Department of Biochemistry and Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University TaiKang Medical School (School of Basic Medical Sciences), Wuhan, Hubei, 430071, China.
Department of Orthopaedic Trauma and Microsurgery, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, 430071, China.
Cell Death Dis. 2022 Sep 7;13(9):774. doi: 10.1038/s41419-022-05220-x.
Ischemia-reperfusion (I/R) induced acute kidney injury (AKI), characterized by excessive mitochondrial damage and cell apoptosis, remains a clinical challenge. Recent studies suggest that regulator of calcineurin 1 (RCAN1) regulates mitochondrial function in different cell types, but the underlying mechanisms require further investigation. Herein, we aim to explore whether RCAN1 involves in mitochondrial dysfunction in AKI and the exact mechanism. In present study, AKI was induced by I/R and cisplatin in RCAN1 mice and mice with renal tubular epithelial cells (TECs)-specific deletion of RCAN1. The role of RCAN1 in hypoxia-reoxygenation (HR) and cisplatin-induced injury in human renal proximal tubule epithelial cell line HK-2 was also examined by overexpression and knockdown of RCAN1. Mitochondrial function was assessed by transmission electron microscopy, JC-1 staining, MitoSOX staining, ATP production, mitochondrial fission and mitophagy. Apoptosis was detected by TUNEL assay, Annexin V-FITC staining and Western blotting analysis of apoptosis-related proteins. It was found that protein expression of RCAN1 was markedly upregulated in I/R- or cisplatin-induced AKI mouse models, as well as in HR models in HK-2 cells. RCAN1 deficiency significantly reduced kidney damage, mitochondrial dysfunction, and cell apoptosis, whereas RCAN1 overexpression led to the opposite phenotypes. Our in-depth mechanistic exploration demonstrated that RCAN1 increases the phosphorylation of mitochondrial fission factor (Mff) by binding to downstream c-Jun N-terminal kinase (JNK), then promotes dynamin related protein 1 (Drp1) migration to mitochondria, ultimately leads to excessive mitochondrial fission of renal TECs. In conclusion, our study suggests that RCAN1 could induce mitochondrial dysfunction and apoptosis by activating the downstream JNK/Mff signaling pathway. RCAN1 may be a potential therapeutic target for conferring protection against I/R- or cisplatin-AKI.
缺血再灌注（I/R）诱导的急性肾损伤（AKI）的特征是线粒体损伤和细胞凋亡过度，这仍然是一个临床挑战。最近的研究表明，钙调神经磷酸酶 1 调节子（RCAN1）调节不同类型细胞中的线粒体功能，但潜在机制需要进一步研究。在此，我们旨在探讨 RCAN1 是否参与 AKI 中的线粒体功能障碍及其确切机制。在本研究中，通过 I/R 和顺铂在 RCAN1 敲除小鼠和肾近端小管上皮细胞（TEC）特异性 RCAN1 敲除小鼠中诱导 AKI。通过 RCAN1 的过表达和敲低，还研究了 RCAN1 在人肾近端小管上皮细胞系 HK-2 中的缺氧再氧合（HR）和顺铂诱导损伤中的作用。通过透射电子显微镜、JC-1 染色、MitoSOX 染色、ATP 产生、线粒体裂变和线粒体自噬来评估线粒体功能。通过 TUNEL 测定、Annexin V-FITC 染色和凋亡相关蛋白的 Western blot 分析检测细胞凋亡。结果发现，RCAN1 蛋白表达在 I/R 或顺铂诱导的 AKI 小鼠模型以及 HK-2 细胞中的 HR 模型中均明显上调。RCAN1 缺乏显著减轻肾脏损伤、线粒体功能障碍和细胞凋亡，而 RCAN1 过表达则导致相反的表型。我们深入的机制探索表明，RCAN1 通过与下游 c-Jun N 末端激酶（JNK）结合，增加线粒体裂变因子（Mff）的磷酸化，然后促进动力相关蛋白 1（Drp1）向线粒体迁移，最终导致肾 TEC 过度的线粒体裂变。总之，我们的研究表明，RCAN1 通过激活下游 JNK/Mff 信号通路，引起线粒体功能障碍和细胞凋亡。RCAN1 可能是一种潜在的治疗靶点，可用于对抗 I/R 或顺铂-AKI。
