高尿酸通过TLR4/NF-κB信号通路诱导肾小管上皮细胞上皮-间充质转化的作用研究

High uric acid induces epithelial-mesenchymal transition of renal tubular epithelial cells via TLR4/NF-κB signaling pathway

目的探讨尿酸诱导肾小管上皮细胞上皮-间充质细胞转化(epithelia-mesenchymal transition,EMT)的作用及机制。方法采用不同质量浓度的尿酸预孵育肾小管上皮细胞(HK-2)48 h,通过倒置显微镜观察尿酸对HK-2细胞形态的影响,Western blot检测Fibronectin、α-SMA和E-cadherin蛋白表达变化。Real-time PCR检测炎症因子IL-1β、IL-6以及TNF-αmRNA的表达变化,免疫荧光检测上皮细胞标志物cytokeratin和间充质细胞标志物vimentin的表达变化。结果与正常组相比,15 mg/d L的尿酸处理HK-2细胞48 h后,HK-2细胞间隙增大,呈长梭形改变。间充质细胞标志物Fibronectin、α-SMA表达明显升高,而上皮细胞标志物E-cadherin的表达明显下降(P<0.05)。同时,高浓度尿酸诱导了HK-2细胞炎症因子IL-1β、TNF-α的表达升高(P<0.05),TLR4/NF-κB信号通路活化,而靶向抑制NF-κB信号通路活化可以显著抑制尿酸诱导的EMT。结论高尿酸可以通过活化TLR4/NF-κB信号通路诱导肾小管上皮细胞EMT的发生,靶向干预NF-κB信号通路可以显著抑制尿酸诱导的肾间质纤维化。

Objective To determine the effect of epithelial-mesenchymal transition （EMT） of renal tubular epithelial cells induced by uric acid, and investigate the underlying mechanism. Methods Human renal tubular epithelial HK-2 cells were cultured in vitro and incubated with different concentrations of uric acid for 48 h. The effects of uric acid on the morphology of HK-2 cells were observed and recorded with the aid of inverted microscope. The protein levels of fibroneetin, a-smooth muscle actin （a-SMA） and E-cadherin were detected by Western blotting, while the mRNA expression levels of IL-115, IL-6 and TNF-a were detected by real-time PCR. Immunofluorescence assay was used to detect the changes of epithelial cell marker cytokeratin and mesenchymal cell marker vimentin. Results Compared with untreated HK-2 cells, treatment of 15 mg/dL uric acid for 48 h induced the HK-2 cells turned into long-spindled shape, with markedly increased intercellular space. The protein levels of mesenchymal marker fibronectin and a-SMA were significantly increased, while that of the epithelial cell marker E-cadherin was significantly decreased （P 〈 0.05 ）. High dose of uric acid enhanced the expression levels of IL-115 and TNF-α （ P 〈 0.05 ） and induced activation of TLR4/NF-KB signaling pathway in HK-2 cells （ P 〈 0. 05 ）. Targeted inhibition of NF-KB signaling pathway significantly suppressed EMT induced by uric acid in HK-2 cells. Conclusion High uricacid induces EMT in renal tubular epithelial cells by activation of the TLR4/NF-KB signaling pathway, and the targeted intervention of NF-KB signaling pathway can significantly suppress uric acid induced renal interstitial fibrosis.