A total of 32 male C57BL/6 mice (age, 6–8 weeks; body weight, 20–25 g) was procured from the Experimental Animal Center of Nanjing Medical University (Nanjing, China) and maintained under specific pathogen-free conditions with ad libitum access to food and water (22±2°C, Relative Humidity: 40–60%, Light/Dark Cycle: 12/12-h light/ dark cycle). Following a 7-day acclimation period, mice were randomly allocated into four experimental groups (n=6 per group), including control, SFN (10 mg/kg) (26), cisplatin (20 mg/kg, CAS No. 15663-27-1, Sigma-Aldrich) (27) and cisplatin + SFN groups. Starting from day 7 of the experiment, SFN was administered daily to mice in the relevant groups, whereas control and cisplatin-only groups received equivalent volumes of saline. After 7 days of pre-treatment with SFN or saline, mice in the cisplatin and cisplatin + SFN groups received a single intraperitoneal injection of cisplatin (20 mg/kg in saline). Euthanasia was performed 72 h after cisplatin administration via intraperitoneal injection of 5% sodium pentobarbital (150 mg/kg).

To confirm mortality following euthanasia, mice underwent checks for: i) Cardiac and respiratory arrest; ii) pain responses; and iii) pupillary responses to light stimuli. Blood samples were immediately collected via terminal cardiac puncture. A total of 0.8–1.0 ml of blood was extracted per mouse into serum-separating tubes. Serum was obtained by centrifugation of blood samples at 3,000 g for 15 min at 4°C and samples were stored at −80°C for subsequent analysis. During the study, the following humane endpoints were established and strictly observed: i) Severe lethargy or unresponsiveness to stimuli; ii) loss of >20% body weight; iii) inability to access food or water; and iv) signs of severe distress, for example labored breathing or hunched posture (28). No mice were euthanized prematurely due to reaching predetermined humane endpoints. All procedures and were approved by the Ethics Committee of Nanjing Medical University (approval no. IACUC 2024-0811).

The human proximal tubular epithelial cell line HK-2 was obtained from the American Type Culture Collection. Cells were maintained in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS; both Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin solution comprising 100 U/ml penicillin and 100 µg/ml streptomycin under standard culture conditions (37°C; 5% CO₂ humidified atmosphere). Upon achieving 70% confluence, cells were serum-starved by switching to FBS-free medium. Prior to experimental treatments, cells were pre-treated with one or both of 5 µM SFN and 1.9 µM ML385, an NRF2 inhibitor, dissolved in dimethyl sulfoxide for 4 h (37°C). Following pre-treatment, cellular models were established by exposing HK-2 cells to 10 µg/ml cisplatin for 24 h (37°C) (27). For the control group, cells were cultured in PBS-free medium without pre-treatment or cisplatin.

Serum samples from mice in each treatment group were analyzed to quantify specific biomarkers, namely blood urea nitrogen (BUN; Jianglai Biotechnology Co., Ltd. (Cat. No. JL12567) with 50 µl of serum per well, serum creatinine (Scr) from Nanjing Jiancheng Bioengineering Institute (Cat. No. C011-2-1) with 20 µl of serum per well, MDA from Xitang Biotechnology Co., Ltd. (Cat. No. XT-MDA-001) with 10 µl serum per well, SOD from Abcam (Cat. No. ab65354) using 15 µl of serum per well and GSH from Kaiji Biotechnology Co., Ltd. (Cat. No. KGA7305) with 50 µl of serum per well. All biochemical analyses followed established protocols provided by the manufacturer's instructions.

Serum and cell supernatant samples underwent centrifugation at 3, 000 × g for 10 min at 4°C. The levels of IL-1β, IL-6 and TNF-α were measured using ELISA kits according to the instructions provided by the manufacturer (IL-1β, Elabscience Biotechnology Co., Ltd., No. E-EL-M0059), (IL-6, Elabscience Biotechnology Co., Ltd., No. E-EL-M0044) and (TNF-α, all Elabscience Biotechnology Co., Ltd., No. E-EL-M0059, E-EL-M0049).

Mouse renal tissues were fixed in 4% PFA at 4°C for 24 h, then embedded in paraffin. After dewaxing, tissue sections (4 µm) were rehydrated using gradient ethanol. Sections were subsequently stained with hematoxylin for 10 min, rinsed with tap water for 2 sec and then stained with eosin for 5 min. After rinsing with tap water for 1 sec, the sections were dehydrated in gradient ethanol at 75, 95 and 100%, each for 5 sec, and subsequently air-dried. All procedures were performed at room temperature. Changes to mouse renal structure were observed under a light microscope in five randomly-selected visual fields.

PAS staining was performed on 4 µm-thick paraffin-embedded mouse renal sections. The renal tissues were fixed in 4% paraformaldehyde at 4°C overnight before embedding. After deparaffinization in xylene and hydration in a graded ethanol series, tissue sections were oxidized with 0.5% periodic acid for 10 min and subsequently stained with Schiff reagent for 30 min to detect polysaccharides and glycoproteins at room temperature. Nuclei were counterstained with Mayer's hematoxylin for 1 min and sections were subsequently differentiated in acid alcohol and blued in alkaline solution for 2 min at room temperature. Following dehydration through an ethanol-xylene series, sections were mounted with resin medium. Staining patterns in five randomly selected were visualized using bright-field light microscopy at ×20 magnification.

Briefly, 5-µm paraffin-embedded renal tissue sections and cultured HK-2 cells grown on coverslips were fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature. Samples were subsequently permeabilized using 0.1% Triton X-100 in 0.1% sodium citrate for 30 min on ice. After washing with PBS, samples were incubated with TUNEL reaction (One-Step TUNEL Apoptosis Assay kit, Green Fluorescence, Catalog No. KTA2010, Abbkine, Wuhan, China) mixture for 1 h at 37°C in a humidified chamber. Nuclei were counterstained with 1 µg/ml DAPI for 5 min (22–25°C). Slides were mounted with antifade mounting medium and images of five randomly selected fields of view were captured using a fluorescence microscope.

ROS levels were quantified using the fluorescent probe DCFH-DA. For the HK-2 cell line, cells (1–5×10⁵) were cultured to 70–80% confluence in 6-well plates, washed with PBS and subsequently incubated with 10 µM DCFH-DA in serum-free medium for 30 min at 37°C in the dark. Cells were then washed twice with ice-cold PBS to remove extracellular dye. Finally, ROS levels in different treatment groups were analyzed by flow cytometry.

For mouse renal tissue samples, sections were equilibrated in PBS (pH 7.4) for 10 min at 37°C and incubated with 10 µM DCFH-DA dissolved in serum-free DMEM (Gibco, No. 12634010) for 30 min in the dark at 37°C. Excess probe was removed by gently washing the sections three times with PBS to minimize background fluorescence. Data were analyzed using FACSCelesta^™ and FlowJo^™ Software Version 10.8.1 from BD Biosciences.

Tissue sections (−80°C) were thawed using PBS solution and subsequently dried using absorbent paper. Sections (5–10 µm) underwent fixation with 4% PFA for 20 min at room temperature. Following rinsing with PBS, sections were treated with 0.1% Triton X-100 for 5 min at room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 15 min at room temperature. Sections were then blocked using a solution of 5% bovine serum albumin (BSA) (Sigma-Aldrich, No. A7906) for 1 h at room temperature and incubated with the primary antibodies (F4/80, Abcam, ab300421) 1:500, NRF2 (Abcam, ab313825) 1:100) overnight at 4°C. Following three washes with PBS, sections were incubated with the secondary antibody (Goat Anti-Mouse IgG H&L (HRP)(Abcam, ab205719) 1:2,000) for 1 h at room temperature. Samples underwent DAPI (Vector Laboratories, No. SK-4100) staining for 5 min at room temperature to visualize nuclei and were sealed beneath a coverslip. Finally, images were captured with a light microscope.

HK-2 cells (5×10⁴ to 1×10⁵) in the logarithmic growth phase were lifted and centrifuged (1,000 × g for 5 min at room temperature), and the cell density was adjusted for inoculation in 12-well plates at 37°C with 5% CO₂ overnight, with three replicate wells prepared for each group. Cells were fixed with 4% PFA for 30 min at room temperature, followed by permeabilization with 0.1% (v/v) Triton X-100 (Sigma-Aldrich, No. T8787) in PBS at room temperature for 10 min to disrupt the cell membrane. Following permeabilization, samples were blocked with 5% (w/v) BSA in PBS containing 0.1% Tween-20 at room temperature for 60 min. Then cells were incubated with primary antibodies (Nrf2 (Abcam, ab313825) 1:500) overnight. Samples were washed to remove excess primary antibodies 4°C overnight and incubated with secondary antibodies (Goat Anti-Rabbit IgG H&L (HRP; Abcam, ab6721) 1:2,000) for 2 h at room temperature. Following this incubation, secondary antibodies were washed off and cells were incubated for 15 min with DAPI staining solution at room temperature. Samples were washed again and images of cells were captured using a fluorescence microscope.

To assess the viability of HK-2 cells, a CCK-8 assay was performed (Beyotime Biotechnology) in accordance with the manufacturer's instructions. HK-2 cells (5,000 cells) were seeded into 96-well plates, which were incubated for 48 h. Subsequently, 10 µl CCK-8 reagent was introduced into each well and the plates were incubated for a duration of 2 h. The absorbance of samples was then quantified at a wavelength of 450 nm using a microplate reader (SMR60047 Smart Microplate Reader, manufactured by USCNK Life Science Co. Ltd.). All incubation steps were performed at 37°C with 5% CO₂.

HK-2 cells were harvested following centrifugation at 300 × g for 5 min at 4°C, washed twice with cold PBS and resuspended in 1X binding buffer at a density of 1×10⁶ cells/ml. Subsequently, 100 µl cell suspension was incubated with 5 µl annexin V-FITC and 5 µl propidium iodide in the dark for 15 min at room temperature. Samples were analyzed immediately using a BD FACSCanto^™ II flow cytometer (BD Biosciences) and using FlowJo v10.8.1 (BD Biosciences, USA).

RT-qPCR was utilized to analyze the mRNA expression of NRF2. TRIzol (Thermo Fisher Scientific, Inc.) was used to extract RNA from HK-2 cells and cDNA was synthesized utilizing the PrimeScript^™ RT Reagent Kit (Takara Bio Inc.), according to the manufacturer's instructions. qPCR was conducted using the SYBR-Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) on a GeneAmp^® PCR System 9700 (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primer sequences used were as follows: NRF2, forward 5′-GCCACCGCCAGGACTACAG-3′, reverse 5′-GCAACAAGAGCAGCCACCTC-3′; and GAPDH, forward 5′-CCCTCGTCCCGTAGACAAAATG-3′, reverse 5′-TGAGGTCAATGAAGGGGTCGT-3′. The thermocycling conditions for the qPCR reaction included an initial denaturation step at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 10 sec and annealing and extension at 60°C for 10 sec. GAPDH served as the internal control, and the relative expression levels of NRF2 were assessed using the 2^−ΔΔCq analysis method (29).

Total protein was extracted from HK-2 cells and mouse renal tissues using RIPA buffer (Beyotime Biotechnology) in the presence of protease inhibitors. A BCA Protein Assay Kit (Beyotime Biotechnology) was used to determine the total protein content of the samples. Proteins (20–30 µg/lane) were separated using 10% SDS-PAGE and subsequently transferred via electrophoresis onto a polyvinylidene fluoride membrane. Membranes were blocked for 1 h with 5% skimmed milk powder in Tris-buffered saline + 20% Tween-20 (TBST) at room temperature. Membranes were incubated with primary antibodies (KIM-1 (Proteintech, 30948-1-AP) 1:500, NGAL (Proteintech, 31721-1-AP) 1:500, Bax (Proteintech, 50599-2-Ig) 1:500, Bcl-2 (all Proteintech, 68103-1-Ig) 1:500, Cleaved caspase-3 (Abcam, ab214430), Caspase-3 (Abcam, ab184787) 1:500, NRF2 (Abcam, ab313825) 1:500, GAPDH (all Abcam, ab181602; all 1:500) overnight at 4°C. Membranes were washed with TBST solution and treated with HRP-conjugated goat anti-rabbit secondary antibodies for 1 h at room temperature. Protein bands were visualized utilizing an ECL reagent (Thermo Fisher Scientific, Inc.), and analyzed by ImageJ (Version 1.54, developed by the National Institutes of Health).

All experimental data are presented as the mean ± standard deviation of three independent experimental repeats. Data were analyzed using GraphPad Prism 7.0 (Dotmatics). Data were analyzed using unpaired t-test or one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To explore the role of SFN in the development and progression of CI-AKI, the present study established a mouse model of CI-AKI, and mice were separated into groups for treatment with or without SFN. As shown in Fig. 1A, the levels of Scr and BUN in the CI-AKI model group were significantly higher than the control group, whereas SFN treatment significantly attenuated the heightened levels of Scr and BUN. Mouse kidney markers were measured to determine the extent of pathological damage to the kidneys in different treatment groups. The protein expression of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) was significantly increased in the model group compared with the control group, whereas SFN significantly reduced the expression of KIM-1 and NGAL in the model + SFN group compared with the model group (Fig. 1B). Furthermore, H&E and PAS staining were performed to investigate whether SFN improved the renal pathology of CI-AKI in model mice. As demonstrated in Fig. 1C and D, the results of both H&E and PAS staining indicated that epithelial cells in the control group exhibited intact morphology and were arranged neatly, whereas the renal pathological structure of cisplatin-treated model mice demonstrated notable damage, such as irregular morphology and detachment of renal tubular epithelial cells, marked dilation of renal tubules and vacuolar degeneration of renal tubules. Compared with the model group, kidney damage after prophylactic administration of SFN was markedly reduced. Furthermore, administration of SFN to control mice showed no effects on Scr and BUN, KIM-1 and NGAL and renal pathology (Fig. 1A-D) compared with the control group. These findings indicated that SFN ameliorated CI-AKI in mice.

Apoptosis, inflammation and oxidative stress serve important roles in the progression of CI-AKI (30). As shown in Fig. 2A, the number of TUNEL-positive cells in the renal tubular lumen of cisplatin-treated model mice was notably increased compared with the control group, and treatment with SFN markedly reduced the apoptosis of renal tubular cells in model mice. Furthermore, the levels of apoptosis-related proteins, namely Bax, Bcl-2, cleaved caspase-3 and caspase-3, were detected via western blot assay. Compared with the control group, the expression of the pro-apoptotic protein Bax and the ratio of cleaved caspase-3/caspase-3 in the kidneys of mice treated with cisplatin were significantly increased, whereas the expression level of the anti-apoptotic protein Bcl-2 significantly decreased. SFN treatment in model mice significantly alleviated these changes (Fig. 2B). Furthermore, the levels of inflammatory indicators, such as F4/80, IL-1β, IL-6 and TNF-α, were measured via IHC and ELISA. As shown in the representative immunohistochemistry images in Fig. 2C, F4/80 levels were markedly increased in the model group compared with the control group. However, treatment of model mice with SFN resulted in a notable decrease in F4/80 levels compared with untreated model mice. Similarly, IL-1β, IL-6 and TNF-α were significantly upregulated in the model compared with the control group; however, these levels were significantly reduced in the model + SFN group compared with the model group (Fig. 2D). Furthermore, DCFH-DA staining was used to visualize ROS levels in each treatment group. Staining results revealed that ROS levels were markedly increased in the model group compared with the control group. However, ROS levels were notably attenuated in the model + SFN group compared with the model group (Fig. 2E). Similarly, biochemical analysis was utilized to evaluate the expression levels of MDA, SOD and GSH. As demonstrated in Fig. 2F, compared with the control group, MDA in the model group was significantly upregulated, and the levels of SOD and GSH were significantly decreased. However, compared with the model group, SFN treatment significantly mitigated MDA levels and partially yet significantly restored SOD and GSH levels. These findings indicated that SFN inhibited cisplatin-induced apoptosis, inflammation and oxidative stress.

The NRF2 signaling pathway, as one of the major signal transduction pathways in CI-AKI, plays an important role in the progression and development of CI-AKI (31). IHC and western blotting were performed to determine the effects of SFN on the expression of NRF2. As shown in Fig. 3, SFN upregulated the expression of NRF2 signaling pathway in CI-AKI mice compared with untreated model mice. These findings indicated that SFN ameliorated CI-AKI in mice by upregulating NRF2.

To further investigate the protective effect and mechanisms of SFN in HK-2 cells in vitro, the present study evaluated changes in cell apoptosis, inflammation and oxidative stress. CCK-8 assay indicated that, compared with the control group, cisplatin significantly inhibited the viability of HK-2 cells, whereas treatment of cisplatin-induced cells with SFN mitigated this effect (Fig. 4A). Flow cytometry, DCFH-DA staining and ELISA demonstrated that cisplatin significantly promoted apoptosis, inflammation and oxidative stress in HK-2 cells relative to the control group. Notably, treatment of model cells with SFN significantly mitigated cisplatin-induced apoptosis, inflammation and oxidative stress (Fig. 4B-D). Furthermore, the present study performed RT-qPCR, western blotting and immunofluorescence staining to assess the expression of NRF2. As indicated in Fig. 4E-G, treatment with SFN significantly upregulated NRF2 in cisplatin-induced HK-2 cells compared with the model group. Furthermore, co-treatment with the NRF2 inhibitor ML385 partially yet significantly mitigated the effects of SFN on the viability, apoptosis, inflammation and oxidative stress of HK-2 model cells induced with cisplatin (Fig. 4A-D). These findings indicated that SFN protected HK-2 cells against cisplatin-induced apoptosis, inflammation and oxidative stress in vitro via regulation of the NRF2 signaling pathway.