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. 2026 Feb 2;7(6):1241–1250. doi: 10.34067/KID.0000001150

Arginase-1 Expressing Macrophages Interact with Myofibroblasts to Regulate Insulin-Like Growth Factor-1-Dependent Tubule Repair

Bhavya Bharathan 1, Bismark O Frimpong 2, Leyuan Xu 1, Lloyd G Cantley 1,2,✉
PMCID: PMC13337152  PMID: 41628890

Visual Abstract

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Keywords: AKI, cell signaling, endothelial cells, fibroblast, ischemia-reperfusion, macrophages, proliferation, proximal tubule, tubule cells

Abstract

Key Points

  • Insulin-like growth factor-1 is upregulated in the injured outer stripe of the kidney only if Arginase-1 (Arg1)-expressing macrophages are present.

  • Arg1-expressing macrophages induce myofibroblasts and endothelial cells of the outer stripe to express insulin-like growth factor-1 rather than making it themselves.

  • Myofibroblast-expressed insulin-like growth factor-1 induces proximal tubule insulin-like growth factor-1 receptor activation and subsequent proliferation.

Background

Following AKI, successful regeneration of tubular epithelium is essential to restore normal kidney function. We have previously reported that arginase-1-expressing macrophages in the outer medulla promote tubular proliferation after ischemic injury; however, the mechanism of this effect remained unidentified.

Methods

Arginase-1+ macrophage-dependent proliferative signals and their cellular source were determined by cell sorting and single-cell transcriptional profiling at 2 days postinjury in mice subjected to ischemia-reperfusion injury and in vitro using transwell coculture of macrophages and renal cells.

Results

Quantitative PCR analysis of outer medullary RNA on day 2 post–ischemia-reperfusion injury in wild type and macrophage-specific arginase-1 null (Arg1mko) mice identified insulin-like growth factor-1 (Igf1) as the epithelial growth factor significantly upregulated in an arginase-1-dependent manner after kidney injury. Single-cell RNA sequencing analysis of mouse kidneys, human kidney biopsies, and sorted outer medullary cells identified myofibroblasts and endothelial cells as potential cellular sources of IGF1. In vitro studies showed that myofibroblast and endothelial cell expression of Igf1 was dependent on macrophage arginase-1 expression and that myofibroblast-secreted IGF1-induced epithelial cell IGF1 receptor activation and proliferation. Consistent with this, in vivo activation of the IGF1 receptor on surviving S3 proximal tubule cells after injury was dependent on macrophage Arg1 expression.

Conclusions

Our results demonstrate that alternatively activated macrophages signal cooperatively with myofibroblasts and possibly endothelial cells to coordinate local IGF1 secretion and proliferative tubule repair at sites of epithelial cell loss after kidney injury. These findings support an important role of activated myofibroblasts in effective tubule repair.

Introduction

AKI is linked to substantial morbidity and mortality, frequently causing permanent kidney damage.1–5 Resolution of AKI involves a complex sequence of events, including egress or apoptosis of neutrophils, conversion of proinflammatory macrophages to a reparative state, and proliferation and redifferentiation of surviving tubular cells.4,6 After ischemia-reperfusion kidney injury (IRI), this sequence of events is most prominent in the outer renal medulla where the S3 segment of the proximal tubule suffers the greatest loss of tubular cells.7–10

Macrophages have emerged as critical players in this repair process,11–13 with our group demonstrating that macrophages expressing arginase-1 (ARG1) play a pivotal role in facilitating tubular proliferation after kidney injury in mouse models.9 As ARG1 is an intracellular enzyme that can facilitate protein translation,14,15 we hypothesized that its upregulation in alternatively activated macrophages can induce the secretion of a factor(s) that promotes tubular cell proliferation. Our current findings show that insulin-like growth factor-1 (Igf1 gene name, IGF1 protein name) is selectively upregulated in an Arg1-dependent manner in the outer stripe of the medulla after ischemic injury but that the source of Igf1 is myofibroblasts and possibly endothelial cells rather than macrophages themselves. IGF1 is a growth factor that is well known to promote the growth and division of renal cells by activating the IGF1 receptor (IGF1R), which initiates intracellular signaling pathways such as PI3K/Ak strain transforming, extracellular-signal regulated kinase/mitogen activated protein kinase, and janus kinase/signal transducer and activator of transcription.16–18 These pathways regulate crucial processes including cell proliferation, differentiation, survival, and inhibition of apoptosis, making it particularly important for normal kidney development, glomerular function, and tubular cell proliferation. 19–22 Here, we show that the complex interplay between dedifferentiated tubular cells, alternatively activated Arg1+ macrophages, and activated myofibroblasts regulates local IGF1 production and promotes proliferative tubule repair after kidney injury.

Methods

Animal Models and Surgery

All animal protocols were approved by the Yale University Institutional Animal Care and Use Committee. Eight- to 12-week-old wild-type (WT) and LysM-Cre;Arg1fl/fl mice (Arg1mko) were used in this work. All mice were on the C57BL/6J background. Male mice were used exclusively to minimize the total number of animals required for statistical analysis, as there is a significant difference in susceptibility to IRI between male and female mice. All mice were housed under a 12-hour light/12-hour dark cycle with unrestricted access to standard food and water both before and after surgery. To induce repairable kidney injury, mice were subjected to ischemia-reperfusion with contralateral nephrectomy (IRI-CL/NX), as previously described.9,23 In brief, mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) while positioned on a 37°C warming pad with a rectal temperature probe. A midline abdominal incision was made, and warm renal ischemia was induced by applying a nontraumatic microaneurysm clip (Micro Clamps Cat. #: 00398-02; Fine Science Tools) to the left renal pedicle for 16′45 sec, with nephrectomy of the right kidney. After the procedure, mice received intraperitoneal injection of 1 ml sterile saline to prevent dehydration and buprenorphine for postoperative pain relief. For kidney cell isolation, WT and Arg1mko mice were euthanized on day 2 after IRI. Blood for serum urea estimation was collected before and on day 1 after reperfusion. BUN assays were performed using Stanbio BUN Test Kit (Cat. # SB-0580-250). Mice with BUN levels between 80 and 120 mg/dl on day 1 after IRI were selected for subsequent analyses, as this range reflects consistent and reproducible injury (Supplemental Figure 1A).

Magnetic-Activated Cell Sorting

PDGFRβ-expressing and F4/80-expressing cells were sorted from healthy and IRI kidneys from WT and Arg1mko mice using magnetic activated cell sorting (MACS). For this, we used the EasySep Phycoerythrin (PE) Positive Selection Kit II (Stem Cell Technologies, Cat. # 17684). In brief, kidneys were microdissected to isolate the outer medulla and minced into small pieces in ice-cold Liberase/DNase/MgCl2 mix for 5–8 minutes. The cell mix was then incubated in Liberase at 37°C in a shaking incubator for 30 minutes with intermittent pipetting every 10 minutes. The cell/tubule mix was then passed sequentially through 70 and 40 μm cell strainers to obtain predominantly single cells, washed with 1X PBS, and incubated for 15 minutes at RT with 3 μg/ml of anti-PDGFRβ (clone# APB5, Invitrogen, Cat. #12-1402-81) or 3 μg/ml of anti-F4/80 (clone# BM8, Biolegend, Cat. #123110) following the kit protocol. Magnetic beads were added, and cell-attached beads were washed and collected as per instructions.

Fluorescence-Activated Cell Sorting

Single-cell suspensions of outer medulla were obtained as described above from mouse kidneys on day 2 after injury (n=4 mice). Washed cells underwent Fc receptor blocking and were incubated with fluorophore-conjugated antibodies targeting CD140b using PE anti-PDGFRβ antibody (Invitrogen, Cat. #12-1402-81) or CD31 using PE-Cy7 anti-platelet endothelial cell adhesion molecule-1 antibody (clone# 390, Invitrogen, Cat. #25031182) for 30 minutes at 4°C in the dark. After staining, cells were washed, resuspended in FACS buffer (0.5% BSA, 2 mM EDTA, 0.1% Pluronic F-68 in PBS), and analyzed using a BD FACS Aria flow cytometer, and data were processed using the BD FACSDiva 9.4 software.

Single-Cell RNA Sequencing

Single-cell RNA sequencing (scRNA-seq) of mouse cells was performed on the MACS-sorted PDGFRβ+ cells described above from mouse kidney outer medulla on day 2 after injury (n=1 mouse). The cells were counted and assessed for viability using Trypan Blue exclusion staining. The library preparation and sequencing were performed at the Yale Center for Genome Analysis, as previously described using the 10× Genomics Chromium Single Cell 3′ Gene Expression 3P v4 (GEM-Х) assay.9,23 In brief, single cells were encapsulated with barcoded gel beads using the Chromium Controller (10× Genomics), and reverse transcription was performed in droplets to produce barcoded cDNA. Libraries were constructed according to the manufacturer's protocol and sequenced on Illumina HiSeq 4000 sequencer. Raw data were processed using the Cell Ranger pipeline (v8.0.1). Furthermore, downstream analysis, including normalization, quality control, dimensionality reduction, cell clustering, and differential gene expression, was performed using Seurat (v5.3.0) in R v4.4. Genes expressed in a minimum of three cells were retained in the analysis. Cells expressing <200 or >3000 genes were excluded. Cells expressing >50% mitochondrial genes were also excluded (Supplemental Figure 1, B–D). Uniform Manifold Approximation and Projection analysis of previously reported primary cultured renal cells (PCRC) scRNA-seq (database accession code-GSE188966), healthy mice (database accession code-GSE197626), and the current MACS-sorted PDGFRβ+ cells (database accession code-GSE304245) was used to visualize different cell clusters based on their transcriptional profile in two-dimensional space, and gene expression patterns were represented as heatmaps with dot plots.

Analysis of Publicly Available Single-Cell Sequencing Data from Human Kidney Biopsies Obtained by Kidney Precision Medicine Project

The scRNA-seq data for human kidney cells were obtained from the publicly available Kidney Precision Medicine Project (KPMP) Central Biorepository (https://atlas.kpmp.org/).24 KPMP has obtained biopsies of patients with AKI, CKD, and reference for extensive research analysis. The dataset used includes single-cell transcriptional analysis of kidney biopsy samples from 24 healthy transplant donors (preperfusion biopsies), 19 patients with AKI, and 47 patients with CKD. The analytical pipeline was performed, as previously described.25 Relative gene expression levels were visualized using dot plots.

Transwell Culture and Preparation of Conditioned Media

PCRC and bone marrow–derived macrophages (BMDM) were isolated and cultured, as previously described.9 Macrophage-derived conditioned medium (MՓ CM) was prepared using transwell cocultures with macrophages in the bottom well. Control media (MՓ CM1) was prepared by culturing WT macrophages in both the top and bottom wells for 24 hours at 37°C in 1% FBS DMEM. MՓ CM2 and MՓ CM3 were prepared by culturing either WT macrophages (MՓ CM2) or Arg1mko macrophages (MՓ CM3) in the bottom well with PCRCs in the top well in 1% FBS DMEM containing 20 ng/ml granulocyte-monocyte colony stimulating factor (GM-CSF) at 37°C for 24 hours (Supplemental Figure 2A). Pure Arg1mko cells were generated, as previously described,9 by growing BMDM from Arg1fl/fl;LysMCre/+; Rosa26mTmG mice (containing a mix of tdTomato+ (Arg1WT) and GFP+ (Arg1mko) BMDM (Supplemental Figure 2B), followed by FACS sorting for GFP+, Arg1mko cells (fraction P4 in Supplemental Figure 2C). The conditioned media was collected from the bottom well, and the macrophages from all three conditions were harvested for RNA analysis of Arg1 gene expression. n=3 mice used to prepare each macrophage conditioned media.

Myofibroblast conditioned media (MF CM) was prepared from MACS-sorted PDGFRβ+ cells. These cells were isolated on day 2 after IRI-CL/NX and cultured for 6–7 days to expand PDGFRβ+ cell numbers. Quantitative PCR confirmed that Pdgfrβ mRNA levels increased with 6 days of culture, whereas PECAM1 mRNA levels were decreased (Supplemental Figure 2D). The PDGFRβ enriched cells were seeded into 12-well culture dishes, serum starved overnight, and then cultured in MՓ CM1, MՓ CM2, or MՓ CM3 for 24 hours to generate MF CM1, MF CM2, and MF CM3, respectively. n=7 mice used for preparing each conditioned media.

Immunostaining

Kidneys were perfused with ice-cold PBS, harvested and fixed in 4% paraformaldehyde, processed, and sectioned at 5-μm thickness. Deparaffinized tissue sections were rehydrated using a series of decreasing concentrations of ethanol (100%, 95%, 80%, and 70%) and microwaved in Epitope Retrieval Solution pH 9 (Leica Biosystems, Cat. # RE7119) for 20 minutes. The sections were then blocked in 5% BSA in 1× tris-buffered saline-T for 1 hour at RT and incubated with primary antibodies at 4°C for overnight. The primary antibodies used were as follows: anti–phospho-IGF1R antibody (anti-pTyr 1161, 1:100, Cusabio, Cat. # CSB-PA120059), anti-Ki67 antibody (1:200, Abcam, Cat. # ab15580), and Lotus tetragonolobus lectin (LTL, 1:400, Vector Laboratories Ltd Cat. # FL-1321). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. For staining of cultured cells, PT cells were visualized using anti-AQP1 antibody (1:100, Invitrogen, Cat. # MA1-24915). n=5 mice were used from Arg1 WT healthy, Arg1 day 2 IRI, and Arg1mko day 2 IRI mice for immunostaining.

ELISA

IGF1 ELISA assays were performed using Mouse IGF1 ELISA kit (Cat. # ELM-IGF1-1, RayBiotech), and GH ELISA assays were performed by Human GH ELISA Kit (Cat. # ELH-GH-1, RayBiotech). The assay was performed based on the kit protocol. In brief, the diluted samples and standards were loaded into IGF1 antibody-coated wells of the ELISA microplate and incubated at RT for 2.5 hours for binding. The wells were washed with the wash buffer provided and incubated with biotinylated anti-mouse IGF1 detection antibody for 1 hour at RT. After washing away the unbound antibody, horse radish peroxidase-conjugated streptavidin was added to the wells and the plate was then incubated for 45 minutes at RT. The wells were again washed and incubated with 3,3′,5,5′-tetramethylbenzidine substrate, followed by stop solution. The intensity of the color developed was measured at 450 nm using a microplate reader (Agilent BioTek Synergy HTX).

Quantitative PCR

RNA from cultured cells and kidney tissues was extracted with a RNeasy Mini kit (Qiagen, Cat. #74106), and 1 μg of total RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Cat. #1708890). Gene expression was analyzed using gene-specific primers by quantitative real-time PCR (iTaq Universal SybrGreen Supermix; Bio-Rad, Cat. #1725124) using a CFX96 machine (Bio-Rad) and normalized to hypoxanthine guanine phosphoribosyl transferase. The mRNA ratios were represented by 2−ΔCT. Sequences of primers used for quantitative PCR are presented in Supplemental Table 1.

Immunoblot

Cultured cells were lysed in ice-cold radio-immunoprecipitation assay buffer containing protease, and phosphatase inhibitors and protein concentration were determined using the Pierce BCA Protein Assay Reagent kit (Thermo Fisher Cat. #23228 and Cat. #23222). After sodium dodecyl sulfate-polyacrilymide gel electrophoresis, proteins were transferred to polyvinylidene difluoride membrane for 1 hour at 4°C, blocked with 5% BSA for 1 hour at RT, and then incubated with primary antibodies at 4°C for overnight. After multiple washes in tris-buffered saline with tween-20 and secondary antibody incubation for 1 hour at RT, the membranes were developed using enhanced chemiluminescence to visualize and capture the protein band signals. The primary antibodies used were anti–phosphor-IGF1R (pTyr 1161, 1:1000; Abcam, Cat. #ab39398), antitotal IGF1R antibody (1:1000; Abcam, Cat. # 39–6700), and anti-glyceraldehyde-3-phopshate dehydrogenase (1:1000; Santa Cruz, Cat. # sc-32233).

Statistical Analysis

All data are presented as mean±SEM. Multigroup comparisons were performed using one-way ANOVA with a Tukey multiple comparison test for subgroup comparisons; two-group comparisons were analyzed by t tests. All the analysis was performed using Prism 10.5.0 (GraphPad Software, Inc.). P values < 0.05 were considered statistically significant.

Results

Igf1 Is Upregulated in an Arg1-Dependent Manner in the Outer Medulla after Kidney Injury

To identify the growth factor(s) responsible for Arg1-dependent tubular epithelial proliferation after renal injury, we analyzed the outer medulla of mouse kidneys 2 days after IRI/CL-NX.23 Quantitative PCR revealed that Igf1 was significantly induced postinjury in the outer medulla (Figure 1A), whereas other growth factors remained unchanged relative to healthy controls (Supplemental Figure 3A). Arg1fl/fl;LysMCre/+ mice lacking Arg1 expression in macrophages (Arg1mko) failed to show an increase in Igf1 mRNA expression after IRI/CL-NX in the outer medulla (Figure 1A). Analysis of our previously published scRNA-seq data from day 3 after IRI shows that injured mouse proximal tubule cells express high levels of IGF1 receptor (IGF1R) mRNA compared with other growth factor receptors (Supplemental Figure 3B),23 and immunofluorescent staining demonstrates that the IGF1R is activated in tubular cells in the outer medulla of WT mice on day 2 after IRI compared with the control kidneys and injured Arg1mko kidneys (Figure 1B, quantified in 1C).

Figure 1.

Figure 1

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Igf1 is upregulated in an Arg1-dependent manner in the outer medulla after kidney injury. Arg1 WT and Arg1mko were subjected to IRI/CL-NX and kidneys harvested and microdissected for analysis on day 2. (A) Igf1 mRNA levels in the outer medulla of healthy kidneys and Arg1mko kidneys, n=8 mice per group (B). Immunofluorescence staining of outer medullary sections of Arg1 WT healthy, Arg1 WT injured, and Arg1mko injured kidneys with phospho-IGF1R (red), LTL (green), and DAPI (blue). (C) MFI of phospho-IGF1R signals as shown in (B) were quantified using ImageJ, with five high power fields/kidney section analyzed/group (n=4 mice). (D) Analysis of mRNA expression of Igf1 in MACS sorted F4/80+ macrophages; (n=5 mice). (E) Analysis of mRNA expression of Igf1 by naïve, Arg1low BMDM (transwell coculture of BMDM with BMDM) versus alternatively activated, Arg1high BMDM (transwell coculture of BMDM with PCRC+GM-CSF, [n=3]). (F) IGF1 protein concentrations determined by ELISA in the 24 hours conditioned media collected from the conditions described in (E) showing the secreted IGF1 protein levels in MՓ CM1 (naïve, Arg1 low macrophages) and MՓ CM2 (alternatively activated, Arg1 high macrophages) (n=3/group). P values are shown, and data are presented as mean±SEM. Arg1, arginase-1; Arg1mko, macrophage-specific Arg1 null; BMDM, bone marrow-derived macrophages; DAPI, 4′,6-diamidino-2-phenylindole; GM-CSF, granulocyte-monocyte colony stimulating factor; IGF1R, IGF1 receptor; IRI, ischemia-reperfusion kidney injury; IRI-CL/NX, ischemia-reperfusion with contralateral nephrectomy; LTL, Lotus tetragonolobus lectin; MACS, magnetic activated cell sorting; MFI, mean fluorescence intensity; MՓ CM, macrophage-derived conditioned medium; PCRC, primary cultured renal cells; pIGF1R, phosphorylated IGF1R; WT, wild type

To determine whether Arg1+ macrophages are the source of IGF1 in injured kidneys, we microdissected the outer medulla from day 2 IRI mice and uninjured controls followed by magnetic cell sorting with anti-F4/80 to enrich outer medulla macrophages. Quantitative PCR confirmed enrichment for both Adgre (F4/80) and Arg1 in the day 2 IRI F4/80 enriched cell population (Supplemental Figure 3, C and D). However, Igf1 mRNA levels were not increased in the macrophages isolated from injured kidneys versus healthy kidneys (Figure 1D), and bone marrow–derived macrophages induced to express Arg1 in vitro by exposure to cultured tubular cells +GM-Csf as previously described (Supplemental Figure 3E)9,11 did not significantly upregulate Igf1 mRNA or protein secretion as compared with control BMDM (Figure 1, E and F). Together, these results suggest that macrophage-expressed Arg1 is required for Igf1 expression, but macrophages themselves are not the predominant source of IGF1 in the injured outer medulla.

Igf1 Is Expressed by Outer Stripe Myofibroblasts and Endothelial Cells

Analysis of publicly available single-cell RNA sequencing data from human kidney biopsy samples obtained by the KPMP (Figure 2A) and from healthy mice (Figure 2B) demonstrates that the cells with highest Igf1 expression in the kidney are Pdgfrβ positive myofibroblasts. MACS enrichment of Pdgfrβ+ cells from the outer stripe of the mouse kidney (Supplemental Figure 4A) confirmed the presence of higher levels of Igf1 expression in PDGFRβ enriched cells from the outer medulla 2 days after IRI (Figure 2C). To more precisely identify the cellular source of Igf1, we performed scRNA-seq of the MACS-sorted PDGFRβ enriched cells from the day 2 injured outer stripe and found that eleven cell populations were present in the mixture (Figure 2D). Of these eleven cell types, Igf1 mRNA expression was greatest in endothelial cells and myofibroblasts, with lower expression detectable in macrophages (Figure 2E). Detailed transcriptional analysis of these macrophages revealed that the Igf1-expressing macrophages were predominantly Arg1 negative (Supplemental Figure 4B). FACS sorting of PDFGRβ+and CD31+ (PECAM+) cells from the injured outer stripe (Supplemental Figure 4, C–F) confirmed high Igf1 mRNA expression by both cell types (Figure 2, F and G).

Figure 2.

Figure 2

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Igf1 is expressed by outer medullary myofibroblasts and endothelial cells. (A) Igf1 expression is greatest in human kidney myofibroblasts based on scRNA-seq of human kidney biopsy samples from publicly available KPMP datasets. (B) scRNA-seq from healthy mouse kidneys confirms that Igf1 expression is highest in myofibroblasts. (C) Igf1 mRNA levels in MACS sorted PDGFRβ- and PDGFRβ+ cell populations from the outer stripe of healthy and injured kidneys, n=5 mice per group. (D) UMAP visualization of identified cell clusters present in freshly isolated PDGFRβ+ cells from mouse outer stripe 2 days post-IRI. (E) Heatmap showing Igf1 expression across different clusters with highest expression in endothelial cells and myofibroblasts. (F and G) Igf1 levels in FACS-sorted PDGFRβlo and PDGFRβhi cells (F) and PECAMlo and PECAMhi cells (G) isolated from day 2 IRI kidneys, n=4 mice per group. P values are shown, and data are presented as mean±SEM. IRI, ischemia-reperfusion injury; KPMP, Kidney Precision Medicine Project; PDGFR, platelet derived growth factor receptor; PECAM, platelet endothelial cell adhesion molecule-1; scRNA-seq, single-cell RNA sequencing; UMAP, Uniform Manifold Approximation and Projection

Conditioned Media from Arg1+ Macrophages Induces Myofibroblasts and Endothelial Cells to Produce IGF1 In Vitro

We have previously reported that PCRCs proliferate in response to conditioned medium from Arg1 expressing BMDM.9 The published scRNA-seq analysis of those PCRC demonstrated that Pdgfrβ+myofibroblasts comprise approximately 5% of the PCRC cell population, whereas endothelial cells were not detected.9 Further analysis of that scRNA-seq data shows that Igf1 mRNA is expressed specifically by the myofibroblast population in PCRC and is highly upregulated after coculture with macrophages (Figure 3A), while the Igf1R is most highly expressed by proximal tubular epithelial cells in coculture with macrophages (Figure 3B).

Figure 3.

Figure 3

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Arg1+ macrophage conditioned media induces myofibroblasts and endothelial cells to produce Igf1 in vitro. (A) scRNA-seq analysis of cultured PCRC cells demonstrates that myofibroblasts increase Igf1 mRNA expression in coculture with WT macrophages. (B) PTEC express the highest level of Igf1r after coculture with macrophages. (C) quantitative PCR analysis shows that cultured MACS-enriched PDGFRβ+ cells express high levels of Igf1 mRNA in response to culture in MՓ CM2 compared with MՓ CM1 and MՓ CM3, n=5. (D) PDGFRβ-enriched cells secrete high levels of IGF1 protein in response to culture in MՓ CM2, n=7. (E and F) HUVEC endothelial cells significantly increase Igf1 mRNA expression (E) and IGF1 protein secretion (F) when treated with MՓ CM2 compared with MՓ CM1 and MՓ CM3, n=5. P values are shown, and data are presented as mean±SEM. HUVEC, human umbilical vein endothelial cells; MAC, macrophages; PTEC, proximal tubular epithelial cells; TEC, other tubular epithelial cells.

To investigate whether Arg1+ macrophages secrete a factor(s) to induce Igf1 expression by myofibroblasts, we generated macrophage conditioned medium (MՓ CM) from naïve, Arg1low WT BMDM (MՓ CM1); alternatively activated, Arg1high WT macrophages (MՓ CM2); or alternatively activated Arg1mko macrophages that fail to express ARG1 despite coculture with PCRC+GM-Csf (MՓ CM3; Supplemental Figure 2A). RNA isolated from the macrophages used in the three coculture conditions confirmed the selective induction of Arg1 in the cells that generated MՓ CM2 (Supplemental Figure 3E). This medium was used to stimulate MACS-sorted PDFGRβ-enriched cells that were isolated from day 2 injured mouse kidney outer stripe and cultured for 6–7 days to expand the PDGFRβ+myofibroblast cell population (Supplemental Figure 2D). Of note, endothelial cell markers were markedly reduced in MACS-sorted PDGFRβ-enriched cells after 6 days of culture (Supplemental Figure 2D). PDGFRβ-enriched cells exposed to MՓ CM2 for 24 hours showed a significant increase in Igf1 mRNA as compared with those exposed to MՓ CM1 or MՓ CM3 (Figure 3C). Furthermore, the resulting media contained significantly more IGF1 protein than was seen after culture in MՓ CM1 or MՓ CM3 (Figure 3D), with an approximately six-fold increase in IGF1 protein compared with that seen before PDGFRβ-enriched cell culture (936.52 pg/ml versus 138.82 pg/ml, Figure 1F), confirming that myofibroblasts secrete significantly more IGF1 than do Arg1+ macrophages. To determine if endothelial cells can also increase Igf1 expression in response to Arg1+ macrophages, cultured human umbilical vein endothelial cells were exposed to MՓ CM1, 2, or 3 for 24 hours and analyzed as above. Exposure of these endothelial cells to MՓ CM2 resulted in comparable upregulation of Igf1 mRNA, with concentrations of secreted IGF1 protein that reached approximately 50% those seen from myofibroblasts (Figure 3, E and F). These results demonstrate that Arg1+ macrophage-derived secreted factor(s) can induce IGF1 production by both myofibroblasts and endothelial cells.

Myofibroblast-Expressed IGF1 Activates the IGF1 Receptor and Tubular Cell Proliferation

The conditioned medium that was generated as described above by culture of PDGFRβ-enriched myofibroblasts for 24 hours in MՓ CM1, MՓ CM2, or MՓ CM3 (termed MF CM1, MF CM2, and MF CM3, respectively) was used to stimulate cultured PCRC for 15 minutes. This time point was chosen because PCRCs stimulated in vitro with 10 ng/ml IGF1 demonstrated peak Igf1R phosphorylation at 15 minutes (Supplemental Figure 5). Immunofluorescence staining of PCRC stimulated with MF CM2 (containing the highest IGF1 levels) revealed increased phospho-IGF1R colocalized with the proximal tubular marker AQP1 as compared with that seen with MF CM1 stimulation (Figure 4A, quantified in 4B). Immunoblot analysis confirmed that stimulation of PCRC with MF CM2 resulted in an increase in phosphorylated IGF1R as compared with stimulation with MF CM1 (Figure 4, C and D). Furthermore, Ki67 staining 24 hours after stimulation showed that MF CM2 induced a significant increase in Ki67+ cells as compared with MF CM1 or MF CM3 (Figure 4E, quantified in 4F). Consistent with our published findings,9 Ki67+ tubular cells were significantly higher in the outer medullary LTL+proximal tubules of WT injured mice as compared with Arg1mko-injured mice (Figure 4G, quantified in Figure 4H).

Figure 4.

Figure 4

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Myofibroblast conditioned media activates the IGF1 receptor and tubular proliferation. (A) Immunofluorescence staining of cultured PCRCs exposed to myofibroblast CM1 (MF CM1, induced by Arg1low macrophages) or MF CM2 (induced by Arg1high macrophages) for 15 minutes. 10 ng/ml IGF1 stimulation was used as positive control. The activated IGF1 receptor (green) was detected using anti–phospho-IGF1R antibody and proximal tubule cells identified using AQP1 (red) and nuclei by DAPI staining (blue). (B) Representative densitometry of the images as in (A) showing mean fluorescence intensity, n=4. (C) Representative immunoblot performed on lysates from PCRCs treated with the indicated CM showing pIGF1R, with total IGF1R and GAPDH as loading controls, and (D) corresponding bar graph showing the quantification normalized to total IGF1R, n=3. (E) Ki67 staining of PCRCs exposed to the indicated myofibroblast CM for 24 hours (MF CM3=induced by Arg1MKO macrophages). 10 ng/ml IGF1 was used as the positive control. (F) Quantification of the percentage of Ki67 positive cells as in (E), n=3 (G) Immunofluorescence costaining of Ki67 and LTL in the outer medulla of WT control kidneys (Arg1 WT healthy), WT kidneys on day 2 post-IRI (Arg1 WT Injured), and Arg1mko kidneys on day 2 post-IRI (Arg1mko Injured). DAPI staining of nuclei is shown (blue). Arrows indicate Ki67+ nuclei of tubular cells from LTL+proximal tubules. (H) Quantification of numbers of Ki67+ nuclei adjacent to LTL+proximal tubules as in (G), n=5. P values are shown, and data are presented as mean±SEM. GAPDH, glyceraldehyde-3-phopshate dehydrogenase; MF CM, myofibroblast conditioned media.

Discussion

Previous studies in mice subjected to ischemic kidney injury have established that cross-talk between injured tubular cells and nearby macrophages, including tubular cell GM-Csf expression, leads to macrophage alternative activation. These alternatively activated, Arg1-expressing macrophages, in turn, are required for normal tubular cell proliferative repair and improvement in kidney function seen after kidney injury.9,11,13 However, the mechanism by which Arg1+ macrophages promote proliferative tubular repair has remained unclear.

Arginase is a cytosolic enzyme responsible for converting arginine into ornithine, leading to increased levels of multiple intracellular polyamines including putrescine, spermidine, and spermine. Recently, several groups have shown that increased production of these polyamines in macrophages can support the translation of specific populations of mRNA transcripts into their protein products and thus regulate macrophage responses such as polarization and cellular metabolism 14,15 Since our discovery of the connection between macrophage Arg1 expression and tubular cell proliferation, we have examined cultured bone marrow macrophages and isolated kidney macrophages for the expression of multiple growth factors including Egf, Hbegf, Igf1, and Hgf and found no significant differences in their mRNA or secreted protein products in the setting of alternative activation (Figure 1, D–F, and data not shown). This suggested the possibility that additional cell types beyond surviving tubular cells and macrophages may participate in the repair niche. In this study, we used microdissection of the outer medulla on day 2 after injury to focus specifically on mRNA transcript expression at the site of greatest tubular injury and subsequent proliferation and found that Igf1 was the most strongly induced growth factor in this region and that Igf1 expression was dependent on the presence of Arg1+ macrophages (Figure 1A).

Our results align with previous studies showing that IGF1 can regulate proximal tubular regeneration.20,21,26–29 Specifically, Wu et al. showed that IGF1 exerts its protective effects through the activation of the extracellular-signal regulated kinase/mitogen activated protein kinase signaling pathway in tubular epithelial cells after renal injury.21 However, the source of intrarenal IGF1 has been unclear, with studies suggesting that it can be made by the collecting duct, macrophages, mesenchymal stem cells, or fibroblasts.19,21,27,30 Using single-cell RNA sequencing analysis of both human and mouse kidneys, we found that myofibroblasts are the cells that express the highest levels of Igf1 mRNA in the kidney (Figure 2, A and B). We therefore used magnetic beads to isolate PDGFRβ+ cells from healthy and injured mouse outer medulla and confirmed that Igf1 mRNA coenriched with Pdgfrβ (Figure 2C) but found that the PDGFRβ-enriched cells actually contained eleven cell types overall, of which myofibroblasts, endothelial cells, and some macrophages had detectable Igf1 expression in the injured outer medulla (Figure 2C). To obtain more pure cell populations, we used FACS sorting of injured outer medulla and confirmed that Igf1 mRNA expression is increased in both PDGFRβ+ and PECAM+ cells (Figure 2, F and G). Although our sequencing data from healthy kidneys only showed Igf1 expression by myofibroblasts, the ability of endothelial cells to express Igf1 in the setting of kidney injury has been previously described.31 Overall, these data support an important role of activated myofibroblasts in normal repair pathways and suggest caution in developing strategies to deplete these cells in the setting of AKI.

Our in vitro studies used conditioned medium from naïve and alternatively activated macrophages to determine if Igf1 message and protein induction was dependent on direct cell-cell contact or a paracrine secreted factor. We found that while myofibroblasts, endothelial cells, and macrophages at baseline expressed low levels of Igf1 mRNA and secreted protein, myofibroblasts and endothelial cells alone expressed higher levels of Igf1 mRNA and protein in response to the conditioned medium from alternatively activated macrophages. Importantly, Arg1mko macrophages that lack Arg1 but still express other genes associated with alternative activation when cocultured with PCRC and GM-Csf9 fail to induce Igf1 expression. Together, these findings show that arginase-1 expressing, alternatively activated macrophages secrete a factor that can induce Igf1 mRNA and protein expression by both myofibroblasts and endothelial cells.

Although our study did not identify the macrophage-secreted factor that induces Igf1 expression following kidney injury, work from many groups has shown that growth hormone (GH) is the most common regulator of Igf132,33 and that GH is a potential mediator of injury-induced responses in the kidney.20,32,34 GH has been studied in the context of renal inflammation and regeneration, normal kidney development, and glomerular hemodynamic regulation20,34 but has not to date been found to be expressed by alternatively activated macrophages. Analysis of our existing single-cell RNA sequencing data and of cultured macrophages does not demonstrate upregulation of GH mRNA or protein by macrophages in culture (Supplemental Figure 6) or any other cell type in the injured kidney, so additional studies will be required to determine whether locally produced GH is the actual Igf1 inducer. In addition, to limit the number of animals needed for the in vivo studies, all IRI experiments were performed in male mice as this gender exhibits an increased susceptibility to tubular injury with a shorter duration of ischemia. Although the macrophages used for in vitro culture came from both female and male mice, the in vivo cross-talk between macrophages and myofibroblasts in female mice will need to be confirmed in subsequent studies.

In conclusion, these findings support a role of at least four cell types cooperatively regulating tubule repair in a paracrine signaling model: the surviving tubular cell that secretes both M-Csf to promote macrophage proliferation and survival and GM-Csf to promote alternative macrophage activation; the alternatively activated macrophage that expresses ARG1 in response to the injured tubular cell signals; and myofibroblasts and endothelial cells that secrete IGF1 in response to the ARG1-dependent macrophage-derived factor, leading to tubular cell IGF1R activation and reparative proliferation. The complexity of this repair niche underscores the need to fully understand the cross-talk between all cells in the zone of injury before therapeutically inhibiting or depleting any of these pathways.

Acknowledgments

The authors acknowledge Blue Lake, Sanjay Jain, and all KPMP researchers and participating patients who contributed to the single-cell RNA sequencing data analyzed to generate Figure 2A. The authors also acknowledge the University of Michigan Medical School Central Biorepository (RRID: SCR_026845) for providing biospecimen storage, management, and distribution services in support of the research reported in this publication.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The KPMP is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) through the following grants: U01DK133081, U01DK133091, U01DK133092, U01DK133093, U01DK133095, U01DK133097, U01DK114866, U01DK114908, U01DK133090, U01DK133113, U01DK133766, U01DK133768, U01DK114907, U01DK114920, U01DK114923, U01DK114933, U24DK114886, UH3DK114926, UH3DK114861, UH3DK114915, and UH3DK114937. The authors gratefully acknowledge the essential contributions of our patient participants and the support of the American public through their tax dollars.

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/KN9/B521.

Author Contributions

Conceptualization: Lloyd G. Cantley.

Data curation: Bhavya Bharathan, Lloyd G. Cantley, Bismark O. Frimpong, Leyuan Xu.

Formal analysis: Lloyd G. Cantley, Leyuan Xu.

Investigation: Bhavya Bharathan, Bismark O. Frimpong.

Project administration: Lloyd G. Cantley.

Writing – original draft: Bhavya Bharathan.

Writing – review & editing: Lloyd G. Cantley, Bismark O. Frimpong, Leyuan Xu.

Funding

L.G. Cantley: National Institute of Diabetes and Digestive and Kidney Diseases (DK093771). L. Xu: National Institute of Diabetes and Digestive and Kidney Diseases (DK135689), National Institutes of Health (R01DK135689).

Declarative Statements

All animal experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals or an equivalent standard that meets or exceeds the ethical and welfare requirements outlined in the NIH Guide. All protocols were approved by the appropriate institutional animal care and use committee.

Data Availability Statements

Original data generated for the study will be made available upon reasonable request to the corresponding author. Data Type: Research Protocols. Reason for Restricted Access: Access is not restricted. The single cell sequencing data was already published and deposited by KPMP and Leyuan Xu. The new data in this paper is from individual animal experiments and will be made available on request.

Supplemental Material

This article contains the following supplemental material online at http://links.lww.com/KN9/B522.

Supplemental Table 1. Primer sequences used for quantitative PCR.

Supplemental Figure 1. BUN levels in mice kidneys and quality control metrics for scRNA-seq of mouse kidney post-IRI.

Supplemental Figure 2. Schematic representation of transwell coculture set up and isolation of GFP+ cells from Arg1fl/fl; LysMCre/+; Rosa26mTmG BMDM.

Supplemental Figure 3. Analysis of growth factor mRNA expression in the outer medulla and cellular source of Igf1 and its receptor.

Supplemental Figure 4. Overview of MACS and FACS cell enrichment.

Supplemental Figure 5. IGF1R phosphorylation time course optimization.

Supplemental Figure 6. Expression levels of GH by macrophages.

Supplemental Figure 7. Full blot images for all the Western blots shown in the Figures.

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Associated Data

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

Data Availability Statement

Original data generated for the study will be made available upon reasonable request to the corresponding author. Data Type: Research Protocols. Reason for Restricted Access: Access is not restricted. The single cell sequencing data was already published and deposited by KPMP and Leyuan Xu. The new data in this paper is from individual animal experiments and will be made available on request.

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