Department of Pediatrics, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, People's Republic of China; Department of Pediatrics, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning 121001, People's Republic of China.
Department of Pediatrics, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning 121001, People's Republic of China.
Acta Histochem. 2019 Aug;121(6):695-703. doi: 10.1016/j.acthis.2019.06.005. Epub 2019 Jun 22.
Renal fibrosis is a common mechanism that leads to all kidney diseases and Epithelial-mesenchymal transition (EMT) is considered as one of the potential mechanisms of renal fibrosis. Inhibitor of growth 4 (ING4) was reported to involve in several diseases; especially it was negatively correlated with lung fibrogenesis parameters. However, the role of ING4 and underlying mechanisms in EMT are still unknown. In this study, we used a UUO rat model to mimic renal fibrosis, which was examined by Masson and HE staining analysis. To explore the effects of ING4 on hypoxia-induced EMT, HK2 cells were treated with hypoxia to induce EMT and ING4 was over-expressed in hypoxia-treated HK2 cells by transfection of pEGFP-N1-ING4. MTT assay was used to describe the cell viability of HK2 cells under the hypoxic condition. The expression levels of ING4, hypoxia-inducible factor-1α (HIF-1α), and EMT markers (E-cadherin, N-cadherin and vimentin) were examined in vivo and in vitro by western blot, qRT-PCR, immunohistochemical staining or Immunofluorescence. Our results showed that, in a UUO rat model, ING4 was decreased and EMT was developed with reduction in E-cadherin and increase in N-cadherin and vimentin, suggesting a significant association between ING4 expression and EMT. Under hypoxia, E-cadherin was down-regulated and N-cadherin and vimentin were up-regulated, indicating that hypoxia induced EMT in HK2 cells. Nonetheless, changes in the expression of EMT biomarkers were inhibited by over-expression of ING4. Moreover, over-expressing ING4 decreased the expression of HIF-1α and snail in HK2 cells. These findings suggest that ING4 may inhibit hypoxia-induced EMT via decreasing HIF-1α and snail in HK2 cells, indicating the potential of ING4 as a therapeutic target for renal fibrosis.
肾纤维化是导致所有肾脏疾病的常见机制，上皮-间充质转化（EMT）被认为是肾纤维化的潜在机制之一。生长抑制因子 4（ING4）被报道涉及多种疾病；特别是它与肺纤维化参数呈负相关。然而，ING4 在 EMT 中的作用及其潜在机制尚不清楚。在这项研究中，我们使用 UUO 大鼠模型模拟肾纤维化，通过 Masson 和 HE 染色分析进行检查。为了探讨 ING4 对缺氧诱导的 EMT 的影响，我们用缺氧处理 HK2 细胞以诱导 EMT，并通过转染 pEGFP-N1-ING4 使缺氧处理的 HK2 细胞过表达 ING4。MTT 测定法用于描述 HK2 细胞在缺氧条件下的细胞活力。通过 Western blot、qRT-PCR、免疫组织化学染色或免疫荧光，在体内和体外检测 ING4、缺氧诱导因子-1α（HIF-1α）和 EMT 标志物（E-钙粘蛋白、N-钙粘蛋白和波形蛋白）的表达水平。我们的结果表明，在 UUO 大鼠模型中，ING4 减少，EMT 发展，E-钙粘蛋白减少，N-钙粘蛋白和波形蛋白增加，表明 ING4 表达与 EMT 之间存在显著关联。在缺氧条件下，E-钙粘蛋白下调，N-钙粘蛋白和波形蛋白上调，表明缺氧诱导 HK2 细胞发生 EMT。然而，通过过表达 ING4 抑制了 EMT 生物标志物表达的变化。此外，过表达 ING4 降低了 HK2 细胞中 HIF-1α 和 snail 的表达。这些发现表明，ING4 可能通过降低 HK2 细胞中的 HIF-1α 和 snail 来抑制缺氧诱导的 EMT，表明 ING4 作为肾纤维化治疗靶点的潜力。
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