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IF 7.9 1区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Clinical and Translational Medicine Pub Date : 2024-09-17 DOI:10.1002/ctm2.70024

Shangting Han, Jiayu Guo, Chenyang Kong, Jun Li, Fangyou Lin, Jiefu Zhu, Tianyu Wang, Qi Chen, Yiting Liu, Haochong Hu, Tao Qiu, Fan Cheng, Jiangqiao Zhou

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引用次数: 0

摘要

背景肾缺血再灌注损伤（IRI）是急性肾损伤（AKI）的主要原因。迄今为止，延迟肾脏 IRI 和延长患者生存期的有效疗法仍然缺乏。安基蛋白重复结构域 1（ANKRD1）与某些病理生理过程有关联，但它在肾脏 IRI 中的作用尚未得到探讨。方法利用 IRI-AKI 小鼠模型和体外模型研究 ANKRD1 的作用。采用免疫沉淀-质谱法鉴定潜在的与 ANKRD1 相互作用的蛋白质。分别使用免疫沉淀和近接试验以及免疫印迹法检测了蛋白质与蛋白质之间的相互作用和蛋白质泛素化。使用生化和细胞技术评估了细胞活力、损伤和脂质过氧化。结果首先，我们发现 ANKRD1 在肾脏 IRI 模型中显著升高。通过重组腺相关病毒（rAAV9）在小鼠肾脏的所有细胞类型中全面敲除 ANKRD1 可减轻缺血/再灌注诱导的肾损伤和衰竭。在暴露于缺氧复氧或过氧化氢的人肾近曲小管细胞中，沉默ANKRD1可提高细胞活力并减轻细胞损伤，而过表达ANKRD1则产生相反的效果。其次，我们发现 ANKRD1 在肾脏 IRI 期间的有害功能包括通过直接与酰基辅酶 A 合成酶长链家族成员 3（ACSL3）结合并降低其水平来促进脂质过氧化和铁变态反应。此外，通过药物方法和体外 RNA 干扰减弱体内 ACSL3 可减轻 ANKRD1 敲除的抗铁锈作用。最后，我们发现 ANKRD1 通过调节 E3 连接酶含三方基序 25（TRIM25）催化 ACSL3 的 K63 链接泛素化，促进了 ACSL3 的翻译后降解，从而扩大了脂质过氧化和铁变态反应，加重了肾损伤。结论我们的研究揭示了 ANKRD1 在肾脏 IRI 中以前未知的功能。通过驱动 ACSL3 泛素化和降解，ANKRD1 加剧了铁蛋白沉积，并最终加剧了 IRI-AKI，这凸显了 ANKRD1 作为肾脏 IRI 治疗靶点的潜力。要点/亮点 Ankyrin repeat domain 1（ANKRD1）在体内和体外肾缺血再灌注损伤（IRI）模型中被迅速激活。敲除 ANKRD1 可减轻肾损伤并保护肾功能。在肾脏 IRI 中，ANKRD1 的功能恶化与铁蛋白沉积有关。 ANKRD1通过泛素-蛋白酶体途径促进酰基辅酶A合成酶长链家族成员3（ACSL3）降解。 E3连接酶含三方基序25（TRIM25）负责ANKRD1介导的ACSL3泛素化。

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ANKRD1 aggravates renal ischaemia‒reperfusion injury via promoting TRIM25-mediated ubiquitination of ACSL3

Background

Renal ischaemia‒reperfusion injury (IRI) is the primary cause of acute kidney injury (AKI). To date, effective therapies for delaying renal IRI and postponing patient survival remain absent. Ankyrin repeat domain 1 (ANKRD1) has been implicated in some pathophysiologic processes, but its role in renal IRI has not been explored.

Methods

The mouse model of IRI-AKI and in vitro model were utilised to investigate the role of ANKRD1. Immunoprecipitation-mass spectrometry was performed to identify potential ANKRD1-interacting proteins. Protein‒protein interactions and protein ubiquitination were examined using immunoprecipitation and proximity ligation assay and immunoblotting, respectively. Cell viability, damage and lipid peroxidation were evaluated using biochemical and cellular techniques.

Results

First, we unveiled that ANKRD1 were significantly elevated in renal IRI models. Global knockdown of ANKRD1 in all cell types of mouse kidney by recombinant adeno-associated virus (rAAV9)-mitigated ischaemia/reperfusion-induced renal damage and failure. Silencing ANKRD1 enhanced cell viability and alleviated cell damage in human renal proximal tubule cells exposed to hypoxia reoxygenation or hydrogen peroxide, while ANKRD1 overexpression had the opposite effect. Second, we discovered that ANKRD1's detrimental function during renal IRI involves promoting lipid peroxidation and ferroptosis by directly binding to and decreasing levels of acyl-coenzyme A synthetase long-chain family member 3 (ACSL3), a key protein in lipid metabolism. Furthermore, attenuating ACSL3 in vivo through pharmaceutical approach and in vitro via RNA interference mitigated the anti-ferroptotic effect of ANKRD1 knockdown. Finally, we showed ANKRD1 facilitated post-translational degradation of ACSL3 by modulating E3 ligase tripartite motif containing 25 (TRIM25) to catalyse K63-linked ubiquitination of ACSL3, thereby amplifying lipid peroxidation and ferroptosis, exacerbating renal injury.

Conclusions

Our study revealed a previously unknown function of ANKRD1 in renal IRI. By driving ACSL3 ubiquitination and degradation, ANKRD1 aggravates ferroptosis and ultimately exacerbates IRI-AKI, underlining ANKRD1's potential as a therapeutic target for kidney IRI.

Key Points/Highlights

Ankyrin repeat domain 1 (ANKRD1) is rapidly activated in renal ischaemia‒reperfusion injury (IRI) models in vivo and in vitro.
ANKRD1 knockdown mitigates kidney damage and preserves renal function.
Ferroptosis contributes to the deteriorating function of ANKRD1 in renal IRI.
ANKRD1 promotes acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) degradation via the ubiquitin‒proteasome pathway.
The E3 ligase tripartite motif containing 25 (TRIM25) is responsible for ANKRD1-mediated ubiquitination of ACSL3.

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来源期刊

Clinical and Translational Medicine Multiple-

CiteScore

15.90

自引率

1.90%

发文量

450

审稿时长

4 weeks

期刊介绍： Clinical and Translational Medicine (CTM) is an international, peer-reviewed, open-access journal dedicated to accelerating the translation of preclinical research into clinical applications and fostering communication between basic and clinical scientists. It highlights the clinical potential and application of various fields including biotechnologies, biomaterials, bioengineering, biomarkers, molecular medicine, omics science, bioinformatics, immunology, molecular imaging, drug discovery, regulation, and health policy. With a focus on the bench-to-bedside approach, CTM prioritizes studies and clinical observations that generate hypotheses relevant to patients and diseases, guiding investigations in cellular and molecular medicine. The journal encourages submissions from clinicians, researchers, policymakers, and industry professionals.