in the urine.
This effect was blocked by coperfusion with anti-MCP-1 (Fig. 6).
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Expression of MCP-1 RNA and protein in the isolated perfused
kidney.
After demonstrating that TGF- is a downstream target, we then tested
a potential feedback effect on MCP-1 expression by neutralizing TGF-. Northern blot analysis of total RNA obtained from isolated glomeruli after perfusions revealed a robust increase in MCP-1 transcripts (Fig. 7). This increase was
stimulated further by application of a neutralizing
anti-TGF-1-3 antibody, whereas coperfusion with
recombinant TGF-1 slightly reduced stimulated MCP-1 mRNA
expression [Fig. 7; controls: 1.0, ATS-RS + rec-TGF-: 0.8 ± 0.2, ATS-RS: 2.8 ± 0.4 (P < 0.05 vs.
controls; n = 2), ATS-RS + anti-TGF- Ab:
3.5 ± 0.7 (P < 0.01 vs. controls;
n = 3), controls + 
-TGF- Ab: 1.50 ± 0.3 relative changes in mRNA expression normalized to 18S].
Quantitative RT-PCR confirmed these changes (Fig. 6). Although MCP-1
was hardly detectable in control perfusions, its mRNA expression was
increased ~2.5-fold in ATS- and RS-perfused kidneys (Fig.
8). This induction was enhanced further
by additional infusion of anti-TGF-1-3 antibody.
Perfusion with the anti-TGF-1-3 antibody alone
almost doubled MCP-1 transcripts (Fig. 8). When ATS-RS perfusion was
combined with the administration of recombinant TGF-1
protein, the increase in MCP-1 expression was slightly decreased
compared with ATS-RS alone (Fig. 8). Infusion of recombinant TGF-1 protein alone almost completely blocked basal
MCP-1 mRNA expression (Fig. 8).
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1-3 antibody (Fig. 9). In contrast, no
MCP-1 protein expression was detected after infusion of recombinant
TGF- protein, whereas anti-TGF-1-3 antibody
treatment alone resulted in a small but significant increase in MCP-1
protein [Fig. 9; controls: 1.0, ATS-RS: 7.3 ± 1.04 (P < 0.05 vs. controls), ATS-RS + anti-TGF-1-3: 10.5 ± 0.7 (P < 0.01 vs. controls), controls + rec-TGF-1:
0.9 ± 0.2, controls + anti-TGF- 1-3:
1.6 ± 0.2 (P < 0.05 vs. controls) relative changes in MCP-1 expression normalized to -actin, n = 2].
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1-3 antibody did not further
increase maximal urinary MCP-1 levels, the increase was sustained
compared with perfusion without the antibody (Fig. 10). When the
anti-TGF-1-3 antibody was replaced by nonspecific
IgG, no alterations were seen (data not shown). Perfusion with only the
anti-TGF-1-3 antibody moderately stimulated MCP-1
formation with a short delay (Fig. 10). Additional treatment of ATS-
and RS-perfused kidneys with recombinant TGF- protein significantly
reduced the urinary MCP-1 excretion compared with ATS or RS alone (Fig.
10). Treatment of perfused kidneys with only recombinant TGF-
protein resulted in undetectable levels of MCP-1 protein in the urine
(data not shown).
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expression. We used
low-cycle cDNA amplification of reverse-transcribed RNA from glomeruli
isolated after control and ATS-RS perfusion as well as different
cultured renal cells to test for the presence of CCR2 transcript. As
demonstrated in Fig. 11, A
and B, CCR2 RNA is clearly present in isolated glomeruli (Fig. 11A) and cultured rat mesangial cells but not in a
previously established rat glomerular endothelial cell line (Fig.
11B). However, we failed to demonstrate CCR2 receptor
expression by immunohistochemistry in renal sections obtained from
perfused rat kidneys with commercially available antibody (data not
shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We have previously found that application of an anti-MCP-1
antiserum reduced glomerular TGF- synthesis and collagen type IV
deposition in rats with experimental glomerulonephritis
(26). Because inflammatory cells may release profibrogenic
cytokines such as TGF- (7, 29), this study left open
whether the beneficial effects of anti-MCP-1 antiserum in nephritic
animals was because of a decrease in glomerular recruitment of such
cells. Consequently, we were interested in the present study to learn
whether there is a potential interrelationship between MCP-1 and
TGF- expression in resident glomerular cells in the absence of
infiltrating immune-competent cells and applied the isolated perfused
rat kidney as a tool for studying this relationship in glomerular
immune injury (11).
We have previously described in detail that perfusion of isolated kidneys with ATS-RS induces glomerular injury and that neither ATS nor RS alone was effective (11). In addition, we clearly demonstrate in the present study that perfusion with ATS and RS as a complement source is necessary for the induction of chemokines. Perfusion with RS alone did not exert glomerular injury.
We could show with different methodological approaches that glomerular
formation of both TGF- RNA and protein is strongly enhanced after
the application of ATS and RS as a complement source. The stimulated
glomerular synthesis of TGF- leads to an increased excretion in the
urine during perfusion. This effect was attenuated significantly by coperfusion with a neutralizing anti-MCP-1 antibody. On the other hand, the effect of the glomerular immune injury on
TGF- expression could be, at least to some extent, mimicked by
infusion of recombinant MCP-1 protein. These findings strongly suggest
that MCP-1 does not only have chemoattractant properties on circulating
leukocytes but also regulates TGF- expression in glomerular cells.
TGF- is an important mediator of extracellular matrix deposition and
plays a role in the development of fibrosis in several models of
glomerular injury (4, 29). Since the onset of increased
TGF- protein excretion in the urine was observed very early after
immune injury, it is tempting to suggest that the release and
activation of latent TGF- from cellular stores before upregulation
of de novo synthesis. However, we did not measure latent
TGF- secretion in the present study. The increase in TGF-
secretion 60 min after ATS-RS likely represents the beginning of the
glomerular de novo synthesis of TGF- because glomerular transcripts
and protein excretion were increased. The neutralization of MCP-1 by
coperfusion with the antibody prevented both the early release and the
subsequent upregulation of TGF- RNA in the later phase of the
experiments. How neutralization of MCP-1 interferes with the early
urinary secretion of TGF- remains unclear and requires further studies.
Perfusion of control kidneys with the neutralizing anti-MCP-1 antibody
also resulted in a downregulation of basal TGF- RNA and protein
expression. These findings suggest that even TGF- expression in the
basal state may be regulated by MCP-1. However, one could argue that
reduction of TGF- expression by the anti-MCP-1 antibody alone in the
absence of ATS-RS is the primary reason for the attenuation of TGF-
expression after perfusion with ATS-RS and anti-MCP-1 antiserum.
Although we believe that the magnitude of reduction of TGF-
expression is greater by the anti-MCP-1 antibody after ATS-RS
application compared with the control situation, we could not rule out
that other cytokines besides MCP-1 may play a role in ATS- and
RS-mediated induction of TGF-. This is a limitation of our study
using the isolated kidney, and one should be cautious to extrapolate
these findings to the whole organism. Further studies are necessary to
test whether TGF- expression is regulated exclusively by MCP-1.
Although a relationship between MCP-1 and TGF- has been described
previously (8), it remained unclear whether resident glomerular cells play a role in this mechanism in the absence of
infiltrating immune cells. For example, Lloyd and co-workers (17) reported that an elevated MCP-1 expression induced
crescent formation and interstitial fibrosis in a model of crescentic
glomerulonephritis. It was assumed, but not really studied, that the
function of MCP-1 in the fibrotic process might be mediated by
fibrogenic agents such as TGF- (17). A possible role of
MCP-1 in the progression of interstitial fibrosis and in the
stimulation of collagen deposition in the tubulointerstitium has also
been suggested (18), but the mediation of these effects
remained to be determined. Gharaee-Kermani et al. (8)
found in an in vitro approach using lung fibroblasts that treatment
with MCP-1 resulted in an increased formation of procollagen I, which
could be completely inhibited by antisense oligonucleotides directed
against TGF-. On the other hand, MCP-1-mediated TGF- induction
may also underlie the surprising observation that MCP-1 protects mice
in lethal endotoxemia by exerting anti-inflammatory effects in vivo
(35). TGF- has anti-inflammatory properties by way of a
variety of different mechanisms, including inhibition of cell adhesion
molecules and suppression of T cells among many other functions
(16, 21). TGF-1 null mice exhibit an
excessive inflammatory phenotype that results in severe autoimmune
disease (5), clearly suggesting a role of TGF- in
preventing inflammation.
Chemokines, including MCP-1, exert their putative effects through
activation of specific G protein-coupled seven-transmembrane receptors
(2, 24). MCP-1 binds exclusively to the so-called CCR2
receptor (2, 14). In accordance with our findings, CCR2 receptor knockout mice reveal an increased severity of
glomerulonephritis (3). Although TGF- was not studied
in this system, it is intriguing to speculate that a lack of TGF-
induction by MCP-1 contributed to this more malign course of
nephrotoxic nephritis (3).
Immunohistochemical studies and analysis of glomerular suspensions
obtained from human renal biopsies as well as various models of renal
disease induced in mice failed to detect CCR2 expression on intrinsic
resident renal cells (19, 27, 28). In contrast, this
receptor was solely expressed on infiltrating immune cells (27,
28). However, mRNA for CCR2 has been reported on cultured human
umbilical vein endothelial cells and in the mouse kidney (14,
32). We have performed limited studies to address CCR2 receptor
expression on glomerular resident cells. Using low-cycle cDNA
amplification after reverse transcription of RNA, we could clearly
detect CCR2 transcripts in cultured rat mesangial cells (primary
cultures and a cell line) and rat glomeruli isolated from control and
ATS- and RS-perfused kidneys. In contrast, a previous established and
characterized rat glomerular endothelial cell line (34)
did not express CCR2 mRNA. However, we failed using commercially
available anti-human CCR2 receptor antibodies to demonstrate expression
by immunohistochemistry (data not shown). Analysis of CCR2 protein
expression in the rat is presently hampered by the absence of specific
antibodies and a failure of anti-human antiserum to cross-react with
the rat receptor. It is possible that only a few CCR2 receptors are
expressed on the surface of renal cells that could not be detected with
the presently used antibodies. Alternatively, these antisera are
generated against CCR2 epitopes that are expressed on leukocytes but
that may be structurally different on solid organs, including the
kidney. This is apparently a more generous phenomenon with chemokine
receptors, and other investigators could not study CCR1 receptor
expression in mice because of a lack of appropriate antibodies against
murine CCR1 (1). However, earlier functional studies
demonstrated that MCP-1 influences the growth of cultured rat vascular
smooth muscle cells, suggesting that CCR2 expression is not limited to leukocytes (9). Our present study clearly suggests that
MCP-1 induces TGF- expression on intrinsic renal cells because no
infiltrating immune cells are present in the isolated kidney. Further
studies, including investigations using cultured glomerular cells, are necessary to address whether the regulatory loop between MCP-1 and
TGF- could be also reconstituted in an in vitro environment. Moreover, it remains presently unclear how activation of CCR2 receptors
may ultimately lead to an increase in TGF- expression
The amount of MCP-1 formation appears to be controlled by TGF- in
our system because blockade of TGF- by a monoclonal antibody resulted in a further increase in MCP-1 expression, whereas the administration of recombinant TGF- protein reduced MCP-1 production. This demonstrates a feedback loop between MCP-1 and TGF-, as outlined in Fig. 12. Our present
experiments do not characterize the exact mechanisms by which TGF-
reduces MCP-1 formation, and further studies are necessary to address
this important issue. Yet, an inhibitory action of TGF- on MCP-1
expression has also been described previously. Kitamura and
Sütö (13) found an inhibition of MCP-1
production after treatment with conditioned medium derived from
cultured rat mesangial cells. They identified in elegant studies
TGF- as the active substance that was able to block MCP-1 formation
in macrophages even after stimulation with LPS (12).
|
What could be the teleological function of a feedback mechanism between
MCP-1 and TGF- as depicted in Fig. 12? Chemokines may be originally
evolved as part of the innate immune system playing a role in
orchestrating the immune response toward exogenous intruders such as
bacteria by recruiting leukocytes to the region of local infection. It
is intuitively understandable that there should exist a local feedback
mechanism mediated by TGF- to turn off an overshoot in inflammation.
TGF--mediated synthesis of extracellular matrix components may
further serve to prevent spreading of the infection through gaining
vascular access and to protect intact tissue parts (13).
However, in chronic autoimmune diseases, such as chronic
glomerulonephritis with continuous activation of immune responses,
activated TGF- expression paves the way to glomerulosclerosis
(13).
In summary, our data suggest that MCP-1 plays a role in the increased
formation of TGF- by intrinsic glomerular cells in the ATS model in
the absence of infiltrating immune cells. This process is
controlled by a negative-feedback mechanism through newly synthesized
TGF- protein. Although glomerular induction of TGF- may limit
inflammation, it may also play an important role in the development of
irreversible renal scarring.
| |
ACKNOWLEDGEMENTS |
|---|
This work was supported by Deutsche Forschungsgemeinschaft Grant Sta193/6-4/5.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. Wolf, Div. of Nephrology and Osteology, Dept. of Medicine, Univ. of Hamburg, Univ. Hospital Eppendorf (UKE), Martinistr. 52, Pav.61, 20246 Hamburg, Germany (E-mail: Wolf{at}uke.uni-hamburg.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00349.2001
Received 27 November 2001; accepted in final form 1 June 2002.
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REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Anders, HJ,
Vielhauer V,
Frink M,
Linde Y,
Cohen CD,
Blattner SM,
Kretzler M,
Strutz F,
Mack M,
Gröne HJ,
Onuffer J,
Horuk R,
Nelson PJ,
and
Schlöndorff D.
A chemokine receptor CCR1 antagonist reduces renal fibrosis after unilateral ureter ligation.
J Clin Invest
109:
251-259,
2002[Web of Science][Medline].
2.
Arai, H,
Tsou CL,
and
Charo IF.
Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: evidence that directed migration is mediated by 
dimers released by activation of G
i-coupled receptors.
Proc Natl Acad Sci USA
94:
14495-14499,
1997
3.
Bird, JE,
Giancarli MR,
Kurihara T,
Kowala MC,
Valentine MT,
Gitlitz PH,
Pandya DG,
French MH,
and
Durham SK.
Increased severity of glomerulonephritis in C-C chemokine receptor 2 knockout mice.
Kidney Int
57:
129-136,
2000[Web of Science][Medline].
4.
Border, WA,
Noble NA,
and
Ketteler M.
TGF-: a cytokine mediator of glomerulosclerosis and a target for therapeutic intervention.
Kidney Int
47, Suppl49:
59-61,
1995.
5.
Böttinger, EP,
Letterio JJ,
and
Roberts AB.
Biology of TGF- in knockout and transgenic mouse models.
Kidney Int
51:
1355-1360,
1997[Web of Science][Medline].
6.
Chirgwin, JM,
Przybyla AE,
MacDonald RJ,
and
Rutter WJ.
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18:
5294-5299,
1979[Medline].
7.
Diamond, JR,
Kees-Folts D,
Ding G,
Frye JE,
and
Restrepo NC.
Macrophages, monocyte chemoattractant peptide-1 and TGF-1 in experimental hydronephrosis.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F926-F933,
1994
8.
Gharaee-Kermani, M,
Denholm EM,
and
Phan SH.
Costimulation of fibroblast collagen and transforming growth factor 1 gene expression by monocyte chemoattractant protein-1 via specific receptors.
J Biol Chem
271:
17779-17784,
1996
9.
Ikeda, U,
Okada K,
Ishikawa SE,
Saito T,
Kasahara T,
and
Shimada K.
Monocyte chemoattractant protein 1 inhibits growth of rat vascular smooth muscle cells.
Am J Physiol Heart Circ Physiol
268:
H1021-H1026,
1995
10.
Jocks, T,
Freudenberg J,
Zahner G,
and
Stahl RAK
Platelet-activating factor mediates monocyte chemoattractant protein-1 expression in glomerular immune injury.
Nephrol Dial Transplant
13:
37-43,
1998
11.
Jocks, T,
Zahner G,
Helmchen U,
Kneissler U,
and
Stahl RAK
Antibody and complement reduce renal hemodynamic function in the isolated perfused rat kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F179-F185,
1996
12.
Kitamura, M.
Identification of an inhibitor targeting macrophage production of monocyte chemoattractant protein-1 as TGF-1.
J Immunol
159:
1404-1411,
1997[Abstract].
13.
Kitamura, M,
and
Sütö TS.
TGF- and glomerulonephritis: anti-inflammatory versus prosclerotic actions.
Nephrol Dial Transplant
12:
669-679,
1997
14.
Kurihara, T,
and
Bravo R.
Cloning and functional expression of mCCR2, a murine receptor for the C-C chemokines JE and FIC.
J Biol Chem
271:
11603-11606,
1996
15.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4 (Abstract).
Nature
227:
680,
1970[Medline].
16.
Letterio, JJ,
and
Roberts AB.
Regulation of immune responses by TGF-.
Annu Rev Immunol
16:
137-161,
1998[Web of Science][Medline].
17.
Lloyd, CM,
Minto AW,
Dorf ME,
Proudfoot A,
Salant DJ,
and
Gutierrez-Ramos J-C.
Role of MCP-1 and Rantes in inflammation and progression to fibrosis during murine crescentic nephritis.
J Leukoc Biol
62:
676-680,
1997[Abstract].
18.
Lloyd, CM,
Minto AW,
Dorf ME,
Proudfoot A,
Wells TNC,
Salant DJ,
and
Gutierrez-Ramos J-C.
Rantes and monocyte chemoattractant protein-1 (MCP-1) play an important role in the inflammatory phase of crescentic Nephritis, but only MCP-1 is involved in crescent formation and interstitial fibrosis.
J Exp Med
185:
1371-1380,
1997
19.
Mack, M,
Cihak J,
Simonis C,
Luckow B,
Proudfoot AEI,
Plachy J,
Brühl H,
Frink M,
Anders HJ,
Vilehauer V,
Pfirstinger J,
Stangassinger M,
and
Schlöndorff D.
Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice.
J Immunol
166:
4697-9704,
2001
20.
Okuda, S,
Languino LR,
Ruoslahti E,
and
Border WA.
Elevated expression of transforming growth factor beta and proteoglycan production in experimental glomerulonephritis.
J Clin Invest
86:
453-462,
1990[Web of Science][Medline].
21.
Park, SK,
Yang WS,
Lee SK,
Ahn H,
Park JS,
Hwang O,
and
Lee JD.
TGF-1 down-regulates inflammatory cytokine-induced VCAM-1 expression in cultured human glomerular endothelial cells.
Nephrol Dial Transplant
15:
596-604,
2000
22.
Qian, SW,
Kondaiah P,
Roberts AB,
and
Sporn MB.
cDNA cloning by PCR of rat transforming growth factor -1 (Abstract).
Nucleic Acids Res
18:
3059,
1990
23.
Rollins, BJ,
Morrison ED,
and
Stiles CD.
Cloning and expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties.
Proc Natl Acad Sci USA
85:
3738-3742,
1988
24.
Rossi, D,
and
Zlotnik A.
The biology of chemokines and their receptors.
Annu Rev Immunol
18:
217-242,
2000[Web of Science][Medline].
25.
Rovin, BH.
Chemokine blockade as a therapy for renal disease.
Curr Opin Nephrol Hypertens
13:
225-232,
2000.
26.
Schneider, A,
Panzer U,
Zahner G,
Wenzel U,
Wolf G,
Thaiss F,
Helmchen U,
and
Stahl RAK
Monocyte chemoattractant protein-1 mediates collagen deposition in experimental glomerulonephritis by transforming growth factor-.
Kidney Int
56:
135-144,
1999[Web of Science][Medline].
27.
Segerer, S,
Cui Y,
Eitner F,
Goodpaster T,
Hudkins KL,
Mack M,
Catron JP,
Colin Y,
Schlondorff D,
and
Alpers CE.
Expression of chemokines and chemokine receptors during human renal transplant rejection.
Am J Kidney Dis
37:
518-531,
2001[Web of Science][Medline].
28.
Segerer, S,
Cui Y,
Hudkins KL,
Goodpaster T,
Eitner F,
Mack M,
Schlöndorff D,
and
Alpers CE.
Expression of the chemokine monocyte chemoattractant protein-1 and its receptor chemokine receptor 2 in human crescentic glomerulonephritis.
J Am Soc Nephrol
11:
2231-2242,
2000
29.
Sharma, K,
and
Ziyadeh FN.
The emerging role of transforming growth factor- in kidney diseases.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F829-F842,
1994
30.
Stahl, RAK,
Helmchen U,
Paravicini M,
and
Schollmeyer P.
Glomerular prostaglandin formation in two-kidney-one-clip hypertensive rats.
Am J Physiol Renal Fluid Electrolyte Physiol
247:
F975-F981,
1984
31.
Stahl, RAK,
Thaiss F,
Disser M,
Helmchen U,
Hora K,
and
Schlöndorff D.
Increased expression of MCP-1 in anti-thymocyte antibody-induced glomerulonephritis.
Kidney Int
44:
1036-1047,
1993[Web of Science][Medline].
32.
Weber, KSC,
Nelson PJ,
Gröne HJ,
and
Weber C.
Expression of CCR2 by endothelial cells. Implications for MCP-1 mediated wound injury repair and in vivo inflammatory activation of endothelium.
Arterioscler Thromb Vasc Biol
19:
2085-2093,
1999
33.
Wenzel, UO,
Schneider A,
Valente AJ,
Abboud HE,
Thaiss F,
Helmchen UM,
and
Stahl RAK
Monocyte chemoattractant protein-1 mediates monocyte/macrophage influx in anti-thymocyte antibody-induced glomerulonephritis.
Kidney Int
51:
770-776,
1997[Web of Science][Medline].
34.
Wolf, G,
Ziyadeh FN,
Zahner G,
and
Stahl RAK
Angiotensin II is mitogenic for cultured rat glomerular endothelial cells.
Hypertension
27:
897-905,
1996
35.
Zisman, DA,
Kunkel SL,
Strieter RM,
Tsai WC,
Bucknell K,
Wilkowski J,
and
Standiford TJ.
MCP-1 protects mice in lethal endotoxemia.
J Clin Invest
99:
2832-2836,
1997[Web of Science][Medline].
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