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The Atypical Chemokine Receptor 2 Limits Progressive Fibrosis after Acute Ischemic Kidney Injury

Open ArchivePublished:November 15, 2018DOI:https://doi.org/10.1016/j.ajpath.2018.09.016
      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.
      • Chawla L.S.
      • Kimmel P.L.
      Acute kidney injury and chronic kidney disease: an integrated clinical syndrome.
      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.
      • Rabb 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.
      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.
      • Rabb 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.
      • Anders H.J.
      • Vielhauer V.
      • Schlöndorff D.
      Chemokines and chemokine receptors are involved in the resolution or progression of renal disease.
      • Bonventre J.V.
      • Yang L.
      Cellular pathophysiology of ischemic acute kidney injury.
      In addition, ongoing inflammation increases and activates fibrogenic cells in the renal interstitium, including phagocytes, fibroblasts, and myofibroblasts.
      • Bonventre J.V.
      • Yang L.
      Cellular pathophysiology of ischemic acute kidney injury.
      • Wynn T.A.
      • Ramalingam T.R.
      Mechanisms of fibrosis: therapeutic translation for fibrotic disease.
      • Lovisa S.
      • Zeisberg M.
      • Kalluri R.
      Partial epithelial-to-mesenchymal transition and other new mechanisms of kidney fibrosis.
      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).
      • Rabb 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.
      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.
      • Graham G.J.
      • Locati M.
      Regulation of the immune and inflammatory responses by the “atypical” chemokine receptor D6.
      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.
      • Graham G.J.
      • Locati M.
      Regulation of the immune and inflammatory responses by the “atypical” chemokine receptor D6.
      • Bonavita O.
      • Mollica Poeta V.
      • Setten E.
      • Massara M.
      • Bonecchi R.
      ACKR2: an atypical chemokine receptor regulating lymphatic biology.
      ACKR2 is present in many parenchymal organs, including barrier tissues like the skin, gut, lung, and placenta,
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      • Wylie S.M.
      • Yang J.
      • Landau N.R.
      • Graham G.J.
      Cloning and characterization of a novel promiscuous human β-chemokine receptor D6.
      • Nibbs R.J.
      • Wylie S.M.
      • Pragnell I.B.
      • Graham G.J.
      Cloning and characterization of a novel murine β chemokine receptor, D6: comparison to three other related macrophage inflammatory protein-1α receptors, CCR-1, CCR-3, and CCR-5.
      with the major site of ACRK2 expression being on lymphatic endothelial cells.
      • Nibbs R.J.
      • Kriehuber E.
      • Ponath P.D.
      • Parent D.
      • Qin S.
      • Campbell J.D.
      • Henderson A.
      • Kerjaschki D.
      • Maurer D.
      • Graham G.J.
      • Rot A.
      The β-chemokine receptor D6 is expressed by lymphatic endothelium and a subset of vascular tumors.
      Analysis of Ackr2-deficient mice demonstrated that ACKR2 limits inflammatory responses and tissue injury in several disease models, including skin inflammation, myocardial infarction, systemic infection with Mycobacterium tuberculosis, and toxic liver injury.
      • Martinez 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.
      • Cochain 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.
      • Di 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.
      • Berres 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.
      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.
      • Lee 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.
      • McKimmie 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.
      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.
      • Liu 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.
      • Savino 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.
      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.

      Materials and Methods

      Mice

      Ackr2-deficient mice (Ackr2−/−) on the C57BL/6J background have been previously described.
      • Di 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.
      • Jamieson 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.
      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.

      Induction of Renal Ischemia-Reperfusion Injury

      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 after UUO

      Obstructive nephropathy was induced in age-matched groups of female mice by unilateral ureteral ligation, as described previously.
      • Anders 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.
      • Vielhauer 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.
      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

      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).
      • Huang 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.
      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.

      RNA in Situ Hybridization for Ackr2

      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.

      Assessment of Renal Injury by Histology and Immunohistochemistry

      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.
      • Broekema 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.
      Interstitial volume expansion was semiquantitatively assessed, as described.
      • Anders 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.
      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.
      • Anders 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.
      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.
      • Vielhauer 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.
      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.).

      Flow Cytometry of Leukocytes in Kidneys, Peripheral Blood, Spleen, and Bone Marrow

      Preparation of renal single-cell suspensions and antibody staining were performed as previously described.
      • Vielhauer 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.
      • Schwarz 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.
      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.
      • Reich 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.
      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).

      Quantitative RT-PCR

      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-qPCR
      Primer namePrimer sequence
      ACKR2F: 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.

      Analysis of KIM-1 and Chemokine Protein Levels by Enzyme-Linked Immunosorbent Assay

      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.

      Statistical Analysis

      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.

      Results

      Expression of ACKR2 in Healthy and Acutely Injured Murine Kidney

      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.
      • Bideak 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.
      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.
      • Bideak 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.
      Similar to its reported function in skin, lung, and heart,
      • Martinez 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.
      • Cochain 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.
      • Di 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.
      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.
      Figure thumbnail gr1
      Figure 1ACKR2 expression in mouse kidney. A: Analysis of Ackr2 mRNA expression in organs from seven adult male C57BL/6 mice reveals significant baseline Ackr2 mRNA levels in the kidney (dashed line), as well as spleen, lymph nodes, thymus, lung, heart, bladder, and skin. B: Renal Ackr2 mRNA expression is significantly induced in kidneys at 1 day, 5 days, and 5 weeks after ischemia-reperfusion injury (IRI) compared with healthy control kidneys, whereas no significant expression could be detected in Ackr2−/− mice. PCR results were normalized to 18S rRNA as a housekeeping gene. C: In situ hybridization (ISH) of Ackr2 demonstrates Ackr2 mRNA transcripts (red signals indicated by arrows) in interstitial lymphatic endothelial cells of wild-type (WT) control kidneys and more prominently in postischemic kidneys at day 5 after IRI. Images show representative cortical tissue. Data are expressed as mean ± SEM. n = 5 to 10 mice per group (B). P < 0.05, ∗∗P < 0.01 compared with control kidney. Original magnification, ×400 (C).

      Ackr2 Deficiency Does Not Affect Early AKI after Bilateral IRI

      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.
      Figure thumbnail gr2
      Figure 2Ackr2 deficiency does not affect acute renal ischemia-reperfusion injury (IRI). Wild-type (WT) and Ackr2−/− mice underwent bilateral IRI for 30 minutes. A: Baseline levels and increases 24 hours after surgery of serum creatinine and urea are similar in both genotypes. B: Tubular injury, as evaluated by semiquantitative scoring of periodic acid-Schiff (PAS)–stained sections, is comparable at 24 hours after IRI in both groups of mice. C: Similarly, renal protein and mRNA expression levels of kidney injury molecule (KIM)-1 and renal chemokine (C-C motif) ligand (CCL) 2 levels are not different. D: Intrarenal leukocyte accumulation was analyzed by flow cytometry of renal single-cell suspensions prepared from age-matched uninjured control mice (Co) and mice 24 hours after IRI. Renal leukocyte content is similar in healthy kidneys, but significant increases of CD11c+ F4/80+ mononuclear phagocytes are present in ischemic Ackr2−/− kidneys compared with WT kidneys. E: Representative renal sections of WT and Ackr2-deficient mice stained for Ly-6B.2+ neutrophils, F4/80+ interstitial mononuclear phagocytes, and CD3+ T cells. Leukocyte infiltrates were quantified as described in . AE: Data are representative of two independently performed experiments. Data are expressed as means ± SEM. n = 5 to 6 mice per group (AE). P < 0.05. Original magnifications: ×200 (B and E, top and middle rows); ×400 (E, bottom row). hpf, high-power field.

      Ackr2 Deficiency Leads to Persistent Increases in Renal Leukocyte Numbers during the Early Recovery Phase after Unilateral IRI

      It was next investigated whether lack of ACKR2 would persistently elevate intrarenal leukocyte numbers during the early recovery phase after AKI, which could also lead to more extensive injury. To this end, wild-type and Ackr2−/− kidneys were analyzed 5 days after 45 minutes of unilateral renal pedicle clamping. Histology revealed comparable structural injury in both genotypes, as demonstrated by similar tubular injury and interstitial volume scores, and similar staining of tubules with L. tetragonolobulus lectin (Figure 3A). However, renal KIM-1 protein levels and mRNA expression of the tubular injury markers KIM-1 (official name, HAVCR1), neutrophil gelatinase-associated lipocalin (official name, LCN2), and tissue inhibitor of metalloproteinase 2 (TIMP2) were increased in ischemic Ackr2−/− kidneys (Figure 3B). Moreover, renal leukocytes were significantly elevated in Ackr2−/− kidneys 5 days after IRI, including neutrophils and CD11c+ F4/80+ mononuclear phagocytes, but not CD11c+ F4/80 dendritic cells or T cells (Figure 3C). Flow cytometric analysis was confirmed by immunohistochemistry, which localized increased renal infiltrates of neutrophils and F4/80+ phagocytes in Ackr2−/− mice to the corticomedullary junction, which is the major site of ischemic tubular damage (Figure 3D). Consistently, renal levels and mRNA expression of inflammatory chemokines, as well as some M1 and M2 macrophage markers, increased in postischemic Ackr2−/− kidneys (Figure 4). Of importance, baseline expression of these inflammatory mediators in uninjured kidneys of healthy wild-type and Ackr2−/− mice was similar, with the only exception being a significantly induced constitutive expression of inducible nitric oxide synthase in Ackr2-deficient kidneys (Figure 4). Together, these data indicate that Ackr2 deficiency resulted in persistently elevated numbers of renal neutrophils and macrophages during the early recovery phase after IRI. This was associated with increased expression of tubular cell stress markers and inflammatory mediators in postischemic kidneys, but did not affect the extent of tubular structural injury or loss.
      Figure thumbnail gr3
      Figure 3Ackr2 deficiency does not affect tubular injury and loss but leads to persistently elevated intrarenal leukocyte accumulation 5 days after renal ischemia-reperfusion injury (IRI). Wild-type (WT) and Ackr2−/− mice were subjected to unilateral IRI for 45 minutes. Contralateral (CK) and postischemic kidneys (IRI) were analyzed 5 days after surgery. A: Similar tubular injury in postischemic kidneys of both groups of mice was demonstrated by semiquantitative morphometry of periodic acid-Schiff (PAS)–stained sections and comparable staining of postischemic kidneys with Lotus tetragonolobus lectin to identify proximal tubules at day 5 after IRI. B: Renal protein levels of kidney injury molecule (KIM)-1 as well as mRNA expression of the kidney injury markers KIM-1, neutrophil gelatinase-associated lipocalin (NGAL), and tissue inhibitor of metalloproteinase 2 (TIMP2) significantly increase in postischemic Ackr2−/− kidneys, indicating aggravated tubular cell stress. C: Flow cytometry of renal single-cell suspensions reveals significantly increased infiltrates of Ly6G+ neutrophils and CD11c+ F4/80+ mononuclear phagocytes, but not CD11c+ F4/80 dendritic cells in postischemic kidneys of Ackr2−/− mice. D: Representative renal sections of WT and Ackr2-deficient mice stained for neutrophils, F4/80+ interstitial mononuclear phagocytes, and CD3+ T cells. Data are expressed as means ± SEM. n = 5 to 8 mice per group (AD). P < 0.05, ∗∗P < 0.01. Original magnifications: ×100 (A, top row); ×25 (A, bottom row); ×200 (D, top and middle rows); ×400 (D, bottom row). hpf, high-power field.
      Figure thumbnail gr4
      Figure 4Renal chemokine levels and mRNA expression of inflammatory mediators in postischemic Ackr2−/− kidneys 5 days after renal ischemia-reperfusion injury (IRI). A: Renal levels of proinflammatory chemokines, as determined by enzyme-linked immunosorbent assay, increased in IRI kidneys of Ackr2−/− mice at day 5 after IRI. B: Expression of ACKR2 was induced in postischemic wild-type (WT) kidneys compared with uninjured control (Co) and contralateral kidneys (CK) and is absent in Ackr2−/− kidneys. Ackr2-deficient healthy and contralateral kidneys reveal an induced constitutive expression of inducible nitric oxide synthase (iNOS), whereas mRNA levels of other proinflammatory chemokines and macrophage markers were comparable in both genotypes. In contrast, in postischemic Ackr2−/− kidneys, expression of proinflammatory chemokines and the macrophage markers interferon (IFN)-γ, macrophage scavenger receptor (MSR)-1, and arginase (Arg) 1 increases. Data are expressed as means ± SEM. n = 5 mice per group (B, Co); n = 8 mice per group (B, CK and IRI). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. CCL, chemokine (C-C motif) ligand; FIZZ-1, a resistin-like protein markedly induced by IL-4 and IL-13; MRC, mannose receptor; TNF, tumor necrosis factor.

      Persistently Elevated Leukocyte Infiltrates Are Associated with Increased Tubular Injury in Ackr2-Deficient Kidneys 5 Weeks after Unilateral IRI

      To further explore potential roles of ACKR2 in renal regeneration versus progression to CKD, wild-type and Ackr2−/− mice were analyzed 5 weeks after 45 minutes of unilateral renal pedicle clamping. Postischemic Ackr2−/− kidneys displayed significantly more tubular damage and loss, as revealed by a higher tubular injury and interstitial volume scores, and reduced L. tetragonobulus lectin staining (Figure 5A). Consistently, KIM-1 protein expression and mRNA expression of tubular injury markers increased in Ackr2−/− kidneys (Figure 5B). This was associated with increased numbers of intrarenal leukocytes, with enhanced accumulation of CD11c+ F4/80+ phagocytes and CD4+ T cells, as characterized by flow cytometry (Figure 5C). Immunohistochemistry confirmed increased tubulointerstitial infiltrates of F4/80+ macrophages and CD3+ T cells in Acrk2−/− kidneys 5 weeks after IRI (Figure 5D). Thus, lack of Ackr2 results in persistent increases of intrarenal mononuclear leukocyte infiltrates in postischemic kidneys during the regeneration phase, which was associated with more severe tubular injury and atrophy.
      Figure thumbnail gr5
      Figure 5Ackr2 deficiency results in increased tubular injury and loss associated with more extensive intrarenal leukocyte accumulation 5 weeks after renal ischemia-reperfusion injury (IRI). Wild-type (WT) and Ackr2−/− mice were subjected to unilateral IRI for 45 minutes. Contralateral (CK) and postischemic kidneys (IRI) were analyzed 5 weeks after surgery A: Semiquantitative morphometry of periodic acid-Schiff (PAS)–stained sections and staining of postischemic kidneys with Lotus tetragonolobus lectin demonstrate significantly increased tubular injury, interstitial volume expansion, and tubular loss in postischemic Ackr2−/− kidneys at week 5 after IRI. B: Renal protein levels of kidney injury molecule (KIM)-1 and mRNA expression of the kidney injury markers KIM-1, neutrophil gelatinase-associated lipocalin (NGAL), and tissue inhibitor of metalloproteinase 2 (TIMP2) are significantly increased in Ackr2−/− kidneys. C: Flow cytometric analysis reveals significant increases in intrarenal CD45+ leukocytes, CD4+ T cells, and CD11c+ F4/80+ mononuclear phagocytes in postischemic Ackr2−/− kidneys compared with WT kidneys. D: Representative immunohistochemistry of renal tissue obtained from WT and Ackr2-deficient mice 5 weeks after IRI stained for neutrophils, F4/80+ interstitial mononuclear phagocytes, and CD3+ T cells demonstrates significantly enhanced accumulation of phagocytes and T cells in postischemic Ackr2−/− kidneys. Data are expressed as means ± SEM. n = 8 mice per group (AD). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Original magnifications: ×100 (A, top row); ×25 (A, bottom row); ×400 (D, top and bottom rows); ×200 (D, middle row).

      Lack of Ackr2 Promotes Renal Inflammation and Inflammatory Macrophage Infiltrates in Postischemic Kidneys

      Protein levels of the proinflammatory chemokines CCL2, CCL5, and CXCL10 were significantly elevated in postischemic Ackr2−/− kidneys (Figure 6A). Together with our previous findings demonstrating increased CCL2 levels in tumor necrosis factor-α–stimulated Ackr2-deficient tubulointerstitial tissue in vitro,
      • Bideak 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.
      these results are consistent with a defect in chemokine clearance in Ackr2-deficient kidneys. In addition, chemokines expressed by the higher number of attracted leukocytes may subsequently contribute to increased renal levels. Persistent intrarenal leukocyte infiltrates with ongoing inflammation may prevent tubular repair in Ackr2−/− mice after IRI. Therefore, it was investigated whether increased tubular injury was paralleled by the expression of inflammatory mediators in postischemic kidneys 5 weeks after IRI. As expected, ACKR2 expression was absent in Ackr2−/− kidneys. Ackr2 deficiency was associated with significantly elevated renal mRNA expression of the proinflammatory chemokines CCL2, CCL5, CCL22, and CXCL10 (Figure 6B), all known to be expressed by activated proinflammatory macrophages.
      • Mantovani A.
      • Sica A.
      • Sozzani S.
      • Allavena P.
      • Vecchi A.
      • Locati M.
      The chemokine system in diverse forms of macrophage activation and polarization.
      Moreover, mRNA levels of M1 macrophage markers, expressed by classically activated proinflammatory mononuclear phagocytes, were increased in Ackr2−/− kidneys (Figure 6B). Consistently, flow cytometric analysis demonstrated significantly higher numbers of intrarenal CD11b+ Ly6Chigh inflammatory macrophages in postischemic kidneys of Ackr2−/− mice, most of which also expressed the CCL2 receptor CCR2 (Figure 6C). These data suggest that increased renal levels of inflammatory chemokines, including CCL2, augment accumulation of mononuclear leukocytes in postischemic Ackr2−/− kidneys in vivo, including CCR2+ inflammatory macrophages. This results in persistently aggravated tubulointerstitial inflammation and increased tubular injury in Ackr2−/− mice.
      Figure thumbnail gr6
      Figure 6Increased renal chemokine levels are associated with aggravated inflammation in postischemic Ackr2−/− kidneys 5 weeks after renal ischemia-reperfusion injury (IRI). A: Renal levels of proinflammatory chemokines were determined by enzyme-linked immunosorbent assay in contralateral (CK) and postischemic (IRI) kidney tissue of wild-type (WT) and Ackr2−/− mice at week 5 after unilateral IRI for 45 minutes, as described in . B: Expression of Ackr2 is induced in postischemic WT kidneys compared with contralateral kidneys and absent in Ackr2−/− kidneys. Expression of proinflammatory chemokines and M1 macrophage markers is more abundant in postischemic Ackr2−/− kidneys. C: Flow cytometry of renal single-cell suspensions reveals a significantly increased accumulation of CD11b+ Ly6Chigh inflammatory macrophages in postischemic Ackr2−/− kidneys, which expressed the chemokine (C-C motif) ligand (CCL) 2 receptor CCR2. Data are expressed as means ± SEM. n = 5 to 9 mice per group (AC). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. IFN, interferon; iNOS, inducible nitric oxide synthase; TNF, tumor necrosis factor.

      Increased Tubular Injury and Inflammation Is Associated with More Severe Interstitial Fibrosis in Postischemic Kidneys of Ackr2−/− Mice

      CKD after AKI is characterized by fibrotic remodeling of renal interstitial tissue, with loss of nephrons paralleled by progressive accumulation of extracellular matrix and fibrogenic cells.
      • Wynn T.A.
      • Ramalingam T.R.
      Mechanisms of fibrosis: therapeutic translation for fibrotic disease.
      • Lovisa S.
      • Zeisberg M.
      • Kalluri R.
      Partial epithelial-to-mesenchymal transition and other new mechanisms of kidney fibrosis.
      Increased tubulointerstitial inflammation in postischemic Ackr2−/− kidneys may help drive this fibrotic response, in particular when associated with an accumulation of alternatively activated M2 macrophages thought to mediate matrix deposition and tissue remodeling.
      • Mantovani A.
      • Sica A.
      • Sozzani S.
      • Allavena P.
      • Vecchi A.
      • Locati M.
      The chemokine system in diverse forms of macrophage activation and polarization.
      Indeed, mRNA levels of M2 macrophage markers were increased in postischemic kidneys of Ackr2−/− mice 5 weeks after IRI (Figure 7A). Moreover, significantly increased mRNA expression of fibrosis markers, extracellular matrix components, α-SMA, and fibroblast-specific protein 1 suggested enhanced fibrosis in Ackr2−/− kidneys (Figure 7A). Masson trichrome staining revealed significantly enhanced matrix deposition, and immunohistochemistry showed an increased accumulation of α-SMA+ myofibroblasts in postischemic Ackr2−/− kidneys (Figure 7B). By contrast, at day 5 after IRI, the extent of fibrotic remodeling was similar to wild type (Supplemental Figure S1), despite enhanced leukocyte infiltration and inflammation in Ackr2−/− kidneys in the early recovery phase after IRI.
      Figure thumbnail gr7
      Figure 7Ackr2 deficiency results in increased renal fibrosis in postischemic kidneys 5 weeks after renal ischemia-reperfusion injury (IRI). A: mRNA expression levels of M2 macrophage markers, fibrosis-associated molecules, extracellular matrix molecules, and fibroblast markers were determined in contralateral (CK) and postischemic (IRI) kidneys of wild-type (WT) and Ackr2−/− mice. B: Masson trichrome staining reveals increased interstitial matrix deposition in postischemic Ackr2−/− kidneys, and immunohistochemistry confirms an enhanced accumulation of α-smooth muscle actin (α-SMA)+ myofibroblasts in postischemic Ackr2−/− kidneys compared with WT kidneys. C: Increased intrarenal accumulation of CD45+ CD11b+ collagen 1+ fibrocytes in postischemic Ackr2−/− kidneys compared with WT kidneys, as quantified by flow cytometric analysis. Representative fluorescence-activated cell sorting plots gated on CD45+ renal cells are shown for CK and IRI kidneys of WT and Ackr2−/− mice. Numbers shown are the mean percentage of renal fibrocytes per total renal cells in each group. Data are expressed as means ± SEM. n = 5 to 9 mice per group (AC). P < 0.05, ∗∗P < 0.01. Original magnification, ×200 (B). Arg, arginase; CTGF, connective tissue growth factor; FIZZ-1, a resistin-like protein markedly induced by IL-4 and IL-13; FSP, fibroblast-specific protein; hpf, high-power field; MRC, mannose receptor; MSR, macrophage scavenger receptor.
      Bone marrow–derived fibrocytes have been proposed to contribute to renal fibrosis in postischemic kidneys.
      • Reich 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.
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • Denzel A.
      • Schmidbauer K.
      • Göbel N.
      • Talke Y.
      • Schweda F.
      • Mack M.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      Because fibrocytes express CCR2,
      • Reich 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.
      increased abundance of its ligand CCL2 in Ackr2-deficient tubulointerstitial tissue could help foster fibrocyte migration into postischemic Ackr2−/− kidneys. Renal CD45+ CD11b+ collagen 1+ fibrocytes were, therefore, analyzed in kidneys 5 weeks after IRI by flow cytometry, which revealed a significant increase in fibrocytes in Ackr2−/− kidneys (Figure 7C).
      Taken together, Ackr2 deficiency not only results in more severe tubular injury associated with sustained accumulation of intrarenal leukocytes and inflammation, but also in significantly aggravated renal fibrosis in postischemic kidneys 5 weeks after IRI, which is a characteristic finding in CKD after AKI.

      CCL2 Levels and Leukocyte Counts in Blood Increase in Ackr2-Deficient Mice

      Analysis of systemic effects of Ackr2 deficiency revealed significantly elevated CCL2 serum levels in Ackr2−/− mice 5 weeks after IRI (Supplemental Figure S2A). This was paralleled by increased peripheral leukocyte counts in Ackr2-deficient mice, with significant higher numbers of circulating neutrophils, dendritic cells, monocytes, and, particularly, inflammatory CD11b+ Ly6Chigh monocytes expressing CCR2 (Supplemental Figure S2B). No differences in leukocyte numbers were evident in spleen, but a tendency toward reduced leukocyte counts was noted in Ackr2−/− bone marrow, with significantly reduced numbers of CD11b+ Ly6Chigh monocytes expressing CCR2 (Supplemental Figure S2B). These results could indicate increased mobilization of CCR2+ monocytes from bone marrow, potentially mediated by elevated systemic levels of the respective chemokine ligands in Ackr2−/− mice.
      • Serbina N.V.
      • Pamer E.G.
      Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2.
      • Tsou C.L.
      • Peters W.
      • Si Y.
      • Slaymaker S.
      • Aslanian A.M.
      • Weisberg S.P.
      • Mack M.
      • Charo I.F.
      Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites.
      • Li L.
      • Huang L.
      • Sung S.S.
      • Vergis A.L.
      • Rosin D.L.
      • Rose Jr., C.E.
      • Lobo P.I.
      • Okusa M.D.
      The chemokine receptors CCR2 and CX3CR1 mediate monocyte/macrophage trafficking in kidney ischemia-reperfusion injury.
      Thus, higher numbers of circulating inflammatory monocytes may contribute to increased renal leukocyte infiltration and inflammation in Ackr2-deficient mice.

      Ackr2 Deficiency Does Not Affect Renal Fibrosis in Hydronephrotic Kidneys after UUO

      It was further investigated whether impaired renal chemokine clearance in Ackr2−/− kidneys could aggravate inflammatory responses and fibrosis in obstructive nephropathy induced by UUO, a classic model for fibrotic remodeling in CKD.
      • Eddy A.A.
      • Lopez-Guisa J.M.
      • Okamura D.M.
      • Yamaguchi I.
      Investigating mechanisms of chronic kidney disease in mouse models.
      Unilateral ureter ligation for 7 and 14 days resulted in equal tubular dilation, tubular cell injury, and interstitial volume expansion in obstructed wild-type and Ackr2−/− kidneys (Supplemental Figure S3A), with similar intrarenal accumulation of most leukocyte subsets (Supplemental Figure S3, B and C). Significant increases were found only in CD11c+ F4/80 dendritic cells and CD3+ T cells in Ackr2−/− mice by flow cytometry and immunohistochemistry, respectively (Supplemental Figure S3, B and C). Renal mRNA expression of KIM-1 and proinflammatory chemokines CCL2 and CCL5 was comparable, as were numbers of inflammatory CD11b+ Ly6Chigh macrophages in obstructed kidneys (Supplemental Figure S4, A and B). Renal expression of fibrotic markers and the accumulation of α-SMA+ myofibroblasts and fibrocytes were similar in obstructed wild-type and Ackr2−/− kidneys (Supplemental Figure S4, C and D). Despite lacking a renal phenotype, Ackr2−/− mice displayed increased serum CCL2 levels and higher numbers of circulating inflammatory Ly6Chigh monocytes at day 14 after UUO (Supplemental Figure S5). Thus, in contrast to progressive renal fibrosis after IRI, and despite similar systemic effects, Ackr2 deficiency did not accelerate renal leukocyte accumulation, inflammation, and tubulointerstitial fibrosis in the UUO model of CKD, which is characterized by severe tubular injury and rapid fibrotic remodeling.

      Ackr2 Deficiency Aggravates Renal Injury, Inflammation, and Interstitial Fibrosis after Aristolochic Acid–Induced Nephropathy

      To clarify whether anti-inflammatory and antifibrotic functions of ACKR2 are specific to chronic IRI, or can be extended to other forms of AKI that lead to CKD, aristolochic acid–induced nephropathy was induced in wild-type and Ackr2−/− mice. After the aristolochic acid–induced tubulotoxic injury, wild-type mice developed progressive renal failure accompanied by leukocyte infiltration and inflammation (Figure 8). Despite comparable initial injury, Ackr2 deficiency significantly aggravated renal functional impairment and tubular injury in the chronic phase of the model, which was associated with increased renal leukocyte accumulation and the expression of inflammatory mediators (Figure 8). More important, fibrotic remodeling of injured kidneys was more severe in Ackr2−/− mice, as revealed by increased renal expression of extracellular matrix molecules and fibrosis markers and more extensive accumulation of Masson trichrome–stained interstitial matrix and α-SMA+ myofibroblasts (Figure 9). These data support the contention that ACKR2 plays an important role in limiting fibrotic remodeling and progression to CKD after various forms of AKI, including ischemic and tubulotoxic injury.
      Figure thumbnail gr8
      Figure 8Renal injury, leukocyte accumulation, and inflammation in wild-type (WT) and Ackr2−/− mice with aristolochic acid–induced nephropathy (AAN). A: Increases in serum creatinine and urea were comparable in WT and Ackr2−/− mice during the induction phase of the model, indicating similar initial renal injury in both genotypes. At day 14 of AAN, renal function was significantly worse in Ackr2−/− mice compared with WT mice. The dashed lines separate data of the induction and chronic phases. B: Tubular injury, as evaluated by semiquantitative scoring of periodic acid-Schiff (PAS)–stained sections. C: Renal mRNA expression of tubular injury markers is aggravated in Ackr2−/− kidneys compared with WT kidneys at day 14. D: Representative renal sections illustrating increased infiltration of F4/80+ interstitial mononuclear phagocytes and CD3+ T cells in Ackr2−/− mice. Leukocyte infiltrates were quantified as described in . E: Increased renal mRNA expression of proinflammatory chemokines and several M1 and M2 macrophage markers in Ackr2−/− kidneys after AAN. Data are expressed as means ± SEM. n = 5 to 6 mice per group (A–E). P < 0.05, ∗∗P < 0.01. Original magnification, ×400 (B and D). Arg, arginase; CCL, chemokine (C-C motif) ligand; FIZZ-1, a resistin-like protein markedly induced by IL-4 and IL-13; hpf, high-power field; IFN, interferon; iNOS, inducible nitric oxide synthase; KIM, kidney injury molecule; MRC, mannose receptor; MSR, macrophage scavenger receptor; NGAL, neutrophil gelatinase-associated lipocalin; TNF, tumor necrosis factor.
      Figure thumbnail gr9
      Figure 9Renal fibrosis in wild-type (WT) and Ackr2−/− mice with aristolochic acid–induced nephropathy (AAN). A: Increased renal mRNA expression of fibrosis-associated molecules, extracellular matrix, and fibroblast markers in Ackr2−/− mice at day 14 after AAN. B: Increased interstitial matrix deposition and accumulation of α-smooth muscle actin (α-SMA)+ myofibroblasts in injured Ackr2−/− kidneys, as revealed by Masson trichrome staining and immunohistochemistry, respectively. Data are expressed as means ± SEM. n = 5 to 6 mice per group (A and B). P < 0.05, ∗∗P < 0.01. Original magnifications: ×400 (B, top row); ×200 (B, bottom row). CTGF, connective tissue growth factor; FSP, fibroblast-specific protein; hpf, high-power field.

      Discussion

      After AKI, persistent inflammatory injury instead of a resolution of inflammation promotes nephron loss, fibrotic remodeling, and progression to CKD. Therefore, factors controlling local inflammatory responses are essential for tubular repair and functional recovery after acute renal insults, including IRI.
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      • Vielhauer V.
      • Schlöndorff D.
      Chemokines and chemokine receptors are involved in the resolution or progression of renal disease.
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      • Shah J.V.
      • Bonventre J.V.
      Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.
      • Mulay S.R.
      • Thomasova D.
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      • Anders H.J.
      MDM2 (murine double minute-2) links inflammation and tubular cell healing during acute kidney injury in mice.
      ACKR2 promotes resolution of inflammation by chemokine clearance from inflamed sites,
      • Graham G.J.
      • Locati M.
      Regulation of the immune and inflammatory responses by the “atypical” chemokine receptor D6.
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      • Rot A.
      • Graham G.J.
      • Nibbs R.J.
      The chemokine receptor D6 constitutively traffics to and from the cell surface to internalize and degrade chemokines.
      and an anti-inflammatory role of ACKR2 has been demonstrated in various disease models.
      • Martinez de la Torre Y.
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      • Dupor J.
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      • Mantovani A.
      Increased inflammation in mice deficient for the chemokine decoy receptor D6.
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      • Levy B.
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      • Locati M.
      • Combadiere C.
      • Silvestre J.S.
      The chemokine decoy receptor D6 prevents excessive inflammation and adverse ventricular remodeling after myocardial infarction.
      • Di Liberto D.
      • Locati M.
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      • Vecchi A.
      • Meraviglia S.
      • Salerno A.
      • Sireci G.
      • Nebuloni M.
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      • 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.
      • Berres 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.
      Herein, it was shown that ACKR2 also limits inflammation after renal IRI. In wild-type mice, renal ACKR2 expression was significantly induced after IRI and was localized to endothelial cells of the tubulointerstitium. Previously, increased CCL2 levels have been noticed in the supernatants of Ackr2-deficient tubulointerstitial tissue but not glomeruli on stimulation with tumor necrosis factor-α,
      • Bideak 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.
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      which in the kidney are present in the tubulointerstitium, but not in glomeruli. In vivo, increased intrarenal chemokine levels in Ackr2−/− mice were associated with more severe tubular injury and enhanced accumulation of mononuclear phagocytes in postischemic Ackr2−/− kidneys at early (day 5) but more prominently at late (week 5) time points after IRI. Studies in other disease models have reported similar findings of increased leukocyte numbers and tissue damage in affected organs of Ackr2−/− mice.
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      Moreover, Ackr2 deficiency resulted in more severe inflammation in postischemic kidneys, as revealed by increased renal expression of proinflammatory chemokines and M1 macrophage markers. CCL2 promotes infiltration of CD11b+ Ly6Chigh inflammatory monocytes into postischemic kidneys, which is dependent on the CCL2 receptor CCR2.
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      Along this line, a significant increase in CCR2-expressing CD11b+ Ly6Chigh macrophages was observed in Ackr2−/− kidneys 5 weeks after IRI. This suggests that increased tubulointerstitial CCL2 levels in postischemic Ackr2−/− kidneys may enhance the CCL2/CCR2-dependent recruitment of inflammatory macrophages during the regeneration phase after IRI. Moreover, Ackr2 deficiency increased systemic inflammation that was characterized by high CCL2 levels and increased levels of neutrophils and CD11b+ Ly6Chigh inflammatory monocytes in the peripheral circulation. This may additionally enhance early and persistent inflammatory leukocyte infiltrates into postischemic kidneys of Ackr2−/− mice. Thus, when resolution of renal inflammation is needed to help shift the balance from ongoing inflammatory injury to regeneration, Ackr2 deficiency promotes persistent inflammation with associated aggravated tubular injury in postischemic kidneys, which drives AKI toward CKD.
      The development of CKD after AKI is characterized not only by tubular loss but also interstitial fibrosis.
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      Ackr2 deficiency promoted fibrosis in postischemic kidneys. Five weeks after IRI, an increase in mRNA expression levels was found for M1 and M2 macrophage markers in Ackr2−/− kidneys. Renal phagocytes with an M2 phenotype mediate tissue repair and also promote matrix deposition and fibrotic remodeling.
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      Fibrocytes express CCR2.
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      Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model.
      Therefore, increased levels of the CCR2 ligand CCL2 in Ackr2−/− kidneys may enhance renal fibrocyte recruitment after IRI. Using flow cytometry, a significant increase in fibrocytes in postischemic Ackr2−/− kidneys 5 weeks after IRI was confirmed. Interestingly, an activated CCL2/CCR2 axis was also found to enhance fibrocyte recruitment in a model of IL-10–induced lung fibrosis.
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      Thus, in addition to promoting persistent inflammatory injury, Ackr2 deficiency may directly contribute to fibrotic tissue remodeling after renal IRI by facilitating CCL2-dependent fibrocyte recruitment into postischemic kidneys.
      Prominent tubulointerstitial expression of CCL2 and concomitant infiltration of CCR2+ mononuclear leukocytes and fibrocytes also occur during progressive tubular injury in UUO kidneys after complete ureter ligation.
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      Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model.
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      This suggested that a potential aggravation of interstitial inflammation and fibrosis could occur in obstructed Ackr2−/− kidneys. However, Ackr2 deficiency was not found to significantly affect tubular injury, leukocyte accumulation, inflammation, or progressive fibrosis in UUO kidneys, despite increased serum CCL2 levels and circulating inflammatory monocytes in Ackr2−/− mice at day 14 after UUO. This suggests that a lack of ACKR2-dependent chemokine scavenging does not influence the severe inflammation and tubular injury seen in UUO kidneys. Because permanent ureter ligation leads to progressive damage of obstructed kidneys, the model lacks a repair phase after the AKI event, in which ACKR2 activity could limit ongoing inflammation and facilitate tissue regeneration.
      To further explore the potential function of ACKR2 in limiting the progression toward CKD after AKI, aristolochic acid–induced nephropathy was analyzed as an additional model of AKI-induced CKD, in which a regenerative phase follows the initial renal injury. Similar to postischemic kidneys, Ackr2 deficiency resulted in more severe renal damage, inflammation, and fibrotic remodeling in this model. Comparable anti-inflammatory and antifibrotic effects of ACKR2 were recently reported in mice with immune complex glomerulonephritis that was induced by injection of nephrotoxic serum.
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      Taken together, these data suggest an important general role for ACKR2 in limiting ongoing inflammation and fibrotic tissue remodeling in the kidney after an episode of renal injury. However, a recent study in diabetic OVE mice found that Ackr2 deficiency resulted in renal protection, reduced leukocyte infiltration, and decreased renal fibrosis at 6 months.
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      Reasons for these discrepant results are unclear, but may again lie in the progressive nature of diabetic kidney injury. Similar to ureter ligation in the UUO model, persisting hyperglycemia leads to ongoing tissue damage without subsequent recovery periods. Moreover, by diversely affecting chemokine-mediated accumulation of immunosuppressive versus proinflammatory leukocyte subsets in chronic disease, ACKR2 may, depending on disease type and chronicity, mediate complex and not solely anti-inflammatory effects.
      • Savino B.
      • Castor M.G.
      • Caronni N.
      • Sarukhan A.
      • Anselmo A.
      • Buracchi C.
      • Benvenuti F.
      • Pinho V.
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      Control of murine Ly6Chigh monocyte traffic and immunosuppressive activities by atypical chemokine receptor D6.
      In summary, our findings identify ACKR2 as an endogenous regulator limiting tubular injury, persistent inflammation, and fibrotic remodeling after renal IRI and tubulotoxic damage. This study suggests that ACKR2-mediated control of local inflammatory responses facilitates tubular repair and recovery after episodes of acute renal injury. Therefore, ACKR2 may be a suitable target for innovative therapeutic approaches to inhibit progression to CKD after AKI.

      Acknowledgments

      We thank Dan Draganovici and Jana Mandelbaum for excellent technical assistance.

      Supplemental Data

      • Supplemental Figure S1

        Similar extent of renal fibrosis in postischemic wild-type (WT) and Ackr2−/− kidneys 5 days after renal ischemia-reperfusion injury (IRI). A: mRNA expression levels of fibrosis-associated molecules and extracellular matrix molecules were determined in contralateral (CK) and postischemic kidneys (IRI) of WT and Ackr2−/− mice. B: Immunohistochemistry demonstrates similar accumulation of α-smooth muscle actin (α-SMA)+ myofibroblasts in postischemic WT and Ackr2−/− kidneys. Data are expressed as means ± SEM. n = 8 mice per group (A and B). Original magnification, ×200 (B). CTGF, connective tissue growth factor.

      • Supplemental Figure S2

        Increased serum chemokine (C-C motif) ligand (CCL) 2 levels and mobilization of CCR2+ inflammatory monocytes from bone marrow into the circulation in Ackr2−/− mice 5 weeks after renal ischemia-reperfusion injury (IRI). A: CCL2 serum levels are significantly elevated in Ackr2−/− mice compared with wild-type (WT) mice. B: Flow cytometric analysis of leukocyte subsets in whole blood, spleen, and bone marrow reveals a significant increase in blood CD45+ leukocytes in Ackr2-deficient mice, including CD11b+ Ly6G+ neutrophils, CD11b+ CD11c+ dendritic cells, CD11b+ F4/80+ monocytes, and CD11b+ Ly6Chigh inflammatory monocytes, which mainly expressed CCR2. Leukocyte numbers in spleen are similar in WT and ACKR2−/− mice. In bone marrow, a significant decrease in CD11b+ Ly6Chigh monocytes expressing CCR2 is noted. Data are expressed as means ± SEM. n = 9 mice per group (A); n = 5 mice per group (B). P < 0.05, ∗∗P < 0.01.

      • Supplemental Figure S3

        Renal injury and leukocyte accumulation in wild-type (WT) and Ackr2−/− mice with obstructive nephropathy at days 7 and 14 after unilateral ureteral obstruction (UUO). A: Representative periodic acid-Schiff (PAS)–stained sections illustrating comparable tubular dilation after UUO and similar increases in tubular injury and interstitial volume expansion in obstructed kidneys of WT and Ackr2−/− mice. Morphometric analysis of contralateral control kidneys (CK) and obstructed kidneys (UUO) at days 7 and 14 was performed, as described in Materials and Methods. B: Renal leukocyte infiltrates were quantified by flow cytometric analysis of renal single-cell suspensions, which revealed comparable numbers for most leukocyte subsets in UUO kidneys of WT and Ackr2−/− mice at days 7 and 14, with a tendency toward higher CD4+ T-cell numbers and significantly increased CD11c+ F4/80- dendritic cells in obstructed Ackr2−/− kidneys at day 7 but not day 14 after UUO. C: Immunohistochemistry confirmed similar accumulation of F4/80+ phagocytes in the tubulointerstitium of obstructed WT and Ackr2−/− kidneys and significantly increased CD3+ T-cell infiltrates in ACKR2−/− kidneys with UUO at day 7, but not day 14. Data are expressed as means ± SEM. n = 8 to 9 mice per group (AC). P < 0.05, ∗∗P < 0.01. Original magnifications: ×200 (A); ×400 (C). hpf, high-power field.

      • Supplemental Figure S4

        Expression of tubular injury markers, proinflammatory chemokines, markers of renal fibrosis, and accumulation of inflammatory macrophages and fibrocytes in wild-type (WT) and Ackr2−/− mice with obstructive nephropathy after unilateral ureteral obstruction (UUO). A: At day 14 after UUO, mRNA expression of the tubular injury marker kidney injury molecule (KIM)-1 and proinflammatory chemokines was comparable in obstructed kidneys of WT and Ackr2−/− mice. B: Flow cytometry demonstrated similar increases in intrarenal CD11b+ Ly6Chigh inflammatory phagocytes in obstructed kidneys (UUO) at days 7 and 14 after UUO compared with contralateral kidneys (CKs). C: At day 14 after UUO, mRNA expression of extracellular matrix molecules and fibroblast markers was comparable in obstructed kidneys of both genotypes. D: Immunohistochemistry revealed similar interstitial accumulation of α-smooth muscle actin (α-SMA)+ myofibroblasts in obstructed kidneys of WT and Ackr2−/− mice at days 7 and 14 after UUO. Numbers of CD45+ CD11b+ collagen 1+ fibrocytes in obstructed kidneys of both genotypes were comparable, as quantified by flow cytometry. Data are expressed as means ± SEM. n = 8 to 9 mice per group (AD). CCL, chemokine (C-C motif) ligand; FSP, fibroblast-specific protein.

      • Supplemental Figure S5

        Serum chemokine (C-C motif) ligand (CCL) 2 levels and circulating inflammatory monocytes increase in Ackr2−/− mice 14, but not 7 days after unilateral ureteral ligation (UUO). A: CCL2 serum levels are significantly elevated in Ackr2−/− mice compared with wild-type (WT) mice at day 14 after UUO. B: Flow cytometry demonstrates increases of circulating CD11b+ F4/80+ monocytes and CD11b+ Ly6Chigh inflammatory monocytes in the peripheral blood of Ackr2−/− mice compared with WT mice at day 14 after UUO. Data are expressed as means ± SEM. n = 8 to 9 mice per group (A and B). P < 0.05.

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