Preparation of ASCs

ASCs were isolated from superficial and deep subcutaneous abdominal adipose tissues collected from patients (age range, 44–57 years) who underwent breast reconstruction, herein referred to as “Sup-ASCs” and “Deep-ASCs,” respectively. These cells were cultured in low glucose Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO) containing 10% FBS (Sigma-Aldrich) and were used in all experiments up to five passages. The Medical Ethics Committee of Hiroshima Graduate School of Biomedical Science permitted collection of the adipose tissue (Permit number: E-1516, registered on January 29, 2019). Each patient had provided written informed consent.

Flow cytometric analysis

Flow cytometric analysis was performed in accordance with previously described methods [15]. An anti-human CD44 IgG antibody (BioLegend, San Diego, CA, 338804, 1:20), anti-human CD73 IgG antibody (BioLegend, 344004, 1:20), anti-human CD90 IgG antibody (BioLegend, 328108, 1:20), anti-human CD11b IgG antibody (BioLegend, 301404, 1:20), anti-human CD34 IgG antibody (BioLegend, 343504, 1:20), anti-human CD45 IgG antibody (BioLegend, 304006, 1:20), anti-human HLA-A–C IgG antibody (BioLegend, 311404, 1:20), and anti-human HLA-DR IgG antibody (BioLegend, 307604, 1:20) were used. Stained ASCs were analyzed by a BD FACSVerse (Becton, Dickinson and Company, Franklin Lakes, NJ). The data were evaluated by FlowJo software (FlowJo, LLC, Ashland, OR).

Cell proliferation assay

Proliferative activity of ASCs was examined by a water-soluble tetrazolium salt (WST)-1 assay (Takara Bio, Shiga, Japan). ASCs (5 × 103/100 μL) were seeded in 96-well microplates and cultured in DMEM containing 10% FBS. After incubation for 0, 24, and 48 h, 10 μL WST-1 reagent was added to each well, followed by culture for 2 h. Absorbance was measured using a microplate reader at a wavelength of 450 nm and reference wavelength of 620 nm.

Differentiation experiments

ASCs were cultured in adipogenic differentiation medium (Takara Bio) or osteogenic differentiation medium (Sigma-Aldrich) for 14 days in accordance with the manufacturers’ protocols. Oil Red O (Sigma-Aldrich) and Alizarin Red S (FUJIFILM Wako Pure Chemical, Osaka, Japan) were used to assess adipogenic and osteogenic differentiation, respectively. The stained area was estimated using ImageJ software by examining five randomly selected fields.

Preparation of conditioned medium

To generate conditioned medium from Sup-ASCs and Deep-ASCs (Sup-ASC-CM and Deep-ASC-CM, respectively), ASCs (5 × 105/dish) were seeded in 10 cm dishes and incubated in DMEM containing 10% FBS. At 80% confluence, the culture medium was replaced with DMEM containing 0.1% FBS, followed by culture for 24 or 48 h. Then, each medium was collected.

Enzyme-linked immunosorbent assay (ELISA)

ELISAs of prostaglandin E2 (PGE2) (Enzo Life Science, Farmingdale, NY), vascular endothelial growth factor (VEGF) (R&D Systems, Minneapolis, MN), and hepatocyte growth factor (HGF) (R&D Systems) were performed in accordance with the manufacturers’ protocols. Concentrations were normalized to the total protein content.

Cell culture and treatments

HK-2 cells were purchased from the American Type Culture Collection (Manassas, VA) and cultured in accordance with a previously described method [15]. Cells at 70% confluence were exposed to serum starvation in DMEM containing 0.1% FBS or conditioned medium from ASCs for 24 h, and then 10 ng/ml recombinant human transforming growth factor (TGF)-β1 (R&D Systems) was added to the cells directly. HK-2 cells were collected 30 min after the addition of TGF-β1 (to examine the p-Smad2 protein levels) and 24 h after the addition of TGF-β1 (to investigate α-SMA protein levels), and then used for in vitro experiments.

THP-1 cells were also purchased from the American Type Culture Collection and cultured following previously described methods [29]. To induce differentiation of THP-1 cells into macrophages, THP-1 cells were exposed to 160 nM phorbol 12-myristate 13-acetate (Sigma-Aldrich) for 48 h. Subsequently, the medium was changed to conditioned medium from ASCs. After 24 h, the cells were collected and subjected to in vitro experiments.

Animals

Male Sprague Dawley (SD) rats (8 weeks old) for IRI model establishment were obtained from Charles River Laboratories Japan (Yokohama, Japan). A total of 20 male SD rats were used in this study. All rats were reared in standard cages under a 12-h light–dark cycle at approximately 25 °C and 40–60% humidity, and were provided with free access to food and water. They were randomly divided into four groups (n = 5 in each group): sham, PBS, Sup-ASC, and Deep-ASC groups. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Hiroshima University (Hiroshima, Japan) (Permit number: A16-83) and conducted following the “Guide for the Care and Use of Laboratory Animals, 8th ed, 2010” (National Institutes of Health, Bethesda, MD). The study results were reported in accordance with ARRIVE guidelines 2.0.

Experimental animal model

Renal IRI was induced by transiently clamping the unilateral renal artery. Rats were anesthetized by an intraperitoneal injection of three types of mixed anesthetic agents: medetomidine (0.15 mg/kg), midazolam (2 mg/kg), and butorphanol (2.5 mg/kg). After performing a laparotomy, the left kidney was exposed. Subsequently, the renal pedicle was clamped by atraumatic vascular clamps for 1 h, followed by reperfusion on a heating blanket. After reperfusion, PBS only or ASCs (2.5 × 105 cells/rat) in 0.2 ml PBS were administered through the abdominal aorta clamped above and below the left renal artery bifurcation. At 21 days post-injection, the rats were sacrificed by exsanguination under anesthesia (medetomidine 0.15 mg/kg, midazolam 2 mg/kg, butorphanol 2.5 mg/kg, intraperitoneally) and their left kidneys were collected to assess fibrosis.

Western blot analysis

Sample collection and western blotting were carried out following previously described methods [15]. A rabbit monoclonal anti-p16INK4a antibody (Abcam, Cambridge, UK, ab51243, 1:2500), rabbit monoclonal anti-p21WAF1/CIP1 antibody (Abcam, ab109199, 1:1000), rabbit monoclonal anti-tissue factor (TF) antibody (Abcam, ab228968, 1:1000), rabbit monoclonal anti-thrombomodulin (THBD) antibody (Abcam, ab109189, 1:5000), rabbit monoclonal anti-CD163 antibody (Abcam, ab182422, 1:1000), rabbit polyclonal anti-CD68 antibody (Abcam, ab125212, 1:1000), mouse monoclonal anti-α-SMA antibody (Sigma-Aldrich, A2547, 1:5000), mouse monoclonal anti-GAPDH antibody (Sigma-Aldrich, G8795, 1:5000), rabbit monoclonal anti-phosphorylated Smad2 (p-Smad2) antibody (Cell Signaling Technology, Danvers, MA, #3108, 1:1000), mouse monoclonal anti-Smad2 antibody (Cell Signaling Technology, #3103, 1:1000), and mouse monoclonal anti-α-tubulin antibody (Sigma-Aldrich, T9026, 1:5000) were used as primary antibodies. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Dako, Glostrup, Denmark) or goat anti-mouse immunoglobulin G (Dako) were used as secondary antibodies. SuperSignal West Dura or Pico system (Thermo Fisher Scientific, Rockford, IL) was used to detect signals. The intensity of each band was quantified by ImageJ software (version 1.47v; National Institutes of Health) and standardized to the level of either GAPDH or α-tubulin.

Histological and immunohistochemical analyses

Sections of formalin-fixed, paraffin-embedded tissues were stained with hematoxylin and eosin (HE) and Masson trichrome for morphological and fibrotic evaluation in accordance with previously described protocols [15]. Areas of interstitial fibrosis were evaluated using a Lumina Vision (Mitani, Osaka, Japan) by examining five randomly selected fields (× 200) of the cortex. Immunohistochemical staining was also performed in accordance with previously described methods [15]. A mouse monoclonal anti-α-SMA antibody (Sigma-Aldrich, A2547, 1:5000) was used as the primary antibody. The positive area was estimated using ImageJ software by examining five randomly selected fields (× 200) of the cortex.

Quantitative real-time reverse transcription-PCR

RNA extraction and real-time reverse-transcription PCR were performed in accordance with previously described methods [15]. Specific oligonucleotide primers and probes for human TF (assay ID: Hs00175225_m1), human THBD (assay ID: Hs00264920_s1), human tumor necrosis factor-α-induced protein 6 (TSG-6) (assay ID: Hs00200180_m1), rat collagen type I (assay ID: Rn01463848_m1), rat collagen type III (assay ID: Rn01437681_m1), human β-actin (assay ID: Hs99999903_m1), and rat GAPDH (assay ID: Rn01775763_g1) were obtained as TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA). mRNA levels were normalized to the mRNA level of either β-actin or GAPDH.

RNA-sequencing

RNA extraction from ASCs was performed following previously described methods [15]. Three biological replicates were prepared for each ASC type. RNA-sequencing was performed by Macrogen Japan (Tokyo, Japan). The total RNA concentration was calculated by Quant-IT RiboGreen (Invitrogen, Waltham, MA). To assess the integrity of total RNA, samples are run on the TapeStation RNA screentape (Agilent, Santa Clara, CA). Only high-quality RNA preparations with RIN < 7.0 were used for RNA library construction. A library was independently prepared with 1 µg total RNA for each sample using the Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA). The qualified libraries were sequenced using an Illumina NovaSeq (Illumina, Inc.) with paired-end (2 × 100 bp) reads. After raw reads were trimmed and quality controlled, the processed reads were aligned to Homo sapiens (hg38) using HISAT v2.1.0 [30]. Then, transcript assembly of known transcripts was processed by StringTie v1.3.4d [31, 32]. On the basis of the results, expression abundances of transcripts and genes were calculated as the read count or fragments per kilobase of exon per million mapped read value per sample.

Enrichment analysis of differentially expressed genes

Transcripts with fold-change values > 2.0 with P ≤ 0.05 were included in the analysis as differentially expressed genes (DEGs). Gene Ontology (GO) functional enrichment analysis of the significant gene list was performed using g:Profiler tool (https://biit.cs.ut.ee/gprofiler/).

Statistical analysis

All experiments were performed three times and similar results were obtained. Results are expressed as means ± standard deviations (S.D.). For multiple group comparisons, one-way ANOVA followed by Tukey–Kramer’s post-hoc test was applied. Comparisons between two groups were analyzed by Student’s t-test. P < 0.05 was considered statistically significant.