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Published online before print November 30, 2007
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From the Department of Internal Medicine,* University of Turin, Turin; and the Department of Cardionephrology, University of Genoa, Genoa, Italy
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Abstract |
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In the adult kidney, podocytes are in a quiescent state; however, both proliferation and acquisition of a migratory phenotype have been reported in pathological conditions. In crescentic glomerulonephritis (GN), podocytes detach from the glomerular basement membrane (GBM), assume a migratory phenotype, and trigger crescent formation by establishing bridges between the tuft and the Bowman’s capsule.2 In addition, cells derived from migrated podocytes proliferate and participate in crescent formation.3 Furthermore, in nephrotic conditions, podocyte effacement, which requires cytoskeleton remodeling, foot process movement over the GBM, and slit diaphragm reconstruction, may also be considered a migratory event aimed to compensate for podocyte loss by covering areas of bare GBM.4
Monocyte chemoattractant protein-1 (MCP-1) is a potent mononuclear cell chemoattractant produced by a variety of mesenchymal cells, including glomerular cells.5-7 Within the glomeruli, there is MCP-1 overexpression in both crescent GN8 and nephrotic conditions.9-11 Furthermore, immunohistochemistry studies have shown that glomerular podocytes are the predominant glomerular cell type overexpressing MCP-1 in various proteinuric conditions, such as diabetic, hypertensive, and membranous nephropathies.9,12,13 Finally, recent studies have shown that blockade/loss of MCP-1 has antiproteinuric and renoprotective effects in both experimental diabetic nephropathy14 and crescentic GN,15,16 suggesting a role of MCP-1 in the pathogenesis of the glomerular damage.
MCP-1 binds and signals through a seven-transmembrane protein-coupled receptor, the cognate CC chemokine receptor 2 (CCR2), which is predominantly expressed by monocytes.17 Local recruitment of monocytes is considered the predominant mechanism by which MCP-1 contributes to the renal damage; however, CCR2 expression has been demonstrated in other cell types, both in vitro18-23 and in vivo,24-27 indicating that the MCP-1/CCR2 system has other effects beyond monocyte accrual. Notably, MCP-1 binding to the CCR2 receptor induces both chemotaxis and proliferation in epithelial, endothelial, and vascular smooth muscle cells,19-22,27 suggesting a role of the MCP-1/CCR2 system in conferring a proliferative and migratory phenotype in cells other than monocytes.
In vitro data on CCR2 expression by podocytes are conflicting,28,29 but a recent study in the Alport mouse model has shown overexpression of the CCR2 receptor in glomerular podocytes in vivo,29 suggesting a potential pathophysiological relevance of the MCP-1/CCR2 system in this cell type. The present study was designed to test first whether cultured human podocytes express the CCR2 receptor and whether MCP-1 binding to CCR2 can induce a migratory/proliferative response in this cell type. Second, to determine whether these in vitro findings were relevant in vivo, we have assessed CCR2 expression in renal biopsies from patients with crescentic GN.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Cell Culture
Primary cultures of human podocytes were established as previously described.30 Cells were cultured in Dulbecco’s modified Eagle’s medium containing L-glutamine, 6.8 mmol/L glucose, 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified 5% CO2 incubator at 37°C. Studies were performed at passages 20 to 30. Cell viability was determined by trypan blue exclusion test.
Monocytes were isolated from total blood samples of human healthy volunteers using Lymphoprep density gradient centrifugation followed by adhesion separation in Dulbecco’s modified Eagle’s medium containing 10% FCS. Cell number was determined using a standard counting chamber after cell harvesting with 0.25% trypsin and 0.5% EDTA.
Podocyte Characterization
The cells were identified by positive staining for nephrin, synaptopodin, Wilm’s tumor antigen, podocalyxin, zonula occludens-1, cytokeratin, vimentin, and laminin; negative staining for smooth muscle-type myosin, FVIIIr:Ag, and CD45; and cytotoxicity in response to puromycin aminonucleoside (10 to 50 µg/ml).30 Immunofluorescence staining for podocyte cell markers was performed on cells fixed in 3.5% paraformaldehyde containing 2% sucrose for 15 minutes at 4°C and incubated with polyclonal primary antibodies and then with FITC-conjugated secondary antibodies. To assess cell morphology, scanning electron microscopy was performed on samples postfixed in 2.5% glutaraldehyde, dehydrated in alcohol, dried, and coated with gold by sputter coating. The specimens were examined in a scanning Jeol T300 electron microscope (JEOL, Tokyo, Japan), and images were obtained via secondary electron at a working distance of 15 to 25 mm and at an accelerating voltage of 20 to 25 kV.
RT-PCR Analysis
Total RNA was extracted using RNeasy Mini spin columns and freed from contaminant DNA by treatment with DNase I. RNA was reverse transcribed (1 µg) according to standard protocols. The PCR analysis was performed with oligonucleotide primers specifically designed to amplify human CCR2 (patented by R&D Systems) yielding a PCR product of 406 bp. After an initial denaturation at 94°C for 9 minutes, the cDNA was amplified for 32 cycles with the following setting: denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, and elongation at 72°C for 45 seconds with a final elongation at 72°C for 10 minutes.18 Total RNA obtained from human monocytes, the cell type predominantly expressing CCR2, was used as positive control. PCR products were resolved in a 1.5% agarose gel containing ethidium bromide, and a digital image of the gel was captured using the Gel Doc XR system (Bio-Rad, Milan, Italy).
Cytofluorimetric Analysis
Cells were detached from plates using a nonenzymatic cell dissociation solution and washed with PBS containing 4% heat-inactivated human serum. After blocking with whole heat-inactivated human serum for 15 minutes, cells were incubated with either a mouse anti-human CCR2 or an IgG1 control antibody diluted in PBS containing 4% serum for 45 minutes at 4°C. After washing in PBS containing 4% serum, cells were incubated with a FITC-conjugated rabbit anti-mouse antibody. Cells were then washed twice and analyzed by flow cytometry (FACscan; Becton-Dickinson, Mountain View, CA). For each determination, 10,000 cells were analyzed.
Chemotaxis Assay and Checkerboard Analysis
Chemotaxis assays were performed in standard Boyden chambers with 8-µm pore-size polycarbonate filters precoated with collagen type IV for 2 hours. A podocyte cell suspension in serum-free medium was placed in the upper chamber. Serum-free media containing either rh-MCP-1 (0.1, 1, 10, and 100 ng/ml) or vehicle was added to the lower compartment of the Boyden chamber. In a subset of experiments, the assay was performed on podocytes preincubated with either the specific CCR2 inhibitor RS102895 (RS, 6 µmol/L) or vehicle. After 5 hours of incubation at 37°C, the filters were removed; the migrated cells were fixed in methanol, stained with 2% Giemsa solution, and counted in 10 fields under light microscopy. All conditions were evaluated in duplicate for each experiment. To differentiate between chemokinesis and chemotaxis, checkerboard analysis was performed by adding rh-MCP-1 to both the lower and upper compartment of the Boyden chamber.
Haptotaxis Assay
A haptotaxis assay was performed in triplicate using a Boyden chamber. Briefly, uncoated polycarbonate filters were floated on serum-free media containing either rh-MCP-1 (10 ng/ml) or control (1 mg/ml bovine serum albumin) overnight at 37°C. After washing in PBS, the filters were placed in the Boyden chamber with the coated surface of the filter facing toward the lower well, which was filled with serum-free media.31 Podocytes were then seeded into each upper chamber in serum-free media and incubated for 5 hours at 37°C. At the end of the incubation period, filters were removed, fixed, and stained, and migrated cells were counted as described above.
In Vitro Wound Healing Assay
Podocytes were seeded into 96-well tissue culture plates and allowed to grow to confluence. After a 24-hour quiescent period in medium containing 0.5% FCS, cells were incubated for 1 hour with RS (6 µmol/L) or vehicle, then the medium was removed, and monolayers were wounded using a single pass with a sterile yellow pipette tip. The medium containing either vehicle or rh-MCP-1 (10 ng/ml), with or without RS (6 µmol/L) addition, was returned to the wells, and wound closure was monitored over time with a x4 objective on a Leica DFC320 phase contrast inverted microscope (Leica, Wetzlar, Germany). Images of the entire wounded area were captured using a Leica DMIL digital camera, and the area of the wound measured in arbitrary units using the Image J 1.32 software (available for free download from http://rsb.info.nih.gov/ij/). This method of injury led to a consistent baseline wounded area (91,273 ± 2853 arbitrary units, n = 21). Each condition was examined in triplicate.
Agarose Strip Method
A modified agarose strip method was used as previously described.32 In brief, a 2.5% agarose and 1% glycerol solution was pipetted as a thin strip on the base of 6-cm plates and allowed to dry. A podocyte cell suspension was added and cultured to form a confluent monolayer interrupted by the agarose strip. The agarose strip was carefully removed, RS (6 µmol/L) or vehicle was added to the wells, and the wound closure was analyzed as described above. All experiments were performed at least in triplicate.
Proliferation Assay
Cell proliferation was assayed using a colorimetric immunoassay based on the measurement of BrdU incorporation according to the manufacturer’s instructions. Briefly, podocytes were plated on flat-bottom 96-well tissue culture plates (density, 
2500 cells per well) and allowed to adhere overnight. After a 24-hour quiescent period in medium containing 0.5% FCS, rh-MCP-1 (1 to 10 ng/ml) was added to the medium for 12, 24, 48, and 72 hours. Cells were labeled with BrdU during the last 4 hours of the incubation period and fixed, and BrdU incorporation was assayed via colorimetric detection using a plate reader (Bio-Rad 680 Microplate Reader) at 450 nm.
Immunocytochemical Analysis
CCR2 and MCP-1 expression was assessed in podocytes by immunocytochemistry. Cells were fixed in 3.5% paraformaldehyde containing 2% sucrose for 30 minutes. After blocking, cells were incubated with a rabbit anti-human CCR2 antibody, a mouse anti-MCP-1 antibody or isotype-specific irrelevant antibodies. After rinsing, specific staining was revealed using the LSAB+ system-HRP system. Briefly slides were incubated with a biotinylated anti-rabbit secondary antibody, followed by incubation with peroxidase-labeled streptavidin and development using a 3,3'-diaminobenzidine chromogen solution. After counterstaining, slides were mounted and visualized with an Olympus Bx4 I microscope connected to a Leica DMIL camera.
MCP-1 Protein Measurement
Culture supernatants from both wounded and control monolayers were collected, centrifuged to remove cell debris, and stored at –70°C for analysis. MCP-1 protein concentration was measured by a quantitative sandwich enzyme-linked immunosorbent assay using a mouse monoclonal and a rabbit polyclonal anti-human MCP-1 antibody (range, 5 to 2000 pg/ml; intra- and interassay CV, 4.9 and 4.8%, respectively). Results were corrected for cell numbers.
Analysis of Renal Biopsies from Patients with Crescent Glomerulonephritis
The study was performed on renal biopsies of eight patients (five males and three females; age, 63 ± 15 years) with clinical features of rapidly progressive GN and severe renal failure that required hemodyalitic treatment in two patients (serum creatinine 8.2 ± 2.7 mg/ml). Biopsies included in the study presented classic histological features of diffuse crescentic GN. Histological and immunopathological diagnosis was pauci-immune crescentic glomerulonephritis in six cases (that was idiopathic in four cases and secondary to necrotizing vasculitis in two cases), type I membranoproliferative GN in one case, and membranous GN in one case. No patients received steroids or immunosuppressive drugs before renal biopsy. Normal portions of kidneys from patients who underwent surgery for hypernephromas were used as control. The study was approved by the Ethical Committee of the Department of Internal Medicine of the University of Genoa, procedures were in accordance with the Helsinki Declaration, and informed consent was obtained from all subjects.
Immunohistochemical staining was performed on 4-µm paraffin sections of formalin-fixed tissue. Briefly, sections were dewaxed, rehydrated, and immersed in 0.01 mol/L citrate buffer at 100°C for retrieval of antigen sites masked by formalin fixation. Endogenous peroxidase activity was quenched by incubation with 3% H2O2. Endogenous avidin-binding activity was inhibited by sequential treatment with avidin-biotin and nonspecific binding sites blocked with 3% bovine serum albumin. For immunodetection, sections were incubated for 1 hour at room temperature with a rabbit monoclonal anti-CCR2 antibody, and the specific staining was detected using the LSAB+ system-HRP. Sections were counterstained and visualized with an Olympus-Bx4I microscope connected to a Leica DMIL camera. The specificity of the anti-CCR2 antibody was confirmed by replacing the primary antibody with a nonimmune isotypic control antibody. Evaluation of glomerular staining was performed by a pathologist in a blinded fashion.
Double immunofluorescent staining was performed for CCR2 and synaptopodin, a specific podocyte marker. After blocking with 3% bovine serum albumin, sections were incubated with a mouse monoclonal anti-synaptopodin antibody for 18 hours at 4°C, washed in PBS, and then incubated with a FITC-conjugated rat anti-mouse antibody for 1 hour. After washing and further blocking in avidin-biotin, sections were incubated with a monoclonal rabbit anti-CCR2 antibody for 1 hour at room temperature, washed in PBS, incubated with a biotinylated swine anti-rabbit IgG for 1 hour and then with Alexa 555-conjugated streptavidin. Sections were examined using an Olympus epifluorescence microscope (Olympus Bx4 I) with photographic attachment (Leica DMIL). The images were color-combined and assembled into photomontages by using Adobe Photoshop (Universal Imaging Corporation, West Chester, PA).
Data Presentation and Statistical Analysis
The number of experiments, performed at least in triplicate, is reported in the figure legends. All data are presented as mean ± SEM. Data are expressed as fold change over control. Student’s t-test was used for the comparison between two groups. When more than two groups were studied, data were analyzed by analysis of variance, and if significant, the Newman-Keuls was used for post hoc comparisons. Values for P < 0.05 were considered significant.
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Results |
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Our cells displayed positive staining for podocyte markers, including peripheral expression of the podocyte-specific marker nephrin. Furthermore, in subconfluent monolayers, scanning electron microscopy demonstrated the presence of the typical cytoplasmatic projections (Figure 1)
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Human podocytes constitutively express the mRNA encoding the CCR2 receptor, as determined by RT-PCR (Figure 2A)
. A single PCR product of 406 bp was obtained as predicted from the known cDNA sequence for CCR2. Contamination of genomic DNA was excluded by DNase I treatment of total RNA. The specificity of the band was confirmed by the absence of signal in the negative control and by the presence of a band of identical molecular weight in the positive control obtained by amplification of cDNA from human monocytes. Immunocytochemical staining for CCR2 showed positive staining in the majority of podocytes (Figure 2B)
. Furthermore, surface CCR2 protein expression was demonstrated by cytofluorimetry using an antibody recognizing the external domain of CCR2. CCR2 expression was present in approximately 94% of the podocyte population (Figure 2C)
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To investigate whether podocytes, besides expressing CCR2, display a functional response to MCP-1, cells were exposed to increasing rh-MCP-1 concentrations (0.1, 1, 10, and 100 ng/ml), and their migration was tested in a chemotaxis assay.
Addition of rh-MCP-1 to the lower chamber enhanced podocyte migration with a significant increase at a dose as low as 1 ng/ml. Maximal induction was observed at 10 ng/ml without further increases at 100 ng/ml (Figure 3A)
. Because the maximum response was observed at 10 ng/ml, this concentration was used in all subsequent experiments.
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. CCR2 blockade did not alter basal podocyte random movement of control cells, suggesting that MCP-1 specifically affects podocyte directional migration. A checkerboard analysis was also performed to determine whether podocyte migration was due to either chemotaxis or chemokinesis. As shown in Table 1
, in the absence of MCP-1 in the upper well, podocytes migrated toward MCP-1 placed in the lower well. On the contrary, when equal concentrations of MCP-1 were added to both upper and lower wells to destroy the gradient, very little podocyte migration was observed, indicating that the migratory activity of MCP-1 was directional rather than random in nature.
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Given that podocyte motility is an adhesion-dependent event, we also investigated the ability of substrate-bound MCP-1 to induce podocyte migration. As shown in Figure 4
, rh-MCP-1 bound to the polycarbonate filters of the chemotaxis chamber and induced a significant threefold increase in podocyte migration in the absence of a soluble chemoattractant. Furthermore, MCP-1-induced podocyte haptotaxis occurred via the CCR2 receptor because it was completely blocked by podocyte preincubation with RS.
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To test whether the MCP-1-induced migratory phenotype could be implicated in the healing of a wounded podocyte monolayer, we used an in vitro scrape-wound assay model.34
In preliminary experiments, we found that wounded podocyte monolayers underwent almost complete closure within 24 hours as assessed by light microscopy. The addition of the CCR2 inhibitor RS significantly delayed podocyte wound healing compared with vehicle at 24 hours (control versus RS, 12 ± 3 versus 38.1 ± 2.4%; percent wound area compared with baseline; P < 0.001) (Figure 5)
, indicating that CCR2 participates in the normal closure of mechanical wounds in podocyte cultures. This effect was not due to RS cytotoxicity because there were no differences in cell viability between podocytes exposed to RS and control cells (control versus RS: 24 hours, 98.8 ± 0.021 versus 98.7 ± 0.91%; 48 hours, 99.2 ± 0.81 versus 99.7 ± 0.28%; percent viable cells; P = NS). To assess whether podocyte damage was important for inducing CCR2-dependent healing, experiments were also performed using an agarose strip method that introduces gaps into the monolayers without causing significant perturbation to cells. In the presence of RS, a comparable 25% difference in the residual wounded area was observed at 24 hours also in this model (P < 0.05), suggesting that podocyte loss rather than podocyte damage was involved. MCP-1 concentration was similar in the conditioned media from wounded and control monolayers (63.7 ± 1.4 versus 49 ± 6.5 pg/ml; P = NS), and the addition of exogenous rh-MCP-1 to wounded monolayers did not further increase the rate of closure of the mechanical wound (control versus MCP-1, 12 ± 3 versus 18 ± 0.2%; P = NS).
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Because MCP-1 has mitogenic activity in both vascular smooth muscle and endothelial cells, we tested in podocytes whether MCP-1 causes an increase in proliferation that could contribute to CCR2-dependent accelerated wound closure. Podocytes were exposed to rh-MCP-1 (1 to 10 ng/ml) for various time periods (12, 24, 48, and 72 hours), and then proliferation was assessed by both cell counting and BrdU incorporation. As depicted in Figure 6, a
significant 25% increase in the number of counted cells was observed in podocytes exposed to 10 ng/ml MCP-1 for 48 hours. Similarly, podocyte exposure to rh-MCP-1 (10 ng/ml) for 48 hours induced a significant 29% increased in BrdU incorporation. No changes in cell proliferation were seen at other time points or MCP-1 concentrations.
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To clarify the mechanism whereby CCR2 blockade delays wound closure, wounded podocyte monolayers were stained for CCR2 and MCP-1 by immunocytochemistry. As shown in Figure 7
, podocytes adjacent to the wounded area stained positive for CCR2, whereas the intensity of the staining was lower in cells positioned in areas away from the site of injury. No differences were seen in the staining for MCP-1. In these experiments, we used immunocytochemistry conditions (ie, shorter duration of substrate incubation) aimed to enhance differences in staining distribution/intensity, and this explains the lower basal staining for CCR2 in comparison with cells shown in Figure 2B
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To assess the in vivo relevance of our findings and to exclude that CCR2 receptor expression was solely related to in vitro culture conditions, we studied by immunohistochemistry glomerular CCR2 expression in renal sections from control subjects and patients with crescentic GN. We found that only a few glomerular cells, predominantly podocytes, stained positively for CCR2 in the normal glomeruli (Figure 8B)
. CCR2 staining was greatly enhanced in patients with crescentic GN and localized to the glomerular podocytes, Bowman’s capsule, and the cellular crescents (Figure 8, C–F)
. The specificity of the antibody binding in immunohistochemistry was confirmed by the disappearance of the signal when an isotype-matched control antibody was used as primary antibody (Figure 8A)
. Double-labeling immunofluorescence for the podocyte marker synaptopodin (Figure 8G)
and CCR2 (Figure 8H)
showed colocalization (Figure 8I)
, confirming CCR2 receptor by glomerular podocytes. CCR2-positive and synaptopodin-negative cells were present in the crescents.
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Discussion |
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Our results demonstrate that podocytes express the CCR2 receptor at both the mRNA and protein level. A previous report failed to demonstrate mRNA encoding for CCR2 in podocytes,28 whereas a recent study has shown the CCR2 receptor in murine podocytes.29 Our data confirm this latter finding in cells of human origin; furthermore, they provide evidence that CCR2 is functionally active because exposure to MCP-1 induced a significant sixfold increase in podocyte migration. Although originally believed to be a chemoattractant solely for monocytes, there is now evidence that MCP-1 induces migration of several other cell types,19,21,22 and our data extend this to human podocytes.
The peak effect of MCP-1 on podocyte migration was seen at a dose of 10 ng/ml. This concentration is within the higher physiological range because it is comparable with that measured both in vitro in podocytes exposed to high glucose concentrations35 and in vivo at sites of inflammation.36 Addition of exogenous MCP-1, however, is superimposed to the MCP-1 endogenously produced by podocytes and thus mimics a state of MCP-1 excess, suggesting that superphysiological MCP-1 levels are required to induce migration. MCP-1-induced chemotaxis declined at 100 ng/ml, a phenomenon also reported for MCP-1-induced migration of murine mesangial cells.37 A bell-shaped dose-response curve is often noted with chemokine responses and presumably reflects desensitization of the response after treatment with high chemokine concentrations.
The complete inhibition of MCP-1-induced podocyte chemotaxis by RS102895 indicates a specific MCP-1 effect occurring via the CCR2 receptor. The CCR2 receptor exists in two isoforms, CCR2A and CCR2B.17 The latter is responsible for all of the known biological effects of MCP-1, whereas the significance of the CCR2A variant has not yet been elucidated.38 In this study, CCR2 was inhibited using RS102895, a potent and specific antagonist of the CCR2B receptor33 ; thus the isoform B is likely to be implicated.
Assessment of migration in the presence and the absence of a concentration gradient of MCP-1 showed that the migration was directed and not solely due to an increase in random movement. In addition, in the haptotaxis assay, podocyte migration was significantly enhanced when MCP-1 was bound to the filter. This may suggest that MCP-1 secretion followed by binding to matrix components, laid down by the cell front, creates a natural concentration gradient to direct migration.
Covering areas of denudated GBM is a critical event in podocyte pathophysiology, and an MCP-1-induced migratory phenotype could be implicated in this repairing process. Our results provide evidence that closure of a wounded podocyte monolayer is significantly delayed by the addition of RS102895. Monolayers underwent almost complete healing within 24 hours, but there was a significant 26% reduction in the rate of closure in the presence of RS102895. Similar results were obtained using a method that introduces gaps into the cell monolayers without causing significant perturbation to cells, indicating that podocyte damage is not necessary for the induction of motility but rather that the induction of movement is the result of the sudden availability of tissue culture surface area on which the cells can move. Previous studies have reported a role of the MCP-1/CCR2 system in wound closure in other cell types, such as type II alveolar and bronchial epithelial cells19,22 ; however, to our knowledge, this is the first report implicating the MCP-1/CCR2 system in directing the migration to efficient wound healing in podocytes. Of interest, the addition of exogenous rh-MCP-1 did not accelerate wound closure, a finding previously reported in alveolar epithelial cells.19 This is unlikely to be due to saturation of the system by endogenously produced MCP-1 because we found a relatively low MCP-1 concentration in the supernatant of wounded podocyte monolayers.
Repair occurs by both cell proliferation and migration. In our study, the MCP-1 effect on podocyte proliferation was quite modest and required 48 hours to occur. It is thus unlikely that CCR2 blockade interferes with wound closure by preventing podocyte proliferation, and inhibition of podocyte migration is a more likely mechanism. Although podocytes are considered terminally differentiated cells unable to undergo proliferation in vivo, recent studies have documented that podocytes are capable of proliferation in various glomerular diseases,39,40 including crescent GN,41 suggesting a potential pathophysiological relevance of our in vitro finding showing a podocyte proliferative response to MCP-1. Similarly, a mitogenic activity of MCP-1 has been reported in vascular smooth muscle cells and recently implicated in both intimal hyperplasia and accelerated atherosclerosis in vivo.20,42
To clarify the mechanism whereby CCR2 blockade delays wound closure, wounded podocyte monolayers were stained for both CCR2 and MCP-1 by immunocytochemistry. Results showed that the intensity of the staining for CCR2 was greater in podocytes adjacent to the wounded area compared with those located in areas away from the site of injury; whereas no changes were seen in MCP-1 staining. Furthermore, MCP-1 concentrations were similar in the conditioned media from wounded and control podocyte monolayers. Altogether, these data support the hypothesis that a localized CCR2 overexpression is the key determinant of podocyte directional migration rather than changes in MCP-1 expression/ concentration.
To assess whether these in vitro findings are relevant to in vivo pathophysiological conditions, we have studied by immunohistochemistry CCR2 expression in both normal renal cortex and kidney biopsies from patients with crescentic GN. In the normal kidney, we found that only a few glomerular cells stained positively for CCR2 with a predominant podocyte distribution. In keeping with this, mRNA encoding for CCR2 has been detected by in situ hybridization in normal murine podocytes.29 Glomerular CCR2 expression was evident in kidney biopsies from patients with crescentic GN and localized along the glomerular capillary wall, the Bowman’s capsule, and also in the cellular crescents. Both pattern of staining and colocalization with the podocyte marker synaptopodin provide evidence of CCR2 expression by glomerular podocytes.
Cells positive uniquely for CCR2, either in the glomeruli or in the crescents, are likely to be locally recruited monocytes. However, podocytes migrated into crescents are known to lose specific podocyte markers41 ; therefore, the possibility that these cells are, at least in part, dedifferentiated podocytes cannot be excluded.
There is evidence that in early crescentic GN, podocytes assume a migratory phenotype and trigger crescent formation by establishing bridges between the tuft and Bowman’s capsule.2,43,44 Furthermore, podocytes undergo proliferation41 and are believed to participate in cellular crescent formation.41,44 Our in vitro data, showing that MCP-1 binding to CCR2 can induce a migratory and proliferative phenotype in podocytes, taken together with our in vivo demonstration of CCR2 expression by podocyte in crescentic GN suggest that MCP-1 can act directly on podocytes and possibly be involved in the podocyte alterations associated with crescent formation. In line with this hypothesis, neutralization of MCP-1 results in a dramatic decrease in glomerular crescent formation.16,45
On the other hand, podocyte migration in response to the MCP-1 may also be implicated in both covering of areas of denudated GBM and foot process broadening and fusion, which requires acquisition of a migratory phenotype. Further studies are required to assess the in vivo relevance of our results showing MCP-1/CCR2-driven podocyte migration.
In conclusion, our data suggest that the MCP-1/CCR2 system is functionally active in podocytes and overexpressed by this cell type in crescentic GN.
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Footnotes |
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Supported by the European Federation for the Study of Diabetes/Lilly European Diabetes Research Programme and by the Piedmont Region Applied Scientific Research 2004. D.B. is the recipient of a Marie Curie Individual Intra-European Fellowship from the European Community (no. 039574).
Accepted for publication August 21, 2007.
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B and activator protein-1. Arterioscler Thromb Vasc Biol 2002, 22:914-920This article has been cited by other articles:
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174-HaeIII. 2, negative control. 3, positive control human monocytes. 4, human podocytes. B: Representative immunocytochemical staining of podocytes for CCR2 (right). The primary antibody was omitted in the negative control (left). Original magnification, x400. C: Representative histogram showing surface CCR2 expression in podocytes (white histogram). Nonspecific staining (gray histogram) was determined using an isotype-specific control antibody.
) and 10 ng/ml (
) for 12, 24, 48, and 72 hours. Cell proliferation was assessed by cell counting and expressed as fold increase over control (dashed line) (n = 5). *P < 0.05 MCP-1 at 10 ng/ml versus control at 48 hours.
