腹膜透析液通过上调葡萄糖转运体促进腹膜纤维化

doi:10.3785/j.issn.1008-9292.2016.11.07

浙江大学学报（医学版）

2016, Vol. 45

Issue (6): 598-606 DOI: 10.3785/j.issn.1008-9292.2016.11.07

原著

腹膜透析液通过上调葡萄糖转运体促进腹膜纤维化

洪梦琪1(

),聂振禹2,陈争跃2,余雄伟2,包蓓艳2,*(

)

1. 宁波大学医学院, 浙江宁波 315211
2. 宁波市泌尿肾病医院肾内科, 浙江宁波 315192

High glucose dialysate enhances peritoneal fibrosis through upregulating glucose transporters GLUT1 and SGLT1

HONG Mengqi1(

),NIE Zhenyu2,CHEN Zhengyue2,YU Xiongwei2,BAO Beiyan2,*(

)

1. School of Medicine, Ningbo University, Ningbo 315211, China
2. Division of Nephrology, Ningbo Urology and Nephrology Hospital, School of Medicine, Ningbo University, Ningbo 315192, China

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摘要： 目的

研究葡萄糖转运体1（GLUT1）和钠—葡萄糖转运体1（SGLT1）在腹膜纤维化过程中的作用。

方法

体内实验中，取36只SD雄性大鼠随机分为六组：正常对照组、手术对照组、腹膜透析组（PD组）、腹膜透析+根皮素组（PD+T组）、腹膜透析+根皮苷组（PD+Z组）、腹膜透析+根皮素+根皮苷组（PD+T+Z组），每组6只。采用5/6肾脏切除法制作尿毒症模型，腹膜透析采用浓度为2.5%的透析液。停止透析24 h后行腹膜平衡试验评估大鼠腹膜转运功能；HE染色观察壁层腹膜形态；免疫组织化学法检测网膜组织GLUT1、SGLT1、TGF-β1和结缔组织生长因子（CTGF）的表达。体外实验中，体外培养人腹膜微血管内皮细胞（HPEC）并分为五组：正常对照组、腹膜透析组（PD组）、腹膜透析+根皮素组（PD+T组）、腹膜透析+根皮苷组（PD+Z组）、腹膜透析+根皮素+根皮苷组（PD+T+Z组）。采用实时定量PCR和蛋白质印迹法检测网膜组织及微血管内皮细胞内GLUT1、SGLT1、TGF-β1、CTGF mRNA及蛋白表达。

结果

与手术对照组比较，PD组大鼠的腹膜增厚，超滤量增加，GLUT1、SGLT1、TGF-β1、CTGF mRNA及蛋白均表达上升（均P＜0.05）；在使用葡萄糖拮抗剂根皮苷、根皮素后，大鼠腹膜增厚程度减轻，GLUT1、SGLT1、TGF-β1、CTGF mRNA及蛋白表达均下降（均P＜0.05）；相关性分析显示，大鼠腹膜GULT1、SGLT1表达与TGF-β1、CTGF表达均呈正相关（均P＜0.05）。2.5%腹膜透析液可以上调人腹膜微血管内皮细胞GLUT1、SGLT1、TGF-β1、CTGF mRNA及蛋白的表达（均P＜0.05）；使用葡萄糖拮抗剂后，人腹膜微血管内皮细胞GLUT1、SGLT1、TGF-β1、CTGF mRNA及蛋白表达均下降（均P＜0.05）；相关性分析显示，人腹膜微血管内皮细胞GLUT1、SGLT1表达与TGF-β1、CTGF表达均呈正相关（均P＜0.05）。

结论

高糖腹膜透析液可通过上调GLUT1、SGLT1的表达促进腹膜纤维化。

关键词： 腹膜透析; 透析液; 单糖转运蛋白质类; 钠-葡萄糖转运体1; 纤维化/病理生理学; 内皮,血管/细胞学; 腹膜/血液供给; 疾病模型,动物

Abstract: Objective

To investigate the role of glucose transporter 1 (GLUT1) and sodium-glucose cotransporter 1 (SGLT1) in high glucose dialysate-induced peritoneal fibrosis.

Methods

Thirty six male SD rats were randomly divided into 6 groups (6 in each):normal control group, sham operation group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorizin group (PD+Z group), PD+phloretin+phlorizin group (PD+T+Z group). Rat model of uraemia was established using 5/6 nephrotomy, and 2.5% dextrose peritoneal dialysis solution was used in peritoneal dialysis. Peritoneal equilibration test was performed 24 h after dialysis to evaluate transport function of peritoneum in rats; HE staining was used to observe the morphology of peritoneal tissue; and immunohistochemistry was used to detect the expression of GLUT1, SGLT1, TGF-β1 and connective tissue growth factor (CTGF) in peritoneum. Human peritoneal microvascular endothelial cells (HPECs) were divided into 5 groups:normal control group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorezin group (PD+Z group), and PD+phloretin+phlorezin group (PD+T+Z group). Real time PCR and Western blotting were used to detect mRNA and protein expressions of GLUT1, SGLT1, TGF-β1, CTGF in peritoneal membrane and HPECs.

Results

In vivo, compared with sham operation group, rats in PD group had thickened peritoneum, higher ultrafiltration volume, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-β1 were significantly increased (all P＜0.05); compared with PD group, thickened peritoneum was attenuated, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-β1 were significantly decreased in PD+T, PD+Z and PD+T+Z groups (all P＜0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in peritoneum were positively correlated with the expressions of TGF-β1 and CTGF (all P＜0.05). In vitro, the mRNA and protein expressions of GLUT1, SGLT1, TGF-β1, CTGF were significantly increased in HPECs of peritoneal dialysis group (all P＜0.05), and those in PD+T, PD+Z, and PD+T+Z groups were decreased (all P＜0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in HPECs were positively correlated with the expressions of TGF-β1 and CTGF (all P＜0.05).

Conclusion

High glucose peritoneal dialysis fluid may promote peritoneal fibrosis by upregulating the expressions of GLUT1 and SGLT1.

Key words: Peritoneal dialysis Dialysis solutions Monosaccharide transport proteins Sodium-glucose transporter 1 Fibrosis/physiopathology Endothelium, vascular/cytology Peritoneum/blood Disease models, animal

收稿日期: 2016-09-30 出版日期: 2017-08-29

基金资助: 浙江省自然科学基金（Y13H050021）

通讯作者: 包蓓艳 E-mail: 364289549@qq.com;baobeiyan2007@sina.com

作者简介: 洪梦琪(1991-),女,硕士研究生,主要从事腹膜透析治疗的研究; E-mail:364289549@qq.com

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引用本文:

洪梦琪,聂振禹,陈争跃,余雄伟,包蓓艳. 腹膜透析液通过上调葡萄糖转运体促进腹膜纤维化[J]. 浙江大学学报（医学版）, 2016, 45(6): 598-606.

HONG Mengqi,NIE Zhenyu,CHEN Zhengyue,YU Xiongwei,BAO Beiyan. High glucose dialysate enhances peritoneal fibrosis through upregulating glucose transporters GLUT1 and SGLT1. J Zhejiang Univ (Med Sci), 2016, 45(6): 598-606.

链接本文:

http://www.zjujournals.com/xueshu/med/CN/10.3785/j.issn.1008-9292.2016.11.07 或 http://www.zjujournals.com/xueshu/med/CN/Y2016/V45/I6/598

表1 实时定量PCR引物序列

表2 各组腹膜超滤量、D-cr/P-cr和D1/D0比较

图1 各组大鼠腹膜组织横切面(HE染色)

图2 免疫组织化学法检测各组大鼠网膜组织中GULT1、SGLT1、TGF-β1和CTGF蛋白表达

图3 大鼠腹膜GULT1和SGLT1蛋白表达的电泳图

表3 各组腹膜GULT1和SGLT1 mRNA及蛋白表达比较

图4 各组大鼠腹膜TGF-β1和 CTGF蛋白表达的电泳图

表4 各组腹膜TGF-β1、CTGF mRNA及蛋白表达比较

图5 人腹膜内皮细胞GULT1、SGLT1蛋白表达电泳图

表5 各组微血管内皮细胞GULT1和SGLT1 mRNA和蛋白表达水平比较

图6 人腹膜内皮细胞TGF-β1、CTGF蛋白表达电泳图

表6 人腹膜内皮细胞TGF-β1、CTGF mRNA及蛋白表达比较

1	PENG Y M SHU Z J XIAO L et al. A new non-uremic rat model of long-term peritoneal dialysis Physiol Res 2011 60 1 157 164

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