管状上皮细胞分泌的含有 microRNA-216a 的微囊泡通过激活 PTEN/AKT 通路参与肾间质纤维化。
Microvesicles containing microRNA-216a secreted by tubular epithelial cells participate in renal interstitial fibrosis through activating PTEN/AKT pathway.
机构信息
Department of Pediatric Intensive Care Unit, Qindao Women and Children's Hospital, Qindao,
出版信息
Eur Rev Med Pharmacol Sci. 2019 Aug;23(15):6629-6636. doi: 10.26355/eurrev_201908_18552.
OBJECTIVE
The aim of this study was to elucidate the role of microRNA-216a in microvesicles (MVs) during the process of renal interstitial fibrosis and to investigate its underlying mechanism.
MATERIALS AND METHODS
Unilateral ureteral occlusion (UUO) model was first established in mice, and kidney tissues and urine in the obscured kidney were collected. NRK-52E cells were induced with 5 ng/mL transforming growth factor-β1 (TGF-β1) for constructing the renal interstitial fibrosis model in vitro. Subsequently, the expression levels of E-cadherin, α-smooth muscle actin (α-SMA) and fibronectin (FN) in NRK-52E cells induced with or without TGF-β1 were determined, respectively. The culture medium was collected from NRK-52E cells of the control group (without TGF-β1 induction) and the TGF-β1 group (TGF-β1 induction), and MVs were observed. Afterward, NRK-52E cells were treated with MVs isolated from the control group or the TGF-β1 group, followed by detecting the expressions of E-cadherin, α-SMA and FN. Meanwhile, the expression levels of CD63, microRNA-216a, PTEN and p-AKT were determined as well. The microRNA-216 level in kidney tissues and urine of UUO mice were determined. Furthermore, the expressions of PTEN and p-AKT in mouse kidney tissues were accessed by Western blot and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR).
RESULTS
TGF-β1 induction in NRK-52E cells gradually downregulated E-cadherin, whereas upregulated α-SMA and FN with the prolongation of induction time. MVs isolated from the culture medium of the TGF-β1 group downregulated E-cadherin, and upregulated FN and α-SMA. The expression levels of CD63 and microRNA-216a were markedly higher in the TGF-β1 group compared with the control group. Downregulated PTEN and upregulated p-AKT were observed in TGF-β1-induced cells at both mRNA and protein levels. Besides, microRNA-216a expression in mouse kidney tissues and urine from obscured kidney was remarkably increased with the prolongation of UUO. Consistent with those in NRK-52E cells, the protein level of PTEN was significantly decreased, whereas p-AKT was markedly increased with the prolongation of UUO.
CONCLUSIONS
MVs containing microRNA-216a secreted by injured proximal tubular epithelial cells participate in renal interstitial fibrosis by activating the PTEN/AKT pathway.
目的
本研究旨在阐明微小 RNA-216a 在肾间质纤维化过程中微小囊泡（MVs）中的作用，并探讨其潜在机制。
材料与方法
首先建立单侧输尿管梗阻（UUO）模型，收集梗阻肾脏的肾组织和尿液。体外采用 5ng/ml 转化生长因子-β1（TGF-β1）诱导 NRK-52E 细胞构建肾间质纤维化模型。然后，分别检测 TFG-β1 诱导或未诱导的 NRK-52E 细胞中 E-钙黏蛋白、α-平滑肌肌动蛋白（α-SMA）和纤维连接蛋白（FN）的表达水平。收集对照组（未诱导 TGF-β1）和 TGF-β1 组（诱导 TGF-β1）NRK-52E 细胞的培养液，观察 MVs。然后，用对照组或 TGF-β1 组分离的 MVs 处理 NRK-52E 细胞，检测 E-钙黏蛋白、α-SMA 和 FN 的表达水平。同时，测定 CD63、微小 RNA-216a、PTEN 和 p-AKT 的表达水平。检测 UUO 小鼠肾组织和尿液中的微小 RNA-216a 水平。此外，通过 Western blot 和定量实时聚合酶链反应（qRT-PCR）检测小鼠肾组织中 PTEN 和 p-AKT 的表达。
结果
NRK-52E 细胞中 TGF-β1 的诱导逐渐下调 E-钙黏蛋白，而随着诱导时间的延长，α-SMA 和 FN 的表达逐渐上调。从 TGF-β1 组培养上清液中分离的 MVs 下调 E-钙黏蛋白，上调 FN 和 α-SMA。与对照组相比，TGF-β1 组 CD63 和微小 RNA-216a 的表达水平明显升高。TGF-β1 诱导的细胞在 mRNA 和蛋白水平上均观察到 PTEN 下调和 p-AKT 上调。此外，随着 UUO 的延长，小鼠肾脏组织和尿液中的微小 RNA-216a 表达明显增加。与 NRK-52E 细胞一致，随着 UUO 的延长，PTEN 蛋白水平显著降低，而 p-AKT 明显升高。
结论
受损近端肾小管上皮细胞分泌的含有微小 RNA-216a 的 MVs 通过激活 PTEN/AKT 通路参与肾间质纤维化。
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