线粒体Lon蛋白酶在急性肾损伤中的作用及机制研究

Acute kidney injury (AKI) is a common clinical issue with high morbidity and mortality. Although a number of studies related to the AKI have been extensively performed in the past decades, the pathogenic mechanisms of AKI are still elusive, which could contribute to the lack of satisfactory therapies in clinic. Now mitochondrial dysfunction is found to be involved in the pathogenesis of AKI and serves as a potential target of AKI therapy. Lon protease (LONP1), encoded by a nuclear chromosomal gene, is a key molecule in maintaining the mitochondrial homeostasis. Up to date, increasing studies have reported that LONP1 gene mutation, change of activity or expression were closely related to various hereditary or acquired diseases. However, there is little report about LONP1 in kidney diseases. Our preliminary study revealed that LONP1 expression was significantly decreased in renal cortex of AKI mice model and CoCl2-induced tubular epithelial cells. Silencing LONP1 resulted in the cell apoptosis and mitochondrial dysfunction in renal cells. These data suggested a potential role of LONP1 in AKI by targeting mitochondria. Furthermore, bioinformatics analysis indicated two biding sites of transcription factor Nrf2 in the promoter region of LONP1 gene. Meanwhile, similarly as LONP1 expression in AKI, previous studies demonstrated that Nrf2 was significantly decreased in kidneys undergoing AKI. These suggested that Nrf2 might serve as an upstream regulator of LONP1 in AKI. Thus, we speculate that Nrf2/LONP1 could play a protective role in AKI by maintaining the mitochondrial homeostasis. In this proposal, we will fully investigate the role of LONP1 in the pathogenesis of AKI, as well as the underlying mechanisms.

急性肾损伤（AKI）具有高发病率和高死亡率，缺乏满意的治疗手段。线粒体功能障碍参与了AKI的发生，是防治AKI的潜在靶点。线粒体Lon蛋白酶（LONP1）是维持线粒体稳态的关键分子，但其在AKI中的作用尚无报道。我们预实验结果显示LONP1在AKI小鼠肾脏和肾小管上皮细胞显著下降。敲低LONP1可以诱导肾脏细胞凋亡及肾小管上皮细胞线粒体功能异常，提示LONP1可能在AKI过程中发挥了重要作用。进一步，通过生物信息学分析发现LONP1的启动子区存在转录因子Nrf2的结合位点，并且已有研究证实Nrf2在AKI肾脏中表达降低，提示Nrf2可能是LONP1的上游调控因子。由此，我们推测：在AKI过程中，Nrf2表达下调引起LONP1表达下降，引起线粒体稳态失衡，进而参与了AKI发生。我们将在患者、动物、细胞及分子水平系统研究LONP1在AKI中的作用及机制，为防治AKI提供新思路。

我们第一次运用cKO小鼠通过缺血再灌注诱导AKI模型，结果发现肾小管上皮细胞条件性LONP1敲除加重缺血再灌注引起的AKI小鼠肾功能恶化、肾脏病理损伤、肾脏细胞凋亡、线粒体形态损害。但由于第一次进行实验的小鼠数目较少，于是我们再次使用cKO小鼠构建了缺血再灌注诱导的AKI小鼠。但是，与第一次完全相反，此次实验结果表明肾小管条件性LONP1敲除具有减轻缺血再灌注介导的AKI小鼠肾功能恶化的趋势。考虑两次实验建模时缺血时的温度不同，且再灌注的时间也不一样，我们进行了第三次实验。此次实验部分结果与第一次实验结果趋势相一致，即肾小管上皮细胞条件性LONP1基因敲除有加重缺血再灌注损伤诱导的AKI小鼠肾功能恶化的趋势。紧接着，我们进行了第四次实验，此次实验结果与第二次实验结果趋势一致。最后，我们使用cKI 小鼠构建了缺血再灌注诱导的AKI模型。实验结果显示肾小管上皮细胞条件性LONP1过表达能加重缺血再灌注诱导的AKI小鼠肾功能恶化和肾脏病理损害、增加肾脏细胞凋亡。结合四次实验结果均不一致，因此，LONP1在缺血再灌注诱导的AKI中的作用和和机制目前尚不明确，仍需更进一步实验证实。后续我们分别采用cKO小鼠和cKI小鼠建立了顺铂诱导的AKI模型。实验结果显示：肾小管上皮细胞条件性LONP1敲除减轻顺铂诱导的AKI小鼠肾功能下降。而肾小管上皮细胞条件性LONP1过表达能加重顺铂介导的AKI小鼠肾功能恶化、肾脏病理损伤及炎症反应。最后，我们也运用cKO小鼠构建了马兜铃酸（AA）诱导的AKI模型。相关实验结果表明：肾小管上皮细胞条件性LONP1敲除对AA诱导的AKI模型小鼠具有减轻肾功能恶化趋势，同时能增加AA诱导的AKI小鼠的生存时间。