Galectin-3 Expression and Secretion Links Macrophages to the Promotion of Renal Fibrosis
2008; Elsevier BV; Volume: 172; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2008.070726
ISSN1525-2191
AutoresNeil C. Henderson, Alison C. MacKinnon, Sarah L. Farnworth, Tiina Kipari, Christopher Haslett, John P. Iredale, Fu‐Tong Liu, Jeremy Hughes, Tariq Sethi,
Tópico(s)Macrophage Migration Inhibitory Factor
ResumoMacrophages have been proposed as a key cell type in the pathogenesis of renal fibrosis; however, the mechanism by which macrophages drive fibrosis is still unclear. We show that expression of galectin-3, a β-galactoside-binding lectin, is up-regulated in a mouse model of progressive renal fibrosis (unilateral ureteric obstruction, UUO), and absence of galectin-3 protects against renal myofibroblast accumulation/activation and fibrosis. Furthermore, specific depletion of macrophages using CD11b-DTR mice reduces fibrosis severity after UUO demonstrating that macrophages are key cells in the pathogenesis of renal fibrosis. Disruption of the galectin-3 gene does not affect macrophage recruitment after UUO, or macrophage proinflammatory cytokine profiles in response to interferon-γ/lipopolysaccharide. In addition, absence of galectin-3 does not affect transforming growth factor-β expression or Smad 2/3 phosphorylation in obstructed kidneys. Adoptive transfer of wild-type but not galectin-3−/− macrophages did, however, restore the fibrotic phenotype in galectin-3−/− mice. Cross-over experiments using wild-type and galectin-3−/− macrophage supernatants and renal fibroblasts confirmed that secretion of galectin-3 by macrophages is critical in the activation of renal fibroblasts to a profibrotic phenotype. Therefore, we demonstrate for the first time that galectin-3 expression and secretion by macrophages is a major mechanism linking macrophages to the promotion of renal fibrosis. Macrophages have been proposed as a key cell type in the pathogenesis of renal fibrosis; however, the mechanism by which macrophages drive fibrosis is still unclear. We show that expression of galectin-3, a β-galactoside-binding lectin, is up-regulated in a mouse model of progressive renal fibrosis (unilateral ureteric obstruction, UUO), and absence of galectin-3 protects against renal myofibroblast accumulation/activation and fibrosis. Furthermore, specific depletion of macrophages using CD11b-DTR mice reduces fibrosis severity after UUO demonstrating that macrophages are key cells in the pathogenesis of renal fibrosis. Disruption of the galectin-3 gene does not affect macrophage recruitment after UUO, or macrophage proinflammatory cytokine profiles in response to interferon-γ/lipopolysaccharide. In addition, absence of galectin-3 does not affect transforming growth factor-β expression or Smad 2/3 phosphorylation in obstructed kidneys. Adoptive transfer of wild-type but not galectin-3−/− macrophages did, however, restore the fibrotic phenotype in galectin-3−/− mice. Cross-over experiments using wild-type and galectin-3−/− macrophage supernatants and renal fibroblasts confirmed that secretion of galectin-3 by macrophages is critical in the activation of renal fibroblasts to a profibrotic phenotype. Therefore, we demonstrate for the first time that galectin-3 expression and secretion by macrophages is a major mechanism linking macrophages to the promotion of renal fibrosis. Macrophages are pleiotropic inflammatory cells prominent in both acute and chronic inflammation. In the chronic inflammatory milieu, macrophages interact with other cell types including cells of mesenchymal origin (fibroblasts) that transdifferentiate into matrix-secreting myofibroblasts, with resultant scar formation and disruption of tissue architecture. Advanced renal fibrosis with kidney failure is a major health care burden worldwide,1El Nahas AM Bello AK Chronic kidney disease: the global challenge.Lancet. 2005; 365: 331-340Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar and long-term dialysis or transplantation are the only therapeutic options currently available.2Sayegh MH Carpenter CB Transplantation 50 years later—progress, challenges, and promises.N Engl J Med. 2004; 351: 2761-2766Crossref PubMed Scopus (334) Google Scholar Therefore increasing our understanding of the complex interplay between chronic inflammation and progressive fibrosis is a critical step toward the design of rational new treatments. The importance of macrophages in the wound-healing response has been known for some time. In the 1970s studies on skin wound healing by Leibovich and Ross3Leibovich SJ Ross R The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum.Am J Pathol. 1975; 78: 71-100PubMed Google Scholar, 4Leibovich SJ Ross R A macrophage-dependent factor that stimulates the proliferation of fibroblasts in vitro.Am J Pathol. 1976; 84: 501-514PubMed Google Scholar demonstrated that macrophage depletion (with hydrocortisone and anti-macrophage serum) resulted in the delayed appearance of fibroblasts, and their subsequent rate of proliferation was lower than that of controls. More recently, we have shown that selective depletion of macrophages in a model of hepatic inflammation significantly attenuates liver fibrosis.5Duffield JS Forbes SJ Constandinou CM Clay S Partolina M Vuthoori S Wu S Lang R Iredale JP Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair.J Clin Invest. 2005; 115: 56-65Crossref PubMed Scopus (1282) Google Scholar In the kidney there is a striking correlation between tubulointerstitial macrophage infiltration and the severity of fibrosis in human biopsies and the subsequent development and progression of chronic renal failure to end-stage renal failure requiring dialysis.6Bohle A Muller GA Wehrmann M Mackensen-Haen S Xiao JC Pathogenesis of chronic renal failure in the primary glomerulopathies, renal vasculopathies, and chronic interstitial nephritides.Kidney Int. 1996; 54: S2-S9Google Scholar, 7Becker GJ Hewitson TD The role of tubulointerstitial injury in chronic renal failure.Curr Opin Nephrol Hypertens. 2000; 9: 133-138Crossref PubMed Scopus (134) Google Scholar Experimental hydronephrosis induced by unilateral ureteric obstruction (UUO) is a clinically relevant animal model because it mimics congenital obstructive nephropathy (the major cause of end-stage renal disease in children8Roth KS Koo HP Spottswood SE Chan JC Obstructive uropathy: an important cause of chronic renal failure in children.Clin Pediatr. 2002; 45: 309-314Crossref Scopus (41) Google Scholar), with progression through the different stages of obstructive nephropathy leading to tubulointerstitial fibrosis.9Bascands JL Schanstra JP Obstructive nephropathy: insights from genetically engineered animals.Kidney Int. 2005; 68: 925-937Crossref PubMed Scopus (193) Google Scholar Experimental hydronephrosis secondary to UUO is neutrophil- and lymphocyte-independent and is characterized by a marked tubulointerstitial macrophage infiltrate,10Schreiner GF Harris KP Purkerson ML Klahr S Immunological aspects of acute ureteral obstruction: immune cell infiltrate in the kidney.Kidney Int. 1988; 34: 487-493Crossref PubMed Scopus (228) Google Scholar, 11Diamond JR Macrophages and progressive renal disease in experimental hydronephrosis.Am J Kidney Dis. 1995; 26: 133-140Abstract Full Text PDF PubMed Scopus (86) Google Scholar interstitial myofibroblast and tubular epithelial cell proliferation, and progressive scarring with deposition of extracellular matrix early in the course of the disease.12Hughes J Johnson RJ Role of Fas (CD95) in tubulointerstitial disease induced by unilateral ureteric obstruction.Am J Physiol. 1999; 277: F26-F32PubMed Google Scholar, 13Diamond JR van Goor H Ding G Engelmyer E Myofibroblasts in experimental hydronephrosis.Am J Pathol. 1995; 146: 121-129PubMed Google Scholar Furthermore, the inhibition of tubulointerstitial macrophage recruitment reduces the extent and severity of renal fibrosis14Ophascharoensuk V Giachelli CM Gordon K Hughes J Pichler R Brown P Liaw L Schmidt R Shankland SJ Alpers CE Couser WG Johnson RJ Obstructive uropathy in the mouse: role of osteopontin in interstitial fibrosis and apoptosis.Kidney Int. 1999; 56: 571-580Crossref PubMed Scopus (253) Google Scholar, 15Lange-Sperandio B Cachat F Thornhill BA Chevalier RL Selectins mediate macrophage infiltration in obstructive nephropathy in newborn mice.Kidney Int. 2002; 61: 516-524Crossref PubMed Scopus (86) Google Scholar, 16Anders HJ Vielhauer V Frink M Linde Y Cohen CD Blattner SM Kretzler M Strutz F Mack M Grone HJ Onuffer J Horuk R Nelson PJ Schlondorff D A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation.J Clin Invest. 2002; 109: 251-259Crossref PubMed Scopus (209) Google Scholar, 17Eis V Luckow B Vielhauer V Siveke JT Linde Y Segerer S Perez De Lema G Cohen CD Kretzler M Mack M Horuk R Murphy PM Gao JL Hudkins KL Alpers CE Grone HJ Schlondorff D Anders HJ Chemokine receptor CCR1 but not CCR5 mediates leukocyte recruitment and subsequent renal fibrosis after unilateral ureteral obstruction.J Am Soc Nephrol. 2004; 15: 337-347Crossref PubMed Scopus (113) Google Scholar, 18Kitagawa K Wada T Furuichi K Hashimoto H Ishiwata Y Asano M Takeya M Kuziel WA Matsushima K Mukaida N Yokoyama H Blockade of CCR2 ameliorates progressive fibrosis in kidney.Am J Pathol. 2004; 165: 237-246Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar demonstrating that macrophages play a major role in driving fibrosis after UUO. Galectin-3 is a β-galactoside-binding animal lectin of ∼30 kDa19Ho MK Springer TA Mac-2, a novel 32,000 Mr mouse macrophage subpopulation-specific antigen defined by monoclonal antibodies.J Immunol. 1982; 128: 1221-1228PubMed Google Scholar that is highly expressed and secreted by macrophages.20Sato S Hughes RC Regulation of secretion and surface expression of Mac-2, a galactoside-binding protein of macrophages.J Biol Chem. 1994; 269: 4424-4430Abstract Full Text PDF PubMed Google Scholar, 21Liu FT Hsu DK Zuberi RI Kuwabara I Chi EY Henderson Jr, WR Expression and function of galectin-3, a beta-galactoside-binding lectin, in human monocytes and macrophages.Am J Pathol. 1995; 147: 1016-1028PubMed Google Scholar It is up-regulated when monocytes differentiate into macrophages21Liu FT Hsu DK Zuberi RI Kuwabara I Chi EY Henderson Jr, WR Expression and function of galectin-3, a beta-galactoside-binding lectin, in human monocytes and macrophages.Am J Pathol. 1995; 147: 1016-1028PubMed Google Scholar and down-regulated when macrophages differentiate into dendritic cells.22Dietz AB Bulur PA Knutson GJ Matasic R Vuk-Pavlovic S Maturation of human monocyte-derived dendritic cells studied by microarray hybridization.Biochem Biophys Res Commun. 2000; 275: 731-738Crossref PubMed Scopus (127) Google Scholar Furthermore, galectin-3 is a potent mitogen for fibroblasts in vitro,23Moutsatsos IK Wade M Schindler M Wang JL Endogenous lectins from cultured cells: nuclear localization of carbohydrate-binding protein 35 in proliferating 3T3 fibroblasts.Proc Natl Acad Sci USA. 1987; 84: 6452-6456Crossref PubMed Scopus (242) Google Scholar, 24Inohara H Akahani S Raz A Galectin-3 stimulates cell proliferation.Exp Cell Res. 1998; 245: 294-302Crossref PubMed Scopus (171) Google Scholar, 25Sasaki S Bao Q Hughes RC Galectin-3 modulates rat mesangial cell proliferation and matrix synthesis during experimental glomerulonephritis induced by anti-Thy1.1 antibodies.J Pathol. 1999; 187: 481-489Crossref PubMed Scopus (79) Google Scholar, 26Maeda N Kawada N Seki S Arakawa T Ikeda K Iwao H Okuyama H Hirabayashi J Kasai K Yoshizato K Stimulation of proliferation of rat hepatic stellate cells by galectin-1 and galectin-3 through different intracellular signaling pathways.J Biol Chem. 2003; 278: 18938-18944Crossref PubMed Scopus (134) Google Scholar and our previous work has demonstrated that galectin-3 regulates myofibroblast activation and hepatic fibrosis in vivo.27Henderson NC Mackinnon AC Farnworth SL Poirier F Russo FP Iredale JP Haslett C Simpson KJ Sethi T Galectin-3 regulates myofibroblast activation and hepatic fibrosis.Proc Natl Acad Sci USA. 2006; 103: 5060-5065Crossref PubMed Scopus (473) Google Scholar We hypothesized that the major tissue source of galectin-3 driving fibrosis is macrophage-derived, and using a model of hydronephrosis we set out to define whether macrophage-derived galectin-3 is a major mechanism linking macrophages to the promotion of renal myofibroblast activation and fibrosis. Tissue culture reagents were purchased from Life Technologies (Paisley, UK). Tissue culture plastics were obtained from Costar (Loughborough, UK) and Falcon (Runcorn, UK). Cytokines and recombinant mouse galectin-3 were purchased from R&D Systems (Abingdon, UK) and Peprotech EC Ltd. (London, UK). The galectin-3 inhibitor bis- [3-deoxy-3-(3-methoxybenzamido)-β-d-galactopyranosyl]-sulfane was provided by U. Nilsson and H. Leffler, University of Lund, Sweden.28Cumpstey I Sundin A Leffler H Nilsson UJ C2-symmetrical thiodigalactoside bis-benzamido derivatives as high-affinity inhibitors of galectin-3: efficient lectin inhibition through double arginine-arene interactions.Angew Chem Int Ed Engl. 2005; 44: 5110-5112Crossref PubMed Scopus (121) Google Scholar All other reagents were from Sigma-Aldrich Company Ltd. (Poole, UK) unless otherwise stated. Mice were maintained in 12-hour light/12-hour dark cycles with free access to food and water. All procedures were performed in accordance with Home Office guidelines [Animals (Scientific Procedures) Act 1986]. Generation of galectin-3−/− mice by gene-targeting technology has been described previously.29Colnot C Ripoche MA Milon G Montagutelli X Crocker PR Poirier F Maintenance of granulocyte numbers during acute peritonitis is defective in galectin-3-null mutant mice.Immunology. 1998; 94: 290-296Crossref PubMed Scopus (155) Google Scholar As control, age- and sex-matched wild-type littermate mice were used. CD11b-DTR mice were generated and characterized as previously described.5Duffield JS Forbes SJ Constandinou CM Clay S Partolina M Vuthoori S Wu S Lang R Iredale JP Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair.J Clin Invest. 2005; 115: 56-65Crossref PubMed Scopus (1282) Google Scholar Strain-matched controls (FVB/N) were purchased from B and K Ltd. (Hull, UK). All in vivo studies had six mice in each experimental group. UUO was performed by ligation of the left ureter as described previously.12Hughes J Johnson RJ Role of Fas (CD95) in tubulointerstitial disease induced by unilateral ureteric obstruction.Am J Physiol. 1999; 277: F26-F32PubMed Google Scholar Sham-operated control mice underwent an identical surgical procedure to the UUO mice except ligation of the ureter was not performed. Kidneys were harvested at days 3, 7, and 14 after UUO. For macrophage ablation CD11b-DTR mice and strain-matched control FVB/N mice (six mice per group) received three intravenous injections of either diphtheria toxin (DT) (25 ng/g body weight) or phosphate-buffered saline after UUO on days 4, 5, and 6. Kidneys were harvested at day 7 and quartered, and samples were then fixed in either methyl Carnoy's reagent (60% methanol, 30% chloroform, 10% glacial acetic acid) for assessment of macrophage infiltration or neutral buffered formalin for immunohistochemistry. Samples were also snap-frozen in liquid nitrogen for real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Bone marrow-derived macrophages (BMDMs) were prepared from wild-type (WT) and galectin-3−/− mice by maturing bone marrow cells in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 20% L929 conditioned media for 7 to 9 days as described previously.30Duffield JS Erwig LP Wei X Liew FY Rees AJ Savill JS Activated macrophages direct apoptosis and suppress mitosis of mesangial cells.J Immunol. 2000; 164: 2110-2119PubMed Google Scholar Mature BMDMs (5 × 105/well) were added to the wells of 24-well plates (or plated onto glass coverslips for immunofluorescence). After 3 hours the wells were washed to remove nonadherent cells. Wells were treated with lipopolysaccharide (LPS) (100 ng/ml) and murine interferon-γ (IFN-γ) (100 U/ml) in serum-free media. After 24 hours of incubation, the supernatants were harvested and clarified by centrifugation at 10,000 × g for 5 minutes and frozen at −80°C. In vivo-derived peritoneal macrophages were obtained from peritoneal lavage and separated by adhesion onto tissue culture plastic. Cytokine release in macrophage supernatants was determined by cytometric bead array, mouse inflammation kit (BD Biosciences, Oxford, UK). Paraffin-embedded sections of mouse tissue were processed for immunohistochemistry as described previously,5Duffield JS Forbes SJ Constandinou CM Clay S Partolina M Vuthoori S Wu S Lang R Iredale JP Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair.J Clin Invest. 2005; 115: 56-65Crossref PubMed Scopus (1282) Google Scholar and the following primary antibodies were used: mouse monoclonal anti-α-SMA clone 1A4 (Sigma), rat monoclonal anti-mouse galectin-3 clone 8942F (Cedarlane, Ontario, Canada), and rat anti-mouse F4/80 clone CI:A3-1 (Serotec, Oxford, UK). Methyl Carnoy's-fixed paraffin-embedded sections (4 μm) were used to assess macrophage infiltration (F4/80-positive cells), and sections were visualized and quantified as previously described.15Lange-Sperandio B Cachat F Thornhill BA Chevalier RL Selectins mediate macrophage infiltration in obstructive nephropathy in newborn mice.Kidney Int. 2002; 61: 516-524Crossref PubMed Scopus (86) Google Scholar The detection of renal fibrocytes was performed by immunofluorescence using specific antibodies against CD34 (Santa Cruz Biotechnology, Santa Cruz, CA), CD45 (Santa Cruz Biotechnology), and type I collagen polyclonal antibody (Chemicon International, Temecula, CA). CD34, CD45, and collagen I immunofluorescence staining of formalin-fixed sections was performed using species-specific Alexa-568- and Alexa-488-conjugated secondary antibodies and fluorescence microscopy (Carl Zeiss Ltd., Welwyn Garden City, UK). BMDMs plated on glass coverslips were washed and fixed in 3% paraformaldehyde and subjected to indirect immunofluorescence with anti-galectin-3-fluorescein isothiocyanate antibody and then anti-fluorescein isothiocyanate-Alexa-488 antibody. Nuclei were labeled with 4,6-diamidino-2-phenylindole. Renal fibrosis was visualized microscopically and quantified with the use of a picrosirius red stain as described previously.5Duffield JS Forbes SJ Constandinou CM Clay S Partolina M Vuthoori S Wu S Lang R Iredale JP Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair.J Clin Invest. 2005; 115: 56-65Crossref PubMed Scopus (1282) Google Scholar Digital image analysis was used to quantitate the amount of red-stained collagen fibers. Morphometric measurements of 10-μm sections stained with picrosirius red were made using OpenLab software (Improvision, Coventry, UK). Twelve nonoverlapping fields at ×400 magnification from each section (captured with a Leica DMLB microscope, Leica DC300 camera, and Leica image manager; Leica, Milton Keynes, UK) were analyzed in a blinded manner. Each captured field was analyzed by separation into red, green, and blue (RGB) filters, and the red area was mathematically divided by the red, green, and blue (RGB) area and multiplied by 100%. This represents the percentage area staining positively for collagen fibers, providing a quantitative value on a continuous scale. Western blot analysis was undertaken using the following primary antibodies: mouse monoclonal anti-α-smooth muscle actin (SMA) antibody clone 1A4 (Sigma), mouse monoclonal anti-galectin-3 antibody clone A3A12 (Alexis Biochemicals, Nottingham, UK), rabbit polyclonal anti-phospho-Smad2 and anti-phospho-Smad3 (Biosource, Paisley, UK), and goat polyclonal total Smad2/3 antibody (Santa Cruz Biotechnology). Total RNA from whole kidney was reverse-transcribed into cDNA using random hexamers (Applied Biosystems, Warrington, UK). Mouse primers and probes were as follows: galectin-3: forward 5′-TTGAAGCTGACCACTTCAAGGTT-3′, reverse 5′-AGGTTCTTCATCCGATGGTTGT-3′, probe FAM 5′-CGGTCAACGATGCTCACCTACTGCA-3′ TAMRA; α-SMA: forward 5′-TCAGCGCCTCCAGTTCCT-3′, reverse 5′-AAAAAAAACCACGAGTAACAAATCAA-3′; probe FAM 5′-TCCAAA TCATTCCTGCCCA-3′ TAMRA; procollagen(I): forward 5′-TTCACCTACAGCACGCTTGTG-3′, reverse 5′-GATGACTGTCTTGCCCCAAGTT-3′, probe FAM 5′-ATGGCTGCACGAGTCACA-3′ TAMRA; transforming growth factor (TGF)-β: forward 5′-CACCGGAGAGCCCTGGATA-3′, reverse 5′-TGTACAGCTGCCGCACACA-3′, probe FAM 5′-CAACTATTGCTTCAGCTCCACAGAGAAGAACTG-3′ TAMRA. 18S rRNA TaqMan primer probe mix was purchased from Applied Biosystems. Mature BMDMs (day 7) were prelabeled with fluorescent Cell-Tracker Orange as per the manufacturers instructions (Molecular Probes, Eugene, OR). Galectin-3−/− mice underwent UUO surgery at day 0 and received 5 × 106 WT or galectin-3−/− BMDMs at days 1, 3, and 5 intravenously. Tissues were harvested on day 7 including both UUO and contralateral kidney, liver, spleen, and lung. Primary cultures of renal fibroblasts were isolated by trypsin digestion (0.25% for 2 hours at 37°C) of minced normal mouse kidneys, and digests were passed through a 20-μm cell strainer (Becton Dickinson, Oxford, UK) to remove glomeruli. Cells were cultured undisturbed in Dulbecco's modified Eagle's medium containing 15% fetal calf serum for 8 days until cells were confluent. Renal fibroblasts were used at passage 2. Mature BMDMs (1 × 106 cells) were plated in six-well tissue culture dishes and incubated in 1 ml of serum-free media for 48 hours. Conditioned media from BMDMs (0.5 ml) or control media were added to 0.5 ml of renal fibroblasts (4 × 104 cells). Cells were incubated for a further 48 hours (final fetal calf serum concentration, 7.5%) before lysis and Western analysis. Results are presented as means ± SEM. Significance of the differences between means was assessed using one-way analysis of variance or two-tailed Student's t-test. Values of P < 0.05 were considered significant. Unless stated otherwise, studies were performed on three to six independent occasions. Galectin-3 expression was analyzed in a well established experimental model of progressive renal fibrosis (UUO). Galectin-3 expression was markedly increased in the renal interstitium and tubular epithelium after UUO compared with the control sham-operated group (Figure 1, a and b). This increase in galectin-3 expression was confirmed by real-time PCR of whole kidney tissue (Figure 1c, P < 0.001). The significance of the induction of galectin-3 expression in the development of renal fibrosis was examined using the UUO model of progressive renal scarring. Renal collagen deposition was stained with picrosirius red (Figure 2, a and b) and quantified using digital image analysis. Significantly reduced collagen deposition was observed in the galectin-3−/− mice compared with WT (P < 0.05, Figure 2c). Furthermore transcripts for procollagen (I) were also reduced in the galectin-3−/− group compared with WT animals (Figure 2d, P < 0.05). Immunohistochemical examination revealed markedly reduced α-SMA positivity (a marker of activated myofibroblasts, a key cell type involved in extracellular matrix production and scarring in the kidney) in galectin-3−/− compared with WT mice in the UUO model (Figure 2, e and f). α-SMA was quantified using digital image analysis, and significantly less α-SMA staining occurred in the galectin-3−/− mice compared with WT (Figure 2g, P < 0.05). α-SMA mRNA transcripts, as assessed by real-time PCR, were significantly decreased in the galectin-3−/− mice compared with WT animals (Figure 2h, P < 0.01). Therefore absence of the galectin-3 gene protects against renal fibrosis after UUO. Previous studies in which macrophage recruitment to the kidney was inhibited have suggested a role for macrophages in the development of renal fibrosis.14Ophascharoensuk V Giachelli CM Gordon K Hughes J Pichler R Brown P Liaw L Schmidt R Shankland SJ Alpers CE Couser WG Johnson RJ Obstructive uropathy in the mouse: role of osteopontin in interstitial fibrosis and apoptosis.Kidney Int. 1999; 56: 571-580Crossref PubMed Scopus (253) Google Scholar, 15Lange-Sperandio B Cachat F Thornhill BA Chevalier RL Selectins mediate macrophage infiltration in obstructive nephropathy in newborn mice.Kidney Int. 2002; 61: 516-524Crossref PubMed Scopus (86) Google Scholar, 16Anders HJ Vielhauer V Frink M Linde Y Cohen CD Blattner SM Kretzler M Strutz F Mack M Grone HJ Onuffer J Horuk R Nelson PJ Schlondorff D A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation.J Clin Invest. 2002; 109: 251-259Crossref PubMed Scopus (209) Google Scholar, 17Eis V Luckow B Vielhauer V Siveke JT Linde Y Segerer S Perez De Lema G Cohen CD Kretzler M Mack M Horuk R Murphy PM Gao JL Hudkins KL Alpers CE Grone HJ Schlondorff D Anders HJ Chemokine receptor CCR1 but not CCR5 mediates leukocyte recruitment and subsequent renal fibrosis after unilateral ureteral obstruction.J Am Soc Nephrol. 2004; 15: 337-347Crossref PubMed Scopus (113) Google Scholar, 18Kitagawa K Wada T Furuichi K Hashimoto H Ishiwata Y Asano M Takeya M Kuziel WA Matsushima K Mukaida N Yokoyama H Blockade of CCR2 ameliorates progressive fibrosis in kidney.Am J Pathol. 2004; 165: 237-246Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar However, a number of the approaches used do not deplete macrophages specifically, and some deplete neutrophils simultaneously, thereby making interpretation of some of the results more difficult.31Ikezumi Y Hurst LA Masaki T Atkins RC Nikolic-Paterson DJ Adoptive transfer studies demonstrate that macrophages can induce proteinuria and mesangial cell proliferation.Kidney Int. 2003; 63: 83-95Crossref PubMed Scopus (130) Google Scholar, 32Diamond JR Pesek-Diamond I Sublethal X-irradiation during acute puromycin nephrosis prevents late renal injury: role of macrophages.Am J Physiol. 1991; 260: F779-F786PubMed Google Scholar We used the CD11b-DTR mouse5Duffield JS Forbes SJ Constandinou CM Clay S Partolina M Vuthoori S Wu S Lang R Iredale JP Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair.J Clin Invest. 2005; 115: 56-65Crossref PubMed Scopus (1282) Google Scholar, 33Duffield JS Tipping PG Kipari T Cailhier JF Clay S Lang R Bonventre JV Hughes J Conditional ablation of macrophages halts progression of crescentic glomerulonephritis.Am J Pathol. 2005; 167: 1207-1219Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 34Cailhier JF Partolina M Vuthoori S Wu S Ko K Watson S Savill J Hughes J Lang RA Conditional macrophage ablation demonstrates that resident macrophages initiate acute peritoneal inflammation.J Immunol. 2005; 174: 2336-2342PubMed Google Scholar to investigate further the role of macrophages in the evolution of tubulointerstitial scarring. The administration of DT to CD11b-DTR mice specifically ablates monocytes and macrophages.5Duffield JS Forbes SJ Constandinou CM Clay S Partolina M Vuthoori S Wu S Lang R Iredale JP Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair.J Clin Invest. 2005; 115: 56-65Crossref PubMed Scopus (1282) Google Scholar, 34Cailhier JF Partolina M Vuthoori S Wu S Ko K Watson S Savill J Hughes J Lang RA Conditional macrophage ablation demonstrates that resident macrophages initiate acute peritoneal inflammation.J Immunol. 2005; 174: 2336-2342PubMed Google Scholar Immunostaining for macrophages confirmed marked depletion of macrophages in DT-treated mice (Figure 3, a–c) compared to vehicle-treated control mice. We confirmed previous data showing that the numbers of circulating neutrophils, eosinophils, and lymphocytes are not affected by DT treatment in the DTR mouse by flow cytometry (data not shown)5Duffield JS Forbes SJ Constandinou CM Clay S Partolina M Vuthoori S Wu S Lang R Iredale JP Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair.J Clin Invest. 2005; 115: 56-65Crossref PubMed Scopus (1282) Google Scholar, 33Duffield JS Tipping PG Kipari T Cailhier JF Clay S Lang R Bonventre JV Hughes J Conditional ablation of macrophages halts progression of crescentic glomerulonephritis.Am J Pathol. 2005; 167: 1207-1219Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar Macrophage ablation significantly reduced myofibroblast activation and decreased fibrosis as characterized by reduced α-SMA (Figure 3, d–f) and collagen expression (Figure 3, g–i), confirming an important mechanistic role for macrophages in tubulointerstitial scarring after UUO. Circulating fibrocytes derived from the bone marrow have also been shown to contribute to renal fibrosis.35Sakai N Wada T Yokoyama H Lipp M Ueha S Matsushima K Kaneko S Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis.Proc Natl Acad Sci USA. 2006; 103: 14098-14103Crossref PubMed Scopus (220) Google Scholar Therefore we assessed whether administration of diphtheria-toxin (DT) in our macrophage depletion model had any effect on fibrocyte recruitment to the kidney after UUO. Figure 4 demonstrates that DT treatment did not significantly deplete kidney fibrocyte recruitment after UUO compared with non-DT controls. These results demonstrate that the development of tubulointerstitial fibrosis after UUO is macrophage-dependent.Figure 4Macrophage ablation by DT does not affect recruitment of CD34+/ColI+ and CD45+/ColI+ fibrocytes after UUO. a: CD34; b: ColI; c: merged. Arrows indicate CD34+/ColI+ fibrocytes. d: The number of infiltrating fibrocytes (CD34+/ColI+ and CD45+/ColI+) increased at day 7 after UUO, with no significant difference in infiltrating fibrocyte numbers between non-DT-treated (white bars) and DT-treated (black bars) mice. Day 7 UUO DT−CD34+/ColI+, 26.33 ± 2.8 per mm2; day 7 UUO DT+CD34+/ColI+, 29.83 ± 4.7 per mm2 (n
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