C57BL/6 J male mice (8–12 weeks old, 20–25 g) purchased from CLEA (Tokyo, Japan) were used as WT mice. To generate macrophage-specific α7nAChR KO mice, lysozyme M (LysM)-Cre and α7nAChR flox/flox (B6(Cg)-Chrna7tm1.1Ehs/YakelJ, Jackson Laboratory, Bar Harbor, ME, USA) mice were crossbred for several generations. LysM-Cre: α7 flox mice were genotyped using tail PCR based on the protocol provided by the Jackson Laboratory using a MightyAmp Genotyping kit (Takara Bio Inc., Shiga, Japan). The primer sequence used in tail-PCR is listed in Supplementary Table 1. LysM-Cre: α7 flox male and female mice (8–18 weeks old) were used for the experiments. General anesthesia (0.3 mg kg−1 medetomidine, 5 mg kg−1 butorphanol, and 4 mg kg−1 midazolam) was administered for all surgeries and euthanasia.
LPS-induced AKI model and GTS-21 treatment
Septic AKI mouse models were created through intraperitoneal injection of lipopolysaccharides (LPS) from Escherichia coli O111:B4 and purified using phenol extraction (Sigma-Aldrich, Cat#L2630, LPS; 5 mg kg−1). As in our previous study, GTS-21 (20 mg kg−1, Sigma-Aldrich, Cat#SML0326) was injected intraperitoneally 15 min before LPS administration. The vehicle control received an equal volume of normal saline. Four hours after the LPS injection, the mice were euthanized.
Splenectomy was performed under general anesthesia. A small incision was made on the back of the mouse, the splenic vessels were ligated, and the spleen was removed. The sham-operated mice underwent skin incisions only. Seven days after the operation, the mice received an intraperitoneal injection of LPS, followed by a GTS-21 injection. Four hours later, the blood and kidney samples were collected.
CD4 positive T cells depletion
Two groups of WT mice received anti-CD4 (clone: GK 1.5, #BE0003-1, Bio X Cell, Lebanon, NH, USA) antibody or rat IgG2b isotype control (clone: LTF-2, #BE0090, Bio X Cell) treatments (200 μl per mouse, i.p. injection) three days before experiments. The efficacy of CD4 + T cell depletion was confirmed with spleen and whole blood cells by flow cytometry analysis (Attune NxT Flow Cytometer, Thermo Fisher Scientific) (Supplementary Fig. 10).
Validation of CD4 positive T cells depletion
To confirm CD4 positive T cell depletion using Flow cytometry, single cell suspensions were prepared from spleen and whole blood samples. Spleens were mushed using a plunger and passed through a 40 μm filter with 10 mL ice-cold PBS. The cell suspension were centrifuged at 500 × g for 5 min, then the supernatant was discarded. Whole blood cells were subjected to red blood cells (RBC) removal using RBC lysis buffer (5 mL). After blocking nonspecific Fc binding with anti-mouse CD16/32 (2.4G2), the cell suspensions were incubated with the following antibodies: anti-mouse CD4-FITC (GK1.5, Thermo Fisher Scientific), CD8a-eFloro 450 (53-6.7, Thermo Fisher Scientific). 7-AAD (Thermo Fisher Scientific) was used to exclude dead cells. Flow cytometry was performed on Attune NxT (Thermo Fisher Scientific), and the data were analyzed using Flow Jo CE softweare.
RNA extraction and real-time quantitative PCR
Messenger RNA was extracted from a quarter of the left kidney using RNAiso Plus (Takara Bio Inc., Shiga, Japan). RNA was extracted from the cells using the FastGene RNA Premium Kit (NIPPON Genetics, Tokyo, Japan). Reverse transcription was performed using the PrimeScript RT Master Mix (Takara Bio Inc., Shiga, Japan). Synthesized cDNA was used as the template for quantitative real-time PCR, which was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) on a CFX Connect Real-Time PCR Detection System (Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. Furthermore, the relative gene expression levels were calculated using the comparative cycle threshold (CT; 2-ΔΔCt) method. The primer sequences are listed in Supplementary Table 2.
Preparation of peritoneal macrophages
Four days before the experiments, mice were received 3% Thioglycollate (#225710, Becton, Dickinson and Company, Franklin lakes, NJ) 2 mL i.p.. The day of experiments, the mice were euthanized, 10 mL of ice-cold sterile PBS was injected into peritoneal cavity. The injected fluid was collected after the peritoneum was gentry massaged. The collected fluid was centrifuged at 500 × g for 5 min, then the supernatant was discarded. DNA was extracted from the resulting cell pellets using a DNeasy Blood & tissue kit (# 69504, Qiagen, Hilden, Germany).
Measurement of TNF-α
Levels of TNF-α in plasma and culture medium from cell supernatant were measured using ELISA with the TNF alpha Mouse Uncoated ELISA Kit with Plates (88-7324-22, Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s instructions. The Synergy LX (BioTek Instruments) was used for the ELISA plate reader.
Measurement of renal function and apoptosis in kidney tissue
To evaluate renal function, the plasma BUN levels were measured by SRL, Inc. (Osaka, Japan). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to evaluate apoptotic cells in the kidney tissues using Click-iT™ Plus TUNEL Assay Kits for In Situ Apoptosis Detection (C10617, Thermo Fisher Scientific, Waltham, MA). One-quarter of the left kidney was fixed with 4% paraformaldehyde (PFA) and embedded in paraffin. Tissues were sliced into 4-μm sections and stained according to the manufacturer’s protocol, and the slides were examined using a confocal microscope (LSM 800; Zeiss, Oberkochen, Germany).
One-quarter of the left kidney was fixed with 4% paraformaldehyde (PFA) and embedded in paraffin. Tissues were sliced into 3-μm sections and stained with periodic acid-Schiff (PAS) to evaluate the tubular damage. Tubular injury scores were evaluated based on the proportion of injured tubules as follows: 0. None; 1, <25%; 2, 25–50%; 3, 50–75%; and 4, >75%.
Murine macrophage cells, RAW 264 (RIKEN BRC CELL bank, Ibaraki, Japan), were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 50 U mL−1 penicillin, and 50 μg mL−1 streptomycin. U937 cells (JCRB Cell Bank, Osaka, Japan), a human monocyte cell line, were cultured in RPMI-1640 (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% FBS (Sigma-Aldrich). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.
Transwell migration assay
Migration assays were performed using 24 well transwell plates with 8.0 μm pore size inserts. RAW264 cells were cultured in 24 well plates at a density of 4 × 105 cells per well. To identify the migrated cells, RAW264 cells on the upper side were labeled with green fluorescence using the CellTrace CFSE Cell Proliferation Kit (C32554; Thermo Fisher Scientific) according to the manufacturer’s instructions. Carboxyfluorescein succinimidyl ester (CFSE)-labeled cells were seeded into the upper chamber. GTS-21 (100 μM) or α-bungarotoxin (#203980, Sigma-Aldrich) (1 μg ml−1) was added to a medium containing LPS (100 ng ml−1) or PBS as a control, and the number of migrated cells was evaluated after 4 or 24 h. The same treatment medium was then added to the upper and lower chambers. Migrated cells were photographed using a confocal microscopy (C2+ system; Nikon Corporation, Tokyo, Japan), and the images were visualized using IMARIS software (Bitplane, Zurich, Switzerland). The number of CFSE-labeled migrated cells were counted in every three random rows in the field, and the mean value for each group was calculated.
Macrophage co-culture experiment
To investigate the anti-inflammatory effects of macrophage-to-macrophage contact, human macrophages were added to mouse macrophages and co-cultured, and murine TNF-α levels in the medium were measured after administering LPS. Human macrophage-differentiated U937 cells were used. U937 cells were treated with phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 48 h, which allowed the cells to differentiate into macrophages. After washing the cells once with PBS, they were seeded onto pre-seeded RAW264 cells. RAW 264 cells were pre-seeded in 24-well plates at 6 × 104 cells per well, and the same number of differentiated U937 cells were seeded and co-cultured. As a control, RAW264 cells were treated with GTS-21 (100 µM) for 15 min prior to LPS stimulation (100 ng mL−1) for 4 h and the culture medium was collected.
Single-cell RNA-seq and preparation of single cell suspension
A cocktail of 20 U mL−1 DNase (Promega Inc. WI, USA. Cat#M6101) and 2 mg mL−1 collagenase type 1 (Worthington Corp., NJ, USA, Cat#CLS1), 1 mg mL−1 collagenase type 2 (Worthington, Cat#CLS2), and 1 mg mL−1 dispase (Roche, Cat#4942078001) was dissolved in DMEM high glucose (Wako) containing 10% FBS, which was used as a digestion buffer. Whole spleens were harvested and strained using 40 μm filters (SPL Life Sciences Co.Ltd. Pocheon, Korea) with a digestion buffer to obtain single-cell suspensions. The suspensions were subsequently collected in 15 mL tubes with 2.5 mL digestion buffer, pipetted into wells, and incubated in a shaking water bath at 37 °C for 10 min. The supernatant was collected in ice-cold high-glucose DMEM containing 10% FBS to stop enzymatic reactions. Fresh digestion buffer (2.5 mL) was added to the remaining cell pellets and subsequently incubated in a shaking water bath at 37 °C for 10 min. This series of experiments was repeated four times. The single-cell suspensions obtained by collecting the supernatant were passed through a 70-μm cell strainer and further passed through a 40-μm cell strainer. After centrifugation at 300 g for 5 min at 4 °C, the supernatant was discarded. One milliliter of digestion buffer was added to the cell pellets, pipetted well, and diluted up to 10 mL; the solution was then passed through a 40-μm cell strainer thrice. Single-cell suspensions were placed on ice, and the cell numbers were counted. Cell viability and singlets were confirmed using flow cytometry (Attune NxT Flow Cytometer, Thermo Fisher Scientific, Inc., Waltham, MA, USA). This method generated a single-cell suspension with viability greater than 90%. To obtain macrophage-enriched scRNA-seq, macrophages were isolated from spleen single-cell suspension labeling with anti-F4/ 80 Microbeads (130–110–443; Miltenyi Biotec, Bergisch Gladbach, Germany) using the magnetic cell separation method.
Single-cell RNA-seq barcoding and synthesizing cDNA libraries
Seven thousand cells were loaded onto a 10x Genomics Chromium instrument (10x Genomics, Pleasanton, CA, USA) to create single-cell Gel Bead-in-Emulsions (GEMs). cDNA libraries were constructed using 10x Chromium Single cell 3’ Reagent Kits v3.1, following the manufacturer’s instructions. Briefly, amplified cDNA products were cleaned using the SPRI Select Reagent Kit (10x Genomics). Indexed sequencing libraries were constructed and barcoded sequencing libraries were quantified using the Qubit 2.0 ds HS Assay Kit (Invitrogen). The quality of the libraries was checked using an Agilent 2200 Tapestation System (Agilent Technologies,Inc. Santa Clara, CA). Finally, libraries were sequenced using the Illumina NovaSeq platform.
Single-cell RNA-seq data analysis
The fastq files of four treatment groups (veh-veh, veh-GTS, LPS-veh, and LPS-GTS) were processed using the Cell Ranger v.3.1.0 (10× Genomics) count pipeline against the mm10 (v.3.0.0) mouse reference sequence. We used Seurat v.4.0.673 for detailed analysis. Cells with greater than 5% mitochondrial RNA or < 1,000, or > 4,000 detected genes were excluded. The data was subsequently normalized using the “NormalizeData” function, searched for 5,000 highly variable genes using the “FindVariableFeatures” function, and integrated four treatment groups using the “FindIntegrationAnchors” and “IntegrateData” functions. We visualized the integrated data using UMAP58 colored by group identities (Fig. 4b). We also used SingleR v.1.4.157 against the Immunological Genome Project dataset (“ImmGenData”)74 for cell-type recognition. The estimated cell types were visualized using UMAP (Fig. 3c and Fig. 5a, b). Cell–cell communication analysis between cell types was performed using CellPhoneDB75 for each treatment group. The mouse genes were converted into the human genes using the biomaRt package76, and “cellphonedb method statistical_analysis” was run with using the “—threshold=0.2” parameter. The predicted ligand-receptor interactions between cell types in each treatment group were visualized using a heatmap (Fig. 4b). We also visualized the predicted ligand-receptor (LR) interactions related to macrophages using the “cellphonedb plot dot_plot” command (Fig. 4c–e). Macrophage ligand LR pairs and receptors on other immune cells are shown in Fig. 4c Macrophage receptor LR pairs and ligands on other immune cells are shown in Fig. 4d and LR pairs of macrophage ligands and macrophage receptors are shown in Fig. 4e.
Gene set enrichment analysis of top 1,000 genes induced by GTS-21 in Mφ_cluster 1, the largest cluster of macrophage, was performed by Enricher ( and visualized with Enrichr Appyter ( (Fig. 5c).
Analysis of potential ligand-receptor interactions in macrophage-enriched samples was performed using NicheNet R package77 according to the code deposited in GitHub ( linking potential ligands expressed in sender cells to their target genes and their corresponding receptors that are differentially expressed in receiver cells. The ligand-receptor pairs were visualized in a code diagram using the R package circlize78 (Fig. 5f–h).
Data are expressed as mean ± standard error of the mean (SEM). The Student’s t test was used for comparisons between the two groups. One-way, two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used for three or more groups. Statistical significance was defined as P < 0.05. All analyses were performed using the GraphPad Prism 9 software (GraphPad Inc.).
All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals, approved by the University of Tokyo Graduate School of Medicine and Nagasaki University (M-P18-051 for the University of Tokyo and 2006101636 for Nagasaki University).
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.