Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Tokyo, Japan.
PLoS One. 2012;7(7):e41462. doi: 10.1371/journal.pone.0041462. Epub 2012 Jul 30.
Oxidative stress and endoplasmic reticulum (ER) stress play a crucial role in tubular damage in both acute kidney injury (AKI) and chronic kidney disease (CKD). While the pathophysiological contribution of microRNAs (miRNA) to renal damage has also been highlighted, the effect of miRNA on renal damage under oxidative and ER stresses conditions remains elusive.
We assessed changes in miRNA expression in the cultured renal tubular cell line HK-2 under hypoxia-reoxygenation-induced oxidative stress or ER stress using miRNA microarray assay and real-time RT-PCR. The pathophysiological effect of miRNA was evaluated by cell survival rate, intracellular reactive oxygen species (ROS) level, and anti-oxidant enzyme expression in miRNA-inhibited HK-2 or miRNA-overexpressed HK-2 under these stress conditions. The target gene of miRNA was identified by 3'-UTR-luciferase assay.
We identified 8 and 10 miRNAs whose expression was significantly altered by oxidative and ER stresses, respectively. Among these, expression of miR-205 was markedly decreased in both stress conditions. Functional analysis revealed that decreased miR-205 led to an increase in cell susceptibility to oxidative and ER stresses, and that this increase was associated with the induction of intracellular ROS and suppression of anti-oxidant enzymes. While increased miR-205 by itself made no change in cell growth or morphology, cell viability under oxidative or ER stress conditions was partially restored. Further, miR-205 bound to the 3'-UTR of the prolyl hydroxylase 1 (PHD1/EGLN2) gene and suppressed the transcription level of EGLN2, which modulates both intracellular ROS level and ER stress state.
miR-205 serves a protective role against both oxidative and ER stresses via the suppression of EGLN2 and subsequent decrease in intracellular ROS. miR-205 may represent a novel therapeutic target in AKI and CKD associated with oxidative or ER stress in tubules.
氧化应激和内质网（ER）应激在急性肾损伤（AKI）和慢性肾脏病（CKD）的肾小管损伤中起着关键作用。虽然 microRNAs（miRNA）对肾损伤的病理生理作用也已得到强调，但 miRNA 在氧化应激和 ER 应激条件下对肾损伤的影响仍不清楚。
我们使用 miRNA 微阵列分析和实时 RT-PCR 评估缺氧再复氧诱导的氧化应激或 ER 应激下培养的肾小管细胞系 HK-2 中 miRNA 表达的变化。通过 miRNA 抑制的 HK-2 或 miRNA 过表达的 HK-2 在这些应激条件下的细胞存活率、细胞内活性氧（ROS）水平和抗氧化酶表达，评估 miRNA 的病理生理作用。通过 3'-UTR-荧光素酶测定鉴定 miRNA 的靶基因。
我们鉴定出 8 个和 10 个 miRNA，它们的表达分别被氧化应激和 ER 应激显著改变。其中，miR-205 在两种应激条件下的表达均明显降低。功能分析表明，miR-205 表达降低导致细胞对氧化应激和 ER 应激的敏感性增加，而这种增加与细胞内 ROS 的诱导和抗氧化酶的抑制有关。虽然增加的 miR-205 本身不会改变细胞的生长或形态，但在氧化应激或 ER 应激条件下的细胞活力部分恢复。此外，miR-205 与脯氨酰羟化酶 1（PHD1/EGLN2）基因的 3'-UTR 结合并抑制 EGLN2 的转录水平，从而调节细胞内 ROS 水平和 ER 应激状态。
miR-205 通过抑制 EGLN2 并随后降低细胞内 ROS，对氧化应激和 ER 应激均发挥保护作用。miR-205 可能成为与肾小管氧化应激或 ER 应激相关的 AKI 和 CKD 的新型治疗靶点。
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