Tubular GM-CSF Promotes Late MCP-1/CCR2-Mediated Fibrosis and Inflammation after Ischemia/Reperfusion Injury

Leyuan Xu; Diana Sharkey; Lloyd G Cantley

doi:10.1681/ASN.2019010068

. 2019 Jul 17;30(10):1825–1840. doi: 10.1681/ASN.2019010068

Tubular GM-CSF Promotes Late MCP-1/CCR2-Mediated Fibrosis and Inflammation after Ischemia/Reperfusion Injury

Leyuan Xu ^1,^✉, Diana Sharkey ², Lloyd G Cantley ^1,^✉

PMCID: PMC6779361 PMID: 31315923

Significance Statement

Clinical evidence suggests that monocyte chemoattractant protein-1 (MCP-1) may be a useful biomarker to identify patients at risk for kidney fibrosis and CKD progression. Using a mouse model of unilateral ischemia/reperfusion injury (U-IRI), the authors show that sustained GM-CSF expression by renal tubular cells markedly increases Mcp-1 expression in macrophages during the transition from normal kidney repair to maladaptive fibrosis. Eliminating CCR2, the receptor of MCP-1, by using genetic engineering or a drug that blocks CCR2 decreases the accumulation and persistence of macrophages, dendritic cells, and T cells in the kidney, and reduces fibrosis and inflammation after a U-IRI. The results highlight the role of GM-CSF/MCP-1 signaling during the transition from AKI to CKD and suggest that therapeutic inhibition of MCP-1/CCR2 signaling may suppress kidney fibrosis.

Keywords: MCP-1, CCR2, macrophage, renal fibrosis, GM-CSF

Visual Abstract

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Abstract

Background

After bilateral kidney ischemia/reperfusion injury (IRI), monocytes infiltrate the kidney and differentiate into proinflammatory macrophages in response to the initial kidney damage, and then transition to a form that promotes kidney repair. In the setting of unilateral IRI (U-IRI), however, we have previously shown that macrophages persist beyond the time of repair and may promote fibrosis.

Methods

Macrophage homing/survival signals were determined at 14 days after injury in mice subjected to U-IRI and in vitro using coculture of macrophages and tubular cells. Mice genetically engineered to lack Ccr2 and wild-type mice were treated ±CCR2 antagonist RS102895 and subjected to U-IRI to quantify macrophage accumulation, kidney fibrosis, and inflammation 14 and 30 days after the injury.

Results

Failure to resolve tubular injury after U-IRI results in sustained expression of granulocyte-macrophage colony-stimulating factor by renal tubular cells, which directly stimulates expression of monocyte chemoattractant protein-1 (Mcp-1) by macrophages. Analysis of CD45⁺ immune cells isolated from wild-type kidneys 14 days after U-IRI reveals high-level expression of the MCP-1 receptor Ccr2. In mice lacking Ccr2 and wild-type mice treated with RS102895, the numbers of macrophages, dendritic cells, and T cell decreased following U-IRI, as did the expression of profibrotic growth factors and proimflammatory cytokines. This results in a reduction in extracellular matrix and kidney injury markers.

Conclusions

GM-CSF–induced MCP-1/CCR2 signaling plays an important role in the cross-talk between injured tubular cells and infiltrating immune cells and myofibroblasts, and promotes sustained inflammation and tubular injury with progressive interstitial fibrosis in the late stages of U-IRI.

Multiple observational studies have demonstrated that patients who survive an episode of AKI have a significant risk for progression to advanced-stage CKD or ESRD.^1,2 A better understanding of the mechanism by which AKI can lead to CKD is thus important. Both human CKD studies and animal models of the AKI-to-CKD transition suggest that failure to resolve injury after AKI can lead to interstitial infiltration of immune cells, persistent inflammation, and subsequent fibrosis. This has been proposed to involve multiple cell types including tubular epithelial cells, endothelial cells, pericytes, fibroblasts, myofibroblasts, fibrocytes, neutrophils, resident macrophages, infiltrating macrophages, dendritic cells, T cells, and others.^3,4 Of these multiple cell types, myofibroblasts are the most consistently implicated as the effector cell of renal fibrosis.^5,6 Therefore, defining the intercellular signals during tubule-macrophage-myofibroblast activation after AKI can be used to identify novel therapeutic targets to prevent AKI-to-CKD transition.

The cross-talk between endogenous kidney cells and immune cells is complex and remains poorly understood, while the cross-talk between immune cells and myofibroblasts is even less well described. In mouse models of AKI, PMNs and proinflammatory macrophages robustly infiltrate the injured kidney, followed by PMN egress and macrophage transition to an alternatively activated, reparative phenotype.⁷⁻¹² In the event of successful repair, macrophages egress or undergo apoptosis; whereas after more severe or unresolved injury, macrophages persist in the kidney interstitium adjacent to nonrepaired tubules and transition to a profibrotic phenotype, promoting kidney fibrosis directly or indirectly through myofibroblast activation.¹³⁻¹⁷ Thus patrolling/infiltrating macrophages can serve as sensors of the functional health of the tubule-endothelial compartment, and then direct reparative or fibrotic responses accordingly.

The signals that recruit, activate, and retain macrophages in the interstitium are likely to consist of macrophage chemoattractants and/or survival factors.¹⁸ In a mouse model of persistent kidney injury, i.e., unilateral ureteral obstruction (UUO), chemoattractant-to-chemokine receptor signaling such as macrophage chemoattractant protein-1 (MCP-1) and its receptor C-C chemokine receptor type 2 (CCR2) can lead to macrophage persistence.¹⁹ Clinical evidence has also shown that MCP-1 (gene name chemokine [C-C motif] ligand 2, Ccl2) is a potential biomarker for assessment of CKD stage progression in children and for identification of patients at risk for renal fibrosis and worse renal outcomes.^20,21 These observations led us to investigate whether MCP-1/CCR2 signaling can promote macrophage persistence and subsequent profibrotic responses after unresolved AKI. Using a mouse model of maladaptive kidney repair after transient kidney injury (unilateral ischemia/reperfusion injury [U-IRI]), we now show that Ccl2 upregulation occurs in the late stages after IRI and that the dominant source of Ccl2 is the macrophage itself, whereas Ccr2 is expressed on macrophages, dendritic cells, and other immune cells including T cells and PMNs. Using wild-type (WT) and Ccr2^−/− mice, we show that Ccr2 expression is required for macrophage persistence as well as myofibroblast accumulation/activation and maximal kidney fibrosis after U-IRI. In addition, the accumulation of dendritic cells and T cells after U-IRI, as well as sustained expression of proinflammatory chemokines and tubular injury markers, is dependent on Ccr2 expression. Our findings suggest that chronic upregulation of MCP-1 acts to promote a CCR2-dependent profibrotic and inflammatory state that accelerates the AKI-to-CKD transition.

Methods

Animal Surgery and Experimental Protocol

All animal protocols were approved by the Yale University Institutional Animal Care and Use Committee. Both WT and Ccr2^−/− (The Jackson Laboratory) mice (9–10 weeks) were used in this work. All mice were on the C57BL/6J background. Male mice were exclusively used to reduce total numbers of mice required for statistical analysis due to the substantial difference in susceptibility to IRI between male and female mice.²² All mice were maintained on a 12-hour light and 12-hour dark cycle with free access to standard food and water before and after surgery. To establish the U-IRI model, mice were subjected to anesthesia by intraperitoneal injection with ketamine (100 mg/kg) and xylazine (10 mg/kg) on a 37°C warming pad. The abdomen was opened and warm renal ischemia was induced using a nontraumatic microaneurysm clip (FST Micro Clamps) on the left renal pedicle for 27 minutes, leaving the right kidney intact. During surgery, the mice were intraperitoneally injected with normal PBS and buprenorphine to avoid dehydration and postoperative pain, respectively. The design of animal studies was summarized in Supplemental Table 1. Briefly, for the time-course study, WT mice were euthanized on day 0, 1, 7, 14, and 30 after U-IRI (n=6 mice per time point). For kidney cell isolation, WT mice were euthanized on day 14 after U-IRI. The IRI kidneys were subjected to FACS for renal cells, T cells/PMNs, dendritic cells, and macrophages (n=12 mice); myofibroblasts (n=8 mice); as well as infiltrating and resident macrophages (n=6 mice). For the renal fibrosis study, both WT and Ccr2^−/− mice were euthanized on day 14 (n=8 mice per genotype) and on day 30 (n=14 WT mice, n=11 Ccr2^−/− mice) after U-IRI. For in vivo pharmacologic inhibition of CCR2, WT mice were subjected to U-IRI. On day 7, the mice were randomly separated into two groups (n=8 mice per group) and were intraperitoneally injected with either CCR2-antagonist RS102895 (5 mg/kg per injection) or control vehicle, respectively, every 12 hours for 7 days (14 injections in total per mouse) using a modified approach to that previously described.²³ Both vehicle- and RS102895-treated mice were euthanized on day 14 after U-IRI. Blood and kidney tissue samples were obtained at the indicated times after the surgery for further analysis.

ELISA of Serum Kidney Injury Molecule-1 Level

Mouse blood was drawn at the indicated time points. Serum kidney injury molecule-1 (KIM-1) concentrations were measured using the mouse TIM-1/KIM-1/HAVCR Quantikine ELISA Kit (R&D Systems) according to the manufacturer’s instructions.

Histology, Immunohistochemistry, and Immunofluorescence

Kidneys were fixed in 10% formalin and embedded in paraffin. For detection of collagen, deparaffinized kidney sections (5 μm) were rehydrated, stained with Picrosirius red in 1.3% picric acid for 1 hour. F4/80-, CD11c-, and PDGF receptor β (PDGFRβ)–positive cells were detected by immunohistochemistry (IHC) using primary monoclonal antibodies against F4/80, CD11c, and PDGFRβ (#70076, #97585, #3169, respectively; Cell Signaling Technology) as described previously.¹⁴ Citrate buffer antigen retrieval was used for IHC staining. The primary antibodies were omitted as negative controls. All the tissue slides were scanned using Aperio LV1 Real-time slide scanner and processed using ImageScope software. Six independent fields in cortex and four independent fields in outer medulla were analyzed per kidney, and the percent area of Picrosirius red staining and F4/80-, CD11c- or PDGFRβ-positive staining was quantified using ImageJ (National Institutes of Health [NIH]). GM-CSF and F4/80 were detected by immunofluorescence using primary polyclonal antibody against GM-CSF (17762; Proteintech Group) and primary mAb against F4/80 (clone Cl:A3-1; Bio-Rad Laboratories). Proteinase K antigen retrieval was used for immunofluorescence staining. Fluorescent images were taken at ×400 using a Zeiss 710 Confocal Microscope.

FACS Analysis

Kidneys were harvested, minced, incubated with Liberase and DNase-1 for 60 minutes (Roche Diagnostics), and filtered with a 70-mm cell strainer to obtain a single cell suspension. The cells were stained with the following antibodies: anti-PDGFRβ phycoerythrin (PE)-conjugated (clone APB5; eBioscience) or anti-CD45 PE-conjugated (clone 30-F11; eBiosciences), anti-F4/80 FITC-conjugated (clone BM8; eBiosciences), and anti-CD11c allophycocyanin-conjugated (clone N418; BioLegend) or anti-CD11b allophycocyanin-conjugated (clone M1/70; eBiosciences) antibodies with the background set using the appropriate isotype controls (eBiosciences). PDGFRβ⁺, CD45⁺F4/80⁺, CD45⁺CD11c⁺F4/80⁻, CD45⁺CD11c⁻F4/80⁻, CD45⁻CD11c⁻F4/80⁻, CD45⁺CD11b^hiF4/80^int, and CD45⁺CD11c^intF4/80^hi cells were collected and pooled together from two or four individual kidneys for mRNA analysis. Renal cells were defined as CD45⁻CD11c⁻F4/80⁻ cells, macrophages as CD45⁺F4/80⁺ cells, dendritic cells as CD45⁺CD11c⁺F4/80⁻ cells, and T cells/PMNs as CD45⁺CD11c⁻F4/80⁻ cells, as demonstrated in Supplemental Figure 1. Infiltrating (or patrolling) macrophages were defined as CD45⁺CD11b^hiF4/80^int, and resident macrophages as CD45⁺CD11c^intF4/80^hi, as demonstrated in Supplemental Figure 2. Myofibroblasts were defined as PDGFRβ⁺ cells as previously described.¹⁴

In Vitro Cell Culture

Pathogen-free MPT cells (BU-MPT immortalized cells) were plated in a six-well plate in DMEM/F12 media (Gibco) supplied with 10% FBS for 24 hours and then treated with or without serum for 24 hours.

Naive bone marrow–derived macrophages (BMMs) were isolated and cultured for 7 days in the presence of L929 supernatant as previously described.⁷ BMMs were then plated in a six-well plate for 24 hours before initiation of experimental conditions. For in vitro polarization, BMMs were stimulated with 0, 2, 5, or 10 ng/ml mouse recombinant MCP-1 (R&D Systems), 25 ng/ml IL-4 (Sigma-Aldrich) or 25 ng/ml GM-CSF (R&D Systems) in RPMI 1640 media (Thermo Fisher Scientific) supplied with 10% FBS for 24 hours.

For the coculture experiments, MPT cells were plated on 0.4-μm polyester membrane Transwell inserts for a six-well plate (Corning), while BMMs were plated on a six-well plate for 24 hours. The MPT cell–cultured inserts were then placed into the BMM-cultured six-well plate. BMMs were then cocultured with MPT cells in RPMI 1640 without FBS and were treated with recombinant mouse GM-CSF neutralizing antibody (clone MP122E9; R&D Systems) or IgG2A isotype antibody (clone 54447; R&D Systems) for 24 hours.

Quantitative PCR Analysis

Whole-kidney RNA, FACS-sorted cell RNA, or cultured cell RNA was extracted with an RNeasy Mini kit (Qiagen) and reverse transcribed (Bio-Rad). Gene expression analysis was determined by quantitative reverse-transcription PCR using an iCycler iQ (Bio-Rad) as described previously.¹⁴ The primer sequences are provided in Supplemental Table 2. The data were expressed using the comparative threshold cycle (ΔCT) method, and the mRNA ratios were given by 2^−ΔCT or ΔΔCT.

Western Blot Analysis

Kidney lysates were fractioned using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific), and Western blot analysis was completed with antibodies that react selectively with fibronectin, collagen 1 (NBP1-91258, NB600-408, respectively; Novus Biologicals), glyceraldehyde-3-phosphate dehydrogenase (51332; Cell Signaling Technology), and β-actin (sc-69879; Santa Cruz Biotechnology) as described previously.¹⁴

Statistical Analyses

The data were expressed as means±SD. Multigroup comparison was performed using one way ANOVA followed by the Tukey multiple comparison test for subgroup comparison; two-group comparison was analyzed by t test; and the Pearson correlation coefficient, R, was determined using Prism 5 (GraphPad). A value of P<0.05 was considered statistically significant.

Results

Macrophage Accumulation Correlates with Interstitial Fibrosis during AKI-to-CKD Transition

Analysis of the injured kidney after U-IRI in WT mice reveals a parallel increase in Picrosirius red staining/Col1a1 expression and F4/80-positive macrophages from day 1 to 30 after injury (Figure 1A, quantified in B and C) (R=0.97; P=0.006). Analysis of FACS-sorted macrophages from kidneys 14 days after U-IRI (Supplemental Figure 1) revealed low expression of both proinflammatory macrophage markers (inducible nitric oxide synthase 2 [Nos2] and TNFα [Tnfa]) and reparative macrophage markers (arginase 1 [Arg1], mannose receptor 1 [Mrc1], and macrophage scavenger receptor 1 [Msr1]) as compared with the previously described expression of these cytokines in the first week after injury (Figure 1D).^7,8 In contrast, these cells expressed higher levels of the profibrotic macrophage markers (PDGFβ [Pdgfb] and TGFβ [Tgfb1]).^14,24,25

Our observations and others have shown that macrophages are localized adjacent to the tubular basement membrane at sites of tubular cell injury.^7,12,14,26 This suggests that both macrophage homing and survival signals are expressed in the local milieu of the injured tubule. To identify these signals during AKI-to-CKD transition, we examined the expression of chemoattractant and survival receptors using FACS-sorted cells 14 days after U-IRI. The MCP-1 receptor Ccr2 was highly expressed compared with Ccr1, Ccr3, Ccr5 (receptors for MCP-2 and/or macrophage inflammatory protein 1-α [MIP1α]); Cxcr4 (receptor for stromal cell–derived factor 1 [SDF1]); and Cx3cr1 (receptor for fractalkine [CX3CL1]) (Figure 1D). The survival receptors Csfr1 (receptor for macrophage colony-stimulating factor [M-CSF]) and Csf2ra1/Csf2rb1 (receptors for GM-CSF) were expressed at indistinguishably low levels. Interestingly, dendritic cells and CD45⁺CD11c⁻F4/80⁻ cells (predominantly T cells and PMNs, Supplemental Figure 1) that accumulate in the injured kidney also expressed Ccr2 at high levels (Figure 1E). CD45⁺CD11b^hiF4/80^int infiltrating macrophages expressed Ccr2, Pdgfb, and Tgfb1 at the same level as CD45⁺CD11b^intF4/80^hi resident macrophages (Figure 1F, Supplemental Figure 2). In contrast, neither endogenous renal cells nor myofibroblasts express detectable levels of Ccr2 (Figure 1E).

Analysis of whole-kidney mRNA after U-IRI revealed that Ccl2 was significantly upregulated during AKI-to-CKD transition (Figure 1G), correlating with progressive macrophage accumulation (R=0.98; P=0.003) and Col1a1 expression (R=0.99; P=0.001) in the injured kidney. Quantification of Ccl2 expression in flow-sorted cells revealed that macrophages themselves exhibited the greatest expression of Ccl2 (Figure 1H).

Tubular Cell GM-CSF Promotes MCP-1 Expression in Macrophages

We have previously shown that GM-CSF is upregulated in the kidney in the first 5 days after IRI and promotes Janus tyrosine kinase–signal transducer and activator of transcription 5 (STAT5) signaling and tubule cell–mediated reparative macrophage activation.⁸ Analysis of whole-kidney mRNA levels after U-IRI reveals sustained expression of Csf2 in the injured kidney through days 7–14 (Figure 2A), and FACS-sorted cells from day 14 kidneys reveals that CD45⁻ endogenous renal cells are the predominant source of Csf2 (Figure 2B). Immunofluorescence staining confirmed GM-CSF expression by tubular cells that were surrounded by F4/80⁺ macrophages 14 days after U-IRI (Figure 2C). Consistent with the hypothesis that tubular cell stress promotes GM-CSF expression, MPT cells subjected to serum starvation exhibit highly upregulated Csf2 with no change in M-Csf (Csf1) expression (Figure 2D). To determine if macrophage Mcp-1 expression is dependent on tubular cell GM-CSF, we cocultured naive BMMs with serum-starved MPT cells and examined Ccl2 expression (Figure 2E). BMMs robustly increased Ccl2 expression (7.8-fold increase) when they were cocultured with MPT cells for 24 hours (Figure 2F). Addition of the GM-CSF–neutralizing antibody previously shown to prevent GM-CSF–dependent macrophage STAT5 activation⁸ effectively inhibited Ccl2 and Msr1 expression in response to MPT coculture. Furthermore, recombinant GM-CSF directly stimulates macrophage expression of Ccl2 and Ccr2 (7.5- and 1.7-fold, respectively) as well as Msr1 (Figure 2G). In contrast, IL-4 failed to induce Msr1 or Ccr2 expression and provided less stimulation of Ccl2 expression. Together, these results demonstrate that tubule cell–expressed GM-CSF induces macrophage MCP-1 expression.

Figure 2. — Tubular GM-CSF promotes MCP-1 expression by macrophages. (A) Quantitative PCR for *Csf2* was performed on whole-kidney mRNA. ANOVA, P<0.001, F=11.74. **P<0.01, ***P<0.001 versus day 0. n=6 kidneys per time point. (B) Cells freshly isolated from 12 kidneys 14 days after U-IRI were separately FACS sorted for CD45^±CD11c^±F4/80^±, followed by pooling (three pools of four kidneys per pool, 12 kidneys total), RNA extraction, and quantitative PCR analysis of the indicated cell populations for *Csf2*. (C) Kidney sections were immunostained with GM-CSF (red), F4/80 (identifies macrophages, green), and DAPI (identifies nuclei, blue) at day 14 after U-IRI. Original magnification, ×400. (D) MPT cells were cultured with (control) or without serum (starvation) for 24 hours, and cell lysates were harvested for RNA isolation. Quantitative PCR for *Csf1* and *Csf2* was performed on MPT mRNA. **P<0.01 versus control. n=3 per condition. (E) MPT cells and BMMs were cocultured in a Transwell system as described in Methods. (F) BMMs were treated with control, neutralizing (Neut), or isotype (Iso) antibody (Ab) when they were cocultured with MPT cells in a serum starvation media as in (E) for 24 hours. Quantitative PCR for *Ccl2* and *Msr1* on BMM mRNA. *P<0.05, **P<0.01, ***P<0.001. n=3 per condition. (G) BMMs were treated with recombinant mouse GM-CSF or IL-4 for 24 hours, and cell lysates were harvested for RNA isolation. Quantitative PCR for *Ccl2*, *Ccr2*, and *Msr1* was performed on BMM mRNA. *P<0.05, ***P<0.001 versus control. n=3 per condition. BMM, bone marrow–derived macrophage; IRI, ischemia/reperfusion injury; MCP-1, monocyte chemoattractant protein-1; GM-CSF, granulocyte-macrophage colony-stimulating factor; U-IRI, unilateral IRI.

CCR2 Promotes Macrophage Accumulation after Kidney Injury

To determine the functional importance of MCP-1/CCR2 signaling in the kidney, WT and Ccr2^−/− mice were subjected to 27 minutes of U-IRI (Figure 3A). Serum KIM-1 levels, a marker of proximal tubule injury²⁷, were increased to the same degree in both groups 1 day after U-IRI (Figure 3B), suggesting that the initial proximal tubule injury due to IRI was functionally equivalent between Ccr2^−/− and WT mice. At 30 days after injury, the contralateral kidney demonstrated the same degree of hypertrophy and the injured kidney showed the same degree of atrophy in both genotypes (Figure 3C). IHC staining of the U-IRI kidney for F4/80 on day 30 confirmed increased macrophage numbers in WT mice after IRI in both the renal cortex and outer medulla (Figure 3D, quantified in E; compare with Figure 1B), which was significantly attenuated in the Ccr2^−/− mice. Consistent with the IHC staining, mRNA expression of macrophage markers F4/80 and Cd68 was significantly decreased at the whole-kidney level on day 30 in the injured Ccr2^−/− kidneys compared with WT kidneys (Figure 3F), as was the expression of the profibrotic macrophage markers, Pdgfb and Tgfb1 (Figure 3F). Screening other key chemokine/receptor pairs revealed that Ccl2/Ccr2, Ccl3/Ccr1, Cxcl12/Cxcr4, Cx3cl1/Cx3cr1, and Csf1/Csf1r were downregulated in the injured Ccr2^−/− kidneys compared with WT kidneys (Supplemental Figures 3 and 4), consistent with an overall reduction in macrophage accumulation in the injured Ccr2^−/− kidneys. Interestingly, the one ligand/receptor pair that was not downregulated was Ccl8 (Mcp2)/Ccr3, suggesting that this homing/activation pathway might increase as a compensatory response to the loss of MCP-1/CCR2 signaling. Recombinant MCP-1 failed to induce profibrotic macrophage activation in vitro (Supplemental Figure 5), suggesting that MCP-1 serves primarily as a macrophage homing chemokine rather than as a profibrotic activator.

CCR2 Promotes Inflammatory Dendritic Cell and Cytotoxic T Cell Accumulation after Kidney Injury

In light of the expression of Ccr2 by dendritic cells, T cells, and PMNs (Figure 1E), we also examined the effect of whole-body Ccr2 loss on the accumulation of these cells after U-IRI. Quantitative PCR analysis of whole-kidney RNA revealed that Itgax was significantly less upregulated in the injured Ccr2^−/− kidneys compared with the injured WT kidneys (Figure 4A). There was no difference in the level of the PMN marker Ly6G or the general T cell marker Cd3e between injured WT and Ccr2^−/− kidneys; however, there was a modest reduction in Cd8a in the injured Ccr2^−/− kidneys (Figure 4A). Consistent with this, IHC staining for CD11c (gene name Itgax) on day 30 after U-IRI revealed a significant increase in dendritic cells in the cortex and outer medulla in WT mice, which was greatly attenuated in the Ccr2^−/− mice (Figure 4, B and C). These results suggest that both dendritic cells and cytotoxic T cells accumulate in response to MCP-1 homing signals during AKI-to-CKD transition.

Figure 4. — Loss of CCR2 reduces intrarenal dendritic cell and T cell accumulation after U-IRI. (A) Quantitative reverse-transcription PCR analysis for *Itgax*, *Cd3e*, *Cd8a*, and *Ly6G* was performed on whole-kidney mRNA. ***P<0.001. n=14 for WT and 11 for *Ccr2^−/−* mice. (B) Kidney sections were immunostained with anti-CD11c (representative image of cortex and outer medulla shown). Scale bars, 200 µm. (C) Quantitation of CD11c-positive area from cortex and outer medulla of the kidney sections immunostained as in (B). **P<0.01, ***P<0.001 versus WT IRI kidney. n=14 for WT and 11 for *Ccr2^−/−* mice. (D) Cells freshly isolated from 12 kidneys 14 days after U-IRI were separately FACS sorted for CD45^±CD11c^±F4/80^±, followed by pooling (three pools of four kidneys/pool, 12 kidneys total), RNA extraction, and quantitative PCR analysis of the indicated cell populations for *Tnfa*, *Il1b*, and *Havcr1*. (E) Quantitative reverse-transcription PCR analysis for *Tnfa*, *Il1b*, and *Havcr1* was performed on whole-kidney mRNA. **P<0.01, ***P<0.001. n=14 WT and 11 *Ccr2^−/−* mice. IRI, ischemia/reperfusion injury; ns, not statistically significant; U-IRI, unilateral IRI; WT, wild type.

To determine the functional roles of these additional Ccr2-expressing cells, we analyzed the inflammatory cytokine expression levels from FACS-sorted kidney cells 14 days after U-IRI. The results show that Tnfa was expressed by T cells/PMNs and dendritic cells, whereas Il1b was predominantly expressed by dendritic cells alone (Figure 4D). Macrophages failed to express either proinflammatory cytokine at this late time point. At the whole-kidney level, quantitative PCR analysis revealed less expression of Tnfa and Il1b, as well as the tubular injury marker Kim-1 (Havcr1), in the injured Ccr2^−/− kidneys compared with the injured WT kidneys (Figure 4E), suggesting that MCP-1/CCR2 signaling promotes T cell/dendritic cell–dependent interstitial inflammation and tubular injury.

CCR2 Promotes Renal Fibrosis Induced by U-IRI

To determine whether the reduced profibrotic macrophage accumulation and proinflammatory dendritic cell/T cell accumulation seen after injury in Ccr2^−/− mice affects kidney fibrosis and/or myofibroblast accumulation, we analyzed kidney histology, RNA, and protein from both genotypes 30 days after U-IRI. Picrosirius red staining showed that Ccr2^−/− mice had significantly less renal interstitial collagen deposition in the injured kidney in both the cortex and outer medulla compared with WT mice (Figure 5, A and B). Western blot and quantitative PCR analysis revealed that three representative extracellular matrix components—Col1a1, Col3a1, and Fn1—were significantly less upregulated in the injured Ccr2^−/− kidneys compared with injured WT (Figure 5, C–E). IHC staining for the myofibroblast marker PDGFRβ revealed the expected increase in myofibroblasts in the cortex and outer medulla in WT mice after U-IRI compared with the contralateral kidney (Figure 5F, quantified in G). The loss of Ccr2^−/− resulted in a modest effect on myofibroblast numbers with a decrease in PDGFRβ-positive area in the outer medulla but no change in the cortex (Figure 5, F and G). This modest effect was supported by quantitative PCR of whole-kidney mRNA which showed a significant decrease in Pdgfrb but no change in α-smooth muscle actin (Acta2) (Figure 5H). Combined, these data suggest that MCP-1/CCR2 signaling is more important for myofibroblast activation/matrix deposition than for myofibroblast recruitment/retention.

Figure 5. — Loss of *Ccr2* attenuates renal interstitial fibrosis induced by U-IRI. (A) U-IRI and contralateral (CL) kidneys were harvested 30 days after U-IRI and kidney sections stained with Picrosirius red and (B) area of fibrosis quantified in cortex and outer medulla. **P<0.01, ***P<0.001 versus WT IRI kidney. n=14 WT and 11 *Ccr2^−/−* mice. Scale bars, 200 µm. (C) Western blot analysis for fibronectin (FN) and collagen 1 (COL1α1) protein expression was performed on whole-kidney lysates (each lane is from a separate kidney) and (D) quantified and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). **P<0.01, ***P<0.001. n=11 WT and *Ccr2^−/−* mice. (E) Quantitative reverse-transcription PCR for *Col1a1*, *Col3a1*, and *Fn1* was performed on whole-kidney mRNA. ***P<0.001. n=14 WT and 11 *Ccr2^−/−* mice. (F) Kidneys as in (A) were immunostained with anti-PDGFRβ and (G) PDGFRβ-positive area quantified in cortex and outer medulla. *P<0.05. n=14 WT and 11 *Ccr2^−/−* mice. Scale bars, 200 µm. (H) Quantitative reverse-transcription PCR for *Pdgfrb* and *Acta2* was performed on whole-kidney mRNA. ***P<0.001 versus WT IRI kidney. n=14 WT and 11 *Ccr2^−/−* mice. IRI, ischemia/reperfusion injury; ns, not statistically significant; PDGFRβ, PDGF receptor β; U-IRI, unilateral IRI; WT, wild type.

Pharmacologic Inhibition of CCR2 after Kidney Injury Attenuates Kidney Macrophage Accumulation and Renal Fibrosis

Because constitutive loss of Ccr2 might affect the early events of kidney injury and repair,¹⁰ WT mice were subjected to U-IRI, followed for 7 days to allow initial injury and repair events to proceed normally, and then treated with the CCR2 inhibitor RS102895 or vehicle twice daily from day 7 to 14 after injury (Figure 6A). For comparison, additional groups of WT and Ccr2^−/− mice were subjected to U-IRI. All mice were euthanized on day 14 after U-IRI. The initial proximal tubule injury 24 hours after IRI was equivalent among these groups as determined by serum KIM-1 level (data not shown). The number of F4/80⁺ macrophages, CD11c⁺ dendritic cells, and CD8⁺ T cells was significantly decreased in the injured Ccr2^−/− kidneys and WT kidneys treated with RS102895 compared with the injured WT kidneys and ones treated with vehicle, respectively (Figure 6, B–G). Consistent with the importance of these CCR2⁺ cells and profibrotic growth factors in promoting myofibroblast activation, either CCR2 knockout or inhibition led to a significant decrease of kidney fibrosis as shown by Picrosirius red staining, Western blotting for fibronectin, and COL1α1 and quantitative PCR for Col1a1, Col3a1, and Fn1 (Figure 7). Consistent with the modest effect of Ccr2 knockout on myofibroblast accumulation on day 30 (Figure 5), Ccr2 null mice had 34% and 26% reduction of Pdgfrb and Acta2 expression, respectively, on day 14 after U-IRI, whereas pharmacologic inhibition of CCR2 failed to decrease these myofibroblast markers (Figure 7C).

Figure 6. — Pharmacologic inhibition of CCR2 inhibits inflammatory cell accumulation after kidney injury. (A) WT mice were subjected to 27 minutes warm U-IRI, and then injected with RS102895 or control vehicle every 12 hours starting on day 7 after IRI for 7 days. For comparison, another group of WT and *Ccr2^−/−* mice were subjected to 27 minutes warm U-IRI. All mice were euthanized on day 14 after U-IRI. (B) U-IRI kidney sections were immunostained with F4/80 and (C) quantified. ANOVA, P<0.001, F=20.52. *P<0.05, ***P<0.01. n=8 mice per group. Scale bar, 300 µm. (D–F) Quantitative reverse-transcription PCR analysis for *F4/80*, *Itgax*, *Cd8a*, *Pdgfb*, and *Tgfb1* was performed on whole-kidney mRNA. *P<0.05, **P<0.01, ***P<0.001. n=8 mice per group. CL, contralateral; IRI, ischemia/reperfusion injury; KIM-1, kidney injury molecule-1; U-IRI, unilateral IRI; WT, wild type.

Figure 7. — Pharmacologic inhibition of CCR2 suppresses U-IRI induced kidney fibrosis. (A) U-IRI kidneys were harvested at 14 days after IRI, and kidney sections stained with Picrosirius red and (B) fibrosis area quantified. ANOVA, P=0.0196, F=3.871. *P<0.05. n=8 mice per group. Scale bar, 300 µm. Quantitative reverse-transcription PCR for (C) *Pdgfrb* and *Acta2* as well as (D) *Col1a1*, *Col3a1*, and *Fn1* was performed on whole-kidney mRNA. *P<0.05, **P<0.01. n=8 mice per group. (E) Western blot analysis for fibronectin (FN) and collagen 1 (COL1α1) protein expression was performed on whole-kidney lysates (each lane is from a separate kidney) and (F) quantified and normalized to β-actin. **P<0.01, ***P<0.001 versus vehicle-treated IRI kidney. n=8 mice per group. IRI, ischemia/reperfusion injury; CL, contralateral; U-IRI, unilateral IRI; WT, wild type.

Discussion

Chemokine receptors such as CCR2 and CX3CR1 and their cognate ligands MCP-1 and fractalkine (CX3CL1), respectively, play important roles in recruiting bone marrow–derived monocytes to the kidney in response to injury signals after IRI,^10,28 sepsis,²⁹ and diabetic kidney injury.³⁰ These monocytes infiltrate the injured kidney and differentiate into proinflammatory macrophages in response to signals such as danger-associated molecular patterns expressed by injured cells.^13,18 We have previously shown that the subsequent upregulation of tubular cell–expressed GM-CSF induces reparative activation of infiltrating macrophages via Janus tyrosine kinase–STAT5 signaling during days 3–5 after IRI. Together with M-CSF,³¹ GM-CSF is upregulated and secreted by tubular cells and promotes expression of the alternative activation genes Arg1, Mrc1, and Msr1 during kidney repair.⁸ We now show that sustained GM-CSF expression by stressed or nonrepaired tubular cells directly induces the expression of Mcp-1/Ccl2 by peritubular macrophages. Based on the studies by Tanimoto et al.³² we predict that this occurs via STAT5-mediated induction of the Ccl2 promoter. Our data using Ccr2 knockout and inhibition supports the model in which the GM-CSF–mediated Mcp-1 expression leads to recruitment of additional CCR2-positive immune cells including macrophages, dendritic cells, T cells, and PMNs into the kidney interstitium that in turn promote ongoing tubular injury and interstitial fibrosis during AKI-to-CKD transition (Figure 8).

Figure 8. — A proposed model of GM-CSF/MCP-1/CCR2-mediated cell cross-talk during AKI-to-CKD transition. Sustained expression of GM-CSF by nonrepaired tubular cells activates macrophages to express MCP-1, which in turn recruits additional macrophages, dendritic cells, and T cells/PMNs into the injured kidney interstitium. Macrophages are polarized to a profibrotic phenotype and express PDGFβ and TGFβ, which activate PDGFRβ-positive myofibroblasts to express extracellular matrix components such as collagen 1 (COL1α1), COL3α1, and fibronectin. Both dendritic cells and T cells are locally activated to express proinflammatory cytokines such as TNFα and IL-1β, which lead to sustained kidney inflammation and injury. ECM, extracellular matrix; MCP-1, monocyte chemoattractant protein-1; PDGFRβ, PDGF receptor β.

A recent meta-analysis showed that elevated urinary MCP-1 levels were predictive of renal fibrosis on kidney biopsy in 596 patients from four studies with an area under the curve of 0.66, and were independently associated with a more rapid loss of eGFR.²⁰ Our mouse model of renal fibrosis is consistent with these clinical findings, showing that kidney Ccl2 expression highly correlated with kidney fibrosis (R=0.99; P=0.001) and macrophage accumulation (R=0.98; P=0.003). Blockade of MCP-1/CCR2 signaling using either CCR2 inhibitors or Ccr2 knockout has been shown to attenuate renal fibrosis in a UUO model and a reno-vascular hypertension model.^19,33 Our results now show that blockade of MCP-1/CCR2 signaling using pharmacologic inhibition with RS102895 begun 7 days after injury can still reduce renal fibrosis by diminishing late profibrotic macrophage accumulation. Pharmacokinetic analysis of RS102895 has revealed a t_1/2 of approximately 1 hour after subcutaneous injection,²³ suggesting that a multidose treatment regimen would be most effective for optimal blocking of MCP-1/CCR2 signaling.

Currently the signal(s) that polarize macrophages into a profibrotic phenotype remain largely unexplored. Our current in vitro analysis demonstrates that MCP-1/CCR2 signaling does not directly induce profibrotic macrophage polarization, suggesting that the predominant role of MCP-1/CCR2 signaling during the transition to profibrotic macrophages is to induce cell homing/persistence. We have previously shown that breast regression protein-39 (Brp-39 or Chi3l1) expressed by renal cells promotes profibrotic macrophage persistence and interstitial fibrosis using the U-IRI model.¹⁴ In that study, analysis of macrophages isolated from injured kidneys 14 days after U-IRI revealed high-level expression of the proposed profibrotic BRP-39 receptor, PG D2 receptor 2 (Ptgdr2 or Crth2).³⁴ Knockout of BRP-39 reduced expression of profibrotic growth factors including Pdgfb, Tgfb, and Egf in kidney macrophages, suggesting that BRP-39 may serve to directly promote profibrotic macrophage activation. Consistent with this, Chi3l1 was equivalently upregulated in the WT and Ccr2^−/− injured kidney (Supplemental Figure 3), suggesting that tubular cell–derived BRP-39 might serve as a profibrotic macrophage activator independent of the number of macrophages present.

The primary effector cell of matrix deposition and kidney fibrosis is believed to be the PDRGRβ⁺αSMA⁺ myofibroblast.¹⁶ Our analysis of PDRGRβ⁺ myofibroblasts isolated from the injured kidney revealed that they express very low levels of Ccr2 compared with CD45⁺ immune cells, suggesting that macrophage-expressed Mcp1 is unlikely to directly promote myofibroblast homing or activation. Chen and associates have shown that PDGFβ can activate pericyte-to-myofibroblast transformation.³⁵ We have previously shown that deletion of Tgfb1 in myeloid lineage cells results in a modest reduction of isolated fibrosis markers but does not lead to a decrease of interstitial fibrosis in the late stage of U-IRI.³⁶ More recently we have shown that macrophages not only express Tgfb1 but also Pdgfb and Egf,¹⁴ all of which have been demonstrated to induce fibrotic response in various mouse organ models.^35,37–40 In this study, macrophage-expressed Pdgfb and Tgfb1 levels are reduced in the setting of Ccr2 loss or inhibition, however there is only a modest effect of this reduction in Pdgfb on the number of PDRGRβ⁺ myofibroblasts in the injured kidney. In contrast, there is a much more significant reduction in the myofibroblast-expressed matrix components collagen I, collagen III, and fibronectin. Cumulatively, these new data suggest that macrophage-expressed PDGFβ and/or TGFβ are more likely to be important for myofibroblast activation than myofibroblast recruitment.

An important question that these studies raise is what tubular cell stressor induces the sustained upregulation of GM-CSF after U-IRI. Evidence from multiple sources suggests that inflammation plays an important role in the initiation and progression of CKD⁴¹, and so we extended our studies to examine the effects of U-IRI on other immune cells including dendritic cells and T cells/PMNs. It has been shown that CCR2 and CX3CR1 direct migration of dendritic cell precursors to the lung in the setting of infection and inflammation,^42–44 and that after UUO dendritic cells adopt a proinflammatory phenotype that can activate T cells.⁴⁵ Furthermore, 6 weeks after U-IRI, both CD4⁺ and CD8⁺ T cells are found in the injured kidney with a significant upregulation of Il1b, Il6, and Tnfa expression.⁴⁶ In this study, we show that both dendritic cells and T cells/PMNs express high levels of Ccr2 14 days after U-IRI, and that blockade of MCP-1/CCR2 signaling results in reduced dendritic cell and T cell accumulation with diminished Tnfa and Il1b expression, correlating with less expression of the kidney injury marker Havcr1. Thus, we propose that the activated dendritic cells and T cells induce sustained tubular cell injury that in turn promotes the expression of GM-CSF.

Cumulatively, these results demonstrate that GM-CSF–induced MCP-1/CCR2 signaling plays an important role in the cross-talk between injured tubular cells and infiltrating macrophages, dendritic cells, T cells, and myofibroblasts to promote sustained tubular injury and progressive interstitial fibrosis at the time of AKI-to-CKD transition. Several CCR2 antagonists have entered clinical trials for the treatment of CKD due to diabetic kidney disease.^47–49 The current findings suggest that targeting this pathway may also serve to limit kidney fibrosis during the transition from AKI to CKD.

Disclosures

Dr. Cantley reports personal fees from GoldfinchBio, outside of the submitted work. All of the remaining authors have nothing to disclose.

Funding

This work was supported by NIH grants R01 DK093771 (to Dr. Cantley), T32 DK007276, and K01 DK120783 (to Dr. Xu).

Supplementary Material

Supplemental Data

ASN.2019010068SupplementaryData.pdf^{(1.4MB, pdf)}

Acknowledgments

Dr. Xu performed primary experiments and wrote and edited the manuscript. Ms. Sharkey performed in part primary in vitro experiments. Dr. Cantley oversaw the project and contributed to editing the manuscript.

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

Supplemental Material

This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019010068/-/DCSupplemental.

Supplemental Table 1. Mouse experiment design.

Supplemental Table 2. Primer sequences used for quantitative PCR.

Supplemental Figure 1. FACS sorting of macrophages, dendritic cells, T cells/PMNs and renal cells.

Supplemental Figure 2. FACS sorting of resident and infiltrating/patrolling macrophages.

Supplemental Figure 3. Loss of CCR2 reduces chemoattractant receptor expression after unilateral IRI.

Supplemental Figure 4. Loss of CCR2 reduces chemoattractant expression after unilateral IRI.

Supplemental Figure 5. MCP-1 does not polarize bone marrow–derived macrophages (BMMs) in vitro.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Data

ASN.2019010068SupplementaryData.pdf^{(1.4MB, pdf)}

[B1] 1.Coca SG, Singanamala S, Parikh CR: Chronic kidney disease after acute kidney injury: A systematic review and meta-analysis. Kidney Int 81: 442–448, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2.Chawla LS, Kimmel PL: Acute kidney injury and chronic kidney disease: An integrated clinical syndrome. Kidney Int 82: 516–524, 2012 [DOI] [PubMed] [Google Scholar]

[B3] 3.Sato Y, Yanagita M: Immune cells and inflammation in AKI to CKD progression. Am J Physiol Renal Physiol 315: F1501–F1512, 2018 [DOI] [PubMed] [Google Scholar]

[B4] 4.Kinsey GR: Macrophage dynamics in AKI to CKD progression. J Am Soc Nephrol 25: 209–211, 2014 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5.Falke LL, Gholizadeh S, Goldschmeding R, Kok RJ, Nguyen TQ: Diverse origins of the myofibroblast—implications for kidney fibrosis. Nat Rev Nephrol 11: 233–244, 2015 [DOI] [PubMed] [Google Scholar]

[B6] 6.Grgic I, Duffield JS, Humphreys BD: The origin of interstitial myofibroblasts in chronic kidney disease. Pediatr Nephrol 27: 183–193, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Lee S, Huen S, Nishio H, Nishio S, Lee HK, Choi BS, et al.: Distinct macrophage phenotypes contribute to kidney injury and repair. J Am Soc Nephrol 22: 317–326, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8.Huen SC, Huynh L, Marlier A, Lee Y, Moeckel GW, Cantley LG: GM-CSF promotes macrophage alternative activation after renal ischemia/reperfusion injury. J Am Soc Nephrol 26: 1334–1345, 2015 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.Kefaloyianni E, Muthu ML, Kaeppler J, Sun X, Sabbisetti V, Chalaris A, et al.: ADAM17 substrate release in proximal tubule drives kidney fibrosis. JCI Insight 1: e87023, 2016 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10.Li L, Huang L, Sung SS, Vergis AL, Rosin DL, Rose CE Jr, et al.: The chemokine receptors CCR2 and CX3CR1 mediate monocyte/macrophage trafficking in kidney ischemia-reperfusion injury. Kidney Int 74: 1526–1537, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11.Zhang MZ, Yao B, Yang S, Jiang L, Wang S, Fan X, et al.: CSF-1 signaling mediates recovery from acute kidney injury. J Clin Invest 122: 4519–4532, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12.Chiba T, Skrypnyk NI, Skvarca LB, Penchev R, Zhang KX, Rochon ER, et al.: Retinoic acid signaling coordinates macrophage-dependent injury and repair after AKI. J Am Soc Nephrol 27: 495–508, 2016 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Huen SC, Cantley LG: Macrophage-mediated injury and repair after ischemic kidney injury. Pediatr Nephrol 30: 199–209, 2015 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.Montgomery TA, Xu L, Mason S, Chinnadurai A, Lee CG, Elias JA, et al.: Breast regression protein-39/chitinase 3-like 1 promotes renal fibrosis after kidney injury via activation of myofibroblasts. J Am Soc Nephrol 28: 3218–3226, 2017 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15.Lin SL, Castaño AP, Nowlin BT, Lupher ML Jr, Duffield JS: Bone marrow Ly6Chigh monocytes are selectively recruited to injured kidney and differentiate into functionally distinct populations. J Immunol 183: 6733–6743, 2009 [DOI] [PubMed] [Google Scholar]

[B16] 16.Duffield JS: Cellular and molecular mechanisms in kidney fibrosis. J Clin Invest 124: 2299–2306, 2014 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17.Kim MG, Kim SC, Ko YS, Lee HY, Jo SK, Cho W: The role of M2 macrophages in the progression of chronic kidney disease following acute kidney injury. PLoS One 10: e0143961, 2015 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Tubular GM-CSF Promotes Late MCP-1/CCR2-Mediated Fibrosis and Inflammation after Ischemia/Reperfusion Injury

Leyuan Xu

Diana Sharkey

Lloyd G Cantley

Significance Statement

Visual Abstract

Abstract

Background

Methods

Results

Conclusions

Methods

Animal Surgery and Experimental Protocol

ELISA of Serum Kidney Injury Molecule-1 Level

Histology, Immunohistochemistry, and Immunofluorescence

FACS Analysis

In Vitro Cell Culture

Quantitative PCR Analysis

Western Blot Analysis

Statistical Analyses

Results

Macrophage Accumulation Correlates with Interstitial Fibrosis during AKI-to-CKD Transition

Figure 1.

Tubular Cell GM-CSF Promotes MCP-1 Expression in Macrophages

Figure 2.

CCR2 Promotes Macrophage Accumulation after Kidney Injury

Figure 3.

CCR2 Promotes Inflammatory Dendritic Cell and Cytotoxic T Cell Accumulation after Kidney Injury

Figure 4.

CCR2 Promotes Renal Fibrosis Induced by U-IRI

Figure 5.

Pharmacologic Inhibition of CCR2 after Kidney Injury Attenuates Kidney Macrophage Accumulation and Renal Fibrosis

Figure 6.

Figure 7.

Discussion

Figure 8.

Disclosures

Funding

Supplementary Material

Acknowledgments

Footnotes

Supplemental Material

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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