HIF-1α诱导的外泌体tiRNAs对急性肾损伤的保护作用及机制研究

Hypoxia-Inducible Factor 1α(HIF-1α) activation is protective in acute kidney injury(AKI). While exosome mediated cellular communication is involved in AKI. Our previous work has demonstrated that HIF-1α activation could promote exosome production under ischemic condition which is protective in cisplatin induced AKI in vivo with unclear mechanism. Recent researches suggested that stress condition including hypoxia can induce tRNA cleavage into particular fragments(tiRNAs), which may reduce apoptosis by interacting with Cytochrome C. Our pre-experiment also found that HIF-1α activation could up-regulate tiRNA regulator angiogenin and increase exosomal 5’tiRNA-Arg/Val/Gly derived from kidney. Here we proposed that HIF-1α activation may protect acute kidney injury by transcriptionally regulate angiogenin and promote exosomal tiRNAs transfer, which may bind with Cytochrome C and inhibit apoptosis. To validate this hypothesis, we will use ischemic/hypoxia model and pharmacological strategy for HIF-1α activation, followed by an AKI model induced by cisplatin, to elucidate the protective role and mechanism of HIF-1α mediate exosomal tiRNA in AKI. Thus providing new target and strategy for AKI prevention and therapy.

急性肾损伤（AKI）是临床常见急重症，且防治手段有限。课题组前期工作发现缺氧诱导因子HIF-1α可通过外泌体减轻叠氮钠及顺铂诱导的肾小管上皮细胞急性损伤，而具体机制不详。研究显示tRNA衍生片段（tiRNA）可通过作用于细胞色素C减少细胞凋亡。课题组进一步预实验发现HIF-1α活化可上调核糖核酸酶Angiogenin并增加肾组织来源外泌体5’tiRNA-Arg/Val/Gly表达水平。因此，本项目提出HIF-1α可通过转录调控Angiogenin诱导外泌体tiRNAs增加，tiRNAs进一步与细胞色素C结合抑制肾小管上皮细胞凋亡发挥肾脏保护作用。本项目拟通过外泌体功能实验、荧光素酶报告、RIP-qPCR等方法，在已构建的HIF-1α基因敲除小鼠模型、缺血/缺氧小鼠及细胞模型上阐明HIF-1α通过外泌体tiRNA减轻顺铂诱导肾小管上皮细胞损伤的可能机制，为AKI的防治提供新的靶点和依据。

急性肾损伤(AKI)是临床常见的危急重症，而缺血再灌注损伤是临床及实验性AKI的最常见的病因和诱导因素，缺氧因子HIF-1的活化是其过程中关键因素之一。本课题通过高通量测序、外泌体提取、tsRNA检测、tsRNA合成及传染、动物实验、细胞实验等方法及手段进行了一系列的研究。研究显示通过缺血/缺氧或药物手段活化HIF-1可诱导外泌体水平以及细胞水平tRNA衍生片段（tsRNA，tRF/tiRNA)的差异表达。高通量测序检测小鼠缺血模型肾组织外泌体中tsRNAs，共检测出378个差异性表达的tsRNAs，与肾脏组织测序相比共有99个tsRNAs重合。其中HIF-1α诱导的tiRNA-Lys-CTT-003可以抑制细胞凋亡，降低caspase3的表达，促进Bcl2的表达。而tRF-Ala-CGC-023可能通过加重线粒体损伤，降低Mfn2的表达，并且促进细胞凋亡。本项目研究结果提示，肾脏细胞及肾组织外泌体来源的tsRNA参与了缺血再灌注AKI的发生和发展。HIF-1α诱导的tiRNA-Lys-CTT-003可以通过抑制细胞凋亡来减轻顺铂引起的AKI，tRF-Ala-CGC-023可能通过加重线粒体损伤及促进细胞凋亡加重顺铂诱导的AKI。tRNA衍生片段可以通过外泌体运输传递，并通过各种机制参与急性肾损伤过程。tsRNA尤其是外泌体tsRNA有望成为未来诊断和防治AKI的有效潜在靶点。