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Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2021 Jul;32(7):1599–1615. doi: 10.1681/ASN.2020121723

Activation of Sympathetic Signaling in Macrophages Blocks Systemic Inflammation and Protects against Renal Ischemia-Reperfusion Injury

Sho Hasegawa 1,2, Tsuyoshi Inoue 2,3,✉, Yasuna Nakamura 1,3, Daichi Fukaya 2,4, Rie Uni 1,2,✉, Chia-Hsien Wu 1,3, Rie Fujii 1,2, Wachirasek Peerapanyasut 2,5, Akashi Taguchi 6, Takahide Kohro 7, Shintaro Yamada 8,9, Mikako Katagiri 8, Toshiyuki Ko 8,9, Seitaro Nomura 8,9, Atsuko Nakanishi Ozeki 6, Etsuo A Susaki 10,11, Hiroki R Ueda 10,11, Nobuyoshi Akimitsu 6, Youichiro Wada 6, Issei Komuro 8, Masaomi Nangaku 1, Reiko Inagi 2
PMCID: PMC8425643  PMID: 33875568

Significance Statement

The detailed role of neural activity in inflammatory diseases is still unclear because it varies according to the disease situation and responsible cell types. This study shows that activation of β2-adrenergic receptor (Adrb2) signaling in macrophages induces the expression of T cell Ig and mucin domain 3 (Tim3), which contributes to anti-inflammatory phenotypic alterations. Experiments using conditional knockout mice reveal that macrophage Adrb2 signaling directly mitigates LPS-induced systemic inflammation and renal ischemia-reperfusion injury (IRI). The adoptive transfer of Adrb2 signal–activated macrophages also protects against renal IRI, in association with the accumulation of Tim3-expressing macrophages in the renal tissue. These results indicate that macrophage Adrb2 signaling plays critical roles in the severity of AKI.

Keywords: acute renal failure, macrophages, cell biology and structure, cell signaling, cell transfer, clinical immunology, cytokines, immunology, ischemia-reperfusion, kidney

Visual Abstract

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Abstract

Background

The sympathetic nervous system regulates immune cell dynamics. However, the detailed role of sympathetic signaling in inflammatory diseases is still unclear because it varies according to the disease situation and responsible cell types. This study focused on identifying the functions of sympathetic signaling in macrophages in LPS-induced sepsis and renal ischemia-reperfusion injury (IRI).

Methods

We performed RNA sequencing of mouse macrophage cell lines to identify the critical gene that mediates the anti-inflammatory effect of β2-adrenergic receptor (Adrb2) signaling. We also examined the effects of salbutamol (a selective Adrb2 agonist) in LPS-induced systemic inflammation and renal IRI. Macrophage-specific Adrb2 conditional knockout (cKO) mice and the adoptive transfer of salbutamol-treated macrophages were used to assess the involvement of macrophage Adrb2 signaling.

Results

In vitro, activation of Adrb2 signaling in macrophages induced the expression of T cell Ig and mucin domain 3 (Tim3), which contributes to anti-inflammatory phenotypic alterations. In vivo, salbutamol administration blocked LPS-induced systemic inflammation and protected against renal IRI; this protection was mitigated in macrophage-specific Adrb2 cKO mice. The adoptive transfer of salbutamol-treated macrophages also protected against renal IRI. Single-cell RNA sequencing revealed that this protection was associated with the accumulation of Tim3-expressing macrophages in the renal tissue.

Conclusions

The activation of Adrb2 signaling in macrophages induces anti-inflammatory phenotypic alterations partially via the induction of Tim3 expression, which blocks LPS-induced systemic inflammation and protects against renal IRI.

The sympathetic nervous system (SNS) plays important roles in the maintenance of homeostasis. The SNS is known to innervate lymphoid organs, including the spleen and lymph nodes.1,2 This anatomic finding suggests that sympathetic neurotransmitters directly affect immune cell dynamics. Although many previous reports have clarified that SNS activity certainly affects immune systems,3,4 the direction of its immunologic regulation is not straightforward and varies according to the disease situation and responsible cell types. For example, activation of sympathetic signaling enhances retention-promoting signals and consequently inhibits lymphocyte egress from lymph nodes (anti-inflammatory direction).5 In contrast, SNS activation induces the accumulation of pathogenic CD4-positive T cells in the fifth lumbar cord, which is involved in the pathogenesis of experimental autoimmune encephalomyelitis (proinflammatory direction).6 Moreover, adrenergic receptors, which receive sympathetic signaling, are ubiquitously expressed in various cell types in the body, which makes it difficult to precisely understand the sympathetic regulation of inflammatory diseases. Thus, the effect of sympathetic signal activation in vivo is heterogeneous, and “the critical receiver of sympathetic signaling (immune cells, epithelial cells, or other cell types)” may differ depending on the situation.

Therefore, it is important to clarify the detailed role of sympathetic signaling in specific cell types in specific inflammatory situations. Hence, in this study, we focused on sympathetic signaling in macrophages and sought to determine its detailed roles in systemic inflammation (LPS-induced sepsis) and local inflammation (renal ischemia-reperfusion injury).

Methods

Cell Culture and Reagents

RAW 264.7 cells (mouse macrophage cell line) were maintained in DMEM–high-glucose media (D5796; Sigma-Aldrich, St. Louis, MO) containing 10% FBS (F7524, lot #BCBT 3830; Sigma-Aldrich). U937 cells (human monocyte cell line) were maintained in RPMI-1640 media (R8758; Sigma-Aldrich) with 10% FBS. U937 cells were differentiated into macrophages by 48-hour stimulation with 100 nM phorbol 12-myristate 13-acetate (P1585; Sigma-Aldrich). All cells were cultured in a humidified 5% CO2-enriched atmosphere at 37°C. TNF-α was induced by 100 ng/ml LPS (L4391; Sigma-Aldrich). L-NE (25304–31; Nacalai tesque, Kyoto, Japan), salbutamol (S8260; Sigma-Aldrich), butoxamine hydrochloride (B1385; Sigma-Aldrich), and the protein kinase A (PKA) inhibitor 14–22 amide (476485; Sigma-Aldrich) were used in this study.

Measurement of TNF-α and ILs

The TNF-α concentration was measured using TNF-α Mouse Uncoated ELISA Kit with Plates (88–7324–22; Thermo Fisher Scientific, Waltham, MA) or TNF-α Human Uncoated ELISA Kit with Plates (88–7346–86; Thermo Fisher Scientific) according to the manufacture’s protocol. The concentrations of IL-6 and IL-10 were measured using Mouse IL-6 ELISA Kit (KE10007; Proteintech, Rosemont, IL) and Mouse IL-10 ELISA Kit (KE10008; Proteintech).

RNA Sequencing

Total RNA was isolated using the RNeasy Mini Kit (74106; Qiagen, Hilden, Germany). Poly (A)–containing mRNA molecules were isolated from the total RNA and then converted to cDNA with poly A primers using a TruSeq RNA Sample Preparation kit v2 (Illumina). High-throughput sequencing was performed using a Hiseq2500 (Illumina) system. Sequenced paired-end reads were mapped onto the mouse genome build mm10 using hisat2 with the parameter “-q–dta-cufflinks.” The SAM file was converted into BAM format, and the fragments per kilobase of transcript per million mapped fragments were subsequently calculated using cuffdiff and cummerBund. Genes commonly induced by β2-adrenergic receptor (Adrb2) signaling were extracted using the log fold change, which was calculated from log(fragments per kilobase of transcript per million mapped fragments +0.001) counts. The data were deposited in the Genomic Expression Archive (GEA) under accession number E-GEAD-404.

Quantitative Real-Time PCR

Total RNA was isolated using the RNeasy Mini Kit (74106) and reverse transcribed using PrimeScript RT master mix (Takara, Shiga, Japan). The cDNA was then subjected to quantitative real-time PCR (qPCR) using the THUNDERBIRD qPCR Mix (Toyobo, Tokyo, Japan) and a CFX96 Real Time System (Bio-Rad). β-actin was used to standardize the mRNA expression levels. The primer sequences are listed in Supplemental Table 1.

Small Interfering RNA Transfection

Small interfering RNA (siRNA) transfection was conducted using Opti-MEM I Reduced Serum Medium (31985070; Thermo Fisher Scientific) and Lipofectamine RNAiMAX Transfection Reagent (13778150; Thermo Fisher Scientific). T cell Ig and mucin domain 3 (Tim3) knockdown was conducted by Silencer Select Pre-Designed siRNA for mouse Tim3 (s101150 [#1] and s101148 [#2]; Thermo Fisher Scientific). Silencer Select Negative Control No. 1 siRNA (4390843; Thermo Fisher Scientific) was used as the negative control.

Gene Overexpression

Tim3 (Havcr2) plasmid (green fluorescent protein tagged; MG227499; Origene Technologies, Inc., Rockville, MD) or empty plasmid was introduced using a Neon Transfection System (Thermo Fisher Scientific) according to the manufacturer’s protocol. The overexpression of Tim3 was validated by immunocytochemistry.

Animal Experiments

Male mice (8–12 weeks of age, 20–25 g) were used for all experiments. Wild-type (WT) C57BL/6 mice were purchased from Nippon Bio-Supp. Center (Tokyo, Japan). Macrophage-specific Adrb2 conditional knockout (cKO) mice were generated by crossbreeding lysozyme M (LysM)-Cre and Adrb2 flox mice. Genotyping was confirmed by tail PCR using published primers. LysM-Cre mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Adrb2 flox mice7 were provided by Wataru Ogawa (Kobe University, Kobe, Japan) and Gerard Karsenty (Columbia University, New York, NY). All experiments were approved by the University of Tokyo Institutional Review Board (approval nos. P18–051 and H19–164). All animal procedures were performed according to the National Institutes of Health guidelines (Guide for the Care and Use of the Laboratory Animals). BUN and plasma creatinine (Cre) levels were measured by SRL Inc. (Osaka, Japan).

Preparation of Peritoneal Macrophages

After the mouse was euthanized, 8 ml of sterile PBS was injected into the peritoneal cavity. The injected fluid was collected after a gentle massage of the peritoneum. The fluid was centrifuged at 500×g for 5 minutes, and the supernatant was discarded. The cell pellets were resuspended in RPMI-1640 media (R8758; Sigma-Aldrich) with 10% FBS and 1% penicillin-streptomycin, and they were plated in culture dishes. The cells were washed with PBS 1 hour after plating, and the culture medium was refreshed. The next day, the cells were used for experiments.

Renal Bilateral Ischemia-Reperfusion Injury

Mice were anesthetized by the intraperitoneal administration of medetomidine 0.3 mg/kg, butorphanol 5 mg/kg, and midazolam 4 mg/kg. Renal bilateral ischemia-reperfusion injury (bIRI) was performed by clamping the renal pedicles for 26 minutes. The clamps were removed, and the wound was sutured after restoration of blood flow was visually observed. Sham-operated mice underwent the same procedure, without clamping of the renal pedicles.

Splenectomy

Mice were anesthetized by the intraperitoneal administration of medetomidine 0.3 mg/kg, butorphanol 5 mg/kg, and midazolam 4 mg/kg. The splenic vasculature was ligated, and the spleen was removed via a small incision.

Immunohistochemistry

Kidney samples were fixed in Mildform 10 N (133–10311; Wako, Osaka, Japan) before being embedded in paraffin. Tissue sections were subjected to periodic acid–Schiff staining for the histologic examination of tubular necrosis. Semiquantitative tubular injury scores were graded on the basis of the proportion of injured tubules as follows: (0) none, (1) <25%, (2) 25%–50%, (3) 50%–75%, and (4) >75%. The average score of four fields in the outer medulla was calculated for each sample.

For Tim3 immunostaining, anti-Tim3 rabbit mAb (1:200, 83882S; Cell Signaling, Danvers, MA) was used as the primary antibody. The sections were stained with Histofine Simple Stain Mouse MAX PO (R) (414341; Nichirei, Tokyo, Japan) and ImmPACT DAB substrate (SK-4105; Vector, Burlingame, CA), which were then counterstained with Mayer Hematoxylin (Wako).

Three-Dimensional Visualization of the SNS in the Spleen

Three-dimensional (3D) visualization of the SNS in the spleen was performed using Clear, Unobstructed Brain/Body Imaging Cocktails and Computational (CUBIC)8–10 analysis as described in our previous papers.11,12 In brief, the fixed mouse spleen was immersed in CUBIC-L for delipidation and then subjected to immunofluorescence staining. Finally, the refractive index was matched by the placement of the sample in CUBIC-R+. The primary antibody used for staining was antityrosine hydroxylase antibody (sheep polyclonal, 1:100, ab113; Abcam, Cambridge, United Kingdom). The secondary antibody was Alexa Fluor 555–conjugated donkey anti-sheep IgG (1:100, A-21436; Invitrogen, Carlsbad, CA). The raw image data were acquired with a custom-built light sheet fluorescence microscopy (MVX10-LS; developed by Olympus, Tokyo, Japan). The 3D-rendered image was visualized with Imaris (Bitplane).

Adoptive Transfer of Splenic Macrophages

Spleens were harvested from donor mice, and single-cell suspensions were made through a 40-μm cell strainer with sterile PBS. The single-cell suspensions were labeled with anti-F4/80 MicroBeads (130–110–443; Miltenyi Biotec, Bergisch Gladbach, Germany), and F4/80-positive splenocytes were selected by the magnetic cell separation method. The cells were incubated with vehicle or 100 µM salbutamol for 1 hour, and they were washed twice with PBS. The macrophages were intravenously administered to recipient mice.

Single-Cell RNA Sequencing

Digestion buffer was prepared as a mixture of DMEM–high-glucose media (D5796) with 10% FBS (F7524, lot #BCBT 3830), RQ1 RNase-Free DNase (20 U/ml, M6101; Promega, Madison, WI), collagenase type 1 (2 mg/ml, CLS1; Worthington, Columbus, OH), collagenase type 2 (2 mg/ml, CLS2, Worthington), and Dispase II (1 mg/ml, 04942078001; Roche, Mannheim, Germany). Kidneys were harvested, minced into 1-mm3 cubes, and digested using the digestion buffer with shaking at 37°C. The supernatant buffer was collected after shaking for 10 minutes, and fresh digestion buffer was added to the remaining cell aggregations. After this process was repeated four times, the solution was passed through 70- and 40-μm cell strainers successively. The solution was then centrifuged at 300×g for 5 minutes, and the resulting cell pellet was diluted with the digestion buffer again. The solution was passed through 40-μm cell strainers three times, and a single-cell suspension was obtained.

The single-cell suspension was loaded onto a well on the 10× Chromium Single Cell instrument (10× Genomics, Pleasanton, CA). Bar coding and cDNA synthesis were performed using Chromium Single Cell 3’ Reagent Kits v3.1 (10× Genomics) according to the manufacturer’s instructions. High-throughput sequencing was performed using a NovaSeq6000 (Illumina) system at Takara Bio Inc. (Shiga, Japan).

Data Processing and Analysis of Single-Cell RNA-Sequencing Data

The raw data were processed using Cell Ranger v3.1 (10× Genomics) to obtain the filtered feature bar code matrices. Seurat v3 was used for the detailed analysis. We analyzed each sample separately and excluded cells with <200 or >5000 genes detected or with <20,000 unique molecular identifiers detected. We also excluded cells with a relatively high percentage of genes mapped to mitochondrial genes (≥50%). Subsequently, we log normalized the data and obtained 2000 highly variable genes for principal component analysis (PCA) from each dataset with “FindVariableFeatures.” We then merged the list with the standard workflow using “FindIntegrationAnchors” and “IntegrateData.” We subsequently performed PCA for the integrated data using the variable genes and determined significant principal components on the basis of the jackstraw. Clustering was performed using “FindNeighbors” and subsequently, “FindClusters” with a resolution of 0.6. We visualized the data on t-distributed stochastic neighbor embedding (tSNE) using “RunTSNE.” Marker genes in each cluster were identified using “FindAllMarkers” with min.pct=0.25 and logfc.threshold=0.25. The marker genes sorted by the average log fold change are presented in Supplemental Table 2. Macrophage subclustering was performed as follows. First, we extracted the macrophage cluster and changed the default assay from “integrated” to “RNA.” Then, we log normalized the data and obtained 2000 highly variable genes for PCA with “FindVariableFeatures.” Clustering was performed using “FindNeighbors” and subsequently, “FindClusters” with a resolution of 0.8. We visualized the data on tSNE using “RunTSNE.” The data were deposited in the GEA under accession number E-GEAD-405.

Statistical Analyses

All data are presented as means ± SEM. An unpaired two-tailed t test was used to analyze the data for only two groups. For multiplex comparisons, a one-way or two-way ANOVA followed by a post hoc Tukey multiple comparisons test, if appropriate, was applied. P<0.05 was considered statistically significant. All statistical analyses were performed with GraphPad Prism 8 software (GraphPad Software, San Diego, CA).

Results

Activation of Sympathetic Signaling Suppresses the Inflammatory Response of Macrophages via Adrb2

We first examined the effects of sympathetic signaling on the inflammatory response of macrophages using RAW 264.7 cells (mouse macrophage cell line). Norepinephrine (NE), a sympathetic neurotransmitter, suppressed TNF-α induction by LPS in a dose-dependent manner (Figure 1A). This anti-inflammatory effect was mitigated by butoxamine, a selective Adrb2 antagonist (Figure 1B). The dose-dependent anti-inflammatory effect was also induced by salbutamol, a selective Adrb2 agonist (Figure 1C, Supplemental Figure 1). Thus, activation of sympathetic signaling suppresses the inflammatory response of macrophages via Adrb2. Furthermore, we conducted the same experiment using mouse peritoneal macrophages and differentiated U937 cells (human macrophages) and confirmed that the anti-inflammatory effect of Adrb2 signaling was common in macrophages of various origins (Supplemental Figure 2).

Figure 1.

Figure 1.

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Activation of sympathetic signaling suppresses the inflammatory response of macrophages via Adrb2. (A) TNF-α induction by LPS was suppressed by NE treatment in RAW 264.7 cells (n=6). (B) Butoxamine mitigated the anti-inflammatory effect of NE in RAW 264.7 cells (n=8). (C) Salbutamol also suppressed TNF-α induction by LPS in RAW 264.7 cells (n=6). All data are presented as means ± SEM. Statistical comparisons were analyzed by (B) one-way ANOVA or (A and C) two-way ANOVA with a post hoc Tukey multiple comparisons test. n.s., not significant. **P<0.01; ***P<0.001; ****P<0.0001.

Tim3 Is a Mediator of Anti-Inflammatory Effect Induced by Adrb2 Signaling

We attempted to identify the critical gene that mediates the anti-inflammatory effect of Adrb2 signaling. RNA sequencing was conducted for three different in vitro macrophage models. Adrb2 signaling–induced genes were respectively selected in each model: (1) 707 genes, (2) 991 genes, and (3) 1189 genes (Figure 2A, Supplemental Figure 3). Among 37 genes, which were commonly selected in the three models (Supplemental Table 3), we focused on Tim3, given its reported anti-inflammatory role in immune cells. qPCR also confirmed that Tim3 expression was upregulated by the salbutamol treatment in RAW 264.7 cells and peritoneal macrophages (Figure 2B). Moreover, the inhibition of PKA, the canonical Adrb2 signaling downstream pathway component, counteracted the salbutamol-induced upregulation of Tim3 expression in macrophages, confirming that Tim3 is downstream of Adrb2 signaling (Figure 2C).

Figure 2.

Figure 2.

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Tim3 is downstream of Adrb2 signaling and partially mediates its anti-inflammatory effects. (A) RNA sequencing was conducted for three different in vitro macrophage models. The detailed experiment protocols are shown in Supplemental Figure 3: (1) 707 genes were extracted with log fold change (log FC) more than one (NE per vehicle) and log FC less than zero (NE + butoxamine/NE), (2) 991 genes were extracted with log FC more than one (salbutamol per vehicle), and (3) 1189 genes were extracted with log FC more than one (salbutamol per vehicle). A total of 37 genes, including Tim3, were commonly induced by the activation of Adrb2 signaling in the three different models. (B) qPCR confirmed that Tim3 expression was upregulated by the salbutamol treatment in RAW 264.7 cells and peritoneal macrophages (Pmacs; n=3). (C) Tim3 induction by salbutamol was counteracted by the inhibition of PKA in RAW 264.7 cells (n=6). (D) Tim3 knockdown by siRNA partly inhibited the anti-inflammatory effect of salbutamol in RAW 264.7 cells (n=6). All data are presented as means ± SEM. (B) An unpaired two-tailed t test was used to analyze the data for only two groups. For multiplex comparisons, (C) a one-way ANOVA or two-way (D) ANOVA with a post hoc Tukey multiple comparisons test was applied. PKI, protein kinase A inhibitor; siNC, negative control siRNA; siTim3, Tim3 knockdown by siRNA. **P<0.01; ***P<0.001; ****P<0.0001.

Next, we examined the anti-inflammatory role of Tim3 expression in the macrophage inflammatory response. Tim3 knockdown by siRNA partially inhibited the anti-inflammatory effect of salbutamol in RAW 264.7 cells (Figure 2D, Supplemental Figure 4). In contrast, Tim3 overexpression suppressed the inflammatory response of RAW 264.7 cells (Supplemental Figure 5). Thus, Tim3 is downstream of Adrb2 signaling and partially mediates the anti-inflammatory phenotypic alteration, which cannot be simply explained by the conventional macrophage M1/M2 axis (Supplemental Figure 6).

Adrb2 Signaling in Macrophages Plays a Critical Role in the In Vivo Systemic Inflammatory Response

Next, we attempted to clarify the role of macrophage Adrb2 signaling in the systemic inflammatory response using the LPS-induced mouse septic model. First, LPS (5 mg/kg) was intraperitoneally administered to WT mice, immediately after intraperitoneal administration of vehicle or salbutamol (15 mg/kg). We measured plasma cytokine levels as the parameter of the systemic inflammatory response 4 hours after LPS administration (Figure 3A). As a result, plasma TNF-α and IL-6 levels were significantly lower in the salbutamol-treated group than in the vehicle-treated group, whereas the level of IL-10, an anti-inflammatory cytokine, was higher in the salbutamol-treated group. Although renal histologic injury was not apparently observed at this stage, the expression of neutrophil gelatinase–associated lipocalin, one of the AKI biomarkers, was significantly reduced in the salbutamol-treated group, suggesting the protective effects of Adrb2 signaling against kidney injury (Supplemental Figure 7).

Figure 3.

Figure 3.

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Adrb2 signaling in macrophages plays a critical role in the in vivo systemic inflammatory response. (A) LPS was intraperitoneally administered to WT mice immediately after the intraperitoneal administration of vehicle or salbutamol. Plasma TNF-α and IL-6 levels were reduced, whereas plasma IL-10 level was increased by the salbutamol treatment (n=3 or n=5). (B) Macrophage-specific Adrb2 cKO mice were generated by crossbreeding LysM-Cre and Adrb2 flox mice. LPS was intraperitoneally administered to Adrb2 cKO mice and their littermate WT mice immediately after the intraperitoneal administration of vehicle or salbutamol. Deletion of Adrb2 on macrophages partially abolished the salbutamol-induced suppression of the systemic inflammatory response (n=10). All data are presented as means ± SEM. Statistical comparisons were analyzed by a two-way ANOVA with a post hoc Tukey multiple comparisons test. n.s., not significant. *P<0.05; ***P<0.001; ****P<0.0001.

However, we were unable to determine whether the systemic anti-inflammatory effect of salbutamol was associated with macrophages because sympathetic signaling plays various physiologic roles in many cell types in the body. Thus, we generated macrophage-specific Adrb2 cKO mice by crossbreeding LysM-Cre and Adrb2 flox mice and conducted the same experiment (Figure 3B). Deletion of Adrb2 on macrophages then partially abolished the salbutamol-induced suppression of the systemic inflammatory response, showing the importance of macrophages in this context.

Salbutamol Pretreatment Protects the Kidney from bIRI

Thus far, we clarified that Adrb2 signaling on macrophages plays a critical role in the systemic inflammatory response. Next, we aimed to determine the role of macrophage Adrb2 signaling in local acute inflammation. For this purpose, we opted to utilize the renal bIRI model, which is generated by 26 minutes of renal ischemia followed by 24 hours of reperfusion.

Vehicle or salbutamol (15 mg/kg) was intraperitoneally administered 24 hours before renal bIRI. Blood and kidney samples were obtained 24 hours after renal bIRI (Figure 4A). Pretreatment with salbutamol provided strong protection from kidney injury as shown by the lower BUN, lower plasma Cre (Figure 4B), and lesser degree of histologic tubular injury (Figure 4, C and D). Thus, systemic pretreatment with salbutamol protects the kidney from bIRI.

Figure 4.

Figure 4.

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Pretreatment with salbutamol protects against renal ischemia-reperfusion injury. (A) The study protocol is shown. Vehicle or salbutamol (15 mg/kg) was intraperitoneally administered to WT mice 24 hours before renal bIRI. Blood and kidney samples were obtained 24 hours after bIRI. (B) BUN and plasma Cre levels are shown (n=3 or n=6). (C) Representative periodic acid–Schiff staining of the renal outer medulla is shown. Scale bars: 100 μm. (D) Histologic tubular injury scores are shown (n=3 or n=6). (E) The distribution of SNS in the spleen is visualized by tissue clearing and 3D immunofluorescence staining of tyrosine hydroxylase. All data are presented as means ± SEM. Statistical comparisons were analyzed by a two-way ANOVA with a post hoc Tukey multiple comparisons test. n.s., not significant. *P<0.05; **P<0.01; ****P<0.0001.

Splenic Immune Cells May Play Important Roles in the Protective Effect of Salbutamol against Renal bIRI

Next, we tested whether immune systems were involved in the salbutamol-induced protection against renal bIRI. The spleen is central to the immune system, and splenic immune cell dynamics are thought to be influenced by the degree of sympathetic signaling because the sympathetic nerves are densely distributed in the spleen, as visualized by our tissue clearing–based 3D immunofluorescence staining of sympathetic nerves (Figure 4E). We conducted a splenectomy 10 days before the bIRI experiment. As a result, the protective effects of salbutamol against renal bIRI were not observed in splenectomized mice (Supplemental Figure 8), suggesting the importance of splenic immune cells in the renoprotective effects of salbutamol.

Macrophage Adrb2 Signaling Is Critical for the Protective Effect of Salbutamol against Renal bIRI

In order to determine whether macrophages are involved in the salbutamol-induced protection against renal bIRI, we conducted renal bIRI experiments using macrophage-specific Adrb2 cKO and littermate WT mice (Figure 5A). As a result, deletion of Adrb2 on macrophages abolished the salbutamol-induced protection against renal bIRI, as shown by the reversal of plasma Cre levels (Figure 5B) and histologic tubular injury scores (Figure 5, C and D). Thus, the protective effects of salbutamol against renal bIRI are primarily due to the activation of Adrb2 signaling in macrophages.

Figure 5.

Figure 5.

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Macrophage Adrb2 signaling is critical for the protective effect of salbutamol pretreatment against renal ischemia-reperfusion injury. (A) The study protocol is shown. Macrophage-specific Adrb2 cKO and littermate WT mice were used in this experiment. Vehicle or salbutamol (15 mg/kg) was intraperitoneally administered 24 hours before renal bIRI. Blood and kidney samples were obtained 24 hours after bIRI. (B) BUN and plasma Cre levels are shown (n=15–17). (C) Representative periodic acid–Schiff staining of the renal outer medulla is shown. Scale bars: 100 μm. (D) Histologic tubular injury scores are shown (n=12–14). All data are presented as means ± SEM. Statistical comparisons were analyzed by a one-way ANOVA with a post hoc Tukey multiple comparisons test. *P<0.05; **P<0.01; ****P<0.0001.

Adoptive Transfer of Salbutamol-Treated Macrophages Protects the Kidney from bIRI

Given that Adrb2 signaling on macrophages is critical to protection against kidney injury, we pondered whether Adrb2 signal–activated macrophages themselves provide protection against renal bIRI and conducted the adoptive transfer experiment (Figure 6A). Adoptive transfer of 1.6×105 salbutamol-treated (Adrb2 signal–activated) macrophages from donor mice protected the kidneys from bIRI in recipient mice, as shown by the lower BUN, lower plasma Cre (Figure 6B), and lesser degree of histologic tubular injury (Figure 6C).

Figure 6.

Figure 6.

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Adoptive transfer of salbutamol-treated macrophages protects against renal ischemia-reperfusion injury. (A) The study protocol is shown. Salbutamol-treated macrophages from donor mice were adoptively transferred to recipient mice 18 hours before bIRI. Blood and kidney samples were obtained 24 hours after bIRI. (B) BUN and plasma Cre levels are shown (n=3 or n=7). (C) Representative periodic acid–Schiff staining of the renal outer medulla and histologic tubular injury scores are shown (n=3 or n=7). All data are presented as means ± SEM. Statistical comparisons were analyzed by a two-way ANOVA with a post hoc Tukey multiple comparisons test. Mφ, macrophage; n.s., not significant; i.v., intravenous. Scale bars: 100 μm. *P<0.05; **P<0.01; ***P<0.001.

Next, we performed single-cell RNA sequencing (scRNA-seq) of renal tissues to analyze the renoprotective role of salbutamol-treated (Adrb2 signal–activated) macrophages in detail (Figure 7A). We visualized the single-cell datasets using tSNE and identified 19 cell-type clusters (Figure 7B, Supplemental Table 2). For example, the expression of kidney androgen–regulated protein clearly identified proximal tubular cells in the S3 segment, the most vulnerable sections to ischemic injury (Figure 7B, Supplemental Figure 9A). The violin plot of kidney injury molecule 1 (Kim1) expression in this cluster showed that the degree of tubular injury was lower in the bIRI after salbutamol-treated macrophage transfer (Sal_bIRI) condition than in the bIRI after vehicle-treated macrophage transfer (Ctl_bIRI) condition (Figure 7C).

Figure 7.

Figure 7.

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Tim3-expressing macrophages are accumulated in the injured kidney after salbutamol-treated macrophage transfer. (A) The study protocol is shown. Four renal tissues from the adoptive transfer experiment were analyzed by scRNA-seq. The BUN and plasma Cre levels of each mouse are shown. (B) tSNE plots of the single-cell data are visualized with sample information (left panel) and cell-type information (right panel). The total numbers of cells in each sample are as follows: 1667 cells (sham operation after vehicle-treated macrophage transfer [Ctl_sham]), 2128 cells (bilateral ischemia-reperfusion injury after vehicle-treated macrophage transfer [Ctl_bIRI]), 1653 cells (sham operation after salbutamol-treated macrophage transfer [Sal_sham]), and 1932 cells (bilateral ischemia-reperfusion injury after salbutamol-treated macrophage transfer [Sal_bIRI]). (C) The violin plot of kidney injury molecule 1 (Kim1) expressions in the PT (S3) cluster is drawn. The total numbers of cells in this cluster are as follows: 128 cells (Ctl_sham), 139 cells (Ctl_bIRI), 93 cells (Sal_sham), and 136 cells (Sal_bIRI). (D) The violin plot of Tim3 expressions in the macrophage cluster is drawn. The total numbers of cells in this cluster are as follows: 30 cells (Ctl_sham), 71 cells (Ctl_bIRI), 45 cells (Sal_sham), and 91 cells (Sal_bIRI). Tim3-expressing macrophages were accumulated in the injured kidney after salbutamol-treated macrophage transfer (17% [Ctl_sham], 21% [Ctl_bIRI], 4% [Sal_sham], and 33% [Sal_bIRI] of total macrophages in the renal tissue). ATL, ascending thin limb; CD_IC, intercalated cell of the collecting duct; CD_PC, principal cell of the collecting duct; DCT, distal convoluted tubular cell; DTL, descending thin limb; EC, endothelial cell; Mφ, macrophage; PT (S1/2), proximal tubular cell in the S1/2 segments; PT (S3), proximal tubular cell in the S3 segment; SMC, smooth muscle cell; TAL, thick ascending limb; i.v., intravenous.

We especially focused on Tim3 expression in the macrophage cluster because high Tim3 expression is a marker of Adrb2 signal–activated macrophages according to our in vitro data (Figure 2). The macrophage cluster was clearly identified in the tSNE plot of single-cell data (Figure 7B) by the expression of two representative markers, Cd68 and LysM (Supplemental Figure 9B). The violin plot of Tim3 expression in this cluster illustrated that Tim3-expressing macrophages (Adrb2 signal–activated macrophages) were particularly accumulated in the renal tissue of the bIRI after salbutamol-treated macrophage transfer condition (Figure 7D), which was also confirmed by immunohistochemistry (Supplemental Figure 10). Furthermore, we investigated the origin of these Tim3-expressing macrophages from scRNA-seq data. Unbiased subclustering of macrophages yielded four subclusters (Figure 8A). Clusters 1 and 2 were mostly composed of macrophages from bIRI groups (circulating macrophages), whereas cluster 3 was composed of macrophages from sham groups, and cluster 4 contained macrophages from both groups (tissue-resident macrophages) (Figure 8B). As Tim3 was preferentially expressed on the left side of the tSNE plots (Figure 8C), the accumulating Tim3-expressing macrophages after bIRI might be circulating macrophages. Therefore, Adrb2 signal–activated macrophages with high Tim3 expression may come from outside the renal tissues and play critical roles in the protection against renal bIRI.

Figure 8.

Figure 8.

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The accumulating Tim3-expressing macrophages after bIRI are mainly composed of circulating macrophages. (A) Unbiased subclustering of macrophages is visualized as the tSNE plots. (B) In the tSNE plots with sample information, clusters 1 and 2 are mostly composed of macrophages from bIRI groups (circulating macrophages), whereas cluster 3 is composed of macrophages from sham groups, and cluster 4 contains macrophages from both groups (tissue-resident macrophages). (C) The expression levels of Tim3 are visualized on the tSNE plots. Tim3-expressing macrophages were preferentially distributed on the left side (circulating macrophages). Ctl_bIRI, bilateral ischemia-reperfusion injury after vehicle-treated macrophage transfer; Ctl_sham, sham operation after vehicle-treated macrophage transfer; Mφ, macrophage; Sal_bIRI, bilateral ischemia-reperfusion injury after salbutamol-treated macrophage transfer; Sal_sham, sham operation after salbutamol-treated macrophage transfer.

Discussion

In this study, we demonstrated that activation of Adrb2 signaling in macrophages blocks LPS-induced systemic inflammation and protects the kidney from IRI. Furthermore, Adrb2 signaling induces Tim3 expression, which contributes to anti-inflammatory phenotypic alterations in macrophages.

Adrb2 agonists are known to exert anti-inflammatory effects in several inflammatory diseases, such as LPS-induced sepsis3,13 and acute lung injury.14 However, these in vivo observations could not precisely identify the cell type responsible for the anti-inflammatory effect. In our data, the anti-inflammatory effect of salbutamol was mitigated in monocyte-derived macrophage-specific Adrb2 knockout mice (Figure 3), demonstrating the importance of macrophage Adrb2 signaling in this process. Although sympathetic signaling is known to affect other immune cells, including CD4-positive T cells4 and tissue-resident macrophages,15,16 our data clearly demonstrated the importance of monocyte-derived macrophages as the critical receiver of sympathetic signaling in LPS-induced systemic inflammation and renal IRI.

Adrb2 signaling has various effects on local inflammation in the kidney (diabetic kidney disease,17 nephrotoxic injury,18 and septic kidney injury19) because adrenergic receptors are ubiquitously expressed in the body. Of note, the Adrb2 agonist formoterol (postinjury administration) was reported to restore kidney function after renal IRI via mitochondrial biogenesis in the proximal tubules.20,21 In contrast, we observed the anti-inflammatory effect of salbutamol pretreatment in the acute phase of renal IRI, which was mainly derived from macrophage Adrb2 signaling (Figures 4 and 5, Supplemental Figure 8). The adoptive transfer of Adrb2 signal–activated macrophages also protected the kidney from IRI (Figures 6–8). Taken together, the critical receiver of Adrb2 signaling in renal IRI may differ depending on the timing: Macrophages are important in the acute injury phase, and proximal tubules are important in the recovery phase. Future studies are needed to clarify the interaction between these receivers of Adrb2 signaling and the renal SNS. Prior denervation of renal SNS is known to alleviate the severity of renal IRI in animal studies.22 In contrast, we have previously shown that the renal SNS is denervated after IRI, which results in NE depletion inside the kidney.11 Thus, determination of the influence of renal SNS activity or damage on the dynamics of macrophages and the proximal tubules may aid elucidation of the SNS’s role in kidney disease progression.

In this study, we also found that Tim3 expression was induced by the activation of Adrb2 signaling in macrophages (Figure 2). Tim3 was included in the 37 genes, which were commonly selected as Adrb2 signal–induced genes from our RNA sequencing data of three in vitro macrophage models (Supplemental Table 3). Tim3 is expressed on many immune cells, including T cells,23,24 natural killer cells,25 and dendritic cells,26 and suppresses the immunologic activity as one of the immune checkpoints. Previous studies have shown that Tim3 is also expressed on macrophages and plays immunosuppressive roles.27–30 In our data, Tim3 knockdown promoted the inflammatory response of macrophages and mitigated the anti-inflammatory effect of salbutamol (Figure 2D, Supplemental Figure 4). In contrast, Tim3 overexpression attenuated the inflammatory response of macrophages (Supplemental Figure 5). Taken together, these findings indicate that Tim3 is downstream of Adrb2 signaling and contributes to its anti-inflammatory effect, which cannot be simply explained by the conventional macrophage M1/M2 axis (Supplemental Figure 6). In our scRNA-seq dataset, Tim3-expressing macrophages were accumulated in the renal tissue of IRI after the adoptive transfer of Adrb2 signal–activated macrophages (Figure 7D, Supplemental Figure 10), in association with neutrophil’s phenotypic change and reduced renal tubular injury (Figure 7C, Supplemental Figure 11–13). Thus, Tim3-expressing macrophages induced by Adrb2 signaling may elicit direct protective effects against renal IRI.

Although the mechanism of Tim3 induction by Adrb2 signaling was not clarified in detail, we at least confirmed that Tim3 expression induced by Adrb2 activation was dependent on PKA signaling (canonical downstream component of Adrb2 signaling) in macrophages (Figure 2C). As cAMP/PKA signaling is reported to induce promoter/enhancer activity of Tim3 in Jurkat T cells,31 the induction of Tim3 in macrophages may also be explained by promoter/enhancer activity.

The spleen is widely innervated by sympathetic nerves, which is also illustrated in our immunofluorescence staining (Figure 4E). Thus, splenic immune cells may be susceptible to sympathetic signaling. In our experiments, prior splenectomy abolished the protective effects of salbutamol against renal IRI (Supplemental Figure 8). In addition, the adoptive transfer of Adrb2 signal–activated splenic macrophages provided strong protection against renal IRI (Figures 6–8). Thus, splenic macrophages may play important roles in the anti-inflammatory effects of systemic salbutamol administration. The critical role of the spleen in neuroimmune interactions was also reported in the cholinergic anti-inflammatory pathway.32–35 The cholinergic anti-inflammatory pathway, which is activated by electrical stimulation of the vagus nerve, is mediated by the spleen.36–38 Vagus nerve induces the activation of splenic sympathetic nerves, leading to the NE-induced activation of CD4-positive T cells in the spleen.39–41 Various types of splenic immune cells, including macrophages and T cells, may interact with one another and alter the immunologic dynamics in responding to sympathetic signaling. Further studies using single-cell analysis are needed to elucidate the immunologic dynamics in the spleen in response to splenic SNS activation.

Finally, it is important to determine whether the anti-inflammatory role of ADRB2 signaling can be applied in clinical treatment for inflammatory diseases. In our study, ADRB2 activation induced an anti-inflammatory response in differentiated U937 cells (human macrophages) as well as mouse macrophages (Figure 1, Supplemental Figure 2), suggesting that our concept can be applied to human inflammatory diseases. Indeed, genetic variation of ADRB2 was associated with increased mortality and more organ dysfunction in septic shock in a clinical study.42 However, a multicenter, randomized controlled trial (BALTI-2) failed to show the benefits of intravenous salbutamol treatment in the course of acute respiratory distress syndrome.43 In this previous study, salbutamol treatment was poorly tolerated due to the problem of hemodynamics, including tachycardia and arrhythmia.43 Thus, cell type–specific activation of ADRB2 signaling may be needed to prevent such cardiac side effects. In our data, macrophages were the critical receiver of Adrb2 signaling in the protection against renal IRI. As demonstrated in our experiment (Figures 6–8), the adoptive transfer of ADRB2 signal–activated immune cells is a promising candidate for clinical application, although infection risks during immune cell harvest and treatment cannot be ignored in the clinical setting.

Our study has several limitations. First, the scRNA-seq data in our study lacked sufficient statistical power with respect to the dispersion of Kim1 expression in proximal tubules and Tim3 expression in macrophages in the renal tissues (Figure 7). Although we need to increase kidney samples to make a conclusion by the scRNA-seq data alone, the results were compatible with in vitro and in vivo data in the rest of our study, providing the potential mechanism for the protection against renal IRI induced by the adoptive transfer of salbutamol-treated macrophages. Second, our study did not examine how the Adrb2 signal–activated macrophages affect other immune cell dynamics, such as T cells, B cells, natural killer cells, and dendritic cells. The renoprotective effect of the adoptive transfer of salbutamol-treated macrophages might be partially induced by the interaction with other immune cells. The analysis of immune cell dynamics in the lymphoid tissues after the adoptive transfer of Adrb2 signal–activated macrophages would provide additional insights concerning the role of macrophages in the whole immune dynamics.

In conclusion, the activation of Adrb2 signaling in macrophages induces anti-inflammatory phenotypic alterations partially via the induction of Tim3 expression, which blocks LPS-induced systemic inflammation and protects against renal IRI. Our data provide important insights concerning neuroimmune interactions in the pathophysiology of inflammatory diseases.

Disclosures

The Division of CKD Pathophysiology, University of Tokyo is financially supported by Kyowa Kirin Co. Ltd. R. Inagi reports research funding from Kyowa Kirin Co. Ltd. and Nipro; honoraria from Alexion, Bayer, Chugai, Kyowa Kirin Co. Ltd., Mitsubishi Tanabe, MSD, Research and Development Support Center, Kowa, and Sumitomo Dainippon; scientific advisor or membership as associate editor of Clinical and Experimental Pharmacology Physiology and K360 and as editorial board member of American Journal of Physiology Renal Physiology, Kidney International, and Kidney Research and Clinical Practice; and other interests/relationships as councilor and secretary of the Japanese Society of Nephrology (international liaison committee) and the International Maillard Reaction Society (IMARS; chief editor of IMARS Highlights). I. Komuro reports research funding from AstraZeneca, Japan, Astellas Pharma Inc., Bayer Yakuhin, Ltd., Daiichi Sankyo Company, Limited, Kowa Pharmaceutical Co. Ltd., Mitsubishi Tanabe Pharma Corporation, ONO PHARMACEUTICAL CO., LTD., Otsuka Pharmaceutical Co., Ltd., Sanofi, K.K., Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited, and Teijin Pharma Limited and honoraria from AstraZeneca, Japan, Daiichi Sankyo Company, Limited, Mitsubishi Tanabe Pharma Corporation, ONO PHARMACEUTICAL CO., LTD., and Takeda Pharmaceutical Company Limited. M. Nangaku reports honoraria from AstraZeneca and Boehringer Ingelheim and scientific advisor or membership with Akebia, Astellas, Bayer, Boehringer-Ingelheim, Daiichi-Sankyo, GlaxoSmithKline, Japan Tobacco Inc., Kyowa Kirin Co. Ltd., and Mitsubishi-Tanabe.E. A. Susaki and H. R. Ueda are coinventors of patents owned by RIKEN.E. A. Susaki reports patents and inventions with Tokyo Chemical Industry, CO, LTD; speakers bureau with Tokyo Chemical Industry, CO, LTD; and other interests/relationships as a senior researcher at CUBICStars Co. H. R. Ueda reports ownership interest in CUBICStars Inc.; research funding from Olympus Corporation; and patents and inventions with CUBICStars Inc. (inventions on CUBIC-HV reagents) and RIKEN (inventions on CUBIC reagents). All remaining authors have nothing to disclose.

Funding

This work was supported by Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Japan Society for JSPS Research Fellow JSPS KAKENHI grant 19J11928 (to S. Hasegawa); MSD Life Science Foundation (S. Hasegawa and T. Inoue); Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (B) JSPS KAKENHI grants 18H02727 (to R. Inagi) and 18H02824 (to M. Nangaku); Kyowa Kirin Co. Ltd. (R. Inagi); Japan Agency for Medical Research and Development grants JP20gm6210013 (to T. Inoue) and JP20gm6210010 (to S. Nomura); Japan Society for the Promotion of Science Grant-in-Aid for Research Activity Start-Up and for Young Scientists JSPS KAKENHI grants 18H06192 (to T. Inoue) and 20K17242 (to T. Inoue); Kidney Foundation grant JKFB18-3 (to T. Inoue); Salt Science Research Foundation grant 1919 (to T. Inoue); Smoking Research Foundation (to T. Inoue); Yukiko Ishibashi Foundation (to T. Inoue); Mochida Memorial Foundation (to T. Inoue); Takeda Science Foundation (to T. Inoue); Astellas Foundation for Research on Metabolic Disorders (to T. Inoue); and Suzuken Memorial Foundation (to T. Inoue). This work was partially carried out under the support of Isotope Science Center, University of Tokyo. Part of this study was performed in collaboration with Olympus Corporation and with kind software support by Bitplane.

Supplementary Material

Supplemental Figure 3
ASN.2020121723.DCSupplemental.html (1KB, html)
Supplemental Data
ASN.2020121723SupplementaryData1.pdf (5.5MB, pdf)
Supplemental Table 1
ASN.2020121723SupplementaryData2.xlsx (11KB, xlsx)
Supplemental Table 2
ASN.2020121723SupplementaryData3.xlsx (396.1KB, xlsx)
Supplemental Table 3
ASN.2020121723SupplementaryData4.xlsx (16.4KB, xlsx)

Acknowledgments

The authors are grateful to Dr. Naoki Kuramoto, Dr. Yu Hirata, Prof. Wataru Ogawa (Kobe University), and Prof. Gerard Karsenty (Columbia University) for providing the Adrb2 flox mice. The authors also thank Ms. Ikumi Okuaki, Ms. Rieko Matsuda, Ms. Kahoru Amitani, Ms. Sayaka Hayashi, and Ms. Nanako Shida for their technical support.

The illustrations of mice, kidneys, and petri dishes were obtained from TogoTV (2016 DBCLS TogoTV).

Sho Hasegawa performed the main experiments, analyzed the data, and wrote the original draft; Nobuyoshi Akimitsu, Rie Fujii, Daichi Fukaya, Tsuyoshi Inoue, Yasuna Nakamura, Atsuko Nakanishi Ozeki, Wachirasek Peerapanyasut, Rie Uni, and Chia-Hsien Wu performed or contributed to the animal experiments; Takahide Kohro, Akashi Taguchi, and Youichiro Wada performed the Hiseq2500 run and data processing in the RNA sequencing analysis; Mikako Katagiri, Toshiyuki Ko, Issei Komuro, Seitaro Nomura, and Shintaro Yamada aided our scRNA-seq analysis; Etsuo A. Susaki and Hiroki R. Ueda aided 3D visualization using tissue clearing; Reiko Inagi, Tsuyoshi Inoue, and Masaomi Nangaku supervised this study and revised the manuscript; and all authors approved the final version of 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.2020121723/-/DCSupplemental.

Supplemental Figure 1. The effect of β2-adrenergic receptor signaling on various inflammatory cytokines.

Supplemental Figure 2. The anti-inflammatory effect of β2-adrenergic receptor signaling is common in macrophages of various origins.

Supplemental Figure 3. The detailed protocols of RNA sequencing experiments.

Supplemental Figure 4. The effect of T cell Ig and mucin domain 3 knockdown using another small interfering RNA on the inflammatory response of macrophages.

Supplemental Figure 5. The effect of T cell Ig and mucin domain 3 overexpression on the inflammatory response of macrophages.

Supplemental Figure 6. The effect of β2-adrenergic receptor signaling on macrophage M1/M2 markers.

Supplemental Figure 7. Assessment of renal damage in the LPS-induced septic model.

Supplemental Figure 8. Prior splenectomy abolishes the protective effect of salbutamol pretreatment against renal ischemia-reperfusion injury.

Supplemental Figure 9. Cell-type marker gene expression levels for unbiased clustering of single-cell RNA sequencing data.

Supplemental Figure 10. Immunostaining of T cell Ig and mucin domain 3 on renal tissues of the adoptive transfer experiment.

Supplemental Figure 11. Assessment of infiltrating neutrophils in the single-cell RNA sequencing data.

Supplemental Figure 12. M1/M2 marker gene expression levels on the macrophage subclustering data in the single-cell RNA sequencing.

Supplemental Figure 13. The adoptive transfer of salbutamol-treated macrophages 2 days before renal ischemia-reperfusion injury does not provide protection.

Supplemental Table 1. Primer sequences for the quantitative real-time PCRs.

Supplemental Table 2. A list of the marker genes of each cluster in the single-cell RNA sequencing.

Supplemental Table 3. A list of the 37 genes commonly selected in the RNA sequencing from the three in vitro models.

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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 Figure 3
ASN.2020121723.DCSupplemental.html (1KB, html)
Supplemental Data
ASN.2020121723SupplementaryData1.pdf (5.5MB, pdf)
Supplemental Table 1
ASN.2020121723SupplementaryData2.xlsx (11KB, xlsx)
Supplemental Table 2
ASN.2020121723SupplementaryData3.xlsx (396.1KB, xlsx)
Supplemental Table 3
ASN.2020121723SupplementaryData4.xlsx (16.4KB, xlsx)

Articles from Journal of the American Society of Nephrology : JASN are provided here courtesy of American Society of Nephrology

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