ANXA2 and NF-κB positive feedback loop promotes high glucose-induced pyroptosis in renal tubular epithelial cells.
10.11817/j.issn.1672-7347.2025.250305
- Author:
Jiayi YANG
1
,
2
;
Yang LUO
3
;
Zixuan ZHU
3
;
Wenbin TANG
4
,
5
Author Information
1. Department of Hospital Infection Control Center, Xiangya Hospital, Central South University, Changsha
2. jiayi2355@csu.edu.cn.
3. Department of General Practice, Xiangya Hospital, Central South University, Changsha
4. National Clinical Research Center for Geriatric Diseases, Xiangya Hospital, Changsha
5. tangwenbin@csu.edu.cn.
- Publication Type:Journal Article
- Keywords:
Annexin A2;
NLRP3;
diabetic kidney disease;
nuclear factor κB;
positive feedback loop;
pyroptosis
- MeSH:
Humans;
Pyroptosis/drug effects*;
Annexin A2/physiology*;
Epithelial Cells/cytology*;
Kidney Tubules/cytology*;
Glucose/pharmacology*;
Diabetic Nephropathies/metabolism*;
NF-kappa B/metabolism*;
Cell Line;
Cell Proliferation;
Transcription Factor RelA/metabolism*;
Feedback, Physiological
- From:
Journal of Central South University(Medical Sciences)
2025;50(6):940-954
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVES:Pyroptosis plays a critical role in tubulointerstitial lesions of diabetic kidney disease (DKD). Annexin A2 (ANXA2) is involved in cell proliferation, apoptosis, and adhesion and may be closely related to DKD, but its specific mechanism remains unclear. This study aims to investigate the role and molecular mechanism of ANXA2 in high glucose-induced pyroptosis of renal tubular epithelial cells, providing new targets for DKD prevention and treatment.
METHODS:Human renal tubular epithelial HK-2 cells were divided into a normal glucose group (5.5 mmol/L), a high glucose group (30.0 mmol/L), and a osmotic control group (24.5 mmol/L mannitol+5.5 mmol/L glucose). ANXA2 expression was modulated by overexpression of plasmids and small interfering RNA (siRNA). Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) assay, apoptosis by flow cytometry, and ANXA2, p50, and p65 subcellular localization by immunofluorescence. Western blotting was employed to detect α-smooth muscle actin (α-SMA), fibronectin (FN), and collagen type IV (Col-IV). Real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting were used to analyze nuclear factor-κB (NF-κB) subunits p50/p65 and the pyroptosis pathway factors NLR family Pyrin domain containing 3 (NLRP3), caspase-1, inferleukin (IL)-1β, and IL-18. Protein interactions between ANXA2 and p50/p65 were examined by co-immunoprecipitation, while chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were used to examine NF-κB binding to the ANXA2 promoter.
RESULTS:High glucose upregulated ANXA2 expression and promoted its nuclear translocation (P<0.01). High glucose reduced cell proliferation, increased apoptosis, and elevated α-SMA, FN, and Col-IV expression (all P<0.05); ANXA2 overexpression aggravated these effects (all P<0.05), while ANXA2 knockdown reversed them (all P<0.05). High glucose activated NF-κB and increased NLRP3, caspase-1, L-1β, and IL-18 mRNA and protein expression (all P<0.05); ANXA2 overexpression further enhanced this, whereas knockdown suppressed NF-κB activation and downstream factors (all P<0.05). Co-immunoprecipitation confirmed ANXA2 directly binds the NF-κB subunit p65. ChIP assays revealed p65 binds specifically to ANXA2 promoter regions (ChIP-2, ChIP-4, and ChIP-6), and luciferase activity in corresponding mutant constructs (M2, M4, and M6) was significantly increased versus controls (all P<0.05), confirming positive transcriptional regulation of ANXA2 by p65.
CONCLUSIONS:ANXA2 and NF-κB form a positive feedback loop that sustains NLRP3 inflammasome activation, promotes pyroptosis pathway activation, and aggravates high glucose-induced renal tubular epithelial cell injury. Targeting ANXA2 or blocking its interaction with p65 may be a novel strategy to slow DKD progression.
