Animals and Ethics

A total of 30 Wistar male rats (6 weeks old, weighed 180–220 g) were supplied by the Second Affiliated Hospital of Harbin Medical University (Harbin, Heilongjiang, China). To separate the probable confounding factor of sex, only male Wistar rats were used in this study. Before experiments, all rats were kept under standard laboratory conditions, with free access to food and water at 21 ± 4 °C for at least 7 days (under a 12 h/12 h light/dark cycle).

This study was approved by the Ethical Committee of Northeast Agricultural University (SRM-11, NEAUEC2023 03 96, China), and experiments were carried out in accordance with the institutional guidelines on the care and use of experimental animals. This study was conducted in accordance with the International Center for Laboratory Animals 3R based on the Uniform Standards for Clinical Trial Reporting and animal research reporting in vivo experiments guidelines (ARRIVE Guidelines) [28].

Experimental Design

All Wistar rats were randomly divided into five groups (n = 6/group): (1) Control group (C), intraperitoneally injected (i.p.) with 0.9% saline solution; (2) DEX group (D), treated with DEX (dissolved in 0.9% saline, 30 μg/kg, i.p.); (3) AS group (AS), forced to swim in water (18–20 °C) for 15 min (immediately removed when the drowning occurred), then fixed on the fixture, exposing their limbs and head for 3 h; (4) AS + DEX group (AS + D), treated with DEX (dissolved in 0.9% saline, 30 μg /kg, i.p.) 30 min before acute stress; (5) AS + A438079 group (AS + Y), treated with A438079 (a P2X7 inhibitor [29], 5 mg /kg, i.p.) 30 min before acute stress [30]. The procedure of the AS-induced AKI model was completely consistent in the AS, AS + D, and AS + Y groups.

Open-Field Test

Open-field test was applied to evaluate the behavior of the rats to verify the successful establishment of the acute stress model. Briefly, a 100 cm × 100 cm × 40 cm rectangular parallelepiped made of black wood without a lid was used for this study and was evenly divided into twenty-five 20 cm × 20 cm squares. Simultaneously, a camera was placed above the box to record the behavior of the rats. Rats were placed in the center of the open field box when the test started. Then, the results including total distance, average speed, number of crossings, and number of rearing (frequency of both forelimbs being off the ground) were continuously recorded for 3 min using the Super Maze software (Xinruan Information Technology, Shanghai, China). Each rat was tested once. All rats were sacrificed by euthanasia with 3% isoflurane after the test. Blood was collected from rats by cardiac puncture using heparinized needles immediately Then the samples including kidney tissues, and urine were collected respectively.

Biochemical Analysis

To assess kidney function, the serum was used to measure for blood urea nitrogen (BUN) and serum creatinine (CREA) levels using a UniCel DxC800 Synchron (Beckman, USA). Kidney tissues were collected for measurement of ROS, malondialdehyde (MDA), and Superoxide Dismutase (SOD) to assess the enzyme acivities of oxidative stress in the kidney. The level of ROS in the kidney was determined as previously described [17] using the ROS Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions. Briefly, 50 μg of proteins were added to a 96-well plate and mixed with 100 μL of 2′,7′-dichlorodihydrofluorescein diacetate. After incubation at 37 °C for 30 min in a dark condition, the fluorescence (Ex485 nm/Em525 nm) was measured using a Model F4500 fluorescence spectrometer (Hitachi, Tokyo, Japan). Levels of MDA and activity of SOD in kidney tissues were measured by commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the manufacturer’s instructions.

In addition, the urine was measured for urinary contents including pH, urobilinogen (URO), occult blood (BLD), white blood cells (WBC), urinary protein (PRO), urinary sugar (GLU), bilirubin (BIL), and ketone body (KET) using a VetLabTM UA (IDEXX laboratories, Beijing, China).

Detection of urinary adenosine triphosphate (ATP)

The level of urinary ATP was determined by a commercially available enzyme-linked immunosorbent (ELISA) assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Histologic Assessment

Kidney tissue was fixed in 10% neutral formalin (pH 7.4) for 48 h, embedded in paraffin, and then cut into 5 μm sections for hematoxylin and eosin (H&E) staining. Images were captured using an Olympus CKX41 microscope (Olympus, Tokyo, Japan) equipped with a Canon EOS 550D camera (Canon, Tokyo, Japan) at a high-magnification field (200).

The kidney histology of rats was scored blindly as the sum of the scores for each of the five variables. Briefly, each parameter was graded on the following five-point scale: 0, 1, 2, 3, and 4 for none, < 10%, 10%-25%, 25%-50%; and > 50% respectively (The percentage of the grid occupied by neutrophil infiltration was the neutrophil infiltration rate). Necrosis, tubular degeneration, neutrophil infiltration, and hemorrhage scores were evaluated semi-quantitatively [31]. The sum scores were averaged to obtain a semiquantitative histological kidney injury score.

Ultrastructural Observation

The kidneys were fixed overnight with 3% glutaraldehyde. The pellet was post-fixed with 1% osmium tetroxide for 90 min. After washing with double distilled water, stained with 1% uranyl acetate for 1 h. Afterward, the sections were dehydrated in a series of gradient alcohol for 10 min each time. Then placed in fresh pure epoxy resin and polymerized at 60 °C for 2 h. The Sects. (60 nm) were finally stained with lead citrate for 5 min. Images were captured using a transmission electron microscope (Tecnai, Hitachi, Tokyo, Japan) at 100 kV Electron Microscopy Film 4489 (Kodak, ESTAR thick base, San Francisco, CA).

Immunohistochemistry (IHC)

The experimental procedure of kidney tissue sections was consistent with that of the histologic assessment. Then the prepared sections were rehydrated, and after antigen retrieval in 10 mM (pH 6) citrate buffer, the peroxidase activity of the sections was quenched with 3% hydrogen peroxide (H2O2) in methanol for 10 min. After washing, the sections were blocked with 5% goat serum in phosphate-buffered saline (PBS) for 1 h and incubated with primary antibody overnight at 4 °C. The primary antibodies for IHC were anti-p-NF-κB (1:200, Bioss Antibodies, China), anti-NLRP3 (1:200, Wanlei Biotechnology, Shenyang, China), and were incubated with Horseradish Peroxidase (HRP)-labeled anti-rabbit IgG (1:500, Zhongshan Golden Bridge Biotechnology Co., Beijing, China) for 15 min at 37 °C. The color reaction of IHC was performed with a coloring kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Images were captured using an Olympus CKX41 microscope (Olympus, Tokyo, Japan) equipped with a Canon EOS 550D camera (Canon, Tokyo, Japan) at a high-magnification field (200). The IHC quantitative analysis and scoring method were implemented using IHC Profiler ImageJ software (National Institutes of Health, Bethesda, MD).

Immunofluorescence (IF)

Briefly, 5 μm thick kidney sections embedded in paraffin were used for IF. After dewaxing, the sections were heated and then incubated with 3% H2O2 for 10 min. Then the sections were blocked with 5% goat serum in PBS for 1 h and incubated with the primary antibody anti‐P2X7 (1:500; Santa Cruz Biotechnology) overnight at 4 °C. After washing 3 times, the sections were incubated with the fluorescent secondary antibody for 1 h at 37 °C in a dark condition. Then the sections were mounted by using 4′,6‐diamidino‐2‐phenylindole (Good Biotechnology, Co., Ltd., Wuhan, China). Images were acquired with a Nikon Eclipse Ni inverted microscope (TE2000; Nikon, Tokyo, Japan). The IF quantitative analysis and scoring method was implemented using IF Profiler ImageJ software.

Quantitative real-time PCR analysis

Total RNA was extracted from the kidney tissues using the TransZol Up system (Promega, Shanghai, China). Then 5 μg of total RNA was reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara, Dalian, China). Special primers including IL-1β, IL-18, TNFα, NLRP3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) (As shown in Table 1). The GAPDH gene was used as an endogenous control. Transcripts were quantified using Light Cycler 480 II (Roche, Basel, Switzerland). To quantify relative mRNA expression, the cycle threshold (Ct) values of the target genes were normalized to the Ct values of reference gene GAPDH, and the results are presented as fold change using the 2−ΔΔCt method. The PCR results were calculated using the following equations: ΔCt = Ct target gene − Ct GAPDH, and ΔΔCt = ΔCt treated group − ΔCt control group.

Table 1 Real-time PCR PrimersWestern Blotting

The kidney tissues were lysed in an ice-cold Radio-Immunoprecipitation Assay (RIPA) buffer (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with a protease inhibitor. Then the tissue lysates were centrifuged at 12,000 g for 10 min at 4 °C to collect supernatants. The total protein concentrations were quantified using the bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blocked in 5% skim milk in TBST (Tris-buffered saline, 0.1% Tween 20) at 37 °C for 2 h. The following primary antibodies were used: anti-P2X7 (1:1000, Santa Cruz Biotechnology, Texas, USA), anti-p-NF-κB (1:1000, Bioss Antibodies, China), anti-NLRP3 (1:1000, Wanlei Biotechnology, Shenyang, China), anti-apoptosis-associated speck-like protein containing CARD (ASC) (1:1000, Santa Cruz Biotechnology, Texas, USA), cleaved caspase-1 (1:500, Wanlei Biotechnology, Shenyang, China), anti-IL-1β (1:500, Santa Cruz Biotechnology, Texas, USA), anti-TNF-α (1:500, Santa Cruz Biotechnology, Texas, USA), anti-IL-18 (1:500, Wanlei Biotechnology, Shenyang, China), and anti-GAPDH (1:2000, Cell Signaling Technology, MA, USA). After washing three times, membranes were incubated with the secondary horseradish peroxidase-conjugated antibodies (1:6000, ZSGB-BIO, Beijing, China). The protein bands were visualized using the Tanon 5200 Multi-image system (Tanon Science & Technology Co., Shanghai, China). Image-Pro Plus 6.0 software (Media Cybernetics, Washington, USA) was used to analyze the density for each band. Results are presented as the ratio of the intensity of the P2X7, p-NF-κB, NLRP3, ASC, cleaved caspase-1, IL-1β, TNFα, and IL-18 band to the intensity of the GAPDH band.

Statistical Analysis

Data are expressed as means ± standard error means (SEM). Statistical analyses were performed by using SPSS Version 25.0 (Chicago, IL, USA). GraphPad Prism Version 10.0 (San Diego, CA, USA) was used for graph generation. All data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Differences were considered statistically significant at p < 0.05 and extremely significant at p < 0.01.