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The Monocyte Chemoattractant Protein-1/Cognate CC Chemokine Receptor 2 System Affects Cell Motility in Cultured Human Podocytes

2007; Elsevier BV; Volume: 171; Issue: 6 Linguagem: Inglês

10.2353/ajpath.2007.070398

1525-2191

Davina Judith Burt, Gennaro Salvidio, Elena Tarabra, Federica Barutta, Silvia Pinach, Patrizia Dentelli, Giovanni Camussi, Paolo Cavallo Perin, Gabriella Gruden,

Complement system in diseases

In crescentic glomerulonephritis (GN), monocyte chemoattractant protein-1 (MCP-1) is overexpressed within the glomeruli, and MCP-1 blockade has renoprotective effects. Adult podocytes are in a quiescent state, but acquisition of a migratory/proliferative phenotype has been described in crescentic GN and implicated in crescent formation. The cognate CC chemokine receptor 2 (CCR2), the MCP-1 receptor, is expressed by other cell types besides monocytes and has been implicated in both cell proliferation and migration. We investigated whether MCP-1 binding to CCR2 can induce a migratory/proliferative response in cultured podocytes. MCP-1 binding to CCR2 enhanced podocyte chemotaxis/haptotaxis in a concentration-dependent manner and had a modest effect on cell proliferation. Closure of a wounded podocyte monolayer was delayed by CCR2 blockade, and CCR2 was overexpressed at the wound edge, suggesting a role for CCR2 in driving podocyte migration. Immunohistochemical analysis of kidney biopsies from patients with crescentic GN demonstrated CCR2 expression in both podocytes and cellular crescents, confirming the clinical relevance of our in vitro findings. In conclusion, the MCP-1/CCR2 system is functionally active in podocytes and may be implicated in the migratory events triggered by podocyte injury in crescentic GN and other glomerular diseases. In crescentic glomerulonephritis (GN), monocyte chemoattractant protein-1 (MCP-1) is overexpressed within the glomeruli, and MCP-1 blockade has renoprotective effects. Adult podocytes are in a quiescent state, but acquisition of a migratory/proliferative phenotype has been described in crescentic GN and implicated in crescent formation. The cognate CC chemokine receptor 2 (CCR2), the MCP-1 receptor, is expressed by other cell types besides monocytes and has been implicated in both cell proliferation and migration. We investigated whether MCP-1 binding to CCR2 can induce a migratory/proliferative response in cultured podocytes. MCP-1 binding to CCR2 enhanced podocyte chemotaxis/haptotaxis in a concentration-dependent manner and had a modest effect on cell proliferation. Closure of a wounded podocyte monolayer was delayed by CCR2 blockade, and CCR2 was overexpressed at the wound edge, suggesting a role for CCR2 in driving podocyte migration. Immunohistochemical analysis of kidney biopsies from patients with crescentic GN demonstrated CCR2 expression in both podocytes and cellular crescents, confirming the clinical relevance of our in vitro findings. In conclusion, the MCP-1/CCR2 system is functionally active in podocytes and may be implicated in the migratory events triggered by podocyte injury in crescentic GN and other glomerular diseases. Podocytes are highly differentiated cells with a complex cellular morphology. The podocyte cell body bulges into the urinary space and gives rise to primary processes that extend toward the capillaries to which they affix by numerous foot processes. The foot process of neighboring podocytes interdigitate, leaving between them filtration slits bridged by an extracellular structure, known as the slit diaphragm, which represents the major restriction site to protein filtration.1Pavenstädt H Kriz W Kretzler M Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307PubMed Google Scholar 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.2Le Hir M Keller C Eschmann V Hähnel B Hosser H Kriz W Podocyte bridges between the tuft and Bowman's capsule: an early event in experimental crescentic glomerulonephritis.J Am Soc Nephrol. 2001; 12: 2060-2071PubMed Google Scholar In addition, cells derived from migrated podocytes proliferate and participate in crescent formation.3Moeller MJ Soofi A Hartmann I Le Hir M Wiggins R Kriz W Holzman LB Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.J Am Soc Nephrol. 2004; 15: 61-67Crossref PubMed Scopus (161) Google Scholar 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.4Reiser J Oh J Shirato I Asanuma K Hug A Mundel TM Honey K Ishidoh K Kominami E Kreidberg JA Tomino Y Mundel P Podocyte migration during nephrotic syndrome requires a coordinated interplay between cathepsin L and alpha3 integrin.J Biol Chem. 2004; 279: 34827-34832Crossref PubMed Scopus (150) Google Scholar Monocyte chemoattractant protein-1 (MCP-1) is a potent mononuclear cell chemoattractant produced by a variety of mesenchymal cells, including glomerular cells.5Kakizaki Y Waga S Sugimoto K Tanaka H Nukii K Takeya M Yoshimura T Yokoyama M Production of monocyte chemoattractant protein-1 by bovine glomerular endothelial cells.Kidney Int. 1995; 48: 1866-1874Crossref PubMed Scopus (46) Google Scholar, 6Gruden G Setti G Hayward A Sugden D Duggan S Burt D Buckingham RE Gnudi L Viberti G Mechanical stretch induces monocyte chemoattractant activity via an NF-kappaB-dependent monocyte chemoattractant protein-1-mediated pathway in human mesangial cells: inhibition by rosiglitazone.J Am Soc Nephrol. 2005; 16: 688-696Crossref PubMed Scopus (89) Google Scholar, 7Gu L Hagiwara S Fan Q Tanimoto M Kobata M Yamashita M Nishitani T Gohda T Ni Z Qian J Horikoshi S Tomino Y Role of receptor for advanced glycation end-products and signalling events in advanced glycation end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytes.Nephrol Dial Transplant. 2006; 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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.17Charo IF Myers SJ Herman A Franci C Connolly AJ Coughlin SR Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails.Proc Natl Acad Sci USA. 1994; 91: 2752-2756Crossref PubMed Scopus (676) Google Scholar 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 vitro18Giunti S Pinach S Arnaldi L Viberti G Perin PC Camussi G Gruden G The MCP-1/CCR2 system has direct proinflammatory effects in human mesangial cells.Kidney Int. 2006; 69: 856-863Crossref PubMed Scopus (45) Google Scholar, 19Christensen PJ Du M Moore B Morris S Toews GB Paine III, R Expression and functional implications of CCR2 expression on murine alveolar epithelial cells.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L68-L72Crossref PubMed Scopus (37) Google Scholar, 20Viedt C Vogel J Athanasiou T Shen W Orth SR Kubler W Kreuzer J Monocyte Chemoattractant Protein-1 induces proliferation and interleukin-6 production in human smooth muscle cells by differential activation of nuclear factor-κB and activator protein-1.Arterioscler Thromb Vasc Biol. 2002; 22: 914-920Crossref PubMed Scopus (172) Google Scholar, 21Weber KS Nelson PJ Grone HJ 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. 1999; 19: 2085-2093Crossref PubMed Scopus (247) Google Scholar, 22Lundien MC Mohammed KA Nasreen N Tepper RS Hardwick JA Sanders KL Van Horn RD Antony VB Induction of MCP-1 expression in airway epithelial cells: role of CCR2 receptor in airway epithelial injury.J Clin Immunol. 2002; 22: 144-152Crossref PubMed Scopus (57) Google Scholar, 23Coughlan CM McManus CM Sharron M Gao Z Murphy D Jaffer S Choe W Chen W Hesselgesser J Gaylord H Kalyuzhny A Lee VM Wolf B Doms RW Kolson DL Expression of multiple functional chemokine receptors and monocyte chemoattractant protein-1 in human neurons.Neuroscience. 2000; 97: 591-600Crossref PubMed Scopus (192) Google Scholar and in vivo,24Banisadr G Queraud-Lesaux F Boutterin MC Pelaprat D Zalc B Rostene W Haour F Parsadaniantz SM Distribution, cellular localization and functional role of CCR2 chemokine receptors in adult rat brain.J Neurochem. 2002; 81: 257-269Crossref PubMed Scopus (166) Google Scholar, 25Warren GL Hulderman T Mishra D Gao X Millecchia L O'Farrell L Kuziel WA Simeonova PP Chemokine receptor CCR2 involvement in skeletal muscle regeneration.FASEB J. 2005; 19: 413-415PubMed Google Scholar, 26Moore BB Kolodsick JE Thannickal VJ Cooke K Moore TA Hogaboam C Wilke CA Toews GB CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury.Am J Pathol. 2005; 166: 675-684Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 27Spinetti G Wang M Monticone R Zhang J Zhao D Lakatta EG Rat aortic MCP-1 and its receptor CCR2 increase with age and alter vascular smooth muscle cell function.Arterioscler Thromb Vasc Biol. 2004; 24: 1397-1402Crossref PubMed Scopus (157) Google Scholar 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,19Christensen PJ Du M Moore B Morris S Toews GB Paine III, R Expression and functional implications of CCR2 expression on murine alveolar epithelial cells.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L68-L72Crossref PubMed Scopus (37) Google Scholar, 20Viedt C Vogel J Athanasiou T Shen W Orth SR Kubler W Kreuzer J Monocyte Chemoattractant Protein-1 induces proliferation and interleukin-6 production in human smooth muscle cells by differential activation of nuclear factor-κB and activator protein-1.Arterioscler Thromb Vasc Biol. 2002; 22: 914-920Crossref PubMed Scopus (172) Google Scholar, 21Weber KS Nelson PJ Grone HJ 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. 1999; 19: 2085-2093Crossref PubMed Scopus (247) Google Scholar, 22Lundien MC Mohammed KA Nasreen N Tepper RS Hardwick JA Sanders KL Van Horn RD Antony VB Induction of MCP-1 expression in airway epithelial cells: role of CCR2 receptor in airway epithelial injury.J Clin Immunol. 2002; 22: 144-152Crossref PubMed Scopus (57) Google Scholar, 27Spinetti G Wang M Monticone R Zhang J Zhao D Lakatta EG Rat aortic MCP-1 and its receptor CCR2 increase with age and alter vascular smooth muscle cell function.Arterioscler Thromb Vasc Biol. 2004; 24: 1397-1402Crossref PubMed Scopus (157) Google Scholar 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,28Huber TB Reinhardt C Exner M Burger JA Kerjaschki D Saleem MA Pavenstadt H Expression of functional CCR and CXCR chemokine receptors in podocytes.J Immunol. 2002; 168: 6244-6252PubMed Google Scholar, 29Rao VH Meehan DT Delimont D Nakajima M Wada T Gratton MA Cosgrove D Role for macrophage metalloelastase in glomerular basement membrane damage associated with Alport syndrome.Am J Pathol. 2006; 169: 32-46Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar but a recent study in the Alport mouse model has shown overexpression of the CCR2 receptor in glomerular podocytes in vivo,29Rao VH Meehan DT Delimont D Nakajima M Wada T Gratton MA Cosgrove D Role for macrophage metalloelastase in glomerular basement membrane damage associated with Alport syndrome.Am J Pathol. 2006; 169: 32-46Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar 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. All materials were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Fetal calf serum (FCS) was from Euroclone (Milan, Italy). Dulbecco's modified Eagle's medium and Alexa 555-conjugated streptavidin were from Invitrogen (San Giuliano Milanese, Italy). RNeasy Mini spin columns, RNase-free DNase I, and Taq polymerase were purchased from Qiagen (Milan, Italy). The reverse transcription system was from Promega (Madison, WI) and the Cell Proliferation Bromodeoxyuridine (BrdU) Colorimetric Assay was from Roche Diagnostics (Basel, Switzerland). Oligonucleotide primers, rh-MCP-1, mouse anti-MCP-1, Quantikine human MCP-1/CCL2 immunoassay, and mouse anti-CCR2 antibodies were obtained from R&D Systems (Minneapolis, MN). The rabbit anti-CCR2 and the isotype-specific control antibodies were from Epitomics (Burlingame, CA). The mouse anti-synaptopodin antibody and the polyclonal guinea pig anti-nephrin antibody were from Progen Biotechnik (Heidelberg, Germany), and the fluorescein isothiocyanate-conjugated rat anti-mouse antibody from BD Biosciences (San Jose, CA). The LSAB+ system-HRP, the FITC-conjugated rabbit anti-mouse antibody, and the biotinylated swine anti-rabbit antibody were from DAKO (Glostrup, Denmark). Lymphoprep was from Nycomed Pharma AS (Oslo, Norway), and the polycarbonate filters were from NTG Srl (Milan, Italy). Primary cultures of human podocytes were established as previously described.30Doublier S Ruotsalainen V Salvidio G Lupia E Biancone L Conaldi PG Reponen P Tryggvason K Camussi G Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome.Am J Pathol. 2001; 158: 1723-1731Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar 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. 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).30Doublier S Ruotsalainen V Salvidio G Lupia E Biancone L Conaldi PG Reponen P Tryggvason K Camussi G Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome.Am J Pathol. 2001; 158: 1723-1731Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar 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. 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.18Giunti S Pinach S Arnaldi L Viberti G Perin PC Camussi G Gruden G The MCP-1/CCR2 system has direct proinflammatory effects in human mesangial cells.Kidney Int. 2006; 69: 856-863Crossref PubMed Scopus (45) Google Scholar 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). 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 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. 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.31Nasreen N Mohammed KA Galffy G Ward MJ Antony VB MCP-1 in pleural injury: cCR2 mediates haptotaxis of pleural mesothelial cells.Am J Physiol Lung Cell Mol Physiol. 2000; 278: L591-L598PubMed Google Scholar 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. 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 ×4 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. A modified agarose strip method was used as previously described.32Block ER Matela AR SundarRaj N Iszkula ER Klarlund JK Wounding induces motility in sheets of corneal epithelial cells through loss of spatial constraints: role of heparin-binding epidermal growth factor-like growth factor signaling.J Biol Chem. 2004; 279: 24307-24312Crossref PubMed Scopus (116) Google Scholar 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. 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. 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. 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. 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). 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. Our cells displayed positive staining for podocyte markers, including peripheral expression of the podocyte-specific marker nephrin. Furthermore, in subconfluent monolayers, scannin