Chemicals

SS (99% purity) was purchased from Sigma-Aldrich (#214,485, Shanghai, China). Indomethacin (HY-14,397), iohexol (HY-B0594), and NG-nitro-L-arginine methyl ester (L-NAME; HY-18,729 A) were purchased from MedChemExpress (Shanghai, China).

Animals

Male Sprague-Dawley rats (6–7 weeks, 180–220 g) were obtained from Cavens (Changzhou, China) and kept under standard conditions (50–60% humidity, 23 ± 1℃, 12-h light/dark cycle) with free access to food and water. The study was approved by the Ethics Committee of Wuhan City Sixth Hospital, Affiliated Hospital of Jianghan University. All experimental procedures were conducted as per the Guide for the Care and Use of Laboratory Animals.

Animal grouping

After one week of acclimatization, 32 SD rats were randomly grouped as: (1) control group; (2) control + SS group; (3) CI-AKI group; and (4) CI-AKI + SS group, with 8 rats per group (shown in Fig. 1). The rat CI-AKI model was established according to previous description [17]. In short, after 12 h of fasting and water deprivation, rats in groups 3) and 4) were injected with 10 mg/kg indomethacin via the tail vein, followed by injection of L-NAME (10 mg/kg) and iohexol (3 g iodine/kg) 15 and 30 min later, respectively. Rats in the control groups received the equal volume of normal saline at the same time point. Rats in control + SS and CI-AKI + SS groups received intragastrical administration of 0.35 mg/kg SS (dissolved in deionized water) 48 h before indomethacin injection. The dose of SS was selected based on previous reports [16, 18]. No rats were sacrificed due to toxicity-related symptoms during the experimental period. Twenty-four hours after iohexol injection, all rats were sacrificed under anesthesia by decapitation. The blood, urine, and bilateral kidneys were collected.

Fig. 1

Animal grouping of the study

Biochemical analysis

The blood samples were centrifuged at 3000 rpm at 4℃ for 15 min to separate the serum, which was then stored at -80℃ before use. The serum levels of serum creatinine (SCr; C011-2-1), neutrophil gelatinase-associated lipocalin (NGAL; H392-1-1), blood urea nitrogen (BUN; C013-2-1), cystatin C (Cys-C; H336-1-1) and urinary level of kidney injury molecule-1 (KIM-1; H436-1-1) were determined using commercially available assay kits (all from Nanjing Jiancheng Bioengineering Institute, Nanjing, China) as per the manufacturer’s protocols.

Hematoxylin-eosin (H&E) staining

Fresh right kidney tissue was fixed in 4% paraformaldehyde, paraffin-embedded and sectioned (5-µm-thick). The sections were deparaffined, rehydrated and stained with H&E (C0105S, Beyotime, Shanghai, China) following the manufacturer’s instructions. A light microscope (Leica Microsystems) was employed for histological observation of the stained sections. For semi-quantitative analysis of renal tubular injury, 10 high-power nonoverlapping fields were randomly selected to evaluate the histopathological changes including interstitial edema, cast formation, cytoplasmic vacuolar changes, and luminal congestion. The degree of tubular injury was scored by two investigators blinded to the animal grouping following the criteria: 0, no injury; 1, 0–25% injury; 2, 25–50% injury; 3, 50–75% injury; and 4, 75–100% injury.

Measurement of oxidative stress-related factors

Left kidney tissue was homogenized in phosphate-buffered saline (1/10 w/v) and centrifuged at 3000 rpm for 10 min. The supernatant was harvested to determine the activities of SOD (A001-3-2), myeloperoxidase (MPO, A044-1-1), and GSH-Px (A005-1-2) using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute) as per the instructions.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA isolation from the kidney was achieved using TRIzol reagent (R0016, Beyotime). The iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) was employed for cDNA preparation. Real-time qPCR was conducted on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using THUNDERBIRD SYBR® qPCR Mix (Toyobo, Japan). The 2−ΔΔCt method was utilized for calculating relative gene expression, with GAPDH as normalization. Table 1 shows the primer sequences.

Table 1 Primers used for RT-qPCR.Western blotting

RIPA lysis buffer (Thermo Scientific) was employed for the tissue protein extraction, and a BCA assay kit (Beyotime) was employed for the quantification. Protein samples were separated by 10% SDS-PAGE, followed by transferring them onto the polyvinylidene fluoride membranes (Thermo Scientific). Next, the membranes were treated with the blocking buffer (Beyotime), cut into corresponding parts according to the molecular weight, and then incubated with primary antibodies (shown in Table 2) overnight at 4℃. After rinsing thrice with TBST, the membranes were incubated with HRP-conjugated secondary antibody (Abcam, Shanghai, China) for 1 h at room temperature. Blots were developed using an ECL detection kit (Thermo Scientific), and ImageJ software was employed for analyzing the band intensities.

Table 2 Primary antibodies used in Western blottingStatistical analysis

Normal distribution of data was assessed with Kolmogorov-Smirnov test. Data are expressed as the mean ± standard deviation. Difference comparisons were performed by one-way ANOVA followed by Tukey’s post hoc analysis using GraphPad Prism software (version 8.0.2; GraphPad, San Diego, CA). p˂0.05 depicted statistical significance.