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M2 Macrophages-Derived Exosomes Inhibited Podocyte Pyroptosis via lncRNA AFAP1-AS1/EZH2 Axis
Qing Zhan1, Huiyun Liu2, Minyang Zhao1
1Department of Endocrinology and Metabolism, the First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, China.
Abstract:
Macrophage infiltration was closely associated with inflammatory injury of podocytes in diabetic nephropathy (DN), while how macrophages affected podocytes remained not entirely clear. Here, we not only investigated the relationship between macrophages and high glucose (HG)-treated podocytes, but the underlying mechanisms were also explored. Transmission electron microscopy, nanoparticle tracking analysis, and western blot of CD9, CD63, CD81, and Calnexin were performed to identify exosomes; QRT-PCR was performed to detect AFAP1-AS1 expression; Western blot was performed to examine NLRP3, Cleaved caspase-1, and GSDMD-N protein levels; Immunofluorescence was performed to assess co-localisation of NLRP3 and ASC; ELISA was performed to detect IL-18 and IL-1β levels; Cytotoxicity LDH Assay Kit was performed to detect LDH level; RNA pulldown was performed to determine the interaction of AFAP1-AS1 and EZH2; ChIP was employed to determine the interaction of EZH2 and H3K27me3 in the NLRP3 promoter region. The results showed that AFAP1-AS1 expression was down-regulated in the peripheral blood of DN patients, and exosomes derived from M2 macrophages transfected with si-AFAP1-AS1 enhanced HG-induced podocyte pyroptosis via significantly elevating NLRP3, Cleaved caspase-1, and GSDMD-N protein levels, immunofluorescence intensity of NLRP3 and ASC, as well as IL-18, IL-1β, and LDH levels. Mechanistically, AFAP1-AS1 interacted with EZH2 to transcriptionally regulate H3K27me3 level in the NLRP3 promoter region, thus epigenetically repressing NLRP3 level to inhibit podocyte pyroptosis. These results may provide an important target for improving kidney injury in DN.
Insights
Macrophage-derived exosomes carrying low AFAP1-AS1 worsen diabetic kidney injury by promoting podocyte pyroptosis. This occurs through epigenetic repression of NLRP3, highlighting AFAP1-AS1 as a potential therapeutic target for diabetic nephropathy.
Area of Science:
- Nephrology
- Cell Biology
- Molecular Biology
Background:
- Macrophage infiltration is linked to podocyte injury in diabetic nephropathy (DN).
- The precise mechanisms by which macrophages influence podocyte damage in DN are not fully understood.
- This study investigates the interplay between macrophages and high glucose-induced podocyte dysfunction.
Purpose of the Study:
- To elucidate the role of macrophage-derived exosomes in high glucose-induced podocyte pyroptosis.
- To explore the underlying molecular mechanisms involving AFAP1-AS1, EZH2, and NLRP3.
- To identify potential therapeutic targets for mitigating kidney injury in DN.
Main Methods:
- Exosome identification (TEM, NTA, Western blot for CD9, CD63, CD81, Calnexin).
- Gene expression analysis (QRT-PCR for AFAP1-AS1).
- Protein analysis (Western blot for NLRP3, Cleaved caspase-1, GSDMD-N; Immunofluorescence for NLRP3/ASC co-localization).
- Functional assays (ELISA for IL-18, IL-1β; LDH assay for cytotoxicity).
- Molecular interaction studies (RNA pulldown for AFAP1-AS1/EZH2; ChIP for EZH2/H3K27me3 at NLRP3 promoter).
Main Results:
- AFAP1-AS1 expression was decreased in peripheral blood of DN patients.
- Exosomes from M2 macrophages with reduced AFAP1-AS1 exacerbated high glucose-induced podocyte pyroptosis.
- This exacerbation involved increased NLRP3 inflammasome activation (NLRP3, Cleaved caspase-1, GSDMD-N, IL-18, IL-1β, LDH).
- AFAP1-AS1 epigenetically repressed NLRP3 expression by interacting with EZH2 to regulate H3K27me3 at the NLRP3 promoter.
Conclusions:
- AFAP1-AS1, downregulated in DN patients, inhibits podocyte pyroptosis.
- Macrophage-derived exosomes carrying low AFAP1-AS1 promote DN-associated kidney injury.
- The AFAP1-AS1/EZH2/H3K27me3 pathway epigenetically regulates NLRP3, offering a novel therapeutic strategy for DN.
