Department of Nephrology, Seventh People's Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, 200137 Shanghai, China.
Front Biosci (Landmark Ed). 2024 Feb 5;29(2):58. doi: 10.31083/j.fbl2902058.
Pyroptosis is a critical form of cell death during the development of chronic kidney disease (CKD). Tripartite motif 6 (TRIM6) is an E3-ubiquitin ligase that participates in the progression renal fibrosis (RF). The aim of this study was to investigate the roles of TRIM6 and Glutathione peroxidase 3 (GPX3) in oxidative stress-induced inflammasome activation and pyroptosis in Ang-II treated renal tubular epithelial cells.
To study its role in RF, TRIM6 expression was either reduced or increased in human kidney-2 (HK2) cells using lentivirus, and Ang-II, NAC and BMS-986299 were served as reactive oxygen species (ROS) inducer, ROS scavenger and NLRP3 agonist respectively. Pyroptosis and mitochondrial ROS were measured by flow cytometry. The levels of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were determined using commercial kits, while the levels of IL-1β, IL-18, IL-6, and tumor necrosis factor-α (TNF-α) were determined by Enzyme-Linked Immunosorbent Assay (ELISA). Co-immunoprecipitation (Co-IP) assay was used to evaluate the interaction between TRIM6 and GPX3. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot were used to measure mRNA and protein expression, respectively.
Treatment with Angiotensin II (Ang II) increased the protein and mRNA levels of TRIM6 in HK2 cells. Ang II also increased mitochondrial ROS production and the malondialdehyde (MDA) level, but decreased the levels of GSH and SOD. In addition, Ang II enhanced HK2 cell pyroptosis, increased the levels of IL-1β, IL-18, IL-6, and TNF-α, and promoted the expression of active IL-1β, NLRP3, caspase-1, and GSDMD-N proteins. These effects were reversed by knockdown of TRIM6 and by treatment with N-acetyl-L-cysteine (NAC), a ROS scavenger. BMS-986299, an NLRP3 agonist treatment, did not affect ROS production in HK2 cells exposed to Ang II combined with NAC, but cell pyroptosis and inflammation were aggravated. Moreover, the overexpression of TRIM6 in HK2 cells resulted in similar effects to Ang II. NAC and GPX3 overexpression in HK2 cells could reverse ROS production, inflammation, and pyroptosis induced by TRIM6 overexpression. TRIM6 overexpression decreased the GPX3 protein level by promoting its ubiquitination, without affecting the GPX3 mRNA level. Thus, TRIM6 facilitates GPX3 ubiquitination, contributing to increased ROS levels and pyroptosis in HK2 cells.
TRIM6 increases oxidative stress and promotes the pyroptosis of HK2 cells by regulating GPX3 ubiquitination. These findings could contribute to the development of novel drugs for the treatment of RF.
细胞焦亡是慢性肾脏病（CKD）发展过程中的一种关键细胞死亡形式。三结构域蛋白 6（TRIM6）是一种 E3 泛素连接酶，参与肾纤维化（RF）的进展。本研究旨在探讨 TRIM6 和谷胱甘肽过氧化物酶 3（GPX3）在血管紧张素 II（Ang-II）诱导的肾小管上皮细胞氧化应激激活和细胞焦亡中的作用。
为了研究其在 RF 中的作用，我们使用慢病毒在人肾细胞-2（HK2）细胞中降低或增加 TRIM6 的表达，并用 Ang-II、NAC 和 BMS-986299 分别作为活性氧（ROS）诱导剂、ROS 清除剂和 NLRP3 激动剂。通过流式细胞术测量细胞焦亡和线粒体 ROS。使用商业试剂盒测定丙二醛（MDA）、谷胱甘肽（GSH）和超氧化物歧化酶（SOD）的水平，通过酶联免疫吸附试验（ELISA）测定白细胞介素-1β（IL-1β）、白细胞介素-18（IL-18）、白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的水平。使用共免疫沉淀（Co-IP）测定 TRIM6 和 GPX3 之间的相互作用。逆转录-聚合酶链反应（RT-PCR）和 Western blot 分别用于测量 mRNA 和蛋白表达。
血管紧张素 II（Ang II）处理增加了 HK2 细胞中 TRIM6 的蛋白和 mRNA 水平。Ang II 还增加了线粒体 ROS 的产生和丙二醛（MDA）的水平，但降低了 GSH 和 SOD 的水平。此外，Ang II 增强了 HK2 细胞的细胞焦亡，增加了白细胞介素-1β（IL-1β）、白细胞介素-18（IL-18）、白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的水平，并促进了活性白细胞介素-1β（IL-1β）、NLRP3、半胱天冬酶-1（caspase-1）和 GSDMD-N 蛋白的表达。TRIM6 的敲低和 ROS 清除剂 N-乙酰-L-半胱氨酸（NAC）的处理逆转了这些作用。NLRP3 激动剂 BMS-986299 处理不会影响 Ang II 联合 NAC 处理的 HK2 细胞中 ROS 的产生，但会加重细胞焦亡和炎症。此外，HK2 细胞中 TRIM6 的过表达导致与 Ang II 相似的作用。HK2 细胞中 NAC 和 GPX3 的过表达可以逆转 TRIM6 过表达诱导的 ROS 产生、炎症和细胞焦亡。TRIM6 通过促进其泛素化降低了 GPX3 蛋白水平，而不影响 GPX3 mRNA 水平。因此，TRIM6 通过调节 GPX3 泛素化促进 ROS 水平升高和 HK2 细胞的细胞焦亡。
TRIM6 通过调节 GPX3 泛素化增加氧化应激并促进 HK2 细胞的细胞焦亡。这些发现可能有助于开发治疗 RF 的新型药物。
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