Institute of Molecular Rhythm and Metabolism, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, China.
Institute of Molecular Rhythm and Metabolism, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, China.
Phytomedicine. 2024 Dec;135:156235. doi: 10.1016/j.phymed.2024.156235. Epub 2024 Nov 9.
Aristolochic acid I (AAI), an emerging biogenic contaminant widely present in Aristolochic plants, has been implicated in the progression of tubulointerstitial disease, known as aristolochic acid nephropathy (AAN). The circadian clock, a vital regulator of organ homeostasis, is susceptible to external chemical cues, including toxins. However, the reciprocal interactions between AAI and the circadian clock remain unexplored.
We initially assessed sex- and time-dependent nephropathy and behavioral responses in C57BL/6J mice exposed to AAI. Subsequently, we evaluated changes in the expression of circadian clock genes following treatment with AAI or its bioactive metabolite, aristolactam I, using real-time quantitative PCR and immunoblotting in renal tissues and cells. Additionally, real-time reporter assays were conducted on kidney explants from PER2::Luc knock-in reporter mice and Per2-dLuc/Bmal1-dLuc reporter cell lines. To further elucidate the regulatory role of circadian clocks in AAI-induced nephropathy, mice with global or kidney-specific knockout of Bmal1, as well as mice subjected to experimental jetlag, were utilized.
Our findings revealed a sex-dependent nephrotoxicity of AAI, with males exhibiting greater vulnerability. AAI-induced nephropathy was accompanied by impaired spatial cognitive function, disruptions in free-running locomotor activity, altered renal expression of multiple core clock genes, and disturbances in the circadian rhythm of renal PER2::Luc activity. Notably, kidney-specific ablation of the core clock gene Bmal1 significantly exacerbated renal injury and inflammation, whereas disruptions to the central clock, either genetically (through conventional knockout of Bmal1) or environmentally (mimicking jetlag), had minimal effects on AAI nephrotoxicity. Furthermore, both AAI and its bioactive metabolite aristolactam I demonstrated the ability to disrupt circadian clocks in human osteosarcoma cells (U2OS) and mouse renal tubular epithelial cells (mRTEC).
Collectively, these findings highlight the detrimental impact of aristolochic acids on local renal circadian clocks, ultimately exacerbating kidney damage. This study provides novel insights into the molecular mechanisms underlying AAI nephrotoxicity, potentially opening avenues for therapeutic interventions aimed at modulating the renal circadian clock to treat AAN.
马兜铃酸 I（AAI）是一种新兴的生物源污染物，广泛存在于马兜铃属植物中，与肾小管间质性疾病（即马兜铃酸肾病，AAN）的进展有关。生物钟是器官稳态的重要调节剂，易受包括毒素在内的外部化学信号的影响。然而，AAI 与生物钟之间的相互作用仍未被探索。
我们首先评估了暴露于 AAI 的 C57BL/6J 小鼠的性别和时间依赖性肾病和行为反应。随后，我们使用实时定量 PCR 和免疫印迹法评估了 AAI 或其生物活性代谢物马兜铃内酰胺 I 处理后肾组织和细胞中生物钟基因表达的变化。此外，在 PER2::Luc 敲入报告基因小鼠的肾脏外植体和 Per2-dLuc/Bmal1-dLuc 报告细胞系上进行了实时报告基因检测。为了进一步阐明生物钟在 AAI 诱导的肾病中的调节作用，我们使用了全局或肾脏特异性 Bmal1 敲除小鼠以及实验性倒班小鼠。
我们的研究结果揭示了 AAI 的性别依赖性肾毒性，雄性更为易感。AAI 诱导的肾病伴随着空间认知功能受损、自由运行运动活动的破坏、多个核心时钟基因在肾脏中的表达改变以及肾脏 PER2::Luc 活性的昼夜节律紊乱。值得注意的是，核心时钟基因 Bmal1 的肾脏特异性缺失显著加剧了肾脏损伤和炎症，而中央时钟的破坏（通过常规的 Bmal1 敲除）或环境（模拟倒班）对 AAI 肾毒性的影响最小。此外，AAI 和其生物活性代谢物马兜铃内酰胺 I 均表现出破坏人骨肉瘤细胞（U2OS）和小鼠肾小管上皮细胞（mRTEC）中生物钟的能力。
总的来说，这些发现强调了马兜铃酸对局部肾脏生物钟的有害影响，最终加剧了肾脏损伤。本研究为 AAI 肾毒性的分子机制提供了新的见解，为靶向调节肾脏生物钟治疗 AAN 的治疗干预提供了可能性。
