中国血液净化

中国血液净化 ›› 2016, Vol. 15 ›› Issue (05): 304-308.doi: 10.3969/j.issn.1671-4091.2016.05.012

• 基础研究 • 上一篇    下一篇

尾加压素Ⅱ在造影剂介导的人肾小管上皮细胞损伤中的作用

王征1,吴菲1,庞欣欣1,陈冠仲1,张爱华1,范敏华1   

  1.  北京大学第三医院肾内科
  • 收稿日期:2016-01-05 修回日期:2016-02-14 出版日期:2016-05-12 发布日期:2016-05-19
  • 通讯作者: 张爱华 zhangaihua0982@sina.com E-mail:zhangaihua0982@sina.com
  • 基金资助:

    国家自然基金委项目资助(项目编号81170706, 81341022, 81570663)

Roles of urotensin II in the injury of human renal tubular epithelial cell induced by contrast medium

  • Received:2016-01-05 Revised:2016-02-14 Online:2016-05-12 Published:2016-05-19

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摘要： 目的既往文献报道造影剂肾病患者血浆尾加压素Ⅱ(urotensin Ⅱ,UⅡ)水平增高，提示UII 在造影剂肾病中可能发挥作用，但具体机制未明。本研究观察尾加压素Ⅱ在造影剂介导的人肾小管上皮细胞氧化应激，凋亡及自噬中的作用。方法人肾小管上皮细胞(HK-2)暴露于不同浓度的造影剂碘普罗胺(25gI/L,200gI/L)，不同浓度的UⅡ(10- 5mol/l～10- 8mol/l)，UII 联合小剂量造影剂碘普罗胺50gI/L 以及大剂量造影剂碘普罗胺200gI/L，不同处理细胞2h 至24h，测各处理组细胞上清液丙二醛
(malondialdehyde，MDA)的含量，细胞凋亡率，用western blot 检测各组自噬标志物微管相关蛋白1 轻链3-Ⅱ(light chain 3-Ⅱ，LC3Ⅱ),Beclin-1 的表达。结果① 和正常对照组MDA 浓度(20.32±3.00)nmol/ml 相比，UⅡ10- 5mol/l～10- 8mol/l 处理组MDA 浓度[分别为(152.65±18.72)nmol/ml、(149.19±1.17)nmol/ml、(151.12±3.05)nmol/ml、(116.81±6.85)nmol/ml]、碘普罗胺的50gI/l 处理组的MDA 浓度[(99.44±12.00)nmol/ml]显著增加(F=41.863，P=0.000)；但UⅡ10-6mol/l+碘普罗胺200gI/l 和碘普罗胺200gI/相比，细胞上清MDA 含量显著减少[(5.95±1.63)nmol/ml 比(41.70±8.46)nmol/ml，P=0.033，95% CI -66.688～-4.798]；②和正常对照组凋亡率(3.65±1.17)%相比，各个浓度的UⅡ10-5mol/l- 10- 8mol/l 处理组HK- 2 细胞晚期凋亡率[分别为(9.33±0.23)%、(8.63±2.10)%、(9.93±2.35)%、
(10.10±1.97)% ]，以及碘普罗胺50gI/L 组细胞凋亡率[(8.20±1.21)% ]和200gI/L 处理组凋亡率[(15.03±0.55)%]显著增加(F=28.322，P=0.000)；UII10-5mol/L+碘普罗胺50gI/L 与单独碘普罗胺50gI/L 相比, 晚期细胞凋亡率显著增加[(15.16 ± 2.27)% 比(8.20 ± 1.21)% ，P=0.031，95% CI 0.681～12.404)；但UⅡ10-6mol/L+碘普罗胺200gI/L，与单独碘普罗胺200gI/L 相比, 晚期细胞凋亡率显著减少[(10.27±0.61)%比(15.03±0.55)%,P =＜0.001，95% CI -6.481～-3.041]；③ 和正常对照相比，小剂量造影剂碘普罗胺50gI/L 处理后LC3II 的表达显著增加[LC3II/actin 灰度值(0.40±0.02)比(0.09±0.02),t=4.989,P =＜0.001]；和正常对照组相比，UII10-8mol/L 单独处理后LC3II 表达受抑制[LC3 II/actin 灰度值0.029±0.009 比0.05±0.01,t=-2.892,P=0.031]。结论造影剂对肾小管上皮细胞氧化应激、细胞凋亡及自噬均有诱导作用；而UⅡ本身也能独立介导肾小管上皮细胞氧化应激和凋亡，而抑制肾小管上皮细胞自噬，但UⅡ协同小剂量造影剂的促凋亡作用，拮抗大剂量造影剂的促凋亡作用，在大剂量造影剂存在时UⅡ还有减轻细胞氧化应激的作用。鉴于UⅡ作用的双向性，UⅡ在造影剂肾病发病中的作用需要近一步深入研究。

关键词: 造影剂肾病, 尾加压素Ⅱ, 氧化应激, 凋亡, 自噬

Abstract: Objective Previous study showed that plasma urotensin II (UⅡ) increased in contrast induced nephropathy. However, its pathogenesis is unclear. The aim of this study is to observe the roles of UⅡ in the injury of human renal tubular epithelial cell induced by contrast medium. Methods Human renal tubular
epithelial cell (HK-2) was exposed in different concentration of UⅡ, contrast medium (Iopromide), and U Ⅱ combined with iopromide for 2～24 hours. Apoptosis rate by annexin V method, malondialdehyde (MDA) level in the supernatant, and autophgy markers including light chain 3-Ⅱ (LC3Ⅱ) and Beclin-1 by western
blot were measured. Results ① Both UⅡ 10- 5 mol/L～10- 8 mol/L and iopromide 50 gI/L could increase MDA level in supernatant. MDA in supernatant was 152.65±18.72 nmol/ml, 149.19±1.17 nmol/ml, 151.12± 3.05 nmol/ml and 116.81±6.85 nmol/ml, respectively from the cell treated with UⅡ10- 5 mol/L～10- 8 mol/L, and was 99.44±12.00 nmol/ml from the cells treated with iopromide 50 gI/L, significantly higher than that from normal control cells (20.32±3.00 nmol/ml, F=41.863, P=0.000). MDA in supernatant was lower from the cells treated with UⅡ10-6 mol/l + iopromide 200gI/l (5.95±1.63 nmol/ml) than from those treated with iopromide 200gI/l (41.70±8.46 nmol/ml; P=0.033, 95% CI -66.688～-4.798 by post hoc test Dunnett T3)； ② Both UⅡ and iopromide could induce apoptosis in Hk-2 cells. The rate of late apoptosis was 9.33±0.23%, 8.63±2.10%, 9.93±2.35%, 10.10±1.97%, respectively in cells treated with UⅡ10- 5～10- 8 mol/L, was 8.20±1.21% in cells treated with iopromide 50gI/L, and was 15.03±0.55% in cells treated with iopromide 200gI/L, significantly higher than that in normal control cells (3.65±1.17%; F=28.322, P=0.000 by ANOVA). The rateof late apoptosis was higher in cells treated with UⅡ10- 6 mol/L + iopromide 50gI/L (15.16±2.27%) than in those treated with iopromide 50gI/L (8.20±1.21%, P=0.031, 95% CI 0.681～12.404). However, the rate of late apoptosis was lower in cells treated with UⅡ10-6 mol/L+iopromide 200 gI/L (10.27±0.61%) than in those treated with iopromide 200 gI/L (15.03±0.55%; P=0.000, 95% CI -6.481～-3.041 by post hoc test Dunnett T3). ③ LC3Ⅱ expression was lower in Hk-2 cell treated with UⅡ10-8 mol/L (LC3Ⅱ/actin = 0.029±0.009) than in untreated Hk-2 cells (LC3Ⅱ/actin=0.05±0.01; t=-2.892, P=0.031), but was higher in Hk-2 cells treated with iopromide 50 gI/L (LC3II/actin = 0.40±0.02) than in untreated Hk-2 cells (0.09±0.02, t=4.989, P=0.000). Conclusions Contrast medium can induce oxidative stress, apoptosis and autophagy renal tubular epithelial cell. UII can also induce oxidative stress and apoptosis but inhibit autophagy in renal tubular epithelial cell. U Ⅱ prevents HK-2 cell from apoptosis induced by high dose of contrast medium. The dual roles of UⅡ in the contrast induced nephropathy need to be further studied.

Key words: Contrast induced nephropathy, Urotensin Ⅱ, Oxidative stress, Apoptosis, Autophagy