miR-21 contributes to renal protection by targeting prolyl hydroxylase domain protein 2 in delayed ischaemic preconditioning

Aim: Upregulation of miR-21 in renal ischaemic preconditioning (IPC) was associated with increased hypoxia inducible factor (HIF)-1 expression. Hypoxic induction of HIF-1 is mediated by inhibition of prolyl hydroxylase domain protein 2 (PHD2) .We hypothesized that miR-21 regulated HIF-1 by targeting PHD2 in the renal IPC. Methods: Luciferase reporter assay examined if miR-21 target the 3'-untranslated region of PHD2. In vitro, human proximal tubular cell line (HK-2) was incubated in hypoxia or hypoxia/ reoxygenation condition. Kidneys of Mice were respectively subjected to ischaemia/reperfusion injury (IRI) and IPC. Locked nucleic acid (LNA) modified anti-miR-21 was used to knockdown miR-21. Serum creatinine and histological changes estimated the renal injury. Levels of HIF-1, PHD2, VEGF and miR-21 were examined by western blot or real-time PCR. Result: miR-21 targeting of PHD2 was confirmed by 3'-untranslated region reporter assay. miR-21 was significantly upregulated by hypoxia/reoxygenation in HK-2 cell, while PHD2 protein level decreased significantly. LNA anti-miR-21 significantly repressed miR-21 levels and increased the abundance of PHD2. In vivo, IPC upregulated miR-21 expression 24h after the second ischaemia, while PHD2 expression decreased significantly with upregulation of HIF-1 protein and VEGF mRNA. MiR-21 induced by delayed IPC was effectively inhibited by the LNA anti-miR-21. With downregulation of miR-21, the protection of delayed IPC was attenuated and PHD2 protein was increased. Furthermore, upregulation of HIF-1 and VEGF were abolished after the LNA anti-miR-21 treatment. Conclusion: miR-21 could protect kidney against IRI via HIF-1 by inhibiting its target PHD2.The study suggested a new relationship between miR-21 and HIF-1. Summary at a Glance Increased levels of hypoxia inducible factor-1alpha (HIF-1alpha) facilitate the protective effects of renal ischaemic preconditioning. This study by Jiao et al has identified that upregulation of miR-21 targets expression of prolyl hydroxylase domain protein 2 (PHD2) resulting in increased HIF-1alpha levels in renal ischemic preconditioning.