Male Sprague-Dawley (SD) rats were purchased from Charles River Laboratories Japan (Yokohama, Japan). Six-week-old rats were used to collect bone marrow, and 8-week-old rats were used in in vivo experiments. All experiments were performed according to the “Guide for the Care and Use of Laboratory Animals, 8th ed., 2010” (National Institutes of Health, Bethesda, MD) and approved by the Institutional Animal Care and Use Committee of Hiroshima University (Permit number: A15-66 and A17-75).
As primary antibodies, we used a rabbit monoclonal anti-HMGB1 antibody (ab79823; Abcam, Cambridge, UK), rabbit monoclonal anti-IL-18 antibody (ab223293; Abcam), rabbit monoclonal anti-IFN-γ antibody (ab133566; Abcam), mouse monoclonal anti-α-smooth muscle actin (α-SMA) antibody (A-2547; Sigma-Aldrich; St. Louis, MO), mouse monoclonal anti-transforming growth factor-β1 (TGF-β1) antibody (sc-130348; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (G8795; Sigma-Aldrich), rabbit monoclonal anti- phosphorylated Smad2 (p-Smad2) antibody (#3108; Cell Signaling Technology, Danvers, MA), mouse monoclonal anti-Smad2 antibody (#3103; Cell Signaling Technology), rabbit polyclonal anti-collagen type I (Col-I) antibody (ab6308; Abcam), rabbit polyclonal anti-collagen type III (Col-III) antibody (ab7778; Abcam), rabbit polyclonal anti-CD3 antibody (IR503; Dako, Santa Clara, CA), rabbit polyclonal anti-CD68 antibody (ab125212; Abcam), and rabbit monoclonal anti CD163 antibody (ab182422; Abcam).
Rat and human IFN-γ were obtained from PEPROTECH (Rocky Hill, NJ). A PGE2 high sensitivity ELISA kit was purchased from Enzo Life Science (ADI-930-001; Villeurbanne, France). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Sigma-Aldrich.
Rat MSCs (rMSCs) were isolated from bone marrow of the SD rat femur and tibia. rMSCs were cultured in DMEM containing 10% fetal bovine serum (FBS; Sigma-Aldrich) for the primary culture. MSCs were passaged four times before used for administration or in vitro experiments. These cells were confirmed as MSCs by promoting their differentiation into osteocytes and adipocytes with specific differentiation media . Furthermore, we confirmed that standard MSC surface markers CD44 and CD90 were positive in these cells by flow cytometry. Human bone marrow MSCs (hMSCs) and human THP-1 monocytes were purchased from Riken BRC (Ibaraki, Japan). HK-2 cells, a human proximal tubular cell line, were obtained from the American Type Culture Collection (Manassas, VA). These cells were cultured as described previously .
Cell Preparation for in vivo Experiments
rMSCs (3 × 105 cells/100-mm dish) were seeded and cultured in DMEM supplemented with 10% FBS. At 80% confluence, the medium was replaced by fresh medium with or without 200 ng/ml IFN-γ, and the cells were cultured for 24 hours. The cells were resuspended in PBS and subjected to in vivo analyses as IFN-γ-treated rMSCs (IFN-γ rMSCs) or untreated rMSCs (control rMSCs).
Preparation of Conditioned Medium (CM)
After hMSCs or rMSCs were grown to 80% confluence in DMEM supplemented with 10% FBS, the medium was replaced by fresh medium with or without 200 ng/ml IFN-γ. Twenty-four hours later, the medium was replaced by DMEM supplemented with 0.1% FBS, which was collected after 24 or 48 hours.
Cell Treatment with TGF-β1
After incubation in DMEM supplemented with 0.1% FBS, CM from control hMSCs, or CM from hMSCs treated with IFN-γ for 24 hours, human HK-2 cells were treated with 10 ng/ml recombinant human TGF-β1 (R&D Systems, Minneapolis, MN). Thirty minutes or 24 hours later, the cells were collected and subjected to analysis of TGF-β1-induced fibrotic changes.
Polarization of M2 Macrophages
To induce differentiation of THP-1 monocytes into M1 macrophages, THP-1 cells were stimulated by 160 nM phorbol 12-myristate 13-acetate (Sigma-Aldrich) for 48 hours. The medium was replaced by DMEM supplemented with 0.1% FBS, control hMSC-CM, or IFN-γ hMSC-CM. After 48 hours, the cells were collected and subjected to western blot analysis of CD163 and CD68.
Transfection with Prostaglandin E Synthase (PTGES) siRNA
rMSCs and hMSCs were transfected with 20 nM siRNA against PTGES (s133104 and s18305; Applied Biosystems, Waltham, MA) or negative control siRNA (4390843; Applied Biosystems) using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific). Cells were cultured in DMEM supplemented with 10% FBS for 5 days and then subjected to in vitro and in vivo analyses.
Preparation of an IRI Model and MSC Administration
A rat IRI model, an acute kidney injury (AKI) model, was used to analyze the progression from AKI to CKD. Rats were anesthetized by intraperitoneal injection of three types of mixed anesthetic agents (butorphanol, medetomidine, and midazolam). The left renal artery was clamped to induce ischemia. Sixty minutes later, the clamp was opened and reperfusion of blood was confirmed. After confirmation of reperfusion, PBS (vehicle), control MSCs, or IFN-γ rMSCs (5 × 105 cells/rat) were injected into the abdominal aorta. After 7 or 21 days, the rats were sacrificed and their kidneys were collected to evaluate inflammation and chronic fibrosis.
Preparation of a UUO Model and MSC Administration
A UUO model, which is an experimental renal fibrosis model, was prepared to investigate the anti-fibrotic effect of MSCs. Rats were anesthetized by the same procedure described for the IRI model. The left ureter was exposed after an abdominal midline incision and ligated to induce UUO. At 4 days after the UUO operation, PBS, control rMSCs, or IFN-γ rMSCs (2.5 × 106 cells/rat) were injected into the tail vein. After 7 days, the rats were sacrificed and examined for renal fibrosis.
Sample preparation and western blotting were performed as described previously . The protein bands were quantified using ImageJ software (version 1.47v; National Institutes of Health) and normalized to GAPDH levels.
Left kidneys of IRI and UUO rats were fixed in 10% formaldehyde for 18 hours. Fixed samples were embedded in paraffin and cut into 4 µm-thick sections. Immunohistochemical staining of kidneys was performed for light microscopic observation. Five fields (× 100) of the renal cortex were selected randomly. CD3-, CD68-, and CD163-positive cells and areas positive for α-SMA, Col-I, and Col-III were assessed using ImageJ software.
Enzyme-linked Immunosorbent Assay (ELISA)
To evaluate the amount of PGE2 in CM, the ELISA was performed according to the manufacturer’s instructions. PGE2 concentrations were normalized to the total protein content of MSCs.
Quantitative Real-time Reverse Transcription-PCR
RNA extraction and quantitative reverse transcription-PCR were performed as described previously . mRNA levels were normalized to the level of 18 s rRNA. Primers and TaqMan probes (TaqMan Gene Expression Assay) were obtained from Applied Biosystems (Foster City, CA). The probe set ID for rat PTGES is Rn00572047_m1 and for human PTGES is Hs00610420_m1.
All results are expressed as the mean ± standard deviation (SD). Statistical analysis for multiple comparisons was performed using one-way ANOVA followed by Bonferroni’s post-hoc test. The Student’s t-test was performed to compare the difference between two groups. p < 0.05 was defined as statistically significant.