Preparation of MSCs

MSCs were derived from adipose tissues obtained from patients who underwent breast reconstruction. MSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum (FBS) (Sigma-Aldrich) or STK (Kanto Reagents, Tokyo, Japan). Cells up to passage 4 were used in all experiments. The Medical Ethics Committee of Hiroshima Graduate School of Biomedical Science permitted the collection of adipose tissue (permit number: E-1516, registered on January 29, 2019). All patients provided written informed consent.

Animals

Eight-week-old male C57BL/6 mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). A total of 45 mice were used in this study. All mice 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 at the Laboratory Animal Center of Hiroshima University (Hiroshima, Japan). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Hiroshima University (Hiroshima, Japan) (permit number: A22-12) and performed in accordance with the “Guide for the Care and Use of Laboratory Animals, 8th ed, 2010” (National Institutes of Health, Bethesda, MD, USA). The study results were reported in accordance with ARRIVE guidelines 2.0.

Experimental protocol

CIN model mice were established as previously described [20]. Briefly, mice were anesthetized with a mixture of medetomidine (0.3 mg/kg; Kyoritsuseiyaku, Tokyo, Japan), midazolam (4.0 mg/kg; Sand, Tokyo, Japan), and butorphanol (5.0 mg/kg; Meiji Seika Pharma, Tokyo, Japan). The skin on the right side of the spine was incised to expose the right kidney; the right kidney was removed, and the incision was closed with sutures. One week after the procedure, mice were anesthetized, and the left kidney was exposed. The left renal artery was clamped for 30 min to induce ischemia–reperfusion injury, and 200 µL of iohexol (Omnipaque, GE Healthcare, Tokyo, Japan) was injected using a retro-orbital injection method, as described previously [21]. The left kidney received 10 Gy of irradiation in an X-ray generator (MBR1520R-3, Hitachi Power Solutions, Ibaraki, Japan). The entire body, except for the left kidney, was shielded from X ray by a lead plate. After irradiation, MSCs (1.0 × 105 cells/mouse) in 100 µL of phosphate-buffered saline (PBS) were immediately injected through the tail vein. In one experiment, mice were randomized into three treatment groups (n = 5 in each group): PBS, MSC, SF-MSC groups. In a second experiment, mice were randomized into three treatment groups (n = 5 in each group): PBS, NC siRNA SF-MSC, EGF siRNA SF-MSC. In a supplemental experiment, 250 µg/kg recombinant human EGF (rhEGF) (R&D Systems; 236-EG-01M) in 100 µL PBS was injected using the same method, and then mice were randomized into two groups (n = 5 in each group): PBS and rhEGF. The incision was then closed with sutures. At 24 h after tail vein injection, mice were sacrificed by exsanguination under anesthesia (medetomidine 0.3 mg/kg, midazolam 4.0 mg/kg, and butorphanol 5.0 mg/kg intraperitoneally) and kidneys and blood were collected.

Histological analysis

Hematoxylin–eosin (HE) and immunohistochemical staining were performed using established methods [20, 22, 23]. Renal tissue samples were formalin-fixed and paraffin-embedded. Sections (2 µm thick) were prepared for HE staining. HE-stained sections were used to estimate tubular injury, as described previously [24]. Ten high magnification fields (×200) of the renal cortex and cortico-medullary junction were randomly selected from each mouse. The extent of degeneration and detachment of tubular cells was scored as follows: 0, normal; 1, involvement of 1–25% of cells; 2, involvement of 26–50%; 3, involvement of 51–75%; and 4, involvement of 76–100%.

Sections (4 µm thick) were also prepared for TdT-mediated dUTP nick end labeling (TUNEL) and γH2AX staining. TUNEL staining was performed using an in situ Apoptosis Detection kit (MK500; Takara, Shiga, Japan), following the manufacturer’s protocol for paraffin-embedded sections. For γH2AX staining, rabbit monoclonal anti-phospho-histone H2AX antibody (1:500; Cell Signaling Technology) was used as primary antibody. The number of TUNEL-positive cells and γH2AX-positive cells were counted in 10 randomly selected fields (at magnification × 200) in the corticomedullary differentiation area, and data were analyzed using ImageJ software.

Western blotting

Western blot analysis was performed on kidney lysates and cultured cell extracts as previously described [25]. The following primary antibodies were used for analysis: rabbit polyclonal anti-phospho-H2AX (9718S; Cell Signaling Technology), rabbit polyclonal anti-cleaved caspase-3 antibody (9664S; Cell Signaling Technology), rabbit polyclonal anti-cleaved PARP (5625 T; Cell Signaling Technology), mouse monoclonal anti-GAPDH antibody (Sigma-Aldrich), and mouse monoclonal anti-α-tubulin antibody (Sigma-Aldrich). The intensity of each band was analyzed using ImageJ software and standardized by the level of either GAPDH or α-tubulin.

Preparation of conditioned medium

To generate conditioned medium from MSCs, cells were seeded in 10-cm dishes and cultured in STK or DMEM containing 10% FBS. Once the cells achieved 70% confluence, the medium was replaced with DMEM containing 0.1% FBS, and the cells were cultured for 48 h. The medium was collected (indicated as SF-MSC-CM and MSC-CM) and used for further experiments. HK-2 cells were cultured in DMEM containing 5% FBS. Conditioned medium was generated using same method as the control for ELISA analysis.

Irradiation experiment

HEK-293 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle Medium (high glucose) (Nacalai Tesque, Kyoto, Japan) containing 10% FBS (Nichirei Bioscience, Tokyo, Japan), and penicillin/streptomycin (Nacalai Tesque).

HEK-293 cells were cultured in SF-MSC-CM, MSC-CM, or DMEM containing 0.1% FBS for 24 h and then exposed to 30 Gy X-ray irradiation (MBR1520R-3, Hitachi Power Solutions, Ibaraki, Japan). After 24 h, HEK-293 cells were collected for further analysis.

Erlotinib hydrochloride (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was used to inhibit EGFR signaling in HEK-293 cells. When the medium of HEK-293 cell cultures was replaced with SF-MSC-CM, erlotinib was added at a concentration of 10 μM.

Enzyme-linked immunosorbent assay (ELISA)

ELISA kits for vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), milk fat globule-EGF factor 8 (MFGE8), epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), and transforming growth factor-alpha (TGF-α) were obtained from R&D Systems. Assays were performed following the manufacturer’s protocols. Concentrations were normalized to the total protein content or the number of cells.

siRNA transfection

SF-MSCs were transfected with 20 nM siRNA against EGF (siRNA ID 146221; Applied Biosystems) or negative control siRNA (Applied Biosystems) using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific). For in vivo experiments, after 24 h transfection, the cells were collected and administered to mice.

Quantitative real‐time reverse transcription PCR

RNA extraction and real-time reverse transcription PCR were performed following the previously described methods [14]. Specific oligonucleotide primers and probes for human EGF (assay ID: Hs01099990_m1) and 18S rRNA (endogenous control) were obtained as TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). mRNA levels were normalized to the level of 18S rRNA.

Statistical analysis

All data 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.