MBD2 upregulates miR-301a-5p to induce kidney cell apoptosis during vancomycin-induced AKI

Abstract

Despite DNA methylation occurred in acute kidney injury (AKI), how it influenced progression of AKI remains unclear. Methyl-CpG-binding domain protein 2 (MBD2), a protein readers of methylation, was used to analyze the impact of DNA methylation on vancomycin (VAN)-induced AKI. Here, in cultured human kidney tubular epithelial cells (HK-2), we show that knockdown of MBD2 by siRNA attenuated VAN-induced apoptosis, caspase activity, and the expression of BAX and cleaved caspase 3. Interestingly, knockdown of MBD2 by siRNA was associated with the suppression of miR-301a-5p. Mechanistic studies confirmed MBD2 binds to these methylated CpG elements of miR-301a-5p promoter, and then activates miR-301a-5p promoter by suppressing methylation. Furthermore, anti-miR-301a-5p significantly blocked VAN-induced apoptosis and caspase activity in HK-2 cells, which was accompanied by downregulation of p53, and upregulation of MITF, HDGF and MDM-4 together. The latter genes were further identified as target genes of miR-301a-5p, and silencing of MDM-4 promoted p53 accumulation. In vivo, mice with MBD2 knockout (MBD2-KO) were counteracted to VAN-induced AKI, indicated by the analysis of renal function, histology, apoptosis and inflammation. MBD2-KO also significantly suppressed the expression of miR-301a-5p, p53, BAX and cleaved caspase 3, and restored the expression of MDM-4, MITF and HDGF. Finally, in vivo inhibition of miR-301a-5p also ameliorated VAN-induced AKI. Together, these results show the novel MBD2/miR-301a-5p/MITF, HDGF and MDM-4/p53 pathway in VAN-induced AKI.

Atg7 mediates renal tubular cell apoptosis in vancomycin nephrotoxicity through activation of PKC‐δ

“…However, a previous study reported that autophagy led to cell death in cisplatin-treated proximal tubular cells (30), whereas another study found that pharmacologic inhibition of autophagy promoted apoptosis in the same cell line and experimental model (40). A possible explanation is that nonspecific pharmacologic inhibitors of autophagy may cause unwanted side effects (18). To overcome these drawbacks, both Atg7-deficient MEF cells and PT-Atg7-KO mice were established in this study.…”

“…TUNEL and quantification were also conducted in accordance with previous studies (15,17). Morphologic analyses and flow cytometry (FCM) of apoptosis and caspase activity were performed by staining the cells with Hoechst 33342 and enzymatic assay, respectively, as previously described (17)(18)(19).…”

Loganetin protects against rhabdomyolysis‐induced acute kidney injury by modulating the toll‐like receptor 4 signalling pathway

“…To detect tissue injury, mice kidney tissues were fixed in 4% paraformaldehyde buffer for 24 hr and embedded in paraffin. Subsequently, paraffin blocks were cut into 2‐μm sections and then subjected to routine H&E, PAS, and TUNEL staining according to previously described protocols (Wang, Li, et al, ; You et al, ; Zhang, Liu, et al, ). Histological differences among the groups were recorded.…”

Vancomycin‐Induced Kidney Injury: Animal Models of Toxicodynamics, Mechanisms of Injury, Human Translation, and Potential Strategies for Prevention

“…Additionally, p53 has been shown to induce apoptosis via miR‐192‐5p in wild‐type mice, which was suppressed in p53‐KO mice 86 . Blocking miR‐301a‐5p 85 or inhibiting miR‐192‐5p 86 with respective antisense oligonucleotides ameliorated VIKI in C57BL/6 mice.…”

“…The miRNA appears to be an important part of this VIKI mechanistic pathway. The miRNAs miR‐301a‐5p and miR‐192‐5p have been implicated in the development of VIKI through methyl‐CpG‐binding domain protein 2 (MBD2) and p53 85, 86 . Methyl‐CpG‐binding domain proteins, are protein readers of methylation and are actively involved in DNA‐methylation‐mediated transcriptional regulation by binding to methylated CpG on the DNA and either blocking other protein factors from binding to DNA or by binding to promoters of actively transcribed genes 87 .…”