A total of 60 adultC57BL/6 mice (male; age, 8–12 weeks; weight, 22–26 g) were obtained from the Animal Experimental Center of Zunyi Medical University. All experimental protocols were approved by the Animal Care Committee of Zunyi Medical University (approval no. zyfy-an-2024-0710/0358) and supervised by the Ethics Committee of the Affiliated Hospital of Zunyi Medical University. Mice were maintained under controlled environmental conditions (temperature, 22–25°C; humidity, 55–60%; 12-h light/dark cycle) with free access to food and water. All procedures adhered to China's guidelines for animal research, with efforts made to minimize animal use and alleviate potential distress, thus complying with ethical standards.

The ACOP mouse model was established as previously described (13). Briefly, mice were placed in a transparent plastic chamber at 22–24°C. To monitor CO levels, two CO detectors were positioned, one at the center and another near the edge of the chamber. The chamber was then gradually filled with CO gas. The exposure protocol involved an initial phase of 1,000 ppm CO for 40 min, followed by a higher concentration of 3,000 ppm CO for 20 min, or until the mice displayed signs of unconsciousness. Following exposure to CO, the animals were promptly transferred to fresh ambient air and observed until they fully regained consciousness. Mice were anesthetized with 2% isoflurane in air (2–3 l/min) on days 1, 3 and 7. Subsequently, ~0.5–1 ml of whole blood was collected via the retro-orbital venous plexus equally divided into anticoagulant and plain tubes for the preparation of plasma and serum. Both plasma and serum were isolated following centrifugation at 1,000 × g for 10 min at 4°C. Following blood collection, deep anesthesia was maintained with 2% isoflurane, and the mice were sacrificed directly. Different euthanasia methods were selected according to different experimental purposes: In the experiments exploring the time course of ACOP-induced renal injury and conducting transcriptome analysis, to minimize animal suffering and preserve the overall morphological structure of tissues, the experimental animals were sacrificed by cervical dislocation under isoflurane anesthesia. The collected renal tissues were mainly used for phenotypic observation studies. In the inhibitor intervention experiments, under the same isoflurane anesthesia, sacrifice was performed by transcardiac perfusion with ice-cold 0.1 M phosphate-buffered saline (PBS) to harvest renal tissues, which was mainly to meet the higher tissue quality requirements for mechanism research.

To explore the temporal changes in kidney injury subsequent to ACOP, a total of 24 mice were randomly divided into four groups (n=6): The control, ACOP 1-day, ACOP 3-day and ACOP 7-day groups. The degree of kidney injury was evaluated at each time point by histopathological and biochemical analyses. Follow-up experiments were performed at the time point exhibiting the most severe injury (determined by the highest tubular injury score). Total RNA was extracted from the renal tissue of both the control and ACOP model groups for RNA sequencing (RNA-seq) analysis. Raw reads were quality-checked and adapter-trimmed using Fastp (v0.23.2, github.com/OpenGene/fastp). The resulting high-quality clean reads were then aligned to the reference genome using HISAT2 (v2.2.1, http://daehwankimlab.github.io/hisat2/). Gene-level read counts were generated from the alignment files using FeatureCounts (v2.0.3, http://subread.sourceforge.net/). Finally, differentially expressed genes (DEGs) between the control and model groups were identified using the DESeq2 (v1.40.2, http://bioconductor.org/packages/release/bioc/html/DESeq2.html) package, with a significance threshold set at an absolute fold change ≥2 and an adjusted P-value <0.05.

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was then performed to identify key signaling pathways involved in kidney injury. Based on the KEGG results, representative upregulated genes were selected as candidate targets and validated using western blotting. An in vivo intervention utilizing the PI3K inhibitor LY294002 was performed to assess the functional role of specific signaling pathways in ACOP-AKI. A total of 36 mice were randomly allocated into four groups (n=9) in the inhibitor experiments. The control group consisted of mice housed in identical transparent plastic cages as those in the experimental groups without CO treatment or injection. The ACOP group consisted of mice exposed to CO to establish an ACOP model.

Subsequently, mice were subject to intraperitoneal injections with varying components depending on their group. For the ACOP + LY294002 group, LY294002 was initially dissolved in DMSO at a concentration of <0.1% and was subsequently diluted in 0.9% NaCl, resulting in a final injection dose of 10 mg/kg body weight. Mice in the ACOP group were treated with an intraperitoneal injection of an equivalent volume of 0.9% NaCl solution without DMSO, matching the injection volume of LY294002 in the ACOP + LY294002 group. In the ACOP + vehicle group, mice were also exposed to CO and then received an intraperitoneal injection of a 0.9% NaCl solution containing 0.1% DMSO. The first injection was administered within 2 h post-modeling via CO exposure, followed by daily injections until the designated experimental endpoint. Mice were then sacrificed for subsequent analysis.

The concentration of CO in blood was measured using CO assay kit (cat. no. A101-3-1; Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's instructions. Total hemoglobin (Hb) concentration was determined. The optical density (OD) was measured at a wavelength of 540 nm (A540) using a microplate reader (Varioskan Flash; Thermo Fisher Scientific, Inc.). The Hb concentration was calculated as follows: Hb=A540×73.54. Subsequently, the percentage of carboxyhemoglobin (COHb) was determined. The ODs of test and control samples were measured at 568 nm (A568) and 581 nm (A581). The ΔOD value was calculated using the following formula: ΔOD=ΔOD of test sample (A568-A581)-ΔOD of control sample (A568-A581). The COHb was then calculated using the following equation: COHb (%)=(0.822× ΔOD + 0.001) ×100. The final blood CO concentration was calculated as follows: CO concentration=[COHb (%) × Hb (g/l) ×10⁶x4]/64456.

Blood urea nitrogen (BUN) and serum creatinine (SCr) were measured with commercially available assay kits (C013-2-1 and C011-2-1; both Nanjing Jiancheng Bioengineer Institute, China), according to the manufacturer's protocols. The OD was measured at a wavelength of 450 nm, and the concentrations of BUN and SCr were calculated based on a standard curve.

Renal tissue samples were fixed in 4% phosphate-buffered formaldehyde for 24–48 h at room temperature and subsequently embedded in paraffin. The embedded tissues were sliced into 5 µm-thick sections and were then subjected to hematoxylin and eosin (H&E) as well as periodic acid-Schiff (PAS) staining. Specifically, hematoxylin staining was performed for 5–8 min at room temperature, followed by eosin staining for 1–2 min under the same conditions. For the PAS staining, sections were oxidized in periodic acid solution for 5–10 min at room temperature and then stained with Schiff's reagent for 15–30 min at room temperature. After staining, the sections were incubated with diaminobenzidine for 5 min at room temperature, counterstained with hematoxylin for 1–2 min at room temperature, and observed under a light microscope. Tubular injury was assessed according to a five-tier grading system that evaluated the extent of brush border loss, tubular dilation, cast formation, and cellular necrosis, as referenced in (14). The specific grading scheme was defined as: 0, no pathological changes; 1, less than 10% involvement; 2, 10–25% involvement; 3, 26–50% involvement; 4, 51–75% involvement; and 5, >75% involvement.

Renal tissue samples were fixed in 4% phosphate-buffered formaldehyde for 24–48 h at room temperature. Paraffin-embedded tissue sections (thickness, 5 µm) were deparaffinized and rehydrated through a descending series of ethanol to water. Antigen retrieval was performed using citrate buffer (pH 6.0) at 95°C for 20 min. Endogenous peroxidase activity was blocked following tissue incubation with 3% hydrogen peroxide for 10 min at room temperature. Subsequently, sections were blocked with 10% normal goat serum (cat. no. AR0009, Boster Biological Technology) for 1 h at room temperature. The sections were then incubated at 4°C overnight with a primary antibody against neutrophil gelatinase-associated lipocalin (NGAL; rabbit; dilution, 1:200; cat. no. ab125075; Abcam). Sections were washed three times with phosphate-buffered saline (PBS; pH 7.4) and incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:500; cat. no. ab6721; Abcam) for 30 min at room temperature. Following secondary antibody incubation, the sections were washed again with PBS. The tissues were then counterstained with hematoxylin for 1–2 min at room temperature. Images were captured under an optical microscope and analyzed with ImageJ software (v1.8. National Institutes of Health).

The kidney tissue samples were fixed in 4% paraformaldehyde at room temperature for 24–48 h prior to paraffin embedding. Paraffin-embedded sections (5 µm) were deparaffinized and rehydrated as aforementioned, followed by antigen retrieval with citrate buffer (pH 6.0) at 95°C for 20 min. The tissue sections were then incubated with a TUNEL reaction mixture (One Step TUNEL Apoptosis Assay Kit, Beyotime, C1089) for 1 h at 37°C. Subsequently, the sections were counterstaining with hematoxylin (0.1% w/v, Sigma-Aldrich, H9627) for 1–2 min at room temperature, and mounted with antifade mounting medium (Beyotime, P0126). Finally, to assess cell apoptosis, images from five randomly selected fields of view/section were captured under a light microscope.

Total RNA was extracted using a TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., cat. no. 15596026). RNA integrity and concentration were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). Subsequently, for library preparation, high-quality RNA samples (RNA integrity number ≥7.0) were further analyzed using the NEBNext Ultra RNA Library Prep Kit (New England BioLabs, Inc., cat. no. E7530L). The final libraries were quantified using the Qubit^™ dsDNA HS Assay kit (Invitrogen; Thermo Fisher Scientific, Inc., cat. no. Q32851) and diluted to a loading concentration of 1.8 nM. Paired-end sequencing (2×150 bp) was performed on the Illumina NovaSeq 6000 platform with the NovaSeq 6000 S4 Reagent kit (300 cycles; Illumina, Inc., cat. no. 20028312). Then the data were processed and analyzed using Fastp (v0.23.2, github.com/OpenGene/fastp). High-quality reads were then aligned to the reference genome using HISAT2 (v2.2.1, http://daehwankimlab.github.io/hisat2/), followed by gene read counting using FeatureCounts (v2.0.3, http://subread.sourceforge.net/). Differential expression analysis was ultimately performed using the DESeq2 (v1.40.2, http://bioconductor.org/packages/release/bioc/html/DESeq2.html) software.

A panel of candidate genes, including cytokine signaling 6 (SOCS6), mesothelin (MSLN), glutathione-specific γ-glutamylcyclotransferase 1 (CHAC1) and C-C motif chemokine ligand 4 (CCL4), was preselected for initial screening based on their previously reported associations with drug-induced organ injury, cell stress response, inflammation or immune regulation pathways (14–17). CCL4 was prioritized for detailed reporting in the present study because it demonstrated the most consistent and significant dysregulation trends in our preliminary assessments (data not shown).

The serum levels of TNF-α, IL-6 and IL-1β were quantified according to the manufacturer's instructions using Mouse TNF-α ELISA Kit (Order NO. D721217, Sangon Biotech, Shanghai, China), Mouse IL-6 ELISA Kit (Order NO. D721022, Sangon Biotech, Shanghai), and Mouse IL-1β ELISA kit (Order NO. D721017, Sangon Biotech, Shanghai, China).

Total proteins were extracted from kidney tissue using RIPA lysis buffer (Beyotime Institute of Biotechnology), supplemented with protease and phosphatase inhibitors. Protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein extracts (50 µg per lane) were separated by 10% SDS-PAGE, depending on the molecular weight of each target protein, and were then transferred onto PVDF membranes (MilliporeSigma). Following blocking with 5% non-fat milk in TBS-Tween-20 for 1 h at room temperature, the membranes were incubated at 4°C overnight with primary antibodies against NGAL (rabbit; dilution, 1:5,000; cat. no. ab125075; Abcam), C-C motif chemokine ligand 4 (CCL4; rabbit; dilution, 1:800; cat. no. 26614–1-AP; Proteintech Group, Inc.), phosphorylated (p)-protein kinase B (p-Akt; rabbit; dilution, 1:3,000; cat. no. 28731-1-AP; Proteintech Group, Inc.), Akt (rabbit; dilution, 1:5,000; cat no. 10176-2-AP; Proteintech Group, Inc.), NF-κB p65 (Ser536) (rabbit; dilution, 1:5,000; cat. no. 80379-2-RR; Proteintech Group, Inc.), NF-κB p65 (rabbit; dilution, 1:3,000; cat no. 10745-1-AP; Proteintech Group, Inc.), IL-6 (mouse; dilution, 1:3,000; cat. no. 66146-1-Ig; Proteintech Group, Inc.), IL-1β (rabbit; dilution, 1:1,000; cat. no. 26048-1-AP; Proteintech Group, Inc.), TNF-α (rabbit; dilution, 1:1,000; cat. no. 17590-1-AP; Proteintech Group, Inc.), Bax (rabbit; dilution, 1:8,000; cat no. 50599-2-Ig; Proteintech Group, Inc.) and Bcl-2 (mouse; dilution, 1:1,000; cat. no. 26593-1-AP; Proteintech Group, Inc.). Membranes were washed three times with TBST (0.05% Tween-20), and incubated with HRP-conjugated goat anti-rabbit (cat. no. SA00001-2) or goat anti-mouse (1:5,000; cat. no. SA00001-1; Proteintech) secondary antibodies (dilution, 1:5,000; Proteintech Group, Inc.) for 1 h at room temperature. GAPDH (rabbit; dilution 1:20,000; cat. no. 10494-1-AP; Proteintech Group, Inc.) and β-actin (rabbit; dilution, 1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) served as internal loading controls. The proteins were visualized using the WesternBright^® ECL detection kit (Advansta Inc.) combined with the ChemiDoc XRS+ imaging system (Bio-Rad Laboratories, Inc.). The signal intensity of each band was subsequently quantified using ImageLab software v6.1 (Bio-Rad Laboratories, Inc.).

All data are expressed as the mean ± SD. All statistical analyses were performed using SPSS 22.0 (IBM Corp.). The differences between two groups were compared by unpaired Student's t-test, while those among multiple groups were compared by one-way ANOVA. For multiple comparisons following ANOVA, Bonferroni correction was applied. P<0.05 was considered to indicate a statistically significant difference.

To evaluate whether ACOP treatment induces kidney injury in mice, a mouse model of ACOP exposure was established, and blood samples were collected at 1, 3 and 7 days following CO exposure to assess blood CO levels, renal function parameters and renal histopathology (Fig. 1A). The results demonstrated that blood CO levels were markedly elevated in ACOP-treated mice compared with control mice, thus verifying the successful establishment of the mouse model (Fig. 1B). In addition, SCr and BUN levels were markedly increased at all time points following ACOP treatment compared with the control group, thus indicating substantial renal dysfunction (Fig. 1C and D). Histological analysis, using H&E and PAS staining, revealed that the glomerular structure remained intact, although mild mesangial matrix expansion and prominent tubular injury were observed in the ACOP group (Fig. 1E). On day 1 post-ACOP exposure, renal tubular epithelial cells displayed marked swelling, peritubular capillary congestion, cytoplasmic dissolution and nuclear loss (red arrows). On day 3, severe vacuolar degeneration, tubular dilation, brush border disruption and extensive nuclear detachment were evident (yellow arrows). However, on day 7, partial tubular regeneration was observed, although vacuolar changes, tubular dilation and brush border damage persisted (blue arrows). To assess tubular injury, the tubular injury scoring method was applied (18), which showed a significant increase in injury scores in ACOP treated mice compared with control mice, with the highest injury scores observed on day 3. This finding was consistent with the morphological one (Fig. 1F). In addition, the expression levels of NGAL, an early biomarker of AKI, were detected. Immunohistochemical staining demonstrated markedly widespread NGAL expression in renal tubules in mice in the ACOP group compared with the control group (Fig. 1E and G). Western blot analysis further verified that NGAL was markedly upregulated in the ACOP group compared with the control group; this was most noticeable on days 1 and 3, followed by a decrease on day 7 (Fig. 1H and I). Collectively, the aforementioned results suggested that exposure of mice to ACOP could successfully induce AKI.

To further explore the molecular mechanism of ACOP-AKI, RNA-seq analysis was carried out on the kidney tissue samples from the ACOP model and control groups on day 3, identified as the peak of kidney injury. Based on the analysis of differential gene expression, the top 20 DEGs with the highest average expression levels were presented in Table SI, including log₂(fold change) and P-value. The volcano plot in Fig. 2A depicts the distribution of DEGs, revealing significant up- and downregulation trends, whereas the heatmap in Fig. 2B focuses on genes functionally linked to the PI3K/Akt pathway. A KEGG pathway enrichment analysis of DEGs revealed that the ‘PI3K/Akt signaling pathway’ was markedly enriched in the ACOP group (Fig. 2C).

Through candidate gene screening and validation, CCL4 exhibited consistent patterns at both the transcriptional and protein levels in the present model (data not shown), indicating its potential role in the mechanisms underlying ACOP-AKI. In addition, the present study observed that in ACOP-exposed mice, the p-Akt/Akt ratio and Bax protein levels were markedly increased, whereas Bcl-2 expression was markedly decreased when compared with control mice (Fig. 3A). TUNEL staining assays further revealed that the number of TUNEL-positive cells was notably increased in renal tissues of mice in the ACOP group compared with the control group. Merged images demonstrated co-localization of TUNEL signals with DAPI-stained nuclei, confirming the induction of cellular apoptosis in the renal tissue following ACOP injury. Furthermore, the apoptosis appeared to be predominantly localized within the renal tubule (Fig. 3B). Furthermore, the ELISA results demonstrated markedly elevated serum levels of the systemic pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in the ACOP group compared with the control group (Fig. 3C). The aforementioned data indicated that ACOP may have upregulated the expression of CCL4, accompanied by activation of the PI3K/Akt signaling pathway. Furthermore, ACOP induced significant inflammatory responses and notable apoptosis.

To investigate the role of the PI3K/Akt signaling pathway in ACOP-AKI, mice were treated with the PI3K inhibitor LY294002, and renal function and histopathological changes were evaluated on day 3 (Fig. 4A). Biochemical analysis showed that mice in the ACOP group exhibited markedly elevated serum levels of SCr and BUN compared with the control group. In addition, LY294002 treatment partially yet markedly ameliorated these increased serum levels (Fig. 4B and C). Morphological assessment revealed pronounced tubular damage in the kidneys of ACOP-treated mice, which was markedly alleviated in the ACOP + LY294002 group (Fig. 4D). Consistently, the tubular injury score was markedly lower in the ACOP + LY294002 group compared with the ACOP group (Fig. 4E). Immunohistochemical analysis demonstrated a significant increase in NGAL expression in the ACOP group compared with the control group, which was markedly reduced following treatment with LY294002 (Fig. 4F). Finally, western blot analysis was performed to assess the NGAL protein expression levels (Fig. 4G), and densitometric quantification verified that LY294002 markedly suppressed NGAL expression in the kidneys of ACOP-treated mice (Fig. 4H). These findings suggested that LY294002 could effectively mitigate ACOP-AKI.

To further investigate the molecular mechanisms underlying the effects of LY294002 on ACOP-AKI, western blot analysis was employed to detect the expression levels of inflammatory factors and apoptosis-related proteins. The results showed that the ratio of p-Akt/Akt was markedly elevated in the ACOP-treated group, compared with the control, accompanied by a significant upregulation of inflammatory factors such as IL-6, TNF-α and IL-1β. By contrast, treatment with LY294002 markedly reduced the p-Akt/Akt ratio and the protein expression levels of IL-6, IL-1β and TNF-α compared with the ACOP group, suggesting an inhibitory effect of LY294002 on the expression of pro-inflammatory cytokines.

Considering that NF-κB is a key mediator of pro-inflammatory cytokine expression, the present study further assessed the ratio of NF-κB p65 (Ser536)/NF-κB p65. The results revealed that treatment with LY294002 also markedly inhibited the elevation of this ratio in the kidneys of ACOP-treated mice. Furthermore, ACOP treatment induced a significant increase in the expression of the pro-apoptotic protein Bax and a significant decrease in the expression of the anti-apoptotic protein Bcl-2 when compared with the control group. However, LY294002 treatment markedly suppressed ACOP-induced Bax expression and restored Bcl-2 levels (Fig. 5). In conclusion, LY294002 effectively suppressed inflammation and apoptosis in the kidneys of ACOP-treated mice by modulating key signaling pathways such as PI3K/Akt and NF-κB, providing a potential pharmacological target for the treatment of ACOP-AKI.