Ni Haiqiang, Ou Zhiyu, Wang Yuchen, Liu Yanna, Sun Kailun, Zhang Ji, Zhang Jiasi, Deng Wenfeng, Zeng Wenli, Xia Renfei, Xu Jian, Gong Nianqiao, Miao Yun
Department of Transplantation, Nanfang Hospital, Southern Medical University, 510515, Guangzhou, China.
Department of Gastroenterology and Hepatology, Beijing Youan Hospital, Capital Medical University, 100069, Beijing, China.
Cell Death Discov. 2023 Feb 17;9(1):69. doi: 10.1038/s41420-023-01360-x.
The functional status of mitochondria and the endoplasmic reticulum are central to renal ischemia/reperfusion injury (IRI). X-box binding protein 1 (XBP1) is an important transcription factor in endoplasmic reticulum stress. NLR family pyrin domain containing-3 (NLRP3) inflammatory bodies are closely related to renal IRI. In vivo and in vitro, we examined the molecular mechanisms and functions of XBP1-NLRP3 signaling in renal IRI, which influences ER-mitochondrial crosstalk. In this study, mice were subjected to 45 min of unilateral renal warm ischemia, the other kidney resected, and reperfusion was performed for 24 h in vivo. In vitro, murine renal tubular epithelial cells (TCMK-1) were exposed to hypoxia for 24 h and reoxygenation for 2 h. Tissue or cell damage was evaluated by measuring blood urea nitrogen and creatinine levels, histological staining, flow cytometry, terminal deoxynucleotidyl transferase-mediated nick-end labeling, diethylene glycol staining, and transmission electron microscopy (TEM). Western blotting, immunofluorescence staining, and ELISA were used to analyze protein expression. Whether XBP1 regulates the NLRP3 promoter was evaluated using a luciferase reporter assay. Kidney damage was reduced with decreasing blood urea nitrogen, creatinine, interleukin-1β, and interleukin-18 levels. XBP1 deficiency reduced tissue damage and cell apoptosis, protecting the mitochondria. Disruption of XBP1 was associated with reduced NLRP3 and cleaved caspase-1 levels and markedly improved survival. In vitro in TCMK-1 cells, XBP1 interference inhibited caspase-1-dependent mitochondrial damage and reduced the production of mitochondrial reactive oxygen species. The luciferase assay showed that spliced XBP1 isoforms enhanced the activity of the NLRP3 promoter. These findings reveal that XBP1 downregulation suppresses the expression of NLRP3, a potential regulator of endoplasmic reticulum mitochondrial crosstalk in nephritic injury and a potential therapeutic target in XBP1-mediated aseptic nephritis.
线粒体和内质网的功能状态是肾缺血/再灌注损伤（IRI）的核心。X盒结合蛋白1（XBP1）是内质网应激中的一种重要转录因子。含NLR家族pyrin结构域蛋白3（NLRP3）炎性小体与肾IRI密切相关。我们在体内和体外研究了XBP1-NLRP3信号在肾IRI中的分子机制和功能，该信号影响内质网-线粒体相互作用。在本研究中，小鼠接受45分钟的单侧肾温缺血，切除另一侧肾脏，并在体内进行24小时再灌注。在体外，将小鼠肾小管上皮细胞（TCMK-1）暴露于缺氧环境24小时，再复氧2小时。通过测量血尿素氮和肌酐水平、组织学染色、流式细胞术、末端脱氧核苷酸转移酶介导的缺口末端标记、二乙二醇染色和透射电子显微镜（TEM）评估组织或细胞损伤。采用蛋白质免疫印迹法、免疫荧光染色和酶联免疫吸附测定法分析蛋白质表达。使用荧光素酶报告基因测定法评估XBP1是否调节NLRP3启动子。随着血尿素氮、肌酐、白细胞介素-1β和白细胞介素-18水平的降低，肾损伤减轻。XBP1缺乏减少了组织损伤和细胞凋亡，保护了线粒体。XBP1的破坏与NLRP3和裂解的半胱天冬酶-1水平降低有关，并显著提高了存活率。在体外TCMK-1细胞中，XBP1干扰抑制了半胱天冬酶-1依赖性线粒体损伤，并减少了线粒体活性氧的产生。荧光素酶测定表明，剪接的XBP1异构体增强了NLRP3启动子的活性。这些发现表明，XBP1下调抑制了NLRP3的表达，NLRP3是肾损伤中内质网-线粒体相互作用的潜在调节因子，也是XBP1介导的无菌性肾炎的潜在治疗靶点。
