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Acute carbon monoxide poisoning induces renal inflammation and apoptosis via CCL4 upregulation and activation of the PI3K/Akt pathway in mice  

  • Authors:
    • Yu Ni
    • Xuheng Jiang
    • Shuhong Wang
    • Tao Pu
    • Tiantian Yan
    • Anyong Yu
    • Qi Liu
  • View Affiliations / Copyright

    Affiliations: Department of Nephrology, Clinical Medical College of Suzhou Medical College, Soochow University, Suzhou, Jiangsu 215123, P.R. China, Emergency Department, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou 563003, P.R. China, Department of Nephrology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou 563003, P.R. China
    Copyright: © Ni et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 344
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    Published online on: October 7, 2025
       https://doi.org/10.3892/mmr.2025.13709
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Abstract

Acute carbon monoxide poisoning (ACOP) is one of the primary causes of poisoning worldwide, leading to multi‑organ dysfunction, particularly affecting the kidneys. However, the molecular mechanisms underlying the effects of carbon monoxide‑induced acute kidney injury (AKI) remain yet to be fully elucidated. Therefore, the present study aimed to identify key genes and signaling pathways involved in AKI following ACOP in a mouse model using the RNA sequencing (RNA‑seq) technology. Renal function was assessed via measuring serum creatinine (SCr) and blood urea nitrogen (BUN) levels. Additionally, histopathological alterations, cell apoptosis and inflammation were evaluated by immunohistochemistry, TUNEL assay, ELISA and western blot analysis. The results revealed significant renal insufficiency and tubular injury, alongside a heightened inflammatory state and apoptosis in ACOP mice. RNA‑seq and mechanistic studies identified the upregulation of C‑C motif chemokine ligand 4 (CCL4) and activation of the PI3K/Akt pathway as key underlying events. Inhibition of PI3K with LY294002 attenuated these pathological changes and alleviated kidney injury. AKT In conclusion, the present study demonstrated that ACOP induced renal inflammation and apoptosis through CCL4 upregulation and activation of the PI3K/Akt pathway, thus conveying a potential therapeutic target for mitigating ACOP‑induced AKI (ACOP‑AKI). Overall, the present study could provide important insights into the molecular mechanisms of ACOP‑AKI.
View Figures

Figure 1

ACOP induces acute kidney injury in
mice. (A) Schematic illustration of the ACOP mouse model and
experimental design. (B) Bar graph showing CO concentrations in the
blood of mice in the control and ACOP groups. (C) SCr and (D) BUN
levels at different time points after ACOP exposure are shown. (E)
Representative H&E, PAS and NGAL immunohistochemical staining
of kidney tissues at different time points after mice exposure to
ACOP exposure. On day 1, tubular epithelial cells displayed marked
swelling, peritubular capillary congestion and cytoplasmic
dissolution with nuclear loss, as indicated by the red arrows. On
day 3, extensive vacuolar degeneration, tubular dilation, brush
border disruption and nuclear shedding were observed (yellow
arrows). By day 7, signs of tubular regeneration, with persisted
vacuolar degeneration, tubular dilation and brush border damage
(blue arrows), were observed. Scale bar, 100 µm. (F) Quantification
of tubular injury scoring. (G) Quantification of NGAL protein
expression in renal tissue detected by immunohistochemistry. (H)
Expression of NGAL based on immunoblot bands. (I) Semi-quantitation
of NGAL expression. Data are expressed as the mean ± SD (n=6).
*P<0.05 and **P<0.01 vs. control. ACOP, acute carbon monoxide
poisoning; NGAL, neutrophil gelatinase-associated lipocalin; SCr,
serum creatinine; BUN, blood urea nitrogen; CO, carbon monoxide; w,
week; ppm, parts per million; H&E, Hematoxylin and Eosin; PAS,
periodic acid-Schiff; IHC, immunohistochemistry; WB, western blot
analysis.

Figure 2

C-C motif chemokine ligand 4 is
upregulated in ACOP-treated mice via the PI3K/Akt signaling
pathway. (A) Volcano plots showing identified DEGs. Red and blue
dots indicate markedly upregulated and downregulated genes,
respectively, while gray dots represent genes that do not meet the
differential expression criteria. (B) Heatmap of DEGs. Red and
green indicate the upregulated and downregulated genes,
respectively. (C) Kyoto Encyclopedia of Genes and Genomes pathway
enrichment analysis of DEGs. DEG, differentially expressed genes;
ACOP, acute carbon monoxide poisoning; AMPK, AMP-activated protein
kinase; ECM, extracellular matrix; AK, acute kidney; SK, sham
kidney Aqp5, aquaporin 5; Clec1b, C-type lectin domain family 1
member B; Adgra1, adhesion G protein-coupled receptor A1; Mef2c,
myocyte enhancer factor 2C; Il11ra2, interleukin 11 receptor
subunit α2; Ccl4, C-C motif chemokine ligand 4; Ccr1, C-C motif
chemokine receptor 1; Selp, selectin P; Trem2, triggering receptor
expressed on myeloid cells 2; Plxdc1, plexin domain containing 1;
Nos2, nitric oxide synthase 2; Tgfb1i1, transforming growth factor
β1 induced transcript 1; Cysltr2, cysteinyl leukotriene receptor
2.

Figure 3

ACOP induces apoptosis in kidney
tissues and triggers systemic inflammatory responses in mice. (A)
Western blot analysis was performed to determine the expression
levels of CCL4, p-Akt/Akt, Bax and Bcl-2 in renal tissues from the
control and model groups. Statistical evaluation was also
performed. (B) Apoptosis in kidney tissues from mice in the control
and model groups was assessed by TUNEL staining. (C) Serum levels
of the inflammatory cytokines TNF-α, IL-6 and IL-1β in mice were
measured using ELISA. Data are expressed as the mean ± SD (n=3).
**P<0.01 vs. control. CCL4, C-C motif chemokine ligand 4; ACOP,
acute carbon monoxide poisoning; p-, phosphorylated.

Figure 4

Effects of LY294002 on kidney injury
in mice exposed to ACOP. (A) Schematic representation of the
establishment of the ACOP + LY294002 mouse model and experimental
design. The experiment was conducted on day 3 after ACOP exposure,
when the injury was most severe. Blood and tissue samples were
subsequently collected for serological, morphological and protein
expression analyses. (B) SCr and (C) BUN levels in control, ACOP,
vehicle-treated ACOP control and LY294002-treated ACOP groups are
shown. (D) Representative H&E, PAS and NGAL immunohistochemical
staining images of kidney tissues in different groups are shown.
Scale bar, 100 µm. (E) Tubular injury scoring is presented. (F)
Quantification of NGAL protein expression in renal tissue detected
by immunohistochemistry. (G) Expression of NGAL based on immunoblot
bands. (H) Semi-quantitation of NGAL expression. Data are expressed
as the mean ± SD (n=3). *P<0.05 vs. control. ACOP, acute carbon
monoxide poisoning; NGAL, neutrophil gelatinase-associated
lipocalin; SCr, serum creatinine; BUN, blood urea nitrogen; CO,
carbon monoxide; w, week; ppm, parts per million; PAS, periodic
acid-Schiff; IHC, immunohistochemistry; WB, western blot analysis;
ip, intraperitoneal.

Figure 5

Treatment with LY294002 inhibits
inflammation and apoptosis in the kidneys of ACOP mice. (A) The
protein expression levels of p-Akt/Akt, NF-κB p65 (Ser536)/NF-κB
p65, IL-6, TNF-α, IL-1β, Bax and Bcl-2 were detected in kidney
tissues of mice in the control, ACOP, vehicle-treated ACOP control
and LY294002-treated ACOP groups by western blot analysis. (B) The
quantified protein expression levels of (A) are shown. Data are
expressed as the mean ± SD (n=3). *P<0.05 and **P<0.01 vs.
control. ACOP, acute carbon monoxide poisoning; p-,
phosphorylated.

Figure 6

Schematic illustration depicting how
the inflammatory response and apoptosis collectively contribute to
kidney injury, and the underlying mechanisms following exposure to
ACOP. ACOP, acute carbon monoxide poisoning; CCL4, C-C motif
chemokine ligand 4; CCR5, C-C chemokine receptor type 5.
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Copy and paste a formatted citation
Spandidos Publications style
Ni Y, Jiang X, Wang S, Pu T, Yan T, Yu A and Liu Q: Acute carbon monoxide poisoning induces renal inflammation and apoptosis via CCL4 upregulation and activation of the PI3K/Akt pathway in mice&nbsp;&nbsp;. Mol Med Rep 32: 344, 2025.
APA
Ni, Y., Jiang, X., Wang, S., Pu, T., Yan, T., Yu, A., & Liu, Q. (2025). Acute carbon monoxide poisoning induces renal inflammation and apoptosis via CCL4 upregulation and activation of the PI3K/Akt pathway in mice&nbsp;&nbsp;. Molecular Medicine Reports, 32, 344. https://doi.org/10.3892/mmr.2025.13709
MLA
Ni, Y., Jiang, X., Wang, S., Pu, T., Yan, T., Yu, A., Liu, Q."Acute carbon monoxide poisoning induces renal inflammation and apoptosis via CCL4 upregulation and activation of the PI3K/Akt pathway in mice&nbsp;&nbsp;". Molecular Medicine Reports 32.6 (2025): 344.
Chicago
Ni, Y., Jiang, X., Wang, S., Pu, T., Yan, T., Yu, A., Liu, Q."Acute carbon monoxide poisoning induces renal inflammation and apoptosis via CCL4 upregulation and activation of the PI3K/Akt pathway in mice&nbsp;&nbsp;". Molecular Medicine Reports 32, no. 6 (2025): 344. https://doi.org/10.3892/mmr.2025.13709
Copy and paste a formatted citation
x
Spandidos Publications style
Ni Y, Jiang X, Wang S, Pu T, Yan T, Yu A and Liu Q: Acute carbon monoxide poisoning induces renal inflammation and apoptosis via CCL4 upregulation and activation of the PI3K/Akt pathway in mice&nbsp;&nbsp;. Mol Med Rep 32: 344, 2025.
APA
Ni, Y., Jiang, X., Wang, S., Pu, T., Yan, T., Yu, A., & Liu, Q. (2025). Acute carbon monoxide poisoning induces renal inflammation and apoptosis via CCL4 upregulation and activation of the PI3K/Akt pathway in mice&nbsp;&nbsp;. Molecular Medicine Reports, 32, 344. https://doi.org/10.3892/mmr.2025.13709
MLA
Ni, Y., Jiang, X., Wang, S., Pu, T., Yan, T., Yu, A., Liu, Q."Acute carbon monoxide poisoning induces renal inflammation and apoptosis via CCL4 upregulation and activation of the PI3K/Akt pathway in mice&nbsp;&nbsp;". Molecular Medicine Reports 32.6 (2025): 344.
Chicago
Ni, Y., Jiang, X., Wang, S., Pu, T., Yan, T., Yu, A., Liu, Q."Acute carbon monoxide poisoning induces renal inflammation and apoptosis via CCL4 upregulation and activation of the PI3K/Akt pathway in mice&nbsp;&nbsp;". Molecular Medicine Reports 32, no. 6 (2025): 344. https://doi.org/10.3892/mmr.2025.13709
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