Elevation of miR-21, through targeting MKK3, may be involved in ischemia pretreatment protection from ischemia-reperfusion induced kidney injury.

Abstract

Ischemia-reperfusion (IR) causes acute kidney injury (AKI), and ischemia pretreatment may exert protection. Mitogen-activated protein kinase kinase 3 (MKK3), which is involved in the signal transduction pathway in IR-induced injury, is a potential target of miR-21. We aimed to verify the targeting regulation of miR-21 on MKK3 and to explore the effects of miR-21-mediated MKK3 expression changes in AKI. Vectors containing the MKK3 3'UTR and mutated MKK3-3U-M were constructed and co-transfected with nonsense miR, miR-21-5p mimics or inhibitor in HEK293 cells. Gene expressions were detected by dual luciferase reporter assay. The effects of miR-21 on mRNA and protein of MKK3 were investigated in HK-2 cells. Male C57BL/6J mice were treated with ischemic preconditioning (IPC) and IR. Kidney functions were assessed through monitoring serum creatinine (Scr) and blood urea nitrogen (BUN). Pathological changes were observed and scored with histological samples of kidney. Expression levels of miR-21, MKK3, interleukin (IL)-6, tumor necrosis factor (TNF)-α before and after IPC and IR were examined by real-time polymerase chain reaction and/or immunohistochemistry. miR-21 regulated the expression of MKK3 via 3'UTR. Following IR, MKK3, IL-6 and TNF-α levels were increased. Scr, BUN and pathological injuries were aggravated, and miR-21 expression was increased. IPC increased miR-21 levels ahead of IR and inhibited the increases in MKK3, IL-6 and TNF-α levels and the aggravation of Scr, BUN and pathological injuries. miR-21 targets MKK3 in vivo and in vitro, inhibiting the downstream factors IL-6 and TNF-α. Therefore, miR-21 might be involved in protection of IPC against IR of the kidney.