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中华肾病研究电子杂志

中华肾病研究电子杂志 ›› 2014, Vol. 03 ›› Issue (06) : 314 -321. doi: 10.3877/cma.j.issn.2095-3216.2014.06.007

论著

Intermedin 及其受体系统在单侧输尿管梗阻大鼠肾组织的表达
赵宁1, 乔晞1,†(), 王利华1, 张瑞婧1, 韩伟霞1   
  1. 1.030001 太原,山西医科大学第二医院肾内科, 山西省肾脏病研究所
  • 出版日期:2014-12-15
  • 通信作者: 乔晞
  • 基金资助:
    国家自然科学基金青年科学基金项目(81100531)

Expression of intermedin and its receptors in renal tissues of rats with unilateral ureteral obstruction (UUO)

Ning Zhao1, Xi Qiao1,†(), Lihua Wang1, Ruijing Zhang1, Weixia Han1   

  1. 1.Department of Nephrology, Second Hospital of Shanxi Medical University, Shanxi Provincial Kidney Disease Institute, Taiyuan 030001, China
  • Published:2014-12-15
  • Corresponding author: Xi Qiao
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引用本文：

赵宁, 乔晞, 王利华, 张瑞婧, 韩伟霞. Intermedin 及其受体系统在单侧输尿管梗阻大鼠肾组织的表达[J/OL]. 中华肾病研究电子杂志, 2014, 03(06): 314-321.

Ning Zhao, Xi Qiao, Lihua Wang, Ruijing Zhang, Weixia Han. Expression of intermedin and its receptors in renal tissues of rats with unilateral ureteral obstruction (UUO)[J/OL]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2014, 03(06): 314-321.

目的

观察大鼠单侧输尿管梗阻(UUO)模型中肾组织intermedin(IMD)及其受体系统-降钙素受体样受体(CRLR)/受体活性修饰蛋白(RAMP)表达的变化。

方法

健康雄性Wistar 大鼠48 只,随机分为假手术组和UUO 组,分别于术后1、3、7、14 d 随机选取6 只,留取梗阻侧及假手术侧肾组织。 HE、Masson 染色观察肾组织病理变化;Real-Time RT-PCR 检测肾组织中IMD、CRLR、RAMP1、RAMP2、RAMP3 的mRNA 表达;免疫组织化学法检测肾组织中IMD、CRLR、RAMP1、RAMP2、RAMP3 的蛋白表达。 采用析因设计的方差分析进行统计学分析。

结果

肾脏组织病理染色结果显示,假手术组术后各时间点肾小球、肾小管均未见明显损伤;与假手术组相比,UUO 组肾脏病理改变明显,肾间质纤维化程度随梗阻时间延长逐渐加重(P＜0.05);Real-Time RT-PCR 及免疫组织化学结果显示,IMD、CRLR 和RAMP1、RAMP2、RAMP3 的mRNA 及其蛋白质表达从UUO 术后1 d 开始升高(与假手术组比较 P＜0.05),分别于术后3d(RAMP2 和RAMP3)和术后7 d(IMD、CRLR 和RAMP1)达到高峰,以后逐渐下降。

结论

IMD 及其受体系统在UUO 大鼠肾组织表达上调,提示IMD 可能在肾脏间质纤维化中发挥重要作用。

关键词: 肾脏, 纤维化, Intermedin, 降钙素受体样受体, 受体活性修饰蛋白

Objective

To investigate the expression changes of intermedin (IMD) and its receptor system, namely calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP1/2/3), in renal tissues of rats with unilateral ureteral obstruction (UUO).

Methods

Forty-eight male Wistar rats were randomly divided into UUO group (n=24) and sham-operated group (n=24). UUO model was made by ligation of left ureter. For sham-operated group, rats were subjected to left flank incisions, and the kidney was exposed, but the ureter was not ligated. Groups of six animals each were killed at 1, 3, 7, and 14 d after operation, respectively. Kidneys were harvested for further analysis. Paraffin-embedded transverse kidney slices were sectioned at 4 μm, and stained with hematoxylin and eosin. For analyzing the degree of tubulointerstitial collagen deposition, sections were stained with Masson trichrome. mRNA expression levels of IMD, CRLR, and RAMPs were detected by real-time reverse transcription-polymerase chanin reaction (RTPCR). Protein expression levels of IMD, CRLR, and RAMPs were detected by immunohistochemical staining.The variance analysis of factorial design was used for data analysis.

Results

Renal morphology showed that no changes were detected in the sham-operated group. In the UUO group, there were significant pathological changes, and the interstitial fibrosis gradually increased with time (P＜0. 05). Real-time RT-PCR and immunohistochemical staining revealed that, after UUO operation, the mRNA and protein expression levels of IMD, CRLR, RAMP1, RAMP2, and RAMP3 increased from day 1 (P＜0.05, compared with sham group),and reached peak value at day 3 (RAMP2 and RAMP3) or at day 7 (IMD, CRLR and RAMP1),respectively, and then gradually declined.

Conclusion

Our results indicated that IMD and its receptors were upregulated after UUO, which might play a role in the renal fibrosis.

Key words: Kidney, Fibrosis, Intermedin, Calcitonin receptor-like receptor, Receptor activity-modifying proteins
表1 Real-time RT-PCR 引物序列
基因 引物序列
IMD forward5'-GGCCCAGTTGCTGATGGT-3'
reverse5'-TGCCCGGGAGCAGGTA-3'
CRLR forward5'-CTCACGGCAGTGGCCAATAA-3'
reverse5'-AGCCCATCAGGTAAAGATGAATGAA-3'
RAMP1 forward5'-AGCTGTGTCTCAGCCGCTTC-3'
reverse5'-AATCTTGTTTGCCACGAGTTTG-3'
RAMP2 forward5'-GAGTCCCTGAATCAATCTCATCCTA-3'　
forward5'-TCAAAGTCCAGTTGCACCAGTC-3'
RAMP3 forward5'-GAAAGCCTTTGCCGAAATGATG-3'
reverse5'-GCCCACGATGTTTGTCTCCA-3'
β-actin forward5'-CCCATCTATGAGGGTTACGC-3'
reverse5'-TTTAATGTCACGCACGATTTC-3'
图1 两组大鼠术后肾组织病理改变图片(HE×400) 注:UUO 为单侧输尿管梗阻;A 为假手术组;B 为UUO 术后 1 d;C 为UUO 术后3 d;D 为UUO 术后7 d;E 为UUO 术后14 d
图2 两组大鼠术后肾组织纤维化程度(Masson×400) 注: UUO 为单侧输尿管梗阻;A 为假手术组;B 为UUO 术后 1 d;C 为UUO 术后3 d;D 为UUO 术后 7 d;E 为UUO 术后14 d;F 为Masson 染色半定量分析结果;与假手术组相比,aP＜0.05
图3 两组大鼠术后不同时间肾组织IMD、CRLR、RAMP1、RAMP2 及RAMP3 mRNA 的表达 注:UUO 为单侧输尿管梗阻;IMD 为Intermedin;CRLR 为降钙素受体样受体;RAMP 为受体活性修饰蛋白;β-actin 为β-肌动蛋白A 为IMD mRNA 的表达;B 为CRLR mRNA 的表达;C 为RAMP1 mRNA 的表达;D 为RAMP2 mRNA 的表达;E 为RAMP3 mRNA 的表达。 与假手术组比较,aP＜0.05。
图4 两组大鼠术后不同时间肾组织IMD 的表达(免疫组化×400) 注:UUO 为单侧输尿管梗阻;A 为假手术组;B 为UUO 术后1 d;C 为UUO 术后3 d;D 为UUO 术后7 d;E;UUO 术后14 d;F 为免疫组化半定量分析结果; 与假手术组相比,aP＜0.05。
图5 两组大鼠术后不同时间肾组织CRLR 的表达(免疫组化×400) 注:UUO 为单侧输尿管梗阻;A 为假手术组;B 为UUO 术后 1 d;C 为UUO 术后3 d;D 为UUO 术后 7 d;E;UUO 术后14 d;F 为免疫组化半定量分析结果;与假手术组相比,aP＜0.05。
图6 两组大鼠术后不同时间肾组织RAMP1 的表达(免疫组化×400) 注:UUO 为单侧输尿管梗阻;A 为假手术组;B 为UUO 术后 1 d;C 为UUO 术后3 d;D 为UUO 术后 7 d;E;UUO 术后14 d;F 为免疫组化半定量分析结果;与假手术组相比,aP＜0.05。
图7 两组大鼠术后不同时间肾组织RAMP2 的表达(免疫组化 ×400) 注:UUO 为单侧输尿管梗阻;A 为假手术组;B 为UUO 术后 1 d;C 为UUO 术后3 d;D 为UUO 术后 7 d;E;UUO 术后14 d;F 为免疫组化半定量分析结果;与假手术组相比,aP＜0.05。
图8 两组大鼠术后不同时间肾组织RAMP3 的表达(免疫组化 ×400) 注:UUO 为单侧输尿管梗阻;A 为假手术组;B 为UUO 术后 1 d;C 为UUO 术后3 d;D 为UUO 术后 7 d;E;UUO 术后14 d;F 为免疫组化半定量分析结果;与假手术组相比aP＜0.05。
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