miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway

Abstract

Background: miR-214 was demonstrated to be upregulated in models of renal disease and promoted fibrosis in renal injury independent of TGF-β signaling in vivo. However, the detailed role of miR-214 in acute kidney injury (AKI) and its underlying mechanism are still largely unknown.

Methods: In this study, an I/R-induced rat AKI model and a hypoxia-induced NRK-52E cell model were used to study AKI. The concentrations of kidney injury markers serum creatinine, blood urea nitrogen, and kidney injury molecule-1 were measured. The expressions of miR-214, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, were detected by RT-qPCR. The protein levels of Bcl-2, Bax, Dickkopf-related protein 3, β-catenin, c-myc, and cyclinD1 were determined by western blot. Cell apoptosis and caspase 3 activity were evaluated by flow cytometry analysis and caspase 3 activity assay, respectively. Luciferase reporter assay was used to confirm the interaction between miR-214 and Dkk3.

Results: miR-214 expression was induced in ischemia-reperfusion (I/R)-induced AKI rat and hypoxic incubation of NRK-52E cells. Overexpression of miR-214 alleviated hypoxia-induced NRK-52E cell apoptosis while inhibition of miR-214 expression exerted the opposite effect. Dkk3 was identified as a target of miR-214. Anti-miR-214 abolished the inhibitory effects of DKK3 knockdown on hypoxia-induced NRK-52E cell apoptosis by inactivation of Wnt/β-catenin signaling. Moreover, miR-214 ameliorated AKI in vivo by inhibiting apoptosis and fibrosis through targeting Dkk3 and activating Wnt/β-catenin pathway.

Conclusion: miR-214 ameliorates AKI by inhibiting apoptosis through targeting Dkk3 and activating Wnt/β-catenin signaling pathway, offering the possibility of miR-214 in the therapy of ischemic AKI.

Keywords: Acute kidney injury; Dkk3; Wnt/β-catenin signaling pathway; miR-214.

Fig. 1

Evaluation of rat AKI model following I/R surgery and NRK-52E cell model following hypoxia treatment. The serum levels of SCr (a), BUN (b), and urine Kim-1 (c) in I/R-induced rat AKI models at 24 h after surgery were measured. d The mRNA expressions of TNF-α, IL-1β, and IL-6 in hypoxia-induced NRK-52E cell I/R model were detected by qRT-PCR. e The protein levels of Bcl-2 and Bax in hypoxia-induced NRK-52E cell I/R models were assessed by western blot. n = 6 rats/group. *P < 0.05

Fig. 2

miR-214 attenuated NRK-52E cell apoptosis under hypoxia. a qRT-PCR analysis of miR-214 expression in kidney cortex of I/R-induced rat AKI models after 12, 48 and 72 h of reperfusion. n = 6 rats/group. b qRT-PCR analysis of miR-214 expression at 0, 3 h, 6 h, and 9 h during hypoxic incubation of cultured NRK-52E cells. c qRT-PCR analysis of miR-214 expression in NRK-52E cells after transfection with miR-214, anti-miR-214, or matched controls. d–e Flow cytometry was used to analyze the apoptotic rate of NRK-52E cells after transfection with miR-214, anti-miR-214, or respective controls during hypoxia incubation. f Western blot was performed to detect the protein levels of Bcl-2 and Bax in NRK-52E cells after introduction with miR-214, anti-miR-214, or corresponding controls under hypoxia. Each experiment was independently repeated 3 times. *P < 0.05

Fig. 3

Dkk3 was a target of miR-214 in NRK-52E cells. a The predictive wild-type miR-214 binding sites in the 3′UTR of Dkk3 and the corresponding mutant binding sites were displayed. b The relative luciferase activity was measured by luciferase reporter assay after NRK-52E cells were cotransfected with Dkk3-WT or Dkk3-MUT and miR-214 or miR-con. The mRNA (c) and protein (d, e) levels of DKK3 were examined by qRT-PCR and western blot in NRK-52E cells introduced with miR-214, anti-miR-214, or matched controls. Each experiment was independently repeated 3 times. *P < 0.05

Fig. 4

Anti-miR-214 abolished the inhibitory effect of DKK3 knockdown on hypoxia-induced apoptosis in NRK-52E cells by inactivation of Wnt/β-catenin pathway. Western blot analysis of DKK3 level in kidney tissues of I/R-induced rat AKI models after 30 min of ischemia and 12 or 48 h of reperfusion (a) and cultured NRK-52E cells after hypoxia treatment (b). NRK-52E cells transfected with either si-DKK3 alone or combined with anti-miR-214 were incubated under hypoxia, followed by detection of DDK3 protein expression (c), apoptosis (d), caspase 3 activity (e), Bcl-2 and Bax protein levels (f), β-catenin, c-myc, cyclin D1 expression (g) mRNA expressions of TNF-α, IL-1β and IL-6 (h), and the concentrations of TNF-α, IL-1β and IL-6 (i). Each experiment was independently repeated 3 times. *P < 0.05

Fig. 5

miR-214 protected against AKI in vivo through targeting Dkk3 and activating Wnt/β-catenin pathway. miR-214 or miR-con was intraperitoneally injected into mice, followed by ischemic surgery. a qRT-PCR analysis of miR-214 expression in I/R-induced rat AKI model and sham mice. The serum levels of BUN (b), SCr (c), and Urine Kim-1 (d) level in I/R-induced rat AKI model and sham mice. e Western blot analysis of Bcl-2, Bax and fibronectin in I/R-induced rat AKI model and sham group. f The protein levels of DKK3, β-catenin, c-myc, and cyclin D1 in I/R-induced rat AKI model and sham group. Each experiment was independently repeated 3 times. *P < 0.05