Li Jianchun, Ni Yufang, Li Qianqian, Kantapan Jiraporn, Dechsupa Nathupakorn, Wang Li
Department of Radiologic Technology, Molecular Imaging and Therapy Research Unit, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand; Research Center of Integrated Traditional Chinese and Western Medicine, The Affiliated Traditional Chinese Medicine Hospital, Southwest Medical University, Luzhou, China.
Research Center of Integrated Traditional Chinese and Western Medicine, The Affiliated Traditional Chinese Medicine Hospital, Southwest Medical University, Luzhou, China.
Tissue Cell. 2025 Jul 25;97:103055. doi: 10.1016/j.tice.2025.103055.
Epithelial membrane protein 3 (Emp3), an N-glycosylated tetraspanin, is known to regulate tumor progression; however, its role in renal fibrosis (RF) remains unclear. This study aims to elucidate the function of Emp3 in RF.
To evaluate Emp3's role, we utilized electroporation to deliver the Emp3-targeting RfxCas13d knockdown plasmid into the kidney cells of C57BL/6 mice, followed by the well-established unilateral ureteral obstruction (UUO) model. Renal pathology and injury were subsequently assessed. Additionally, immunohistochemistry, quantitative real-time PCR, and Western blot analyses were performed to evaluate fibrosis severity. In vitro, knockdown and overexpression of Emp3 were achieved via transfection in renal tubular epithelial cells (TCMK1) to further investigate Emp3's role. Co-immunoprecipitation (Co-IP) assays were employed to determine the interaction between TGF-β receptor 2 (TGFBR2) and Emp3.
Emp3 was found to be significantly upregulated in chronic kidney disease (CKD) patients and animal models. Knockdown of Emp3 using the RfxCas13d system in the UUO rodent model resulted in notable improvements in renal pathology and reduced the progression of renal fibrosis. Furthermore, Emp3 inhibition markedly suppressed TGF-β/Smad3 signaling activation. In TCMK1 cells, Emp3 knockdown produced similar results, while Emp3 overexpression accelerated pro-fibrotic cellular response. Mechanistically, Co-IP assays revealed a direct interaction between Emp3 and TGFBR2, which activates the TGF-β/Smad3 signaling pathway.
Our research demonstrates that Emp3 plays a critical role in the development of renal fibrosis by facilitating the activation of the TGF-β/Smad3 signaling pathway. Targeting Emp3 with RfxCas13d-mediated inhibition presents a promising therapeutic strategy for addressing RF in CKD.
上皮膜蛋白3（Emp3）是一种N-糖基化的四跨膜蛋白，已知其可调节肿瘤进展；然而，其在肾纤维化（RF）中的作用仍不清楚。本研究旨在阐明Emp3在RF中的功能。
为评估Emp3的作用，我们利用电穿孔将靶向Emp3的RfxCas13d敲低质粒导入C57BL/6小鼠的肾细胞，随后采用成熟的单侧输尿管梗阻（UUO）模型。随后评估肾脏病理和损伤情况。此外，进行免疫组织化学、定量实时PCR和蛋白质印迹分析以评估纤维化严重程度。在体外，通过在肾小管上皮细胞（TCMK1）中进行转染实现Emp3的敲低和过表达，以进一步研究Emp3的作用。采用免疫共沉淀（Co-IP）试验确定转化生长因子-β受体2（TGFBR2）与Emp3之间的相互作用。
发现Emp3在慢性肾脏病（CKD）患者和动物模型中显著上调。在UUO啮齿动物模型中使用RfxCas13d系统敲低Emp3可显著改善肾脏病理，并减少肾纤维化进展。此外，Emp3抑制明显抑制TGF-β/Smad3信号激活。在TCMK1细胞中，Emp3敲低产生了类似的结果，而Emp3过表达加速了促纤维化细胞反应。机制上，Co-IP试验揭示了Emp3与TGFBR2之间的直接相互作用，后者激活TGF-β/Smad3信号通路。
我们的研究表明，Emp3通过促进TGF-β/Smad3信号通路的激活在肾纤维化发展中起关键作用。用RfxCas13d介导的抑制作用靶向Emp3为解决CKD中的RF提供了一种有前景的治疗策略。
