Department of Intensive Care Unit, The Eighth Affiliated Hospital, Sun Yat-sen University, No. 4, Huancheng South Road, Longgang District, Shenzhen City, 518003, Guangdong Province, P.R. China.
J Bioenerg Biomembr. 2024 Jun;56(3):285-296. doi: 10.1007/s10863-024-10015-0. Epub 2024 Mar 22.
Acute kidney injury (AKI) is a serious complication of sepsis patients, but the pathogenic mechanisms underlying AKI are still largely unclear. In this view, the roles of the key component of N6-methyladenosine (m6A)-wilms tumor 1 associated protein (WTAP) in AKI progression were investigated. AKI mice model was established by using cecal ligation and puncture (CLP). AKI cell model was established by treating HK-2 cells with LPS. Cell apoptosis was analyzed by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining and flow cytometry analysis. Cell viability was analyzed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The concentrations of inflammatory factors were examined with ELISA kits. Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and Fe levels were detected with related kits. Gene expression was detected by western blot assay or quantitative real-time polymerase chain reaction (qRT-PCR) assay. The relation between WTAP and lamin B1 (LMNB1) was verified by Methylated RNA Immunoprecipitation (meRIP) assay, RIP assay, dual-luciferase reporter assay and Actinomycin D assay. CLP induced significant pathological changes in kidney tissues in mice and promoted inflammation, mitochondrial damage and ferroptosis. LMNB1 level was induced in HK-2 cells by LPS. LMNB1 knockdown promoted LPS-mediated HK-2 cell viability and inhibited LPS-mediated HK-2 cell apoptosis, inflammation, mitochondrial damage and ferroptosis. Then, WTAP was demonstrated to promote LMNB1 expression by m6A Methylation modification. Moreover, WTAP knockdown repressed LPS-treated HK-2 cell apoptosis, inflammation, mitochondrial damage and ferroptosis, while LMNB1 overexpression reversed the effects. Additionally, WTAP affected the pathways of NF-κB and JAK2/STAT3 by LMNB1. WTAP-mediated m6A promoted the inflammation, mitochondrial damage and ferroptosis in LPS-induced HK-2 cells by regulating LMNB1 expression and activating NF-κB and JAK2/STAT3 pathways.
急性肾损伤（AKI）是脓毒症患者的严重并发症，但 AKI 的发病机制在很大程度上仍不清楚。在这种情况下，研究了 N6-甲基腺苷（m6A）-Wilms 肿瘤 1 相关蛋白（WTAP）的关键组成部分在 AKI 进展中的作用。通过使用盲肠结扎和穿刺（CLP）建立 AKI 小鼠模型。通过用 LPS 处理 HK-2 细胞建立 AKI 细胞模型。通过 TdT 介导的 dUTP 末端标记（TUNEL）染色和流式细胞术分析细胞凋亡。通过 3-（4,5-二甲基-2-噻唑基）-2,5-二苯基-2-H-四唑溴盐（MTT）测定分析细胞活力。通过 ELISA 试剂盒检测炎症因子的浓度。用相关试剂盒检测活性氧（ROS）、丙二醛（MDA）、谷胱甘肽（GSH）和 Fe 水平。通过 Western blot 测定或定量实时聚合酶链反应（qRT-PCR）测定检测基因表达。通过甲基化 RNA 免疫沉淀（meRIP）测定、RIP 测定、双荧光素酶报告基因测定和放线菌素 D 测定验证 WTAP 和核纤层蛋白 B1（LMNB1）之间的关系。CLP 在小鼠的肾脏组织中引起了明显的病理变化，并促进了炎症、线粒体损伤和铁死亡。LPS 诱导 HK-2 细胞中 LMNB1 水平升高。LMNB1 敲低促进 LPS 介导的 HK-2 细胞活力，并抑制 LPS 介导的 HK-2 细胞凋亡、炎症、线粒体损伤和铁死亡。然后，通过 m6A 甲基化修饰，证明 WTAP 促进 LMNB1 的表达。此外，WTAP 敲低抑制 LPS 处理的 HK-2 细胞凋亡、炎症、线粒体损伤和铁死亡，而 LMNB1 过表达则逆转了这种作用。此外，WTAP 通过 LMNB1 影响 NF-κB 和 JAK2/STAT3 途径。WTAP 介导的 m6A 通过调节 LMNB1 表达并激活 NF-κB 和 JAK2/STAT3 途径，促进 LPS 诱导的 HK-2 细胞中的炎症、线粒体损伤和铁死亡。
