A total of 24 male wild-type mice (weight, 20–25 g; age, 6–8 weeks) were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). In addition, nine Nrf2-knockout (KO) male mice (weight, 20–25 g; age, 6–8 weeks) were obtained from Junke Biological Co., Ltd. All animal protocols were approved by the Animal Care and Use Committee of General Hospital of Tianjin Medical University (approval no. IRB2020-DW-04; Tianjin, China), and all efforts were made to minimize animal suffering and the number of animals used. The mice were acclimated to the environment for 3 days prior to the experiment at 22°C with 40–60% humidity, a 12-h light/dark cycle and free access to water and rodent chow. After mice were anesthetized with intraperitoneal (i.p.) 50 mg/kg sodium pentobarbital, the bilateral renal pedicles were ligated for 30 min using microaneurysm clamps. Subsequently, the microaneurysm clamps were removed. In the control group, surgery was performed using the same process; however, renal pedicles were not ligated. Following surgery, 0.9% sodium chloride solution was used to help mice recover. Mice were then sacrificed following reperfusion for 24 h. A total of 24 wild-type mice were randomly divided into the following six groups: i) Control (Con) group (n=9); ii) I/R group (n=9); iii) I/R+MCC950 group (n=3); and iv) I/R+NaHS group (n=3). A total of nine Nrf2-KO mice were randomly divided into the following three groups: i) Con group (n=3); ii) I/R group (n=3); and iii) I/R+NaHS group (n=3).

According to previous research and preliminary experiments (21,22), 20 mg/kg MCC950 (Sigma-Aldrich; Merck KGaA) was injected (i.p.) daily for 14 days prior to surgery in the I/R+MCC950 group. A total of 50 µmol/kg sodium hydrosulfide [NaHS; i.p.; diluted in normal (0.9%) saline; Sigma-Aldrich; Merck KGaA] was injected prior to surgery in the I/R+NaHS group. Once all experimental procedures were complete, mice were sacrificed by cervical dislocation and tissue and blood were collected for subsequent analysis.

Kidney tissue was collected from I/R model mice following 24 h reperfusion to analyze protein expression levels. Briefly, total and nucleic protein were extracted using RIPA buffer (Thermo Fisher Scientific, Inc.) and protein concentration was determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). A total of 40 µg protein per lane was separated via 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes, then blocked with 5% milk for 2 h at room temperature. The membranes were subsequently incubated with the following primary antibodies overnight at 4°C: Anti-NLRP3 (1:500; cat. no. ab214185; Abcam), anti-ASC (1:200; cat. no. sc-514414; Santa Cruz Biotechnology, Inc.), anti-caspase-1 (1:200; cat. no. sc-56036; Santa Cruz Biotechnology, Inc.), anti-IL-1β (1:1,000; cat. no. ab200478; Abcam), anti-Nrf2 (1:1,000; cat. no. ab137550; Abcam), anti-heme oxygenase (HO)-1 (1:2,000; cat. no. ab68477; Abcam), anti-β-actin (1:2,000; cat. no. ab8227; Abcam) and anti-histone H3 (1:2,000; cat. no. ab176842; Abcam). Following primary antibody incubation, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies (1:5,000; cat nos. ab6721 and ab6728; both Abcam) for 1 h at room temperature. Protein bands were visualized by enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) using a Bio-Imaging system (Bio-Rad Laboratories, Inc.) and semi-quantified using Quantity One (V4.6) software (Bio-Rad Laboratories, Inc.). Protein expression levels were normalized to β-actin for cytoplasmic protein or histone for nucleic protein.

Kidney tissue was collected from I/R model mice following 24 h reperfusion to analyze mRNA expression levels. Briefly, total RNA was extracted from tissue using TRIzol^® reagent (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using a RevertAid First Strand cDNA Synthesis kit (cat. no. K1621; Thermo Scientific™; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was subsequently performed using SYBR PCR Master Mix (Thermo Fisher Scientific, Inc.) on a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Denaturation at 95°C for 5 min; 40 cycles at 95°C for 15 sec; and extension at 60°C for 20 sec. The gene-specific primer sequences are listed in Table I. The relative expression levels of the target genes were calculated using the 2^−ΔΔCq method (23) and normalized to GAPDH expression levels.

Kidney tissue was collected from I/R model mice following 24 h reperfusion to determine NLRP3 and Nrf2 expression levels. Briefly, kidney tissue was fixed at room temperature for 48 h in 10% formalin, embedded in paraffin and cut into 5 µm sections. The sections were subsequently blocked with 5% milk at 37°C for 30 min and incubated with anti-NLRP3 (1:100; cat. no. ab214185; Abcam) or anti-Nrf2 (1:200; cat. no. ab137550; Abcam) primary antibodies overnight. Following primary antibody incubation, the sections were incubated with HRP-labeled anti-rabbit secondary antibody (1:200; cat. no. ab214880; Abcam) at 37°C for 30 min, developed using diaminobenzidine solution for <3 min and counterstained with 0.5% hematoxylin for 3 min at room temperature. A light microscope was used to visualize IHC staining (magnification, ×200; Biorevo BZ-9000; Keyence Corporation).

A total of 2–3 ml blood was collected following centrifugation at 3,000 × g for 10 min at 4°C from I/R model mice following 24 h reperfusion. The serum was obtained to analyze renal function via ELISA to determine levels of creatinine (cat. no. C011; Nanjing Jiancheng Bioengineering Institute) and blood urea nitrogen (BUN; cat. no. C013; Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's instructions. Kidney injury molecule-1 (KIM-1) levels in serum were measured by ELISA (cat. no. EK0880; Wuhan Boster Biological Technology Co., Ltd.) according to the manufacturer's instructions.

Kidney tissue was collected from I/R model mice following 24 h reperfusion to evaluate renal histopathological changes. Tissue was fixed at room temperature for 48 h in 10% formalin and then embedded in paraffin. Paraffin-embedded sections were cut into 5-µm thick sections and stained with 0.5% hematoxylin for 5 min and eosin for 3 min at room temperature (25°C). The presence of hemorrhage, tubular cell necrosis, tubular dilatation and cytoplasmic vacuole formation in the tissue was scored as follows: 0, normal kidney; 1, minimal damage; 2, moderate damage; and 3, severe damage (24). Renal injury scores were calculated in a blinded manner by two researchers under a light microscope (magnification, ×200; Biorevo BZ-9000; Keyence Corporation).

Blood was collected (1 ml) from I/R model mice following 24 h reperfusion. The serum was obtained by centrifugation at 3,000 × g for 10 min at 4°C. The serum levels of TNF-α (cat. no. MTA00b), IL-1β (cat. no. MLB00C) and IL-6 (cat. no. M6000B) were analyzed using commercial ELISA kits (R&D Systems, Inc.), according to the manufacturer's protocol on a microplate reader (cat. no. CA94089; Molecular Devices, LLC).

Kidney tissue was collected from I/R model mice following 24 h reperfusion to evaluate cell apoptosis using TUNEL staining. Briefly, tissue was fixed at room temperature for 48 h in 10% formalin, embedded in paraffin and cut into 5-µm thick sections. Sections were incubated with TUNEL reagent in a humidified chamber at 37°C for 60 min in the dark (Roche Diagnostics) and stained with DAPI for 5 min at room temperature. Then, 10 high-power microscope fields were randomly picked and observed in each section with a fluorescence microscope (magnification, ×10).

Data are presented as the mean ± SD of three experiments and were analyzed by GraphPad Prism 5 (GraphPad Software, Inc.). Statistical differences between two groups were analyzed using a paired Student's t-test. Statistical differences between more than three groups were analyzed using one-way ANOVA followed by post hoc Tukey's multiple comparisons test. All data were normally distributed and had an equal variance. P<0.05 was considered to indicate a statistically significant difference.

The NLRP3 inflammasome pathway is activated and serves a crucial role in kidney injury (25,26). A previous study also reported that NLRP3 signaling is activated in renal tissue of mice at 24 h post-reperfusion (4). Therefore, 24 h post-reperfusion was selected as the present assessment time point. NLRP3 protein expression levels were upregulated by renal I/R injury in the I/R group compared with the control (Con) group (Fig. 1A and B). A similar trend was observed in NLRP3 mRNA expression levels (Fig. 1C). Furthermore, the number of NLRP3-positive cells increased in the I/R group compared with the Con group, as determined by IHC staining (Fig. 1D and E).

In order to determine the underlying mechanism of NLRP3 inflammasome activation and the effect on the downstream signaling pathway following renal I/R injury, mice were treated with the NLRP3 inhibitor MCC950. Western blotting results demonstrated that I/R upregulated protein expression levels of NLRP3 and its adaptor ASC and promoted maturation from pro-caspase-1 to caspase-1 and pro-IL-1β to and IL-1β in the I/R group compared with the Con group (Fig. 2A-E). These results indicated that renal I/R may induce activation of the NLRP3 inflammasome pathway and treatment with MCC950 may downregulate NLRP3, ASC, caspase-1 and IL-1β expression levels following renal I/R injury.

In order to determine the effect of NLRP3 on renal injury, I/R model mice were treated with the NLRP3 inhibitor MCC950. Renal I/R model mice exhibited significantly increased levels of BUN, serum creatinine and KIM-1, which is a biomarker of renal injury (Fig. 3A-C), indicating successful induction of renal I/R injury. Histological examination of renal tissue from I/R model mice revealed increased histological score, which was evidenced by severe tubular epithelial swelling, tubular dilation and interstitial edema, vacuolar degeneration and loss of the brush border, which suggested that renal I/R may promoted significant renal tissue damage and increased histological score (Fig. 3D and E). Inhibition of NLRP3 by MCC950 treatment significantly decreased I/R-induced increases in BUN, creatinine and KIM-1 levels and decreased histological score, with renal tissue exhibiting fewer severely injured tubules (Fig. 3A-E).

In addition, levels of inflammatory factors and apoptotic cells following MCC950 administration were analyzed in renal I/R model mice. Indicators of inflammation, including TNF-α, IL-1β and IL-6, were significantly upregulated in the I/R group compared with the Con group (Fig. 3F-H). Moreover, compared with the Con group, the percentage of apoptotic cells was significantly increased at 24 h post-reperfusion in the I/R group (Fig. 3I and J). Compared with the I/R group, the secretory levels of cytokines, TNF-α, IL-1β and IL-6, and the percentage of apoptotic cells were decreased by MCC950 treatment in the I/R+MCC950 group (Fig. 3F-J). These results indicated that inhibition of the NLRP3 inflammasome may prevent release of inflammatory factors and apoptosis of renal tissue induced by I/R.

A previous study revealed that Nrf2 is activated and expression levels of Nrf2 target genes are upregulated in kidney tissue following renal I/R injury (27). Similar findings were obtained in the present research. Nucleic, total protein and mRNA expression levels of Nrf2 were analyzed at 24 h post renal I/R injury. The results demonstrated that, compared with the Con group, the nucleic, total protein and mRNA expression levels of Nrf2 were upregulated in the I/R group (Fig. 4A-D). In addition, IHC analysis found that expression levels of Nrf2 in renal tissue were increased, which was demonstrated by increased number of Nrf2-positive cells observed in the I/R group (Fig. 4E and F). These data suggested that activation of the Nrf2 signaling pathway may initiate a protective response in renal I/R model mice.

In order to verify the effect of Nrf2 on the NLRP3 inflammasome pathway in renal I/R, Nrf2-KO mice were used in subsequent experiments. Protein expression levels of Nrf2 and its target gene, HO-1, were significantly downregulated following renal I/R in KO mice compared with wild-type mice (Fig. 5A-C). Conversely, compared with the I/R wild-type group, the expression levels of NLRP3 and its adaptor, ASC, caspase-1 and IL-1β were upregulated in the I/R KO group (Fig. 5A and D-G). These data indicated that renal I/R may induce activation of the NLRP3 inflammasome in Nrf2-KO mice.

NaHS has been demonstrated to exert effects on Nrf2 expression and NLRP3 inflammasome activation in different disease models (28–30). The present study investigated whether the effect of NaHS on activation of the NLRP3 pathway occurs via the Nrf2 signaling pathway by analyzing expression levels of Nrf2 and NLRP3 inflammasome-associated proteins in wild-type and KO mice. In wild-type mice, compared with the Con group, the expression levels of Nrf2, NLRP3, caspase-1 and IL-1β were upregulated by renal I/R injury. In addition, NaHS treatment further upregulated Nrf2 expression and downregulated NLRP3, caspase-1 and IL-1β expression levels in the I/R+NaHS group compared with the I/R group (Fig. 6A-E). In Nrf2-KO mice, no statistically significant differences were observed in Nrf2, NLRP3, caspase-1 and IL-1β expression levels between the I/R and I/R+NaHS groups (Fig. 6A-E). In the I/R+NaHS group, Nrf2 expression levels were significantly downregulated, whereas NLRP3, caspase-1 and IL-1β expression levels were significantly upregulated in Nrf2-KO mice compared with wild-type mice. These results suggested that NaHS may decrease NLRP3 inflammasome activation via the Nrf2 signaling pathway in renal I/R injury.

NaHS serves an important role in renal damage in AKI, I/R and other disease models (31). NaHS alleviated renal I/R-induced histopathological damage, which was demonstrated by decreased tubular epithelial swelling, tubular dilation and interstitial edema and kidney histological scores in the I/R+NaHS group compared with the I/R group in wild-type mice (Fig. 7A and B). Indicators of renal function, including creatinine, BUN and KIM-1, were also decreased by NaHS treatment in the I/R+NaHS group compared with the I/R group in wild-type mice (Fig. 7C-E). However, in Nrf2-KO mice, NaHS was unable to decrease the increased kidney histological score and levels of renal function indicators induced by renal I/R.

Furthermore, the percentage of apoptotic cells and release of cytokines, TNF-α, IL-1β and IL-6, were all decreased following NaHS treatment in the I/R+NaHS group compared with the I/R group in wild-type mice (Fig. 7F-J). However, NaHS was unable to exert a protective effect against apoptosis and excessive release of inflammatory factors following reperfusion of renal tissue in the I/R+NaHS group of Nrf2-KO mice compared with I/R+NaHS wild-type mice (Fig. 7F-J). These results suggested that NaHS may alleviate renal injury, inflammation and apoptosis in renal I/R wild-type mice, but not in Nrf2-KO mice.