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中华肾病研究电子杂志 ›› 2012, Vol. 01 ›› Issue (02) : 109 -115. doi: 10.3877/cma.j.issn.2095-3216.2012.02.010

论著

P2X4介导高糖刺激肾小管上皮细胞分泌白介素-1β的作用
陈客宏1, 张炜炜1, 张锦华1, 杨聚荣1,(), 李开龙1, 张建国1, 何娅妮1   
  1. 1.400042 重庆,第三军医大学大坪医院肾内科
  • 收稿日期:2012-11-23 出版日期:2012-12-18
  • 通信作者: 杨聚荣
  • 基金资助:
    国家自然科学基金(81070575)

P2X4 receptor mediates interleukin-1β secretion of renal tubular epithelial cells under high glucose stimulation

Ke-hong CHEN1, Wei-wei ZHANG1, Jin-hua ZHANG1, Ju-rong YANG1,(), Kai-long LI1, Jian-guo ZHANG1, Ya-ni HE1   

  1. 1.Department of Nephrology, Daping Hospital, Research Institute of Surgery, Third Military Medical University, Chongqing 400042, China
  • Received:2012-11-23 Published:2012-12-18
  • Corresponding author: Ju-rong YANG
引用本文:

陈客宏, 张炜炜, 张锦华, 杨聚荣, 李开龙, 张建国, 何娅妮. P2X4介导高糖刺激肾小管上皮细胞分泌白介素-1β的作用[J/OL]. 中华肾病研究电子杂志, 2012, 01(02): 109-115.

Ke-hong CHEN, Wei-wei ZHANG, Jin-hua ZHANG, Ju-rong YANG, Kai-long LI, Jian-guo ZHANG, Ya-ni HE. P2X4 receptor mediates interleukin-1β secretion of renal tubular epithelial cells under high glucose stimulation[J/OL]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2012, 01(02): 109-115.

目的

探讨P2X4介导高糖刺激肾小管上皮细胞分泌白介素(IL)-1β的作用。

方法

利用不同浓度(5 ~ 50 mmol/L)葡萄糖刺激肾小管上皮细胞株(HK-2)0 ~ 72 h,观察细胞上清液中三磷酸腺苷(ATP)和白介素(IL)-1β的表达。利用细胞外ATP分解酶(Apyrase)与高糖(35 mmol/L)同时刺激HK-2细胞48 h,观察细胞上清液IL-1β的水平。利用小干扰RNA(siRNA)基因沉默HK-2细胞的P2X4基因,观察高糖对细胞内钾离子钙离子浓度变化和细胞上清液中IL-1β水平的影响。利用PBFI-AM染色剂检测细胞内钾离子,利用Fluo-3/AM检测细胞内钙离子。两组间检测均数的比较采用t检验。两组以上检测均数的比较进行单因素方差分析。

结果

高糖刺激下HK-2细胞上清液中IL-1β、ATP水平升高呈剂量和时间依赖性。Apyrase能完全阻断高糖刺激细胞分泌IL-1β(8.2±4.9 μg/L与18.5±4.0 μg/L,P < 0.05)。高糖刺激下HK-2细胞内钾离子浓度降低,钙离子浓度升高。P2X4基因沉默能够显著阻断高糖刺激下IL-1β的分泌(12.5±2.9 μg/L与20.3±5.3 μg/L,P < 0.05)。

结论

P2X4可介导糖尿病肾病肾小管上皮细胞分泌IL-1β,从而促进肾间质炎症反应。

Objective

To investigate the role of P2X4 in interleukin (IL)-1β secretion of renal tubular epithelial cell under high glucose stimulation.

Methods

Human proximal tubular epithelial cell line (HK-2) cells were grown in media with normal glucose concentration (5 mmol/L), high glucose concentration (15, 25, 35 and 50 mmol/L). HK-2 cells were incubated in media with high glucose(35 mmol/L, 48 h) with and without apyrase. HK-2 cells were transfected with P2X4 siRNA. Intracellular K+ concentration was measured by PBFI-AM. Cytosolic free Ca2+ was indicated by Fluo-3/AM. Data from experiments were analyzed by t-test or ANOVA wherever appropriate.

Results

The IL-1β and adenosine triphosphate (ATP) levels of cell supernatants increased in a dose and time dependent manner under high glucose stimulation. Apyrase significantly inhibited IL-1β level elevations caused by high glucose (8.2±4.9 μg/L vs. 18.5±4.0 μg/L, P < 0.05). High glucose decreased intracellular K+ and increased intracellular Ca2+ in HK-2 cell (P < 0.05). The results suggested that high glucose activated P2X receptors of HK-2 cells. Silencing P2X4 with siRNA diminished the high glucose-induced expression of IL-1β (12.5±2.9 μg/L vs.20.3±5.3 μg/L,P < 0.05).

Conclusion

These results demonstrate that the P2X4 mediates the secretion of IL-1β in renal tubular epithelial cells, which may promote the renal interstitial inflammation in diabetic nephropathy

图1 葡萄糖对HK-2细胞分泌白介素(IL)-1β的剂量和时间效应 注:A图为剂量效应;B图为时间效应;IL-1β为白介素-1β;与对照组比较,*P < 0.05
图2 葡萄糖对HK-2细胞上清液和细胞内的ATP水平的剂量和时间效应 注:A图为葡萄糖对细胞外ATP的剂量效应;B图为葡萄糖对细胞外ATP的时间效应;C图为葡萄糖对细胞内ATP的剂量效应;D图为葡萄糖对细胞内ATP的时间效应;与对照组比较, *P < 0.05
图3 ATP水解酶Apyrase抑制高糖刺激HK-2细胞分泌IL-1β 注:IL-1β为白介素-1β;与高糖组比较,*P < 0.05
图4 高糖对HK-2细胞内的钾钙离子浓度的时间效应 注:A图为细胞内钾离子荧光染色;B图为钾离子荧光强度的半定量分析;C图为细胞内钙离子荧光染色;D图为钙离子荧光强度半定量分析;与对照组比较,*P < 0.05
图5 P2X4 siRNA对HK-2细胞P2X4表达的影响 注:A图为siRNA的转染效率;B图为P2X4 siRNA抑制P2X4 mRNA水平;C和D图为P2X4 siRNA抑制P2X4 蛋白水平;与对照组比较,*P < 0.05
图6 P2X4 siRNA干扰抑制高糖诱导HK-2细胞钾钙离子浓度的改变 注:A图为细胞内钾离子蓝色荧光(上排)和钙离子绿色荧光(下排);B图为钾离子荧光强度半定量分析;C图为钙离子荧光强度半定量分析;与高糖组比较,*P < 0.05
图7 P2X4 siRNA干扰抑制高糖诱导HK-2细胞分泌IL-1β 注:IL-1β为白介素-1β;与高糖组比较,**P < 0.01
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