Existence of a regulatory loop between MCP-1 and TGF-beta in glomerular immune injury

Gunter Wolf, Thomas Jocks, Gunther Zahner, Ulf Panzer, and Rolf A. K. Stahl

Division of Nephrology, Department of Medicine, University of Hamburg, 20246 Hamburg, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta (TGF-beta ) 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-beta 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-beta 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-beta antibody further aggravated this increase, whereas application of recombinant TGF-beta protein reduced MCP-1 formation. Our data demonstrate an intrinsic regulatory loop in which increased MCP-1 levels stimulate TGF-beta formation in resident glomerular cells in the absence of infiltrating immune competent cells.

glomerulonephritis; chemokines; monocyte chemotactic protein-1; transforming growth factor-beta ; glomerulosclerosis; isolated perfused kidney


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta (TGF-beta ) 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-beta production (26). This suggested that the effect of MCP-1 on matrix formation may be mediated through TGF-beta . Because a reduction in glomerular infiltration of monocytes/macrophages could account for the reduced TGF-beta formation in this in vivo setting, it remains unclear whether MCP-1 stimulates TGF-beta formation in resident renal cells such as mesangial cells. Therefore, we further characterized a potential interaction between MCP-1 and TGF-beta 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-beta : MCP-1 is an inducer mediator of TGF-beta , whereas increased TGF-beta exerts a negative feedback on MCP-1 and reduces its expression. This interaction between MCP-1 and TGF-beta may have an important effect in reducing local inflammation in glomerulonephritis but may do so at the expense of stimulating extracellular matrix formation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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-beta 1-3 was purchased from Genzyme. Antibodies against MCP-1 and TGF-beta 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-beta 1-3, three bolus infusions of 100 µg anti-TGF-beta 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-beta 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-beta 1-3 antibody (3 × 100 µg as bolus at minutes 30, 50, and 70; n = 3); group 10, ATS-RS + recombinant TGF-beta 1, 3 × 200 ng as a bolus at minutes 30, 50, and 70 (n = 3); group 11, recombinant TGF-beta 1 alone (dosage as in group 10; n = 3).

An overview of the timing of antibody application, urinary collection, and harvesting of kidneys is given in Fig. 1.


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Fig. 1.   Overview of the perfusion process and the application of substances. In general, after an equilibration period of 20 min, anti-thymocyte serum (ATS)-rat serum (RS) was given during 90 s, and urinary collection was started. The anti-rat monocyte chemoattractant protein (MCP)-1 antibody (Ab) was included for the whole perfusion process, whereas the anti-transforming growth factor (TGF)-beta 1-3 antibody was applied in three bolus infusions. Control experiments in the absence of ATS-RS or application of nonimmune sera were done as described in METHODS according to the outlined time scale.

None of the antibodies used for perfusion affected the binding of ATS to renal mesangium cells. This was evaluated by perfusion with biotinylated ATS that was detected by an enzyme-linked immunosorbent assay (ELISA) after isolation and lysis of the glomeruli (data not shown).

We have previously shown that application of either ATS or RS as a complement source alone did not induce renal injury in the isolated perfused kidney (11). To confirm these findings in the present study, kidneys were perfused with ATS alone, RS alone, or ATS-RS in combination, and MCP-1 was measured by RT-PCR. As demonstrated in Fig. 2, the combination of ATS-RS is necessary for renal injury and chemokine synthesis.


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Fig. 2.   RT-PCR for MCP-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using isolated glomeruli from rats perfused either with ATS alone, RS alone, or with the combination of ATS-RS. Only ATS-RS together induced expression of MCP-1. In accordance with our previous studies (10), ATS or RS alone failed to induce glomerular injury.

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-beta 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-beta 1 was performed using the same method as described above. The primer sequences used for TGF-beta 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-beta 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-beta protein, separate gels were run, and the membranes were incubated with a monoclonal mouse anti-human TGF-beta 1-3 antibody (1:1,000 dilution; Genzyme). This antibody cross-reacts with rat TGF-beta . Selected blots were washed and reprobed with a monoclonal antibody against beta -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 beta -actin was calculated. Western blot analysis was performed two times.

ELISA for measurement of urinary MCP-1 and TGF-beta protein. Urinary excretion of MCP-1 and TGF-beta was measured using commercially available ELISA kits [rat MCP-1 (Cytoscreen Elisa; BioSource International, Camarillo, CA); rat TGF-beta (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-beta 1. A second specific biotinylated antibody then binds to a different epitope. We have tested whether addition of anti-TGF-beta 1-3 antibodies directly to standard curves of the rat MCP-1 ELISA and direct administration of anti-MCP-1 antibody to the TGF-beta 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.


    RESULTS
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ABSTRACT
INTRODUCTION
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Expression of TGF-beta RNA and protein in the isolated perfused kidney. Northern blot analysis showed that glomerular TGF-beta 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-beta 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-beta 1 mRNA expression was evaluated additionally with quantitative RT-PCR. In kidneys that were treated with ATS-RS mRNA, expression for TGF-beta 1 was increased significantly compared with controls (Fig. 4). Coperfusion with an anti-rat MCP-1 antibody attenuated this increase, but TGF-beta 1 expression was still above control levels (Fig. 2). In kidneys that received only anti-MCP-1 with the perfusate, TGF-beta 1 transcripts were reduced below controls. A bolus infusion of recombinant rat MCP-1 protein significantly increased TGF-beta 1 mRNA compared with control perfusions (Fig. 4).


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Fig. 3.   Northern blot for TGF-beta 1 of total RNA from isolated glomeruli. ATS-RS infusion strongly increased the expression of TGF-beta 1 transcripts in perfused kidneys. This effect was ameliorated by coperfusion with a neutralizing antibody against rat MCP-1 (alpha -MCP-1 Ab). Application of this antibody to control kidneys almost completely blocked baseline TGF-beta 1 expression.



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Fig. 4.   Quantitative RT-PCR analysis for TGF-beta 1 with an internal standard. ATS- and RS-treated kidneys displayed a strongly increased expression of TGF-beta 1 RNA compared with controls. alpha -MCP-1 antibody coperfusion decrease this stimulation. alpha -MCP-1 antibody alone reduced TGF-beta 1 RNA levels below controls. Recombinant MCP-1 (rec-MCP-1) protein infusion resulted in slightly increased RNA formation for TGF-beta 1. *P < 0.05 vs. controls, +P < 0.05 vs. ATS-RS only, n = 4.

Perfusion with ATS-RS also resulted in a threefold increase in TGF-beta protein expression, mainly as a monomer form of TGF-beta (Fig. 5). This increase was reduced when the anti-MCP-1 antibody was added to the perfusate (Fig. 5). The antibody alone reduced TGF-beta protein expression below control levels. Perfusion with recombinant rat MCP-1 slightly increased TGF-beta 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-beta expression normalized to beta -actin, n = 2].


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Fig. 5.   Western blot analysis of glomerular lysates detected with an antibody against TGF-beta 1-3. A strong increase in TGF-beta monomers was found after perfusion with ATS-RS. This effect was partly prevented by coperfusion with an alpha -MCP-1 antibody but could be mimicked by perfusion with recombinant rat MCP-1. The blot was washed and reincubated with an antibody against beta -actin to control for small variations in protein loading and transfer. This blot is representative of 2 independent experiments with qualitatively similar results.

Urinary TGF-beta 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-beta concentration already after 10 min. Coperfusion with anti-MCP-1 antibody almost completely prevented this increase in urinary TGF-beta ecxretion (Fig. 6). However, the ATS- and RS-induced urinary excretion of TGF-beta 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-beta protein was found in the urine. This effect was blocked by coperfusion with anti-MCP-1 (Fig. 6).


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Fig. 6.   Determination of urinary TGF-beta protein excretion with enzyme-linked immunosorbent assay (ELISA). Strongly increased values were measured 10 min after ATS-RS administration. When additional perfusion with alpha -MCP-1 antibody was performed, the increase was reduced significantly. Infusion of recombinant rat MCP-1 significantly stimulated TGF-beta protein formation 20 min after application. This effect was blocked by coperfusion with alpha -MCP-1 antibody. *P < 0.01 vs. controls, #P < 0.05 vs. ATS-RS only, n = 4-6.

Expression of MCP-1 RNA and protein in the isolated perfused kidney. After demonstrating that TGF-beta is a downstream target, we then tested a potential feedback effect on MCP-1 expression by neutralizing TGF-beta . 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-beta 1-3 antibody, whereas coperfusion with recombinant TGF-beta 1 slightly reduced stimulated MCP-1 mRNA expression [Fig. 7; controls: 1.0, ATS-RS + rec-TGF-beta : 0.8 ± 0.2, ATS-RS: 2.8 ± 0.4 (P < 0.05 vs. controls; n = 2), ATS-RS + anti-TGF-beta Ab: 3.5 ± 0.7 (P < 0.01 vs. controls; n = 3), controls alpha -TGF-beta 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-beta 1-3 antibody. Perfusion with the anti-TGF-beta 1-3 antibody alone almost doubled MCP-1 transcripts (Fig. 8). When ATS-RS perfusion was combined with the administration of recombinant TGF-beta 1 protein, the increase in MCP-1 expression was slightly decreased compared with ATS-RS alone (Fig. 8). Infusion of recombinant TGF-beta 1 protein alone almost completely blocked basal MCP-1 mRNA expression (Fig. 8).


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Fig. 7.   Northern blot analysis of total RNA obtained from isolated glomeruli after perfusions revealed a robust increase in MCP-1 transcripts. This increase was stimulated further by application of a neutralizing anti-TGF-beta 1-3 antibody (alpha -TGF-beta Ab), whereas coperfusion with recombinant TGF-beta 1 (rec-TGF-beta ) only slightly reduced stimulated MCP-1 mRNA expression.



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Fig. 8.   Quantitative RT-PCR for MCP-1 with RNA obtained from isolated glomeruli. Controls displayed low expression of MCP-1 transcripts. ATS-RS treatment strongly stimulated the MCP-1 RNA expression, which was further enhanced by additional treatment with alpha -TGF-beta antibody. The antibody alone increased MCP-1 expression. Compared with ATS-RS administration, coperfusion of kidneys with recombinant TGF-beta 1 protein ameliorated the stimulatory effect on MCP-1 expression. TGF-beta 1 alone reduced the MCP-1 signal below controls. *P < 0.05 vs. controls, n = 5.

These changes were also reflected in MCP-1 protein expression, as measured by Western blotting of glomerular lysates (Fig. 9). MCP-1 protein was undetectable in control perfused kidneys. A strong increase in chemokine expression was found after perfusion with ATS-RS (Fig. 9). MCP-1 protein expression was increased further by additional administration of anti-TGF-beta 1-3 antibody (Fig. 9). In contrast, no MCP-1 protein expression was detected after infusion of recombinant TGF-beta protein, whereas anti-TGF-beta 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-beta 1-3: 10.5 ± 0.7 (P < 0.01 vs. controls), controls + rec-TGF-beta 1: 0.9 ± 0.2, controls + anti-TGF- beta 1-3: 1.6 ± 0.2 (P < 0.05 vs. controls) relative changes in MCP-1 expression normalized to beta -actin, n = 2].


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Fig. 9.   Western blot analysis of glomerular protein showed that MCP-1 was increased strongly in ATS- and RS-perfused kidneys compared with controls. Neutralizing alpha -TGFbeta 1-3 antibody further increased glomerular MCP-1 protein expression. alpha -TGFbeta 1-3 alone also stimulated MCP-1 protein. The blot was washed and reincubated with an antibody against beta -actin to control for small variations in protein loading and transfer. This blot is representative of 2 independent experiments with qualitatively similar results.

Only basal production of MCP-1, close to the detection limit of the ELISA, was measured in urine after 40 and 50 min of perfusion (Fig. 10). ATS-RS strongly increase urinary MCP-1 protein excretion already at 10 min with a maximum at 30 min and reduction at 40 min after application (Fig. 10). Although coperfusion with the anti-TGF-beta 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-beta 1-3 antibody was replaced by nonspecific IgG, no alterations were seen (data not shown). Perfusion with only the anti-TGF-beta 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-beta protein significantly reduced the urinary MCP-1 excretion compared with ATS or RS alone (Fig. 10). Treatment of perfused kidneys with only recombinant TGF-beta protein resulted in undetectable levels of MCP-1 protein in the urine (data not shown).


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Fig. 10.   Measurement of urinary MCP-1 excretion with ELISA. A strong increase was detected after ATS-RS. Although total MCP-1 excretion could not be stimulated further by addition of alpha -TGFbeta 1-3 antibody, the time of MCP-1 excretion was prolonged. The stimulated MCP-1 urinary excretion was inhibited by coperfusion with recombinant TGF-beta 1. *P < 0.01 vs. controls, #P < 0.05 vs. controls, n = 4-5.

Because it is assumed that MCP-1 exerts its action through activation of C-C chemokine receptor 2 (CCR2) (24), one must postulate the presence of this receptor type on intrinsic glomerular cells to understand MCP-1-mediated TGF-beta 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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Fig. 11.   RT-PCR analysis for expression of CCR2 receptor transcripts. A: a strong specific band for CCR2 is present in glomerular RNA obtained after control and ATS-RS perfusion. B: a weaker, but clearly detectable, band for CCR2 receptor is present in RNA obtained from primary rat mesangial cells and a mesangial cell line. No expression was found in a rat glomerular endothelial cell line, but strong expression was observed in a rat renal fibroblast cell line. Isolation of RNA, reverse transcription, and cDNA amplification were independently performed two times. The identity of the amplified product was confirmed by sequence analysis.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

We have previously found that application of an anti-MCP-1 antiserum reduced glomerular TGF-beta synthesis and collagen type IV deposition in rats with experimental glomerulonephritis (26). Because inflammatory cells may release profibrogenic cytokines such as TGF-beta (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-beta 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-beta RNA and protein is strongly enhanced after the application of ATS and RS as a complement source. The stimulated glomerular synthesis of TGF-beta 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-beta 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-beta expression in glomerular cells. TGF-beta 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-beta 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-beta from cellular stores before upregulation of de novo synthesis. However, we did not measure latent TGF-beta secretion in the present study. The increase in TGF-beta secretion 60 min after ATS-RS likely represents the beginning of the glomerular de novo synthesis of TGF-beta 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-beta RNA in the later phase of the experiments. How neutralization of MCP-1 interferes with the early urinary secretion of TGF-beta 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-beta RNA and protein expression. These findings suggest that even TGF-beta expression in the basal state may be regulated by MCP-1. However, one could argue that reduction of TGF-beta expression by the anti-MCP-1 antibody alone in the absence of ATS-RS is the primary reason for the attenuation of TGF-beta expression after perfusion with ATS-RS and anti-MCP-1 antiserum. Although we believe that the magnitude of reduction of TGF-beta 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-beta . 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-beta expression is regulated exclusively by MCP-1.

Although a relationship between MCP-1 and TGF-beta 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-beta (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-beta . On the other hand, MCP-1-mediated TGF-beta induction may also underlie the surprising observation that MCP-1 protects mice in lethal endotoxemia by exerting anti-inflammatory effects in vivo (35). TGF-beta 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-beta 1 null mice exhibit an excessive inflammatory phenotype that results in severe autoimmune disease (5), clearly suggesting a role of TGF-beta 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-beta was not studied in this system, it is intriguing to speculate that a lack of TGF-beta 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-beta 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-beta 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-beta expression

The amount of MCP-1 formation appears to be controlled by TGF-beta in our system because blockade of TGF-beta by a monoclonal antibody resulted in a further increase in MCP-1 expression, whereas the administration of recombinant TGF-beta protein reduced MCP-1 production. This demonstrates a feedback loop between MCP-1 and TGF-beta , as outlined in Fig. 12. Our present experiments do not characterize the exact mechanisms by which TGF-beta reduces MCP-1 formation, and further studies are necessary to address this important issue. Yet, an inhibitory action of TGF-beta 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-beta as the active substance that was able to block MCP-1 formation in macrophages even after stimulation with LPS (12).


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Fig. 12.   Overview of the proposed feedback mechanism between MCP-1 and TGF-beta . Binding of ATS to mesangial cells and complement activation lead to an increase in mesangial MCP-1 synthesis. MCP-1 stimulates recruitment of macrophages/monocytes (M/M) in the glomerulus and additionally induces local synthesis of TGF-beta in intrinsic glomerular cells. M/M also release TGF-beta . TGF-beta itself suppresses the enhanced MCP-1 synthesis and also counteract proinflammatory stimuli. Moreover, it may directly inhibit chemotaxis of M/M. As a consequence, the inflammation may be limited but at the expense of a stimulation in extracellular matrix production. Solid arrows, stimulation; broken arrows, inhibition.

What could be the teleological function of a feedback mechanism between MCP-1 and TGF-beta 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-beta to turn off an overshoot in inflammation. TGF-beta -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-beta expression paves the way to glomerulosclerosis (13).

In summary, our data suggest that MCP-1 plays a role in the increased formation of TGF-beta 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-beta protein. Although glomerular induction of TGF-beta 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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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