The Atypical Chemokine Receptor 2 Limits Progressive Fibrosis after Acute Ischemic Kidney Injury
2018; Elsevier BV; Volume: 189; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2018.09.016
ISSN1525-2191
AutoresMoritz Lux, Alexander Blaut, Nuru Eltrich, Andrei Bideak, Martin B. Müller, John Michael Hoppe, Hermann‐Josef Gröne, Massimo Locati, Volker Vielhauer,
Tópico(s)Immune cells in cancer
ResumoFollowing renal ischemia-reperfusion injury (IRI), resolution of inflammation allows tubular regeneration, whereas ongoing inflammatory injury mediated by infiltrating leukocytes leads to nephron loss and renal fibrosis, typical hallmarks of chronic kidney disease. Atypical chemokine receptor 2 (ACKR2) is a chemokine decoy receptor that binds and scavenges inflammatory CC chemokines and reduces local leukocyte accumulation. We hypothesized that ACKR2 limits leukocyte infiltration, inflammation, and fibrotic tissue remodeling after renal IRI, thus preventing progression to chronic kidney disease. Compared with wild type, Ackr2 deficiency increases CC chemokine ligand 2 levels in tumor necrosis factor–stimulated tubulointerstitial tissue in vitro. In Ackr2-deficient mice with early IRI 1 or 5 days after transient renal pedicle clamping, tubular injury was similar to wild type, although accumulation of mononuclear phagocytes increased in postischemic Ackr2−/− kidneys. Regarding long-term outcomes, Ackr2−/− kidneys displayed more tubular injury 5 weeks after IRI, which was associated with persistently increased renal infiltrates of mononuclear phagocytes, T cells, Ly6Chigh inflammatory macrophages, and inflammation. Moreover, Ackr2 deficiency caused substantially aggravated renal fibrosis in Ackr2−/− kidneys 5 weeks after IRI, shown by increased expression of matrix molecules, renal accumulation of α-smooth muscle actin–positive myofibroblasts, and bone marrow–derived fibrocytes. ACKR2 is important in limiting persistent inflammation, tubular loss, and renal fibrosis after ischemic acute kidney injury and, thus, can prevent progression to chronic renal disease. Following renal ischemia-reperfusion injury (IRI), resolution of inflammation allows tubular regeneration, whereas ongoing inflammatory injury mediated by infiltrating leukocytes leads to nephron loss and renal fibrosis, typical hallmarks of chronic kidney disease. Atypical chemokine receptor 2 (ACKR2) is a chemokine decoy receptor that binds and scavenges inflammatory CC chemokines and reduces local leukocyte accumulation. We hypothesized that ACKR2 limits leukocyte infiltration, inflammation, and fibrotic tissue remodeling after renal IRI, thus preventing progression to chronic kidney disease. Compared with wild type, Ackr2 deficiency increases CC chemokine ligand 2 levels in tumor necrosis factor–stimulated tubulointerstitial tissue in vitro. In Ackr2-deficient mice with early IRI 1 or 5 days after transient renal pedicle clamping, tubular injury was similar to wild type, although accumulation of mononuclear phagocytes increased in postischemic Ackr2−/− kidneys. Regarding long-term outcomes, Ackr2−/− kidneys displayed more tubular injury 5 weeks after IRI, which was associated with persistently increased renal infiltrates of mononuclear phagocytes, T cells, Ly6Chigh inflammatory macrophages, and inflammation. Moreover, Ackr2 deficiency caused substantially aggravated renal fibrosis in Ackr2−/− kidneys 5 weeks after IRI, shown by increased expression of matrix molecules, renal accumulation of α-smooth muscle actin–positive myofibroblasts, and bone marrow–derived fibrocytes. ACKR2 is important in limiting persistent inflammation, tubular loss, and renal fibrosis after ischemic acute kidney injury and, thus, can prevent progression to chronic renal disease. Acute kidney injury (AKI) is a risk factor for the development of chronic kidney disease (CKD) later in life.1Chawla L.S. Kimmel P.L. Acute kidney injury and chronic kidney disease: an integrated clinical syndrome.Kidney Int. 2012; 82: 516-524Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar Loss of nephrons due to insufficient repair and fibrotic tissue remodeling underlies the progression from acute to chronic renal injury. After the extent of initial injury, the associated inflammatory response represents a determinant of AKI outcome.2Rabb H. Griffin M.D. McKay D.B. Swaminathan S. Pickkers P. Rosner M.H. Kellum J.A. Ronco C. Acute dialysis quality initiative consensus XWG: inflammation in AKI: current understanding, key questions, and knowledge gaps.J Am Soc Nephrol. 2016; 27: 371-379Crossref PubMed Scopus (329) Google Scholar In ischemia-reperfusion injury (IRI), a major cause for human AKI, the release of danger-associated molecular patterns, proinflammatory cytokines, and chemokines by the injured tubules triggers an influx of leukocytes into the site of injury, which further mediates tubular damage.2Rabb H. Griffin M.D. McKay D.B. Swaminathan S. Pickkers P. Rosner M.H. Kellum J.A. Ronco C. Acute dialysis quality initiative consensus XWG: inflammation in AKI: current understanding, key questions, and knowledge gaps.J Am Soc Nephrol. 2016; 27: 371-379Crossref PubMed Scopus (329) Google Scholar, 3Anders H.J. Vielhauer V. Schlöndorff D. Chemokines and chemokine receptors are involved in the resolution or progression of renal disease.Kidney Int. 2003; 63: 401-415Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 4Bonventre J.V. Yang L. Cellular pathophysiology of ischemic acute kidney injury.J Clin Invest. 2011; 121: 4210-4221Crossref PubMed Scopus (1326) Google Scholar In addition, ongoing inflammation increases and activates fibrogenic cells in the renal interstitium, including phagocytes, fibroblasts, and myofibroblasts.4Bonventre J.V. Yang L. Cellular pathophysiology of ischemic acute kidney injury.J Clin Invest. 2011; 121: 4210-4221Crossref PubMed Scopus (1326) Google Scholar, 5Wynn T.A. Ramalingam T.R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease.Nat Med. 2012; 18: 1028-1040Crossref PubMed Scopus (2088) Google Scholar, 6Lovisa S. Zeisberg M. Kalluri R. Partial epithelial-to-mesenchymal transition and other new mechanisms of kidney fibrosis.Trends Endocrinol Metab. 2016; 27: 681-695Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar Excessive production of extracellular matrix by these cells drives fibrotic remodeling, which not only replaces irreversibly damaged nephrons but may directly contribute to progressive tubular injury by compromising capillary blood flow and diffusion of oxygen and nutrients. Therefore, limiting renal inflammation after AKI is crucial to prevent ongoing tubular injury and renal fibrosis (ie, the progression from AKI to CKD).2Rabb H. Griffin M.D. McKay D.B. Swaminathan S. Pickkers P. Rosner M.H. Kellum J.A. Ronco C. Acute dialysis quality initiative consensus XWG: inflammation in AKI: current understanding, key questions, and knowledge gaps.J Am Soc Nephrol. 2016; 27: 371-379Crossref PubMed Scopus (329) Google Scholar The atypical chemokine receptor 2 (ACKR2), previously called D6, is a chemokine scavenging receptor that belongs to the subfamily of atypical chemokine receptors. ACKR2 binds with high affinity to proinflammatory CC chemokines and fosters to their intracellular degradation, thereby reducing local chemokine levels.7Graham G.J. Locati M. Regulation of the immune and inflammatory responses by the "atypical" chemokine receptor D6.J Pathol. 2013; 229: 168-175Crossref PubMed Scopus (45) Google Scholar By scavenging chemokines in tissue, ACKR2 plays important roles in limiting local inflammatory responses, in the resolution of inflammation, and in the regulation of adaptive immune responses.7Graham G.J. Locati M. Regulation of the immune and inflammatory responses by the "atypical" chemokine receptor D6.J Pathol. 2013; 229: 168-175Crossref PubMed Scopus (45) Google Scholar, 8Bonavita O. Mollica Poeta V. Setten E. Massara M. Bonecchi R. ACKR2: an atypical chemokine receptor regulating lymphatic biology.Front Immunol. 2016; 7: 691PubMed Google Scholar ACKR2 is present in many parenchymal organs, including barrier tissues like the skin, gut, lung, and placenta,9Nibbs R.J. Wylie S.M. Yang J. Landau N.R. Graham G.J. Cloning and characterization of a novel promiscuous human β-chemokine receptor D6.J Biol Chem. 1997; 272: 32078-32083Crossref PubMed Scopus (194) Google Scholar, 10Nibbs R.J. Wylie S.M. Pragnell I.B. Graham G.J. 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Nebuloni M. Rukavina D. Vago L. Vecchi A. Lira S.A. Mantovani A. Increased inflammation in mice deficient for the chemokine decoy receptor D6.Eur J Immunol. 2005; 35: 1342-1346Crossref PubMed Scopus (120) Google Scholar, 13Cochain C. Auvynet C. Poupel L. Vilar J. Dumeau E. Richart A. Recalde A. Zouggari Y. Yin K.Y. Bruneval P. Renault G. Marchiol C. Bonnin P. Levy B. Bonecchi R. Locati M. Combadiere C. Silvestre J.S. The chemokine decoy receptor D6 prevents excessive inflammation and adverse ventricular remodeling after myocardial infarction.Arterioscler Thromb Vasc Biol. 2012; 32: 2206-2213Crossref PubMed Scopus (67) Google Scholar, 14Di Liberto D. Locati M. Caccamo N. Vecchi A. Meraviglia S. Salerno A. Sireci G. Nebuloni M. Caceres N. Cardona P.J. Dieli F. Mantovani A. Role of the chemokine decoy receptor D6 in balancing inflammation, immune activation, and antimicrobial resistance in Mycobacterium tuberculosis infection.J Exp Med. 2008; 205: 2075-2084Crossref PubMed Scopus (83) Google Scholar, 15Berres M.L. Trautwein C. Zaldivar M.M. Schmitz P. Pauels K. Lira S.A. Tacke F. Wasmuth H.E. The chemokine scavenging receptor D6 limits acute toxic liver injury in vivo.Biol Chem. 2009; 390: 1039-1045Crossref PubMed Scopus (30) Google Scholar Moreover, ACKR2 deficiency on lymphatic endothelium leads to accumulation of inflammatory chemokines and inappropriate clustering of inflammatory leukocytes around lymphatic capillaries, which reduces fluid flow and dendritic cell entry into lymphatics and regional lymph nodes.16Lee K.M. McKimmie C.S. Gilchrist D.S. Pallas K.J. Nibbs R.J. Garside P. McDonald V. Jenkins C. Ransohoff R. Liu L. Milling S. Cerovic V. Graham G.J. D6 facilitates cellular migration and fluid flow to lymph nodes by suppressing lymphatic congestion.Blood. 2011; 118: 6220-6229Crossref PubMed Scopus (61) Google Scholar, 17McKimmie C.S. Singh M.D. Hewit K. Lopez-Franco O. Le Brocq M. Rose-John S. Lee K.M. Baker A.H. Wheat R. Blackbourn D.J. Nibbs R.J. Graham G.J. An analysis of the function and expression of D6 on lymphatic endothelial cells.Blood. 2013; 121: 3768-3777Crossref PubMed Scopus (62) Google Scholar Thus, impaired lymphatic drainage of inflammatory cells, chemokines, and cytokines may contribute to exaggerated inflammation in Ackr2-deficient tissues and may also explain the reduced T-cell priming seen in some disease models.18Liu L. Graham G.J. Damodaran A. Hu T. Lira S.A. Sasse M. Canasto-Chibuque C. Cook D.N. Ransohoff R.M. Cutting edge: the silent chemokine receptor D6 is required for generating T cell responses that mediate experimental autoimmune encephalomyelitis.J Immunol. 2006; 177: 17-21Crossref PubMed Scopus (63) Google Scholar, 19Savino B. Castor M.G. Caronni N. Sarukhan A. Anselmo A. Buracchi C. Benvenuti F. Pinho V. Teixeira M.M. Mantovani A. Locati M. Bonecchi R. Control of murine Ly6Chigh monocyte traffic and immunosuppressive activities by atypical chemokine receptor D6.Blood. 2012; 119: 5250-5260Crossref PubMed Scopus (29) Google Scholar By limiting and resolving inflammatory responses, ACKR2 activity may also be an important determinant of recovery versus subsequent progressive CKD after AKI. Herein, we speculated that ACKR2 reduces renal inflammation and fibrotic remodeling after AKI, which are typical hallmarks of developing CKD. Ackr2-deficient mice subjected to renal IRI with extended follow-up and aristolochic acid–induced nephropathy were analyzed as models for acute to chronic kidney injury to explore this concept. Ackr2-deficient mice (Ackr2−/−) on the C57BL/6J background have been previously described.14Di Liberto D. Locati M. Caccamo N. Vecchi A. Meraviglia S. Salerno A. Sireci G. Nebuloni M. Caceres N. Cardona P.J. Dieli F. Mantovani A. Role of the chemokine decoy receptor D6 in balancing inflammation, immune activation, and antimicrobial resistance in Mycobacterium tuberculosis infection.J Exp Med. 2008; 205: 2075-2084Crossref PubMed Scopus (83) Google Scholar, 20Jamieson T. Cook D.N. Nibbs R.J. Rot A. Nixon C. McLean P. Alcami A. Lira S.A. Wiekowski M. Graham G.J. The chemokine receptor D6 limits the inflammatory response in vivo.Nat Immunol. 2005; 6: 403-411Crossref PubMed Scopus (254) Google Scholar All experiments were performed on 7- to 10-week–old Ackr2−/− mice with wild-type littermate controls. All experimental procedures were conducted according to the German animal care and ethics legislation and approved by local government authorities. Age-matched groups of female mice were anesthetized before both renal pedicles (acute IRI model) or only the left renal pedicle (subacute and chronic IRI model) was clamped for 25 or 45 minutes, respectively, with a microaneurysm clamp (Medicon, Tuttlingen, Germany) via flank incisions. Body temperature was maintained at 37°C throughout the procedure by placing mice on a heat pad. After clamp removal, restoration of renal blood flow was confirmed by reappearance of original color, and wounds were closed with standard sutures. Mice were euthanized 1, 5, or 35 days after renal pedicle clamping. Phosphate-buffered saline–perfused kidney tissue was stored for further analysis. Serum values for creatinine and urea were measured with an Olympus AU-640 auto-analyzer at Synlab.vet (Augsburg, Germany). Obstructive nephropathy was induced in age-matched groups of female mice by unilateral ureteral ligation, as described previously.21Anders H.J. Vielhauer V. Frink M. Linde Y. Cohen C.D. Blattner S.M. Kretzler M. Strutz F. Mack M. Gröne H.J. Onuffer J. Horuk R. Nelson P.J. Schlöndorff 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, 22Vielhauer V. Allam R. Lindenmeyer M.T. Cohen C.D. Draganovici D. Mandelbaum J. Eltrich N. Nelson P.J. Anders H.J. Pruenster M. Rot A. Schlöndorff D. Segerer S. Efficient renal recruitment of macrophages and T cells in mice lacking the duffy antigen/receptor for chemokines.Am J Pathol. 2009; 175: 119-131Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Unobstructed contralateral kidneys served as controls. At day 7 or 14 after unilateral ureteral obstruction (UUO), the mice were euthanized, and tissue of perfused obstructed and contralateral kidneys was stored for further analysis. Aristolochic acid–induced nephropathy was induced in 9-week–old male mice by i.p. injection of aristolochic acid I sodium salt (Sigma-Aldrich, Deisenhofen, Germany).23Huang L. Scarpellini A. Funck M. Verderio E.A. Johnson T.S. Development of a chronic kidney disease model in C57BL/6 mice with relevance to human pathology.Nephron Extra. 2013; 3: 12-29Crossref PubMed Google Scholar Five injections of 5 mg/kg every second day resulted in tubular injury and subsequent development of chronic renal failure and fibrosis. At day 14, mice were euthanized for analysis of renal pathology. Paraffin sections (5 μm thick) were baked in a dry oven at 60°C for 1 hour before RNAscope assay application (Advanced Cell Diagnostic, Hayward, CA). The probe for murine Ackr2 mRNA was developed by Advanced Cell Diagnostic. Ackr2 mRNA was localized using the RNAscope 2.5 HD reagent kit RED (Advanced Cell Diagnostic) as a detection reagent. Renal tissue was embedded in paraffin, and sections (2 μm thick) were used for periodic acid-Schiff and Masson trichrome staining or immunohistochemistry following standard protocols. The extent of tubular injury was determined on periodic acid-Schiff–stained sections by additive scoring of four damage markers of tubules in the corticomedullary junction (ie, tubular dilation, denudation, intraluminal casts, and cell flattening), each in a range of 0 to 3.24Broekema M. Harmsen M.C. Koerts J.A. Petersen A.H. van Luyn M.J. Navis G. Popa E.R. Determinants of tubular bone marrow-derived cell engraftment after renal ischemia/reperfusion in rats.Kidney Int. 2005; 68: 2572-2581Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Interstitial volume expansion was semiquantitatively assessed, as described.21Anders H.J. Vielhauer V. Frink M. Linde Y. Cohen C.D. Blattner S.M. Kretzler M. Strutz F. Mack M. Gröne H.J. Onuffer J. Horuk R. Nelson P.J. Schlöndorff 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 The extent of postischemic tubular loss was quantified by staining for Lotus tetragonolobus lectin to identify proximal tubules with ImageJ software version 1.51p (NIH, Bethesda, MD; https://imagej.nih.gov/ij) in five low-power fields per kidney (original magnification, ×200). Tubular injury and interstitial volume expansion in obstructed kidneys after UUO were scored semiquantitatively, as described.21Anders H.J. Vielhauer V. Frink M. Linde Y. Cohen C.D. Blattner S.M. Kretzler M. Strutz F. Mack M. Gröne H.J. Onuffer J. Horuk R. Nelson P.J. Schlöndorff 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 The extent of interstitial fibrosis was quantified by assessing the fraction of collagen-rich fibrotic matrix visualized by Masson trichrome staining in 10 low-power fields per kidney with ImageJ software. Myofibroblasts were assessed by quantifying the fraction of stained area for α-smooth muscle actin (α-SMA; 1:300, clone 1A4; Dako Agilent, Santa Clara, CA) in five to eight cortical low-power fields per kidney. For evaluation of renal leukocyte infiltrates by immunohistochemistry, paraffin-embedded renal sections were stained with antibodies against neutrophils (Ly-6B.2, clone 7/4, 1:50; Abd Serotec, Oxford, UK), mononuclear phagocytes (F4/80, clone Cl:A3-1, 1:100; Abd Serotec), and CD3+ T cells (CD3, 1:100, clone CD3-12; Abd Serotec), as previously described.22Vielhauer V. Allam R. Lindenmeyer M.T. Cohen C.D. Draganovici D. Mandelbaum J. Eltrich N. Nelson P.J. Anders H.J. Pruenster M. Rot A. Schlöndorff D. Segerer S. Efficient renal recruitment of macrophages and T cells in mice lacking the duffy antigen/receptor for chemokines.Am J Pathol. 2009; 175: 119-131Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Stained cells were counted in 8 to 10 cortical high-power fields per kidney. F4/80-positive tubulointerstitial infiltrates were quantified as fraction of stained area using ImageJ software. All assessments were performed in a blinded protocol (M.L., A.Bl., and V.V.). Preparation of renal single-cell suspensions and antibody staining were performed as previously described.22Vielhauer V. Allam R. Lindenmeyer M.T. Cohen C.D. Draganovici D. Mandelbaum J. Eltrich N. Nelson P.J. Anders H.J. Pruenster M. Rot A. Schlöndorff D. Segerer S. Efficient renal recruitment of macrophages and T cells in mice lacking the duffy antigen/receptor for chemokines.Am J Pathol. 2009; 175: 119-131Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 25Schwarz M. Taubitz A. Eltrich N. Mulay S.R. Allam R. Vielhauer V. Analysis of TNF-mediated recruitment and activation of glomerular dendritic cells in mouse kidneys by compartment-specific flow cytometry.Kidney Int. 2013; 84: 116-129Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Leukocyte subsets were quantified by four-color flow cytometry using fluorochrome-conjugated antibodies directed to CD45 (clone 30-F11), CD11b (M1/70), CD11c (HL3), Ly-6G (1A8), Ly-6C (AL-21), CD3ε (145-2C11), CD4 (RM4-5), CD8 (53-6.7; all from BD Biosciences, Heidelberg, Germany), F4/80 (clone CL:A3-1; Abd Serotec), and rat anti-CCR2 (clone 475301; R&D Systems, Abingdon, UK). Lymphocytes, monocytes, and granulocytes in peripheral blood were identified by light scatter properties. Intrarenal fibrocytes were identified by surface staining for CD45 and CD11b, followed by intracellular staining with biotinylated anticollagen 1 or respective isotype control (Rockland Immunochemicals, Gilbertsville, PA), as published.26Reich B. Schmidbauer K. Rodriguez Gomez M. Johannes Hermann F. Göbel N. Brühl H. Ketelsen I. Talke Y. Mack M. Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model.Kidney Int. 2013; 84: 78-89Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Analysis was performed with a FACSCalibur flow cytometer and Cellquest Pro software version 6.0 (BD Biosciences). The number of stained renal leukocytes was expressed as percentage of total renal cells. Peripheral blood leukocytes, total leukocytes per spleen, and bone marrow prepared from the right femur were quantified by adding counting beads (Molecular Probes, Eugene, OR). Total RNA was extracted from whole kidneys using the Purelink RNA Mini Kit (Invitrogen, Carlsbad, CA). SYBR Green master mix (Invitrogen) was used to perform real-time quantitative RT-PCR on a Light Cycler 480 (Roche, Mannheim, Germany). Gene-specific primers (300 nmol/L; Metabion, Martinsried, Germany) were used, as listed in Table 1. All samples were run in duplicate and normalized to 18S rRNA.Table 1Primers Used for Real-Time RT-qPCRPrimer namePrimer sequenceACKR2F: 5′-CTTCTTTTACTCCCGCATCG-3′R: 5′-TATGGGAACCACAGCATGAA-3′CCL2F: 5′-CCTGCTGTTCACAGTTGCC-3′R: 5′-ATTGGGATCATCTTGCTGGT-3′CCL5F: 5′-CCACTTCTTCTCTGGGTTGG-3′R: 5′-GTGCCCACGTCAAGGAGTAT-3′CCL22F: 5′-TCTGGACCTCAAAATCCTGC-3′R: 5′-TGGAGTAGCTTCTTCACCCA-3′CXCL10F: 5′-GGCTGGTCACCTTTCAGAAG-3′R: 5′-ATGGATGGACAGCAGAGAGC-3′TNF-αF: 5′-CCACCACGCTCTTCTGTCTAC-3′R: 5′-AGGGTCTGGGCCATAGAACT-3′IL-6F: 5′-TGATGCACTTGCAGAAAACA-3′R: 5′-ACCAGAGGAAATTTTCAATAGGC-3′IL-10F: 5′-ATCGATTTCTCCCCTGTGAA-3′R: 5′-TGTCAAATTCATTCATGGCCT-3′IL-12βF: 5′-GATTCAGACTCCAGGGGACA-3′R: 5′-GGAGACACCAGCAAAACGAT-3′IFN-γF: 5′-ACAGCAAGGCGAAAAAGGAT-3′R: 5′-TGAGCTCATTGAATGCTTGG-3′iNOS1F: 5′-TTCTGTGCTGTCCCAGTGAG-3′R: 5′-TGAAGAAAACCCCTTGTGCT-3′CTGFF: 5′-AGCTGACCTGGAGGAAAACA-3′R: 5′-CCGCAGAACTTAGCCCTGTA-3′MRC1F: 5′-ATATATAAACAAGAATGGTGGGCAGT-3′R: 5′-TCCATCCAAATGAATTTCTTATCC-3′MSR-1F: 5′-CCTCCGTTCAGGAGAAGTTG-3′R: 5′-TTTCCCAATTCAAAAGCTGA-3′Arg1F: 5′-AGAGATTATCGGAGCGCCTT-3′R: 5′-TTTTTCCAGCAGACCAGCTT-3′FIZZ-1F: 5′-CCCTTCTCATCTGCATCTCC-3′R: 5′-CTGGATTGGCAAGAAGTTCC-3′FibronectinF: 5′-GGAGTGGCACTGTCAACCTC-3′R: 5′-ACTGGATGGGGTGGGAAT-3′LamininF: 5′-CATGTGCTGCCTAAGGATGA-3′R: 5′-TCAGCTTGTAGGAGATGCCA-3′Collagen 1α1F: 5′-ACATGTTCAGCTTTGTGGACC-3′R: 5′-TAGGCCATTGTGTATGCAGC-3′Collagen 4α1F: 5′-GTCTGGCTTCTGCTGCTCTT-3′R: 5′-CACATTTTCCACAGCCAGAG-3′α-SMAF: 5′-ACTGGGACGACATGGAAAAG-3′R: 5′-GTTCAGTGGTGCCTCTGTCA-3′FSP1F: 5′-CAGCACTTCCTCTCTCTTGG-3′R: 5′-TTTGTGGAAGGTGGACACAA-3′Arg, arginase; CCL, chemokine (C-C motif) ligand; CTGF, connective tissue growth factor; F, forward; FIZZ-1, a resistin-like protein markedly induced by IL-4 and IL-13; FSP; fibroblast-specific protein; IFN, interferon; iNOS, inducible nitric oxide synthase; MRC, mannose receptor; MSR, macrophage scavenger receptor; R, reverse; RT-qPCR, real-time quantitative RT-PCR; SMA; smooth muscle actin; TNF, tumor necrosis factor. Open table in a new tab Arg, arginase; CCL, chemokine (C-C motif) ligand; CTGF, connective tissue growth factor; F, forward; FIZZ-1, a resistin-like protein markedly induced by IL-4 and IL-13; FSP; fibroblast-specific protein; IFN, interferon; iNOS, inducible nitric oxide synthase; MRC, mannose receptor; MSR, macrophage scavenger receptor; R, reverse; RT-qPCR, real-time quantitative RT-PCR; SMA; smooth muscle actin; TNF, tumor necrosis factor. Renal kidney injury molecule (KIM)-1 expression and chemokine levels in kidney lysates and serum were measured using commercially available enzyme-linked immunosorbent assay kits for KIM-1, chemokine (C-C motif) ligand (CCL) 2, CCL5, and CXCL10 (R&D Systems), following the manufacturer's protocols. Protein content of each kidney sample was determined using the Bradford assay. Chemokine protein levels were additionally normalized to the fraction of parenchymal tissue using the percentage of lectin-positive staining. Results are presented as means ± SEM. Differences between two experimental groups were compared with a two-tailed t-test, and P < 0.05 was considered significant. The expression of ACKR2 was first studied in different organs of healthy adult C57BL/6 mice. Significant Ackr2 mRNA baseline expression was present in kidney, with the most prominent expression seen in lung, spleen, and heart, whereas thymus, lymph nodes, bladder, and skin revealed similar expression levels as found in kidneys (Figure 1A). To further characterize the role of ACKR2 after AKI, Ackr2 mRNA expression was analyzed in kidneys at day 1, at day 5, and at 5 weeks after acute renal IRI. Compared with normal kidneys, Ackr2 mRNA levels were increased by 9.8-, 18.1-, and 13.2-fold, respectively, whereas no expression could be detected in Ackr2−/− mice (Figure 1B). In control and ischemic kidneys, in situ hybridization analysis localized the expression of Ackr2 mRNA transcripts specifically to endothelial cells of the tubulointerstitium (Figure 1C), which were recently identified as LYVE-1–positive lymphatic endothelium.27Bideak A. Blaut A. Hoppe J.M. Müller M.B. Federico G. Eltrich N. Gröne H.J. Locati M. Vielhauer V. The atypical chemokine receptor 2 limits renal inflammation and fibrosis in murine progressive immune complex glomerulonephritis.Kidney Int. 2018; 93: 826-841Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Consistently, increased CCL2 levels were demonstrated in supernatants of Ackr2-deficient tubulointerstitial cells, but not glomeruli, compared with wild type on tumor necrosis factor-α stimulation in vitro.27Bideak A. Blaut A. Hoppe J.M. Müller M.B. Federico G. Eltrich N. Gröne H.J. Locati M. Vielhauer V. The atypical chemokine receptor 2 limits renal inflammation and fibrosis in murine progressive immune complex glomerulonephritis.Kidney Int. 2018; 93: 826-841Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Similar to its reported function in skin, lung, and heart,12Martinez de la Torre Y. Locati M. Buracchi C. Dupor J. Cook D.N. Bonecchi R. Nebuloni M. Rukavina D. Vago L. Vecchi A. Lira S.A. Mantovani A. Increased inflammation in mice deficient for the chemokine decoy receptor D6.Eur J Immunol. 2005; 35: 1342-1346Crossref PubMed Scopus (120) Google Scholar, 13Cochain C. Auvynet C. Poupel L. Vilar J. Dumeau E. Richart A. Recalde A. Zouggari Y. Yin K.Y. Bruneval P. Renault G. Marchiol C. Bonnin P. Levy B. Bonecchi R. Locati M. Combadiere C. Silvestre J.S. The chemokine decoy receptor D6 prevents excessive inflammation and adverse ventricular remodeling after myocardial infarction.Arterioscler Thromb Vasc Biol. 2012; 32: 2206-2213Crossref PubMed Scopus (67) Google Scholar, 14Di Liberto D. Locati M. Caccamo N. Vecchi A. Meraviglia S. Salerno A. Sireci G. Nebuloni M. Caceres N. Cardona P.J. Dieli F. Mantovani A. Role of the chemokine decoy receptor D6 in balancing inflammation, immune activation, and antimicrobial resistance in Mycobacterium tuberculosis infection.J Exp Med. 2008; 205: 2075-2084Crossref PubMed Scopus (83) Google Scholar these data suggest that renal ACKR2 expressed by tubulointerstitial lymphatic endothelial cells could scavenge chemokines produced in the tubulointerstitial compartment and, thus, may play an important role in limiting inflammatory responses after AKI. To explore the potential function of ACKR2 during the acute phase of renal injury, bilateral IRI was induced in wild-type and Ackr2−/− mice by clamping both renal pedicles for 30 minutes, and the kidneys were subsequently harvested 24 hours after surgery. Lack of ACKR2 had no obvious effect on renal functional parameters, as evidenced by the similar increases seen in creatinine and urea in wild-type and Ackr2−/− mice (Figure 2A). Tubular injury by histology (Figure 2B) as well as renal protein and mRNA expression of the tubular damage marker KIM-1 (Figure 2C) were comparable in both groups. Renal CCL2 protein content was similar in wild-type and Ackr2−/− mice at this time point (Figure 2C). However, intrarenal leukocyte infiltration, which was comparable in control kidneys of healthy wild-type and Ackr2−/− mice, was moderately increased in Ackr2−/− kidneys at 24 hours after IRI, being significant for CD11c+ F4/80+ mononuclear phagocytes when analyzed by flow cytometry (Figure 2D), with a similar trend seen in immunohistochemistry (Figure 2E). Taken together, these results indicate that Ackr2 deficiency did not affect severity of the initial ischemic kidney injury but enhanced early accumulation of some phagocytic leukocyte subsets in ischemic kidneys during the first 24 hours after IRI. This may relate to potential systemic ACKR2 effects because intrarenal chemokine levels were not increased at this time point. It was next investigated
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