Preparation of cellular material
Murine RTECs were purchased from Zhongqiaoxinzhou Cell Research (Cat. Number M4100; Shanghai, China) and cultured in Dulbecco’ h modified Eagle’ s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin at 37 ° C in a 5% CO 2 atmosphere. MSCs were generated from bone marrow tissue extracted from the femurs of sacrificed mice, plus suspended in sterile phosphate-buffered saline (PBS). The taken out cells were then resuspended in DMEM, filtered by means of 70-μ m mesh lattice, and plated in flasks containing DMEM supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA). After 72 h associated with culture, any nonadherent cells were removed, and the adherent cells were passed at a low density into brand new flasks. Cells that displayed a typical spindle-shaped appearance had been used for subsequent assays.
Induction of the H/R RTEC model and coculture
To detect the effect of MSCs on miR-223/NLRP3 activity, the RTECs were divided into the following 4 groups: 1) RTEC group (normal RTECs); 2) HUMAN RESOURCES RTEC group, (H/R-treated RTECs); 3) HR RTEC + MSC group (H/R-treated RTECs cocultured with MSCs just for 48 h); and 4) HR RTEC + htMSC group (H/R-treated RTECs cocultured with hypoxia-pretreated MSCs (htMSCs) for 48 h).
To detect the role of the miR-223/NLRP3 path in renal I/R injuries, the RTECs were separated into the following two groups: 1) Negative control team (H/R-treated RTECs cocultured with MSCs transfected with harmful control inhibitors); and 2) miR-223 inhibitor group (H/R-treated RTECs cocultured with MSCs transfected with a miR-223-specific inhibitor).
For the H/R treatment, RTECs were plated into six-well plates plus exposed to H/R conditions as previously described [ 23 ]. Briefly, the RTECs were inoculated at concentrations associated with 5 × 10 5 cells per well and incubated in DMEM tradition medium containing 10% FBS. To synchronize cell development, the culture medium waschanged to serum-free DMEM in 24 h before treatment. Next, the ischemia/hypoxia moderate (glucose; 10, 000 mg/L) was added to the cells, as well as the plates were put into double-layered, sealed, self-styled bags by having an anaerobic indicator. The bags were then filled with low-oxygen gas (5% CO 2, 95% N 2 ) to expel the environment. When the purple anaerobic indicator turned red, the handbag was sealed to maintain the anaerobic condition for 2 h, after which oxygen was added to the bag as well as the medium was changed to DMEM with FBS for further incubation. Hypoxia treatment has been reported to improve the therapeutic effectiveness of MSCs [ 13 ]. For hypoxia pretreatment associated with MSCs, the cells were cultured in 12-well plates along with DMEM supplemented with 10% FBS, 50 μ g/mL penicillin, and 50 mg/L gentamicin (Sigma, St . Louis, MO, USA) prior to being cocultured with RTECs. Every parameter examined in the following assays was measured within at least three identical samples. A double-chamber coculture program was used for cell coculture. The system was separated in to upper and lower chambers by a 0. 4-μ m pore-size membrane layer (12-well insert; BD Biosciences. Franklin Lakes, NJ, USA). RTECs (1 × 10 5 cells/well) were cultured in the lower chamber plus MSCs or htMSCs (1 × 10 5 cells/well) were cultured in the top chamber. Cells were harvested after 24 h, 48 h, and 72 h of culture, respectively.
Development of renal I/R mouse models and transplantation associated with MSCs
Twenty-four KM/NIH mice (female, 34 ± 2 . 1 g) were used for in vivo assays and had been randomly assigned to four different groups (six rodents per group): 1) Control group (mice underwent I/R treatment and were abdominally intravenously injected with the exact same volume of normal saline as used for negative control groups); 2) MSC group (15 min before I/R therapy the mice were abdominally intravenously injected with 2 × 10 6 MSCs in a volume of 200 μ L and then transfected having a negative control inhibitor); 3) miR-223 inhibitor group (15 min before I/R treatment the mice were abdominally intravenously injected with 2 × 10 6 miR-223 knockdown MSCs); and 4) htMSC group (15 min before I/R treatment the particular mice were abdominally intravenously injected with 2 × 10 6 hypoxia-pretreated MSCs). For induction of the renal I/R model, mice had been anesthetized with isoflurane and their rectal temperature had been maintained at 37 ° C. After a midabdominal laparotomy, the kidneys were exposed and the renal pedicles had been clamped with atraumatic vascular clamps for 60 minutes. While the clamps were applied, the left carotid artery was cannulated with PE-50 tubing to allow for intraaortic cellular delivery immediately after blood flow was reestablished. All mice had been sacrificed at 24 h after their operation.
Transfections of MSCs
MSCs (1 × 10 5 ) were seeded into six-well plates (18 replications). Transfection or hypoxic stimulation was performed until the tissues reached 70– 80% confluence. A miR-223-specific inhibitor (sequence: 5′ -ACAGUCAAACAGUUUAUGGGUU-3′ ) or negative control (sequence: 5′ -UGCGCUGCUGGUGCCAACCCUAUUCU-3′ ) purchased from Jikai Biosciences (Shanghai, China) was transfected into the MSCs using Lipofectamine 2000 reagent (Thermo Fisher Scientific Waltham, MA, USA) according to the manufacturer’ s instructions. At 24 h posttransfection, the MSCs were harvested and used for coculture with RTECs. The particular knockdown effect in the MSCs lasted for 96 l.
Dual luciferase assay
Full length NLRP3 mRNA (mNLRP3) 3′ -untranslated region (UTR) was inserted into the psiCHECK-2 luciferase media reporter vector to construct psiCHECK-2-mNLRP3-wt. The mutated form of mNLRP3 3′ -UTR was used to construct psiCHECK-2-mNLRP3-mut. ViaFect™ Transfection Reagent (Promega, Fitchburg, WI, USA) was used to cotransfect 1 μ g of psiCHECK-2-mNLRP3-wt or psiCHECK-2-mNLRP3-mut with 50 nm/L miR-223 mimic or miR-223 inhibitor into RTECs. Dual luciferase activity was assayed 48 h afterwards.
The cell counting kit-8 (CCK-8) assay was utilized to detect RTEC proliferation in the different groups. Briefly, RTECs in the coculture system were harvested at 24 they would, 48 h, and 72 h, respectively, after which 100 μ L of CCK8 solution (Dojindo Molecular Systems, Gaithersburg, MD, USA) was added to each well. Right after 4 h of incubation at 37 ° Chemical the optical density of each well at 450 nm was read with a microplate reader (Thermo Plate, Rayto Life and Analytical Science C. Ltd., Shenzhen, Guangdong, China).
Flow cytometry assay
The apoptotic rates of RTECs tested at 48 h in the different groups were dependant on flow cytometry. Cells in different groups were collected right after centrifugation at 600 g for 5 min, plus their apoptotic rates were measured using an Annexin V-FITC Apoptosis Detection Kit (WLA001c, Wanleibio, Shenyang, China) based on instructions provided by the manufacturer. Briefly, 5 μ L associated with Annexin V was added to different wells containing RTECs. After a 10-min incubation with Annexin V at area temperature, the cells were resuspended in 1 × binding buffer, and 5 μ L of propidium iodide was added to each sample. Next, the apoptotic rates were analyzed by flow cytometry (Accuri C6, BD, San Jose, CA, USA). The overall apoptotic price (UR + LR— all apoptosis cell percentage) had been equal to the sum of the late apoptotic rate (UR, top right quadrant— advanced stage apoptosis cell percentage) as well as the early apoptotic rate (LR, lower right quadrant— prophase apoptosis cell percentage).
Enzyme-linked immunosorbent assay
The levels of renal safeguarding cytokines, including hepatocyte growth factor (HGF; Cat. Number E-EL-R0496c, Elabscience-Biotech Co. Ltd., Wuhan, China), transforming development factor beta (TGF-β; Cat. No . CSB-E04727r, Cusabio Biotech, Wuhan, China), insulin-like growth factor-1 (IGF-1; Cat. Number CSB-E04582r, Cusabio Biotech, Wuhan, China), and vascular endothelial growth factor (VEGF; Cat. No . CSB-E04757r, Cusabio Biotech, Wuhan, China) were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers’ instructions. Serum blood urea nitrogen (BUN) levels were detected using an ELISA package manufactured by Bio-Medical Assay, Beijing, China.
Quantitative real-time polymerase chain reaction (qRT-PCR)
The total RNA in different cell samples at 48 h was extracted using an RNA Purified Total RNA Extraction Kit according to the manufacturer’ s instructions (Cat. Number RP1201, BioTeke, Beijing, China). β -actin and U6 were used as inner reference genes. Next, Super M-MLV reverse transcriptase (Cat. No . RP6502, BioTeke, Beijing, China) was used to invert transcribe samples of total RNA into cDNA templates. Every 20 μ L RT-PCR reaction mixture consisted of 10 μ L Bestar® SybrGreen qPCR Master Mix, zero. 5 μ L of each primer (miR-223, forward: 5′ -ACACTCCAGCTGGGTGTCAGTTTGTCAAATAC-3′, universally reverse: 5′ -CTCAACTGGTGTCGTGGA-3′; NLRP3, forward: 5′ -CAGAAGGCTGTGAGGGGAGA-3′, reverse: 5′ -GCAGACCAGGGGGATGAAG-3′; Notch1, forward: 5′ -GAGATTGGCTCCTATCGCTG-3′, reverse: 5′ -GGGCAGTCATCCACATTTTC-3′; Bcl-2, forward: 5′ -GGCATCTTCTCCTTCCAGC-3′, invert: 5′ -CCTCCCCCAGTTCACCC-3′; Bcl-XL, forward: 5′ -TCGCCAGCCTCTCTCAGC-3′, reverse: 5′ -AGACCCCCAGTGCCATCA-3′; Caspase-1, forward: 5′ -CCTCAAGTTTTGCCCTTTAGA-3′, reverse: 5′ -TACGAGTGGGTGTTTTCATTATT-3′; Caspase-3, forward: 5′ -GGGTGCGGTAGAGTAAGCA-3′, reverse: 5′ -GGAACGAACGGACCTGTG-3′; β -actin forward: 5′ -GGAGATTACTGCCCTGGCTCCTA-3′, reverse: 5′ -GACTCATCGTACTCCTGCTTGCTG-3′; U6 forward: 5′ -CTCGCTTCGGCAGCACA-3′, reverse: 5′ -AACGCTTCACGAATTTGCGT-3′ ), 2 μ L cDNA template, and 7 μ T Rnase-free H 2 O. The hyperbole parameters were as follows: denaturation at 95 ° D for 2 min, followed by 40 cycles at 94 ° C for 20 s, 58 ° D for 20 s, and 72 ° C regarding 30 s, after which the reaction was stopped by reducing the temperature to 4 ° C for 5 min. Relative expression levels of the targeted molecules were computed by an Exicycler™ 96 PCR system (Bioneer, Alameda, CA, USA) using the 2 – △ △ ct method.
Western blot assays
The total proteins in different groups had been extracted using a total protein extraction kit according to the manufacturer’ s instructions (Cal. No . WLA019, Wanleibio, China). GAPDH was used as an internal reference. The protein focus in each sample was determined using the BCA technique. A 20-μ L aliquot of protein (40 μ g) was separated by electrophoresis on a 10% salt dodecylsulfate polyacrylamide gel. Following separation, the targeted protein were transferred onto polyvinylidene difluoride (PVDF; BD, San Jose, CA, USA) sheets, which were then washed along with TTBS for 5 min prior to being incubated inside a powdered skim milk solution for 1 h. Principal antibodies against NLRP3 (1: 400, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Notch1 (1: 1500, Santa claus Cruz Biotechnology Santa Cruz, CA, USA), Bcl-2 (1: 500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl-XL (1: 1000, Santa Cruz Biotechnology, Santa Jones, CA, USA), caspase-1 (1: 2000, Abcam, Cambridge, MOTHER, USA), caspase-3 (1: 800, Abcam, Cambridge, MA, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1: 4000, Abcam; Cambridge, MA, USA) were incubated with the membranes at 4 ° C overnight. Following incubation, the membranes had been washed four times with TTBS, after which secondary HRP goat anti-rabbit antibodies (1: 20, 000, Cat. Number BA1054, Wuhan, China) were added to the mixture plus incubated with the membranes for 45 min at 37 ° C. After an additional six washes with TTBS, the blots were developed using Beyo ECL In addition reagent, and the results were detected with a gel imaging program. The relative levels of BDNF expression in the different examples were calculated using Gel-Pro-Analyzer (Media Cybernetics, Rockville, MARYLAND, USA).
RTECs which had received the different respective remedies for 48 h were seeded into 14-well compartments, washed with PBS, and fixed with 4% paraformaldehyde for 15 min. The cells were then permeabilized along with 0. 5% Triton X-100 for 30 min. Following, the cells were washed three times with PBS (5 minutes per wash), and blocked with 10% goat serum for 15 min. Primary rabbit polyclonal antibodies in order to Notch1 (1: 200, Santa Cruz Biotechnology, USA) plus NLRP3 (1: 100, Santa Cruz Biotechnology, USA) had been then added, and the cells were incubated overnight in 4 ° C in 1% goat serum. The pv cells were then incubated and stained with a fluorescein isothiocyanate secondary antibody (goat-anti-rabbit-Alexa 594 conjugated antibodies, Life Technology, USA) for 1 h. Following incubation with the supplementary antibody, the cells were washed and then stained with four, 6-diamino-2-phenyl indole (DAPI; Life Technologies, USA) for 5 min at room temperature. After three 5-min flushes with PBS buffer, the slides were fixed plus imaged with a fluorescence microscope at × a hundred magnification.
Hematoxylin and eosin (H& E) staining
The histological adjustments in sections of kidney tissue from the different groups had been observed using H& E staining. Briefly, the tissue were placed into Bouin’ s solution (4% formaldehyde) for perfusion fixation, after which they were dehydrated using various concentrations of alcohol and vitrified in dimethylbenzene. The particular tissues samples were then embedded in paraffin, sectioned, stained with H& E, and examined under a microscope at × 200 magnification. Following H& Electronic staining, the cell nuclei were stained blue plus cytoplasmic areas were stained red.
Periodic acid-silver methenamine (PASM) staining
PASM staining was used as an optimal method for determining the extracellular matrix in sections of renal tissue. Quickly, tissues sections were stained in 1% acid regular, after which they were treated with an Ag-methenamine solution and discolored with AuCl. After staining, collagen fibers appeared because dark strands against a pink background.
Terminal deoxynucleoitidyl transferase-mediated nick-end labeling (TUNEL) staining
The rates of cellular apoptosis in samples of renal tissue were determined using TUNEL staining. Briefly, tissue sections were permeabilized with 50 μ L 0. 1% Triton X-100 at area temperature for 8 min. Next, the sections had been washed three times with PBS buffer (5 min for each wash) and then incubated in 3% H two O 2 for 10 min at room temperature. After another three 5-min washes in PBS buffer, the sections were protected with TUNEL reaction solution and incubated at 37 ° C for 1 h in a darkened humidified chamber. Following incubation, the sections were washed 3 times with PBS buffer (5 min per wash), then incubated with Converter-POD at 37 ° C pertaining to 30 min. The reaction was then stopped, and the tissue were restained with hematoxylin. The stained renal cells were then examined under a microscope (CX41, Olympus, Shinjuku, Tokyo, Japan) at × 200 magnification.
Immunohistochemical detection methods
Sections prepared from different tissue samples were analyzed independently by senior pathologists, and then immediately frozen with regard to Notch1 detection. Tissue slides to be used for immunohistochemical assays were stored at 60 ° C overnight, and incubated with dimethylbenzene for dewaxing. The slides had been then hydrated with different concentrations of alcohol (100% designed for 5 min, 95% for 5 min, 85% intended for 5 min, and 75% for 5 min) plus washed with H 2 O 2 for 5 min. Slides along with tissue sections from different animals were fixed utilizing a methanol solution containing 3% H 2 O 2 , and then blocked with 1% bovine serum albumin (BSA) for 30 min on 37 ° C. They were then incubated at 37 ° C for 30 min with a primary antibody against Notch1 (1: 200, Abcam, Cambridge, MA, USA), before being incubated at 4 ° C right away. After four cycles of washing in 0. 01 M PBS (5 min per cycle), a secondary antibody (goat-anti-rabbit-Alexa 594 conjugated antibody; Thermo Fisher Scientific, Waltham, MA, USA) was added to the slides, which were after that incubated at 37 ° C for 30 minutes. After another four cycles of washing in PBS, DAB was added to the slides and reacted just for 3– 10 min until the reaction was stopped simply by addition of ddH 2 O. The particular slides were then restained with hematoxylin, dehydrated, plus examined under a microscope at × 200 magnifying.
Creatinine clearance rate test
Urine was collected within 24 l after each operation and the creatinine levels in lcd and urine samples were detected with a creatinine assay kit (BioAssay Systems, Hayward, CA, USA). The creatinine clearance rate (Ccr) was calculated according to the Cockcroft-Gault method: (urinary volume (mL, 24 h) × urinary : creatinine (μ mol/L))/(serum creatinine (μ mol/L) × urine collection length (min)).
All the data are portrayed as means ± SD. One-way analysis associated with variance (ANOVA), ANOVA for repeated measurements, and Duncan’ s post-hoc test were all performed using a common linear model. A G value < 0. 05 pointed out statistical significance. All the statistical analyses were performed making use of SPSS Statistics for Windows, version 19. 0 (IBM, Armonk, NY, USA).