The sequence of CHBP was the same with HBSP, QEQLERALNSS, and it was thioether-cyclized (molecule weight 1416.8). CHBP was synthesized by Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China.
CASP3siRNA, targeting murine caspase-3 mRNA (NCBI CoreNucleotide Accession No. BC038825), were synthesized (Life Technologies, Paisley, UK). The sequences of the CASP3siRNA (Ambion In Vivo, catalogue number: 4457309) were sense 5’-CCUGGUUACUAUUCCUGGAtt-3’ and antisense 5’-UCCAGGAAUAGUAACCAG
Gtg-3’. The negative control siRNA (NCsiRNA) was also provided by Life Technologies (Ambion In Vivo, catalogue number: 4457289), with sequences of sense 5’-UAACGACGCGACGACGUAAtt-3’ and antisense 5’-UUACGUCGUCGCGU
CGUUAtt-3’. Both CASP3siRNA and NCsiRNA were chemically modified by locked nuclei acid.
Renal IR surgery
Male C57BL/6 mice, 8-12 weeks, were purchased from the Experimental Animal Center of Yangzhou University, China. All animal experiments were performed according to the guidelines of the Laboratory Animal Monitoring Committee of Jiangsu Province.
The renal IR surgical procedures were performed under general anesthesia by intraperitoneal (i.p.) injection of pentobarbital sodium at 75 mg/kg body weight (BW). Bilateral kidneys were exposed via dorsal incisions sequentially, and the renal pedicle was carefully isolated and occluded using a non-traumatic vascular clamp for 30 min. The efficacy of occlusion was confirmed by the color change of kidney surface and to dark red eventually. Followed by removing the clamps, patched blanching appeared to the kidney surface and then normal pink, indicating blood reperfusion. Sham operation was performed in the similar manner, except clamping of renal pedicles. Mice were randomly divided into 7 groups (n = 6 in each group): (1) Sham; (2) IR; (3) IR + CASP3siRNA; (4) IR + NCsiRNA; (5) IR + CHBP; (6) IR + CHBP + CASP3siRNA; (7) IR + CHBP + NCsiRNA. The experimental design is shown in Fig. 1a. 0.03 mg/kg BW of siRNA was injected into the tail vein 2 h pre-surgery. 24 nmol/kg BW of CHBP was given through i.p. at 15 min after clamps were released.
At 48 h of renal IR injury, animals were anaesthetized with pentobarbital sodium, followed by cardiac puncture for drawing whole blood. Serum samples were then obtained by centrifuging at 10,000 rpm for 15 min. Kidneys were removed and transversally cut at the midplane, following crosscutting from the middle. One quarter of each kidney was fixed in 10% neutral formalin for 24 h, while two quarters were rapidly frozen in liquid nitrogen and the fourth part was preserved in RNAlater (Life Technologies).
Serum creatinine (SCr) level was determined using a QuantiChromTM Creatinine Assay Kit (BioAssay Systems, Hayward, USA). Briefly, thirty μl of standard or sample serum were transferred into a 96-well plate followed by adding in 200 μl working reagent per well, a mixture of reagent A and B. Absorbance at 510 nm was read immediately and 5 min later. Calculation was performed according to the manufacturer’s instruction.
Hematoxylin & eosin (H&E) staining of kidney tissues was performed to observe and evaluate the degree of tubulointerstitial damage (TID) in the cortex using a scoring system by assessing tubular damage (degeneration and detachment from basement membrane), interstitial expansion (edema or inflammatory cell infiltration), and dilation of tubular lumina. Histological changes were graded based on the percentage of damaged area involved: < 5% area was scored 0; 5% – 25% area was scored 1; 25% – 50% area was scored 2; 50% – 75% area was scored 3; and area exceeding 75% was scored 4. Kidney sections were blindly reviewed by two researchers independently. The scores from three compartments (tubular and interstitial areas, tubular lumina) of each kidney were obtained from 12 fields at 200 magnifications. The average scores per field of three compartments were then summed up for each kidney. The final score of animal was then calculated by averaging the scores from left and right kidneys.
In Situ End-Labeling (ISEL) of apoptotic cells
Apoptotic cells were detected using a TUNEL Apoptosis Detection Kit (Millipore, MA, USA) by ISEL, as previously described . Paraffin-embedded kidney sections were de-waxed and digested by proteinase K at 20 μg/ml for 10 min at 37oC. The sections were then applied with equilibration buffer, terminal deoxynucleotidyl transferase (TdT) and anti-digoxigenin-peroxidase sequentially. The labeling of apoptotic cells was then revealed with 3-amino-9-ethylcarbazole (AEC, dark red color). Apoptotic cells were examined at 400 magnifications in up to 20 fields of tubulointerstitial areas in the cortex. The number of positively stained cells in each animal was calculated by averaging the average number per field from left and right kidneys. This was blindly reviewed by two researchers independently.
Immunostaining of active caspase-3 in kidneys
Active 17 kDa subunit of caspase-3 was stained on kidney paraffin sections using the method described before . Briefly, sections were de-waxed and performed antigen retrieval before incubation with a rabbit-anti-mouse 17 kDa caspase-3 antibody (1:100 dilution, R&D System, Abingdon, United Kingdom). For negative control, normal rabbit immunoglobulin G was applied at the same concentration of primary antibody. 17 kDa caspase-3+ cells were counted at 400 magnifications in up to 20 cortical fields of each kidney by two researchers independently. The number of apoptotic cells for each animal was obtained by averaging the numbers from all fields in both kidneys.
Western blot analysis
Twenty-five μg of kidney homogenate was separated in reduced SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) gels and electroblotted onto a PVDF membrane. The membrane was then blocked in 5% (weight/volume) non-fat milk, following by probing with an anti-full length caspase-3 antibody (CST, Danvers, USA) at 1: 400 dilution, an anti-high mobility group box 1 (HMGB1) antibody (CST) at 1:1000 or an anti-β-actin antibody (Abcam, Cambridge, UK) at 1:8000 dilution for overnight at 4 °C. The corresponding secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, USA) was then applied to the membrane for 2 h at room temperature. Afterwards, antibody binding was revealed using ECL substrate (Thermo Scientific, Waltham, Massachusetts, USA) and a Molecular Imager Chemi Doc XRS+system (Bio-Rad, Berkeley, USA).
The kidney stored in RNAlater was performed microarray analysis to reveal the profile of whole genomic transcripts by Shanghai Biotechnology Corporation, China. The detection was done in 4 groups (n = 3 in each group): IR, IR + CHBP, IR + CHBP + CASP3siRNA and IR + CHBP + NCsiRNA. The RNA integrity and quantity were monitored by the 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) and NanoDrop One (Thermo Scientific), respectively. Two μg RNA with an Integrity Number of no less than 8 was required for the genomic profile analysis. The Agilent Whole Mouse Genome Oligo Microarray was applied to interrogate about 41,174 transcripts targeting 34,000 well-established annotated genes. The criteria of fold change (FC) > 1.414 (up-regulated genes) or FC < -1.414 (down-regulated genes) and P < 0.05 was used for sorting significant differentially expressed genes (DEGs). The cutoff value of FC was based on the fact that 0.5 cycle was the minimum number of polymerase chain reaction (PCR) cycle to distinguish the expressional differences between two samples.
Validation of candidate DEGs by quantitative PCR (qPCR)
Total RNAs were extracted by Trizol reagent from the kidney tissues of the same animals selected for microarray analysis. One μg total RNA was used for reverse transcription in a 20 μl reaction system supplemented with 4 μl 5x HiScript II qRT SuperMix and RNase-free water using a kit of HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China). The temperature setting was 50°C 15 min, followed by 85°C 2 min. One μl of cDNA product was amplified within a SYBR reaction system (Bioline, London, UK) containing 200 nM forward and reverse primers (Table 1, Biomics, Nantong, China) at 95oC for 10 min followed by 40 cycles of 95oC for 15 s and 55oC for 60 s. The level of β-actin mRNA was used as an endogenous control.
Gene function analysis
Functional enrichment analysis of significant DEGs identified between groups was performed using Gene Ontology (GO, http://geneontology.org/) . The resulting GO terms with P value less than 0.05 were considered significantly enriched.
Data was expressed as mean ± standard error of the mean (SEM). Statistical analysis of the data was performed using GraphPad Prism v8.0 software. One-way ANOVA analysis was used to check the homogeneity of variance for more than two groups. Unpaired student TTEST was then carried out to compare between parameters from two groups. P value < 0.05 was considered as statistically significant.