Sprague-Dawley (SD) bone marrow derived mesenchymal stem cells (BMSCs) (Cyagen Biosciences Inc., Guangzhou, China) were propagated at 37℃ under 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Carlsbad, CA) cultured with 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics (50 U mL−1 penicillin and 50 ug mL−1 streptomycin, Gibco). The passage 4(P4) cells were used for the in vitro experiments. SD-derived BMSCs(P3) (Cyagen Biosciences Inc., USA) that stably express GFP were propagated and the P5-GFP-BMSCs were used for in vivo experiments.
Characterization of BMSCs incubated with EPO
BMSCs were trypsinized after incubation with or without EPO (500 IU/ml) for 48 h and washed three times with phosphate-buffered saline (PBS). Cell surface markers were examined by immunostaining with the following antibodies: phycoerythrin (PE)-conjugated anti-CD45 (BD Biosciences, San Jose, CA), fluorescein isothiocyanate (FITC)-conjugated anti-CD44 (BD Biosciences), and FITC-conjugated anti-CD90 (BD Biosciences) antibody. The labeled cells were analyzed using a flow cytometer (BD LSRFortessa™, Piscataway, NJ). Osteogenic and adipogenic differentiation potential of the cells in the two groups was examined according to the manufacturer's protocol (Cyagen, China).
I/R-AKI kidney homogenate supernatant (KHS) preparation
An AKI model was established using SD rats by clamping both renal pedicles for 45 min, followed by clamp-release to allow reperfusion. Both kidneys were harvested 60 min after reperfusion, cut into small pieces, and homogenized in PBS using a glass homogenizer to obtain a 20 g/l homogenate. The homogenate was then centrifuged at 20,000 rpm for 15 min at 4 ℃. The supernatant was filtered through a 30-μm mesh-sized disposable sterile filter to obtain AKI-KHS, which was stored at -80 ℃ until further use. Normal KHS (N-KHS) obtained from healthy SD rats was used as a control.
AKI-KHS-induced apoptosis in BMSCs
To study the anti-apoptotic effect of BMSCs pretreated with EPO, we established AKI-KHS-induced in vitro injury model: BMSCs or EPO-BMSCs were incubated with AKI-KHS (or N-KHS). Cells were seeded at 4 x105 cells/well in six-well plates in DMEM/F12 with 2% FBS. Transwell chambers with 0.4 μm pore size polycarbonate filter (Corning Incorporated, NY, USA) were introduced in the wells for the interventions. Three groups were set up: the control group (BMSCs + N-KHS group, BMSCs were plated in the 6-well plates and 1.5 ml N-KHS was added to the upper chamber); the BMSCs+AKI-KHS group (BMSCs were plated in the 6-well plates and 1.5 ml AKI-KHS was added to the upper chamber); and the EPO-BMSCs+AKI-KHS group (EPO-BMSCs were plated in the 6-well plates and 1.5 ml AKI-KHS was added to the upper chamber). All groups were incubated at 37℃ for 24 h in a humidified atmosphere with 5% CO2. Then, apoptosis of the BMSCs and EPO-BMSCs was analyzed by flow cytometry, the Annexin V-FITC apoptosis detection kit (Invitrogen, Frederick, MD, USA) was used to detect apoptotic cells according to the manufacturer’s protocol. Then cells were harvested subsequently for western blot analysis.
After each treatment, the cells were washed twice with PBS, harvested, and the proteins were extracted. The following antibodies were used to analyze protein expression: anti-BCL-2 (1:1000, Santa Cruz, CA), p53 (1:1000, Santa Cruz), SIRT1 (1:1000, Santa Cruz), and anti-β-actin (1:2000, Bioworld, Shanghai, China). Protein bands were quantified by densitometry using an Alpha Innotech imaging system. Protein levels were normalized to β-actin expression using Image J analysis software.
Cells were transiently transfected with SIRT1 siRNA or control siRNA using Lipofectamine RNAiMAX reagent (Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. Cells were analyzed 36 h after siRNA transfection. The SIRT1 siRNA and control siRNA were synthesized by RiboBio technologies (RiboBio, Guangzhou, China). The sequence of SIRT1 siRNA was as follows: 5¢-CACCUGAGUUGGAUGAUAUTT-3¢ (sense) and 5¢-AUAUCAUCCAACUAGGUGTT-3¢ (antisense).
Anti-apoptotic effect of BMSCs pretreated with EPO
To study the anti-apoptotic effect of BMSCs pretreated with EPO, we incubated BMSCs and EPO-BMSCs with AKI-KHS (or N-KHS). Five groups were set up: the BMSCs+N-KHS group was used as a control(BMSCs were plated in 6-well plates and 1.5 ml N-KHS was added to the upper chamber); the BMSCs+AKI-KHS group (BMSCs were plated in 6-well plates and 1.5 ml AKI-KHS was added to the upper chamber); the EPO-BMSCs+AKI-KHS group (EPO-BMSCs were plated in 6-well plates and 1.5 ml AKI-KHS was added to the upper chamber); the EPO-BMSCs+AKI-KHS+SIRT1 siRNA group (EPO-BMSCs transfected with SIRT1 siRNA were plated in 6-well plates and 1.5 ml AKI-KHS was added to the upper chamber); and the EPO-BMSCs+AKI-KHS+Con siRNA group (EPO-BMSCs transfected with control siRNA were plated in 6-well plates and 1.5 ml AKI-KHS was added to the upper chamber). All groups were incubated at 37 ℃ for 24 h in a humidified atmosphere of 5% CO2. Then cells were harvested for western blot analysis. After that, we established AKI-KHS-induced in vitro injury model again, the apoptosis of the BMSCs and EPO-BMSCs was analyzed by flow cytometry.
Adult female SD rats (200–250 g), purchased from the animal house of the Faculty of Medicine at Southern Medical University (Guangzhou, China), were used for the study. Animals were housed under specific pathogen-free conditions with a 12 h dark-light cycle and supplied with pelleted food and tap water ad libitum. Animals were allowed to acclimatize to the housing conditions for one week. All procedures were performed in strict accordance with the Guidelines for Animal Experimentation of Southern Medical University (Guangzhou, China).
Animal grouping and treatment
AKI models were established using SD rats. The animals were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg/kg). Abdominal incisions were made, and the two renal pedicles were bluntly separated. A microvascular clamp was used to clamp both renal pedicles for 45 min, followed by clamp-release to allow reperfusion. Then, the abdominal incision was closed. Intervention treatments were administered to rats through tail vein injection after reperfusion.
To determine the effects of BMSCs and EPO-BMSCs treatment, we randomly divided the rats (n = 50) into the following five groups (10 rats per group): sham group (kidneys of the SD rats were exposed for 45 min and 1 ml low-glucose DMEM was injected), model group (both renal pedicles were clamped for 45 min and 1 ml low-glucose DMEM was injected), EPO group (both renal pedicles were clamped for 45 min and 1 ml EPO (500 IU/ml) was injected), BMSCs group (both renal pedicles were clamped for 45 min and 1 x 106 BMSCs were injected), and EPO-BMSCs group (both renal pedicles were clamped for 45 min and 1 x 106 EPO-BMSCs were injected). All rats were housed at a favorable temperature and humidity (a temperature of 21 °C ±2°C, and a humidity of 55 % ±5 %) with an unlimited supply of water and food post-surgery. Rats were sacrificed one and five days after treatment (five rats at each time point). Blood samples and tissues from the kidneys, lung, spleen, and liver were collected for subsequent experiments. Blood samples were collected through the inferior vena cava, and serum was separated and stored at -80 °C until use. Both kidneys of each rat were immediately excised and cut into two coronal sections. The sections were fixed in 4% paraformaldehyde at room temperature. In the BMSCs and EPO-BMSCs group, the lung, spleen, liver, and the remaining part of the kidney were immediately analyzed.
GFP fluorescence in frozen tissue sections
In our previous study, we demonstrated that pretreatment with 500 IU/ml EPO for 48 h prior to infusion markedly increased the homing abilities of BMSCs. To verify the in vivo distribution of BMSCs after intravenous infusion, we prepared fast-frozen sections from the kidney, lung, spleen, and liver 24 h after GFP-BMSCs and GFP-EPO-BMSCs infusion. We immediately observed the sections under a fluorescence microscope.
Renal functional analysis
Renal function was estimated using diagnostic kits. SCr was measured using a colorimetric microplate assay based on the Jaffe reaction (Quantichrom Creatinine Assay; BioAssay Systems). BUN was measured using a colorimetric assay kit according to the manufacturer's instructions (Quantichrom Urea Assay; BioAssay Systems).
Serum concentrations of the inflammatory factors IL-1β and TNF-α and the anti-inflammatory cytokine IL-10 were measured by ELISA (R&D Systems, Minneapolis, MN, USA). The assay was performed according to the manufacturer's instructions.
Renal histological analysis
To detect kidney injuries, we fixed the samples in 4% neutral-buffered paraformaldehyde for histological assessment, embedded them in paraffin, and cut them in 3-μm-thick slices. The sections were then stained with HE (Servicebio, China). Histological examinations were performed in a blinded manner for acute tubular necrosis (ATN) scores regarding the grading of tubular necrosis, cast formation, tubular dilation, and loss of brush border. Ten non-overlapping fields (×200) were randomly selected and scored as follows: 0, no damage; 1, patchy isolated necrosis ≤ 10%; 2, tubular necrosis between 10% and 25%; 3, tubular necrosis between 25% and 50%; 4, tubular necrosis > 50% .
Results are expressed as mean ± standard deviation. Student's t test was performed to analyze the differences between two groups. Multiple-group comparison was performed using one-way analysis of variance (ANOVA) test. SPSS 19.0 statistical software was used for statistical analysis. P < 0.05 was considered statistically significant.