Chemokine Receptor CX3CR1 Regulates Renal Interstitial Fibrosis after Ischemia-Reperfusion Injury
2006; Elsevier BV; Volume: 169; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2006.060043
ISSN1525-2191
AutoresKengo Furuichi, Ji‐Liang Gao, Philip M. Murphy,
Tópico(s)Phagocytosis and Immune Regulation
ResumoTransient renal ischemia induces both inflammatory and fibrotic processes and is a major cause of acute and chronic renal insufficiency. Study of ischemia-reperfusion injury in gene-targeted mice has identified multiple factors responsible for inflammation, whereas mechanisms underlying fibrosis remain poorly defined. Here we demonstrate by both gene inactivation and target protein blockade that a single chemokine receptor subtype, the fractalkine receptor CX3CR1, is able to reduce both inflammation and fibrosis after ischemia-reperfusion injury in the mouse, leading to partially preserved renal function after injury. The mechanism involves selective effects in the outer medulla, including reduced accumulation of macrophages and reduced expression of the macrophage and platelet-derived fibrogenic protein platelet-derived growth factor-B. CX3CR1 is the first chemokine receptor shown to contribute to fibrogenesis in renal ischemia-reperfusion injury. Transient renal ischemia induces both inflammatory and fibrotic processes and is a major cause of acute and chronic renal insufficiency. Study of ischemia-reperfusion injury in gene-targeted mice has identified multiple factors responsible for inflammation, whereas mechanisms underlying fibrosis remain poorly defined. Here we demonstrate by both gene inactivation and target protein blockade that a single chemokine receptor subtype, the fractalkine receptor CX3CR1, is able to reduce both inflammation and fibrosis after ischemia-reperfusion injury in the mouse, leading to partially preserved renal function after injury. The mechanism involves selective effects in the outer medulla, including reduced accumulation of macrophages and reduced expression of the macrophage and platelet-derived fibrogenic protein platelet-derived growth factor-B. CX3CR1 is the first chemokine receptor shown to contribute to fibrogenesis in renal ischemia-reperfusion injury. Acute ischemia of the kidney, a major cause of renal failure, occurs most commonly in the setting of renal artery stenosis, renal transplantation, and shock because of hemorrhage or sepsis. Even when a kidney that has been rendered ischemic regains normal perfusion, pathological changes may persist and progress to chronic functional insufficiency resulting in end-stage renal disease.1Forbes JM Hewitson TD Becker GJ Jones CL Ischemic acute renal failure: long-term histology of cell and matrix changes in the rat.Kidney Int. 2000; 57: 2375-2385Crossref PubMed Scopus (146) Google Scholar Although immunosuppressive drugs have been highly successful in preventing acute kidney allograft rejection, they have not had an impact on late graft failure, which may result in part from early ischemic injury.2Pascual M Theruvath T Kawai T Tolkoff-Rubin N Cosimi AB Strategies to improve long-term outcomes after renal transplantation.N Engl J Med. 2002; 346: 580-590Crossref PubMed Scopus (739) Google Scholar, 3Hariharan S Johnson CP Bresnahan BA Taranto SE McIntosh MJ Stablein D Improved graft survival after renal transplantation in the United States, 1988 to 1996.N Engl J Med. 2000; 342: 605-612Crossref PubMed Scopus (1622) Google Scholar Late allograft failure afflicts a growing number of patients, fed by the large increase in patients living with end-stage renal disease since the advent of hemodialysis.4US Renal Data System, Reference Section A: Incidence of reported ESRD. 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Rodent models of renal ischemia-reperfusion injury have been developed in an effort to develop insights into pathogenesis at the molecular level. Recent studies using such models have succeeded in delineating many factors that are involved in inflammation8Bonventre JV Zuk A Ischemic acute renal failure: an inflammatory disease?.Kidney Int. 2004; 66: 480-485Crossref PubMed Scopus (621) Google Scholar; however, osteopontin is the only molecular determinant of fibrosis identified to date.9Persy VP Verhulst A Ysebaert DK De Greef KE De Broe ME Reduced postischemic macrophage infiltration and interstitial fibrosis in osteopontin knockout mice.Kidney Int. 2003; 63: 543-553Crossref PubMed Scopus (122) Google Scholar Transforming growth factor (TGF)-β and platelet-derived growth factor (PDGF) are well-characterized factors that promote fibrosis in many diseases and organs, including the kidney.10Border WA Noble NA Transforming growth factor beta in tissue fibrosis.N Engl J Med. 1994; 331: 1286-1292Crossref PubMed Scopus (2999) Google Scholar, 11Hugo C The thrombospondin 1-TGF-beta axis in fibrotic renal disease.Nephrol Dial Transplant. 2003; 18: 1241-1245Crossref PubMed Scopus (53) Google Scholar PDGF, which stimulates fibroblast proliferation and production of extracellular matrix, is actually a family of four molecules, PDGF-A and -B and the newly discovered PDGF-C and -D.12Bonner JC Regulation of PDGF and its receptors in fibrotic diseases.Cytokine Growth Factor Rev. 2004; 15: 255-273Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar PDGF-B has been implicated in renal fibrosis based on the effects of direct injection of the factor into rat kidney in vivo.13Tang WW Ulich TR Lacey DL Hill DC Qi M Kaufman SA Van GY Tarpley JE Yee JS Platelet-derived growth factor-BB induces renal tubulointerstitial myofibroblast formation and tubulointerstitial fibrosis.Am J Pathol. 1996; 148: 1169-1180PubMed Google Scholar Infiltrating inflammatory cells are rich sources of these and potentially other fibrogenic mediators.14Hirschberg R Wang S Proteinuria and growth factors in the development of tubulointerstitial injury and scarring in kidney disease.Curr Opin Nephrol Hypertens. 2005; 14: 43-52Crossref PubMed Scopus (78) Google Scholar For this reason we and others have focused on molecular mechanisms responsible for inflammatory leukocyte trafficking to the kidney after ischemia-reperfusion injury. Chemokines, a major class of leukocyte chemoattractants, are particularly relevant in this regard and have been implicated in the pathogenesis of numerous fibrotic disorders; however, none has been linked to fibrosis resulting from renal ischemia-reperfusion injury.7Anders HJ Vielhauer V Schlondorff D Chemokines and chemokine receptors are involved in the resolution or progression of renal disease.Kidney Int. 2003; 63: 401-415Crossref PubMed Scopus (221) Google Scholar In the present report we focus on the chemokine fractalkine (CX3CL1 in standard nomenclature) and its 7-transmembrane domain G protein-coupled receptor CX3CR1. Fractalkine is the only known member of the CX3C chemokine subclass, one of the four major structural divisions of the chemokine family. In addition to functioning as a leukocyte chemoattractant, fractalkine is unusual in that it is also able to function as an adhesion molecule. CX3CR1 is expressed on NK cells, monocytes, T cells, mast cells, and platelets.15Imai T Hieshima K Haskell C Baba M Nagira M Nishimura M Kakizaki M Takagi S Nomiyama H Schall TJ Yoshie O Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion.Cell. 1997; 91: 521-530Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar, 16Fong AM Robinson LA Steeber DA Tedder TF Yoshie O Imai T Patel DD Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow.J Exp Med. 1998; 188: 1413-1419Crossref PubMed Scopus (591) Google Scholar, 17Haskell CA Cleary MD Charo IF Molecular uncoupling of fractalkine-mediated cell adhesion and signal transduction. 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Here we test this hypothesis in a mouse model of ischemia-reperfusion injury of the kidney. Male wild-type and CX3CR1−/− C57BL/6 mice were purchased from Taconic (Germantown, NY). CX3CR1−/− mice, generated as previously described,20Combadiere C Potteaux S Gao JL Esposito B Casanova S Lee EJ Debre P Tedgui A Murphy PM Mallat Z Decreased atherosclerotic lesion formation in CX3CR1/apolipoprotein E double knockout mice.Circulation. 2003; 107: 1009-1016Crossref PubMed Scopus (406) Google Scholar had been backcrossed onto the C57BL/6 background for 10 generations and maintained under specific pathogen-free housing conditions. No significant differences in growth or weight were found between CX3CR1−/− and wild-type C57BL/6 mice. All animals were used at 6 to 8 weeks of age under the auspices of a protocol approved by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee. After general anesthesia was established with xylazine, ketamine, and isoflurane, the left renal artery and vein of CX3CR1−/− and wild-type control mice were exposed by flank incision and clamped for 60 minutes. Kidneys that did not completely recover after unclamping, as assessed by restoration of normal color, were not used for analysis. After releasing the clamp the flank incision was closed in two layers with silk sutures. The animals received warm saline instilled into the peritoneal cavity during the procedure and were allowed to recover with free access to food and water. Sham surgery was performed in a similar manner, except that the renal vessels were not clamped. To evaluate function of the injured kidney, the uninjured kidney was removed on day 12 in a subset of mice. Renal tissues were removed for pathological examination 24 hours, 48 hours, 7 days, and 14 days after ischemia-reperfusion from 14 mice at each time point. Blood samples were taken from the abdominal aorta just before sacrifice to evaluate renal function. Purified polyclonal rabbit anti-CX3CR1 antibodies or control rabbit IgG (Torrey Pines Biolabs, San Diego, CA) were used to evaluate the therapeutic effects of CX3CR1 blockade in ischemia-reperfusion injury as described previously.21Robinson LA Nataraj C Thomas DW Howell DN Griffiths R Bautch V Patel DD Feng L Coffman TM A role for fractalkine and its receptor (CX3CR1) in cardiac allograft rejection.J Immunol. 2000; 165: 6067-6072PubMed Google Scholar Seventy-five μl of anti-CX3CR1 antibodies or control rabbit IgG at a concentration of 20 μg/ml were injected intraperitoneally 1 day before or 1 day after ischemic injury and each day thereafter for a total of 8 or 6 days, respectively. To evaluate effects of anti-CX3CR1 antibodies or control IgG on blood cell counts, we collected whole blood 8 days after injection of the animals (five per group). Effects of anti-CX3CR1 antibodies or control IgG on complement were evaluated by serum C5a concentration by enzyme-linked immunosorbent assay using rat anti-mouse complement component C5a monoclonal antibody (clone 295103), goat biotinylated anti-mouse complement component C5a antibody, and recombinant mouse complement component C5a protein (all from R&D Systems, Minneapolis, MN). Platelets were prepared and labeled for injection using a previously described method with some modifications.22Rand ML Wang H Mody M Chu I Treutiger I Nguyen A Packham MA Freedman J Concurrent measurement of the survival of two populations of rabbit platelets labeled with either two PKH lipophilic dyes or two concentrations of biotin.Cytometry. 2002; 47: 111-117Crossref PubMed Scopus (15) Google Scholar, 23Baker GR Sullam PM Levin J A simple, fluorescent method to internally label platelets suitable for physiological measurements.Am J Hematol. 1997; 56: 17-25Crossref PubMed Scopus (57) Google Scholar Briefly, peripheral blood was drawn from anesthetized wild-type mice or CX3CR1-deficient mice by cardiac puncture using citrate and disodium ethylenediaminetetraacetic acid as anti-coagulants. Fresh anti-coagulated blood was centrifuged at 600 × g for 3 minutes, and platelet-rich plasma was then collected. Centrifugation of the platelet-rich plasma at 1300 × g for 10 minutes produced a platelet pellet. Platelets were labeled with PKH26 red fluorescent cell linker mini kit (Sigma, St. Louis, MO) using the method of Michelson and colleagues24Michelson AD Barnard MR Hechtman HB MacGregor H Connolly RJ Loscalzo J Valeri CR In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.Proc Natl Acad Sci USA. 1996; 93: 11877-11882Crossref PubMed Scopus (504) Google Scholar with minor modifications. Platelets were resuspended in Diluent C at 4 × 109/ml to which 10 μmol/L prostaglandin I2 (PGI2) was added. An equal volume of Diluent C containing freshly prepared 4 μmol/L PKH26 was added, and the suspension was mixed and incubated for 8 minutes at room temperature with occasional inversion. An equal volume of citrate-albumin PGE1 buffer (11 mmol/L dextrose, 128 mmol/L NaCl, 4.3 mmol/L NaH2PO4, 7.5 mmol/L Na2HPO4, 4.8 mmol/L trisodium citrate, 2.4 mmol/L citric acid, 0.35% bovine serum albumin, 0.33 μmol/L PGE1, pH 6.5) was added. The mixture was incubated for 1 minute and centrifuged. The pellet was resuspended in 5 ml of citrate-albumin-PGE1 buffer, incubated for 10 minutes, centrifuged, and resuspended in Tyrode's solution (Sigma) with 0.35% albumin and 3 U/ml apyrase at a platelet count of 2.0 × 109/ml. To evaluate the role of CX3CR1 in the accumulation of platelets in the injured kidney, 2 × 108 PKH26-labeled platelets from wild-type mice or CX3CR1-deficient mice in a total volume of 100 μl were injected in the tail vein of wild-type mice just before ischemia-reperfusion injury. One portion of the renal tissue was fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with periodic acid-Schiff reagent, naphthol AS-D chloroacetate esterase, Gomori's trichrome, or indicated antibodies. Another portion of fresh renal tissue was embedded in OCT compound (Sakura Finetek, Torrance, CA) and snap-frozen on dry ice. Frozen sections were used to detect PKH26-labeled platelets and for immunohistochemistry using antibodies directed against CX3CR1 and F4/80. Deparaffinized sections were treated with Target river solution (DAKO, Carpinteria, CA) before staining of fractalkine and α-smooth muscle actin (α-SMA), with 10 mmol/L Tris buffer and 1 mmol/L ethylenediaminetetraacetic acid for TGF-β staining, or with proteinase K (DAKO) for staining of PDGF-B. Endogenous peroxidase activity and nonspecific binding in the sections was blocked by peroxidase-blocking reagent (DAKO), biotin-blocking system (DAKO) and protein block, serum-free (DAKO). Sections were then incubated with the following primary antibodies and conditions: goat anti-rat fractalkine antiserum (R&D Systems), which cross-reacts with mouse fractalkine, at 1 μg/ml overnight at 4°C; rabbit anti-human PDGF-B antiserum (Calbiochem, San Diego, CA), which cross-reacts with mouse PDGF-B,25Taneda S Hudkins KL Topouzis S Gilbertson DG Ophascharoensuk V Truong L Johnson RJ Alpers CE Obstructive uropathy in mice and humans: potential role for PDGF-D in the progression of tubulointerstitial injury.J Am Soc Nephrol. 2003; 14: 2544-2555Crossref PubMed Scopus (64) Google Scholar at 10 μg/ml for 2 hours at room temperature; rabbit anti-human TGF-β antiserum (Abcam, Cambridge, MA), which cross-reacts with mouse TGF-β, at 4 μg/ml for 2 hours at room temperature; or rabbit anti-mouse CX3CR1 antiserum (kind gift from Dr. T. Imai, Kyoto University) at 5 μg/ml for 2 hours at room temperature. Normal goat or rabbit IgG was used as a negative control. Thereafter, the sections were incubated with biotinylated secondary antibody, followed by incubation with peroxidase-conjugated streptavidin or the EnVision+/HRP kit (DAKO). For α-SMA, polymer-immunocomplex methods were used. Briefly, a mixture of diluted primary antibody, mouse α-SMA monoclonal antibody clone 1A4, 10 μg/ml (DAKO),26Skalli O Ropraz P Trzeciak A Benzonana G Gillessen D Gabbiani G A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation.J Cell Biol. 1986; 103: 2787-2796Crossref PubMed Scopus (1406) Google Scholar and EnVision+/HRP for mouse were incubated at room temperature for 60 minutes. After the incubation, normal mouse serum was added, and the mixture was incubated for 60 minutes. Tissues were incubated with the final mixture at room temperature for 60 minutes. Normal mouse IgG was used as a negative control. After washing with phosphate-buffered saline containing 0.01% Tween, the sections were stained with 3,3′-diaminobenzidine solution and then counterstained with hematoxylin. Tubular necrosis was quantitated in a blinded manner as the percentage of tubules in the outer medulla in which epithelial necrosis or necrotic debris was observed in periodic acid-Schiff-stained sections. Interstitial renal fibrosis was quantitated as the area staining blue in sections stained with Gomori's trichrome. The area of tubular necrosis and interstitial fibrosis in the outer medulla was evaluated by NIH image and expressed as the percentage of total area imaged. Infiltrating leukocytes were quantitated in the outer medulla of the injured kidney, where cell migration was maximal, and the data were expressed as leukocytes/mm2 at magnification ×320. Neutrophils were identified by naphthol AS-D chloroacetate esterase staining (Sigma).27Miyaji T Hu X Star RA Alpha-melanocyte-simulating hormone and interleukin-10 do not protect the kidney against mercuric chloride-induced injury.Am J Physiol. 2002; 282: F795-F801Google Scholar, 28Fernandez M Medina A Santos F Carbajo E Rodriguez J Alvarez J Cobo A Exacerbated inflammatory response induced by insulin-like growth factor I treatment in rats with ischemic acute renal failure.J Am Soc Nephrol. 2001; 12: 1900-1907PubMed Google Scholar T cells and macrophages were detected immunohistochemically with rabbit anti-CD3 antibody (DAKO)29Mallat Z Gojova A Marchiol-Fournigault C Esposito B Kamate C Merval R Fradelizi D Tedgui A Inhibition of transforming growth factor-beta signaling accelerates atherosclerosis and induces an unstable plaque phenotype in mice.Circ Res. 2001; 89: 930-934Crossref PubMed Scopus (411) Google Scholar and rat anti-mouse F4/80 monoclonal antibody (clone BM8; eBioscience, San Diego, CA), respectively. Rabbit anti-CD3 antibody was detected by EnVision+ System (peroxidase) (DAKO), and rat anti-F4/80 antibody was detected by biotinylated polyclonal rabbit anti-rat immunoglobulin antibodies (DAKO) and the LSAB+ system (peroxidase) (DAKO). To identify F4/80 and CX3CR1 double-positive cells, the sections were first incubated for 2 hours at room temperature with rabbit anti-mouse CX3CR1 antibody and then with Alexa Fluor 568-conjugated donkey anti-rabbit IgG (Molecular Probes, Eugene, OR) at 20 μg/ml for 1 hour at room temperature. For staining of F4/80, sections were incubated with rat anti-mouse F4/80 monoclonal antibody. Subsequently, the samples were incubated with Alexa Fluor-488-conjugated donkey anti-rat IgG (Molecular Probes) at 20 μg/ml for 1 hour at room temperature. Alexa Fluor 568-labeled CX3CR1-positive cells and Alexa Fluor 488-labeled F4/80-positive cells were observed by fluorescent microscopy. Tissue homogenates were used to determine TGF-β and PDGF-B expression using murine Quantikine immunoassay kits (R&D Systems) following the manufacturer's directions. All enzyme-linked immunosorbent assay samples were run in duplicate. The amount of TGF-β and PDGF-B was standardized by tissue weight. Total RNA was extracted from the kidney using RNAqueous kit (Ambion Inc., Austin, TX). RNA was treated with deoxyribonuclease and purified with DNA-free (Ambion, Inc.). The RNA was quantified by absorbance at 260 nm in a spectrophotometer (BioPhotometer; Eppendorf AG, Hamburg Germany). cDNA was reverse-transcribed from 1 μg of total RNA using a RETROscript kit (Ambion, Inc.). Fractalkine and CX3CR1 mRNA were detected by 28 cycles of polymerase chain reaction (PCR). PCR was found to be linear between 20 and 35 cycles. β-actin was used as internal standard (QuantumRNA β-actin internal standards, Ambion). The following primers were used for fractalkine: forward (5′-TGGTCCAGAGCTGGCAATAA) and reverse (5′-TGGCTTCCTCACTCTCAGGA). Mouse/rat CX3CR1 PCR primer pair (R&D Systems) was used to detect CX3CR1 expression. The mRNA expression of cytokines, chemokines, chemokine receptors, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed using an MPCR kit (Maxim Biotech, Inc., San Francisco, CA) according to the manufacturer's protocol. Images of UV-illuminated agarose gels were captured, and the density of cDNA bands was analyzed using NIH Image software. PCR was found to be linear between 24 and 36 cycles, and 33 cycles were chosen for all results shown. Cytokine, chemokine, and chemokine receptor mRNA levels were standardized by GAPDH signals and are expressed relative to the levels found in sham-operated mice. Hydroxyproline content was measured as an index of collagen accumulation in the injured kidney, as described previously30Eitzman DT McCoy RD Zheng X Fay WP Shen T Ginsburg D Simon RH Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene.J Clin Invest. 1996; 97: 232-237Crossref PubMed Scopus (525) Google Scholar with some modification. Briefly, a portion of the kidney was weighed, dried for 16 hours at 110°C, then hydrolyzed in 6 N HCl at 110°C for 12 hours. Fifty-μl aliquots were added to 1 ml of a solution containing 1.4% chloramine T (Sigma), 10% n-propanol, and 0.5 mol/L sodium acetate, at pH 6.0. After 20 minutes of incubation at room temperature, 1 ml of Erlich's solution (1 mol/L p-dimethylaminobenzaldehyde in 70% n-propanol, 20% perchloric acid) was added, and the mixture was incubated at 65°C for 15 minutes. Absorbance was measured at 550 nm, and the amount of hydroxyproline was determined against a standard curve generated using known concentrations of reagent hydroxyproline (Sigma), and expressed as the amount (μg) per mg of kidney. Serum creatinine was measured as a marker of renal function. Blood was collected from each mouse at the time of sacrifice and stored at −80°C until use. Creatinine concentration was measured by colorimetric microplate assay (Oxford Biomedical Research, Oxford, MI). The mean and SE (SEM) were calculated on all of the parameters determined in this study. Statistical analyses were performed using unpaired Student's t-test, Kruskal-Wallis test, and analysis of variance test. P < 0.05 was accepted as statistically significant. Fractalkine mRNA was constitutively expressed under homeostatic conditions in normal mouse kidney (data not shown); however, as assessed by PCR analysis of total kidney RNA, the level did not increase significantly in sham-operated mice or with time after ischemia-reperfusion injury in either wild-type or CX3CR1-deficient mice (Figure 1, A and B). Nevertheless the protein level, as assessed by immunohistochemistry, and the spatial distribution did change after injury (Figure 1C). Anti-fractal-kine immunoreactivity was detected mainly on endothelial cells throughout the kidney in sham-operated mice (Figure 1C, i) but by 24 hours after ischemia-reperfusion was redistributed to the outer medulla where it could also be detected in association with infiltrating cells and tubular epithelial cells (Figure 1C, ii). Increased expression persisted throughout the course of experiment but began to wane by day 7 after injury (data not shown). The difference between RNA and protein induction in the model may reflect infiltration of inflammatory cells that constitutively express fractalkine. In contrast to fractalkine's temporal and spatial pattern of expression, constitutive expression of CX3CR1 mRNA was extremely low in sham-operated mice but was markedly increased after ischemia-reperfusion injury, peaking at ∼48 hour in wild-type mice and remaining high throughout the 2-week course of the experiment (Figure 1, D and E). The increase in mRNA corresponded with an increase in anti-CX3CR1 immunoreactivity of wild-type kidney subjected to ischemia-reperfusion injury that was particularly intense in the outer medulla. This co-localized with infiltrating cells, which were mainly macrophages (Figure 1F) as demonstrated by dual staining with anti-F4/80 and anti-CX3CR1 antibodies (Figure 1G). CX3CR1 was expressed by 86.7 ± 1.5% of infiltrating macrophages. This suggested that CX3CR1 might mediate macrophage trafficking to the kidney and promote the inflammatory response after ischemia-reperfusion injury. To test this we examined CX3CR1-deficient mice in greater detail. Like fractalkine and CX3CR1, severe acute tubular necrosis was found consistently most intensely in the outer medulla of mouse kidney 24 to 48 hours after ischemia-reperfusion injury (Figure 2, A–D). With time, tubular epithelial cells proliferated, and tubules were regenerated in both wild-type and CX3CR1-deficient mice, as shown for days 7 and 14 after injury in Figure 2, E–H. Little if any evidence of acute tubular necrosis was detectable in the kidneys of sham-operated mice at 24 hours after surgery (Figure 2I). Quantitation of the area of acute tubular necrosis failed to reveal a significant difference between wild-type and CX3CR1-deficient mice at any time point (Figure 2J). Collagen fibers, which stain blue with trichrome, are normally present at low levels in the renal interstitium and did not increase in the first 48 hours after injury (Figure 3). However, by day 7 large increases were observed in the interstitium of the outer medulla, and this continued to increase through day 14 after injury, the latest time point tested. Unlike acute tubular necrosis, the extent of fibrosis in the outer medulla of the injured kidney was significantly diminished in CX3CR1-deficient mice compared with wild-type mice (Figure 3, A–C). This was confirmed independently and more quantitatively with assays for hydroxyproline content, an index of collagen accumulation in the interstitial space (Figure 3D), and α-SMA, a marker of activated fibroblasts (Figure 4). α-SMA immunoreactivity, which localized with spindle-shaped cells in the interstitium, was also mainly detected in the outer medulla where fibrosis progressed most intensely. Both of these factors were significantly increased in the late phase of injury, eg, 7 and 14 days after reperfusion, but to a significantly lower level in CX3CR1-deficient mice compared to wild-type mice (Figure 3, Figure 4). In sham-operated mouse kidney, α-SMA expression was detected in the blood vessel wall (data not shown).Figure 4α-SMA is induced during ischemia-reperfusion injury of the kidney in a CX3CR1-dependent manner. A: Histopathological analysis of outer medulla. i: CX3CR1-deficient mice stained with anti-α-SMA; ii and iii: wild-type mice stained with anti-α-SMA and control IgG, respectively. B: Quantitation of histopathological data. −/−, CX3CR1 knockout mice; +/+, wild-type control mice; sham, sham-operated wild-type mice tested 24 hours after surgery. Values are mean ± SEM and represent data from three independent experiments with four to five animals in each experiment. *P < 0.05, comparing CX3CR1-deficient mice versus wild-type mice. Original magnifications, ×320.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although fibroblasts are the direct source of collagen in fibrotic lesions, many of the fibrogenic mediators likely to regulate this process come from inflammatory cells. The number of neutrophils increased massively and rapidly after reperfusion, peaking at 31-fold over sham-operated mice 24 hours after injury and then gradually dec
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