Isolation and culture of BMSCs
BMSCs were isolated by the whole bone marrow adherent method from Sprague-Dawley rats (male, 60–80 g) as previously described [32]. Briefly, the rats were sacrificed by injecting overdose pentobarbital, and then soaked in 75% ethanol alcohol for 15 min. BMSCs were isolated from the femur and tibia, then suspended with 5 ml of the BMSCs complete medium (Cyagen, Guangzhou, China) and seeded in T25 culture flasks (Corning, NY, USA) at 37°C in 5% CO2 and 95% humidity. After 48 h, the culture medium was replaced. The adherent cells were passaged when 80% confluent. BMSCs from passage 3–5 were used in the experiments. The animals were obtained from the experimental animal center of Xi’an Jiaotong University, Xi’an, China. The protocol was approved by the Xi’an Jiaotong University Committee on Animal Care regulations.
The construction of BMSCs-ISL1 or BMSCs-Scrambled
The detailed description of the construction of BMSCs-ISL1 or BMSCs-Scrambled has been reported in our prior study [29]. In brief, Adenovirus expressing ISL1 and an empty adenoviral vector were constructed from Hanbio Technology Ltd (Shanghai, China). BMSCs were transfected with adenoviruses harboring EGFP or ISL1 according to the manufacturer’s instructions. The titers of adenoviruses used in this study were 1.8 × 1010 PFU/ml. The culture medium was replaced 24 h after infection. 72 h after the transduction, the cells were selected by 2.5 µg/ml puromycin (Sigma-Aldrich, St. Louis, USA). The green fluorescence could be detected by a fluorescence microscope (Zeiss, Jena, Germany). BMSCs-ISL1 or BMSCs-Scrambled could differentiate into adipocytes and osteoblasts, identified by Oil Red O staining (Cyagen, Guangzhou, China) and Alizarin Red S staining (Cyagen, Guangzhou, China), respectively. BMSCs-ISL1 and BMSCs-Scrambled were identified by staining with allophycocyanin (APC)-conjugated anti-CD29, phycoerythrin (PE)- conjugated anti-CD90, APC-conjugated anti-CD45 and PE-conjugated anti-CD11b/c monoclonal antibodies (eBioscience, CA, USA). Isotype IgG antibodies (eBioscience, CA, USA) were employed as negative controls. Flow cytometry analyses were performed using a FACS Calibur flow cytometer (BD Biosciences, New Jersey, USA). The data were analyzed using FlowJo software (version 10; Treestar, OR, USA).
Preparation of conditioned medium (CM) of BMSCs-ISL1 or BMSCs-Scrambled
Upon 80% confluence of BMSCs-ISL1 or BMSCs-Scrambled, the culture medium was removed and then the cells were washed with phosphate-buffered saline (PBS; Servicebio) twice before treated with Iscove’s Modified Dulbecco Medium (IMDM; Procell, Wuhan, China) for 24 h at 37°C in a humidified atmosphere with 5% CO2. The conditioned medium (CM) was then collected, centrifuged at 3000 rpm for 10 min at 4°C, filtered with 0.22‐µm sterile filters (Millipore, MA, USA) and stored at − 80°C for further use. Both CM of BMSCs-ISL1 (ISL1-CM) and BMSCs-Scrambled (Scrambled-CM) were diluted with the culture medium of HKC cells at ratio of 1:9 before use.
Renal IRI model and in vivo treatment
Male Sprague-Dawley rats (140–160 g) were obtained from the experimental animal center of Xi'an Jiaotong University, Xi'an, China and raised in a temperature-controlled and pathogen-free environment with a 12 h light/dark cycle. All animal experiments conducted in this study were in compliance with the regulations by the Xi'an Jiaotong University Committee on Animal Care. Rat IRI model was then established. Briefly, rats were food-deprived for 12 h before the surgery and anesthetized with intraperitoneal injection of pentobarbital (50 mg/kg). The right unilateral nephrectomy was then performed. The left renal pedicle was exposed by lumbodorsal incision and then clamped with a nontraumatic vascular clamp for 60 min. In the Sham group, the right unilateral nephrectomy was performed and the left renal pedicle was exposed but without clamped. During the procedure, the body temperature of rats was maintained at 37°C using a rectal probe and heat pad. The nontraumatic vascular clamp was removed to restore blood flow, and the kidney was inspected to confirm reperfusion. At the timepoint of reperfusion, rats were administered intravenously via the tail vein with PBS (200 µl), BMSCs-Scrambled (2 × 105 cells in 200 µl PBS) or BMSCs-ISL1 (2 × 105 cells in 200 µl PBS). All rats were euthanized after 24 h of reperfusion, and the blood samples and kidneys were collected for further investigation. Serum levels of creatinine (SCr) and blood urea nitrogen (BUN) were determined by commercial kit reagents (Jiancheng Bioengineering Institute, Nanjing, China).
Cell culture and treatment
Human kidney proximal tubular cells (HKC) were purchased from the Chinese Academy of Medical Sciences cell bank. The cells were cultured in Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12 (Ham) (DMEM/F12 (1:1), Gibco, USA) containing 10% fetal bovine serum (Gibco), penicillin (100 U/mL, Gibco), and streptomycin (0.1g/mL, Gibco) at 37°C in a humidified atmosphere with 5% CO2. HKC cells were treated with IMDM, Scrambled-CM or ISL1-CM for 24 h and then stimulated with 250 µM H2O2 for 4 h. In the subsequent experiments, haptoglobin (MCE, Shanghai, China) was used to treat HKC cells. ERK phosphorylation agonist (S)-2-(4-fluorophenyl)-1-(toluene-4-sulfonyl)-pyrrolidine (Ro 67-7476) was purchased from TargetMol (Massachusetts, USA).
Cell viability assay and cell death analysis
HKC cells (5,000 cells/well) were seeded into 96-well plates and incubated overnight. HKC cells were then treated with CM or Hp at different concentrations for 24 h before being exposed to H2O2. The viability of cells was determined through Cell Counting Kit‐8 (CCK-8) assay (Beyotime Biotechnology, China) by following the manufacturer's instruction. Optical density values were measured at 450 nm on a microplate reader (BioTek, CA, USA). Cell viability was calculated as relative values compared to the control cells.
The apoptosis of HKC cells was measured using an Annexin V-APC and 7-AAD kit (Multisciences, Beijing, China) and detected using flow cytometry, according to manufacturer’s instructions. The percentages of dead cells were analyzed and quantified with FlowJo software (version 10; Treestar, OR, USA).
Histology analysis and immunohistochemical staining
Kidney tissues were fixed with 4% paraformaldehyde solution, followed by paraffin embedding and slicing (4 µm). The sliced sections were deparaffinized and stained with Periodic Acid-Schiff (PAS) staining. The tubular injury was scored as follows: 0, no damage; 1, < 25%; 2, 25 ~ 50%; 3, 50 ~ 75%; 4, > 75%. The tubular injury score was calculated as the average score from 10 random high-power fields (× 20 objective). The tubular injury was evaluated in a blinded manner. For immunohistochemistry staining, renal sections were incubated in 3% H2O2 for 10 min, blocked with 5% bovine serum albumin for 1 h at 37°C and then incubated with primary antibodies against CD68 (GB113109, Servicebio, 1:100) overnight at 4°C. Then the sections were washed and incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody for 1 h at room temperature. DAB staining (G1212, Servicebio, China) was performed and the nuclei were counterstained using hematoxylin (Servicebio, China). The sections were observed by an optical microscopy (NikonInstruments, Melville, NY) and 5 random fields (× 40 objective) were selected and measured.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay
TUNEL staining was performed on the kidney sections to evaluate cell apoptosis, using one-step TUNEL assay kit (Beyotime Biotechnology, Jiangsu, China). The sections were observed and photographed under a fluorescence microscope (Zeiss, Jena, Germany). 5 fields of vision (× 40 objective) were randomly selected to count the number of TUNEL-positive cells.
Dihydroethidium (DHE) staining of kidney tissues
Cryosections from frozen kidney tissues (4 µm) were stained with DHE solution (5 µM) (S0063, Beyotime Biotechnology, China) for 60 minutes in the dark at 37°C, then washed 3 times with PBS. The sections were counterstained with DAPI (Invitrogen) to label the nucleus. 5 fields of vision (× 20 objective) were randomly selected and then the fluorescence intensity of DHE was quantified by ImageJ software (version 1.53; National Institutes of Health).
Dichlorodihyfrofluorescein diacetate (DCFH-DA) staining of HKC cells
Reactive Oxygen Species Assay Kit (S0033S, Beyotime Biotechnology, China) was used to measure intracellular level ROS. In brief, HKC cells were incubated with fresh culture medium containing 10 µM DCFH-DA for 15 min, and then washed three times with PBS. The ROS levels were observed using a fluorescence microscope (Zeiss, Jena, Germany) and analyzed by ImageJ software (version 1.53; National Institutes of Health).
Measurement of oxidative stress
Kidney tissues (100 µg) were homogenized in saline into 10% homogenate. Cell lysates were extracted by radioimmunoprecipitation assay buffer. Superoxide dismutase (SOD) activities and malondialdehyde (MDA) levels were measured in kidney tissues and HKC cells to evaluate oxidative stress using commercial reagent kits (A001-1, Jiancheng Bioengineering Institute, Nanjing, China; G4300-96T, Servicebio, China). In addition, the concentrations of lactate dehydrogenase (LDH) in the serum of rats and supernatants of HKC cells were assessed by the LDH assay kit (S03034, Rayto Life Sciences Co., Ltd, Shenzhen, China).
Enzyme-linked immunosorbent assay (ELISA)
The levels of haptoglobin released in the conditioned medium were measured by the ELISA kits (Elabscience Biotech, Wuhan, China) in accordance with the manufacturer's instructions.
Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from kidney tissues using TRIzol reagent (Thermo Fisher Scientific, USA). Total RNA (1 µg) was reverse-transcribed into cDNA using reverse transcription polymerase (Roche, Mannheim, Germany). RT-qPCR was performed using the 7500 Fast Real-Time PCR System ABI 7500 System (Thermo Fisher Scientific, USA) with quantitative SYBR Green PCR Master Mix (Qiagen, Hilden, Germany). The relative mRNA levels of target genes were calculated using the 2−ΔΔCt method. The expression of each gene was normalized to that of Gapdh. The primers are listed in Table S1.
Western blotting
Proteins were extracted from kidney tissues and HKC cells with RIPA lysis buffer (Beyotime Biotechnology, China) containing protease inhibitor and phosphatase inhibitor (Pierce, IL, USA). To detect haptoglobin in the conditioned medium, methanol–chloroform precipitation method was used [33]. Total protein (25µg) was subjected to 10% SDS-PAGE gels and subsequently transferred onto polyvinylidene difluoride membranes. After blocking with 5% bovine serum albumin (BSA, Gibco, USA) for 1 h at room temperature, membranes were incubated with primary antibodies, including rabbit anti-ISL1 (1:5000, Novus Biologicals, Colorado, USA, #NBP2-14999), mouse anti-Haptoglobin (1:500, Santa Cruz Biotechnology, USA, #sc-376893), mouse anti-Bcl-2 (1:1000, Cell Signaling Technology, USA, #15071S), rabbit anti-Bax (1:1000, Cell Signaling Technology, USA, #2772S), rabbit anti-ERK (1:1000, Cell Signaling Technology, USA, #4695S), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology, USA, #4370S), mouse anti-JNK (1:500, Santa Cruz Biotechnology, USA, #sc-137019), mouse anti- phospho-JNK (1:500, Santa Cruz Biotechnology, USA, #sc-293136) and mouse anti-GAPDH (1:5000, Proteintech, 60004-1-Ig) overnight at 4°C. The membranes were washed with PBS containing Tween 20 and incubated with HRP-conjugated secondary antibodies (1: 5000, Abcam, Cambridge, UK) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence Western blotting detection kit (Tanon, Shanghai, China) and imaged by a chemiluminescence imaging system (Tanon 5200, Shanghai, China). Three independent biological replicates were performed. All protein expression was normalized to GAPDH. The intensity of each band was assessed using the ImageJ software.
Statistical Analysis
The statistical analyses were performed using GraphPad Prism version 9.0 (GraphPad Inc., CA, USA). All data are presented as the means ± standard deviation (SD). Comparisons between groups were tested using Student’s t test or one-way ANOVA with post hoc Tukey’s test where appropriate. A value of P < 0.05 was considered to indicate significance.