IDENTIFICATION OF GLUCOSE TRANSPORTER-1 AND ITS FUNCTIONAL ASSAY IN MOUSE GLOMERULAR MESANGIAL CELLS CULTURED IN VITRO

INTRODUCTION Glomerular mesangial cells play a key role in the development of diabetic glomerular lesions. There is a close correlation between expansion of the mesangium of the glomerulus and the clinical manifestations of diabetic nephropathy(1). Although it has become evident that the accumulation of extracellular matrix (ECM) occurs in the diabetic glomerulus and that mesangial cells in culture demonstrate enhanced production of ECM in response to elevated glucose levels (2), the me…     

IDENTIFICATION OF GLUCOSE TRANSPORTER－1 AND ITS FUNCTIONAL ASSAY IN MOUSE GLOMERULAR MESANGIAL CELLS CULTURED IN VITRO

Objective. To evaluate the role of glucose transporter-1 (GLUT1) in the glucose uptake of glomerular mesanglal cells. Methods. Cultured C57/SJL mouse mesangial cells were used in the study. The expression of GLUT1 mRNA was detected by RT-PCR. The expression of GLUT1 protein was detected by immunofluorescence and flow cytometry. The uptake of glucose and its kinetics were determined by 2-deoxy-[3H] -D-glucose uptake. Results. Both GLUT1 mRNA and protein were found in mouse glomerular mesangial cells. 2-deoxy-D-glucose uptake and kinetics assay showed that this glucose transporter had high affinity for glucose and the glucose uptake specificity was further confirmed by phloretin. Conclusion. Functional GLUT1 did present in mouse mesangial cells cultured in vitro and it might be the predominant transporter mediated the uptake of glucose into mesangial cells.     

半薄SDS-PAGE垂直式平板电泳法检测尿蛋白谱

蛋白尿是肾脏疾患的重要症状之一，其中蛋白的性质与构成（即尿蛋白谱）在诊断肾脏疾病及评估药物疗效等方面有着重要的参考价值。以往采用的蛋白电泳技术，如醋酸纤维薄膜、琼脂糖凝胶、免疫电泳、聚丙烯酰胺凝胶电泳（ＰＡＧＥ）、等电聚集以及ＳＤＳＰＡＧＥ圆盘电泳等方法虽各具优点，但与临床诊断所需的大量尿标本检测，以及蛋白成分高分辨率要求相比还存在着差距。本文介绍的半薄ＳＤＳＰＡＧＥ垂直式平板电泳［２，３］，并结合高敏银染［４］和激光密度扫描的方法，能够快速有效地分析尿液中蛋白质成分。１　材料和方法１．１　…     

大黄酸对系膜细胞葡萄糖摄入的影响及其机制

目的 研究大黄酸对系膜细胞葡萄糖摄入的影响,探讨大黄酸拮抗糖尿病肾脏代谢异常的可能机制。方法 选用体外培养的小鼠肾小球系膜细胞,葡萄糖摄入及其动力学的测定采用２－ｄｅｏｘｙ－［＾３Ｈ］－Ｄ－ｇｌｕｃｏｓｅ摄入法,葡萄糖转运蛋白１（ｇｌｕｃｏｓｅ ｔｒａｎｓｐｏｒｔｅｅｒ,１ＧＬＵＴ１）ｍＲＮＡ表达的检测采用Ｎｏｒｔｈｅｒｎｘ印迹,结果 大黄酸对正常培养条件下系膜细胞的葡萄糖摄入及动力学没有明显影响     

Regulation of the expression and function of glucose transporter-1 by TGF-β1 and high glucose in mesangial cells

Objective To study the effects of high glucose and transforming growth factor-β1 (TGF-β1) on the expression and function of glucose transporter-1 (GLUT1) in mouse mesangial cells.Methods Cultured mouse mesangial cells were used.The expression of GLUT1 mRNA was detected by Northern Blot; glucose uptake and its kinetics were determined with a 2-Deoxy-［(3)H］-D-glucose uptake assay.Results Mesangial cells exposed to enriched glucose medium (20 mmol/L) for 72 hours demonstrated a decrease in both GLUT1 mRNA and V(max) for uptake of the glucose analog, 2-deoxy-D-glucose　(2DOG), as compared to mesangial cells cultured in physiologic glucose concentrations(5.5 mmol/L).In contrast, hypertonic mannitol had no effect on GLUT1 mRNA levels.TGF-β1 treatment for 10 hours stimulated 2DOG uptake, both in 5.5 mmol/L and 20 mmol/L glucose medium, by approximately 4.28-fold in a dose-dependent manner (2 ng/ml maximum).Kinetic analysis of 2DOG uptake revealed an increase in V(max) and a decrease in K(m) in the presence of TGF-β1. TGF-β1 also up-regulated the expression of GLUT1 mRNA in mesangial cells.The addition of anti-TGF-β neutralizing antibody (30 μg/ml) in mesangial cells cultured in enriched glucose medium (20 mmol/L) led to a 40% decrease in 2DOG uptake.Conclusions The expression of GLUT1 can be suppressed by exposure of mesangial cells to high glucose medium, which may serve as a protective mechanism against possible adverse effects of excessive glucose flux into cells.TGF-β1 stimulates glucose uptake by enhancing the expression and function of GLUT1 in mesangial cells.This effect is independent of the glucose milieu in the cultured medium.     

Effect of rhein on glucose transporter-1 expression and its function in glomerular mesangial cells

Ｇｌｕｃｏｓｅｉｓｔｈｅｍａｉｎｆｕｅｌｆｏｒｍｏｓｔｍａｍｍａｌｉａｎｃｅｌｌｓ,ａｎｄｃａｎｂｅｔｒａｎｓｐｏｒｔｅｄｉｎｔｏｃｅｌｌｓｂｙｇｌｕｃｏｓｅｔｒａｎｓｐｏｒｔｅｒｓＧｌｕｃｏｓｅｔｒａｎｓｐｏｒｔｅｒ1(ＧＬＵＴ1)ｈａｓｂｅｅｎｃｏｎｓｉｄｅｒｅｄｏｎｅｏｆｔｈｅｆａｃｉｌｉｔａｔｉｖｅｇｌｕｃｏｓｅｔｒａｎｓｐｏｒｔｅｒｓｒｅｓｐｏｎｓｉｂｌｅｆｏｒｃｏｎｓｔｉｔｕｔｉｖｅｇｌｕｃｏｓｅｔｒａｎｓｐｏｒｔ1　Ａｓｔｈｅｐｒｅｄｏｍｉｎａｎｔｆａｃｉｌｉｔａｔｉｖｅｇｌｕｃｏｓｅｔ…     

转化生长因子及大黄酸对肾小球系膜细胞葡萄糖转运蛋白功能的影响

目的对人肾小球系膜细胞葡萄糖转运蛋白-1（GLUT1）进行鉴定。研究GLUT1的作用特点,TGF-β1对其影响以及大黄酸的干预作用。方法分别用RT-PCR,免疫荧光染色,流式细胞仪和［3H］-2脱氧葡萄糖摄入率,对系膜细胞GLUT1mRNA表达,蛋白质分布和功能进行鉴定。观察不同浓度TGF-β1在加或不加大黄酸的情况下对系膜细胞葡萄糖摄人以及GLUT1mRNA表达的影响。结果人类肾小球系膜细胞上存在功能性的GLUT1。TGF-β1能刺激系膜细胞GLUT1mRNA的表达,并增加系膜细胞葡萄糖的摄入率[TGF-β1（836±35）％,对照（154±12）％、P＜0.01]。大黄酸对正常糖浓度培养条件下系膜细胞糖摄入无影响,但能明显抑制TGF-β1增加系膜细胞糖摄入的作用［TGF-β1为（198±28）％、大黄酸为（576±12）％,P＜0.01]。结论首次报道人系膜细胞上存在GLUT1,TGF-β1能从转录水平上调控GLUT1的功能,使系膜细胞葡萄糖摄入增加,但该作用能够明显地被大黄酸所抑制。     

雷公藤内酯醇降低T淋巴细胞核因子-κB的活性

目的：研究雷公藤内酯醇对Ｔ淋巴细胞核因子－κＢ（ＮＦ－κＢ）活力的影响，进一步研究雷公藤内酯醇免疫抑制作用的机制。方法：利用凝胶迁移率实验（ＥＭＳＡ）检测不同浓度的雷公藤内酯醇对于普通培养状态下和同时使用佛波脂／植物血凝素（ＰＭＡ／ＰＨＡ）激活的Ｊｕｒｋａｔ细胞中ＮＦ－κＢ活力的影响。结果：在普通培养状态下和同时使用ＰＭＡ／ＰＨＡ激活的Ｊｕｒｋａｔ细胞中均存在一定活力的ＮＦ－κＢ，使用ＰＭＡ／ＰＨＡ处理可使Ｊｕｒｋａｔ细胞中ＮＦ－κＢ的活力显著的升高，雷公藤内酯醇可以降低两种培养状况下的Ｊｕｒｋａｔ细胞中ＮＦ－κＢ活力，并以激活状态下更为显著。结论：雷公藤内酯醇降低Ｔ淋巴细胞中ＮＦ－κＢ活力的作用可能是其免疫抑制效应的分子机制之一。     

小鼠肾小球系膜细胞葡萄糖转运蛋白的鉴定及其功能研究

目的：研究葡萄糖转运蛋白１（ｇｌｕｃｏｓｅｔｒａｎｓｐｏｒｔｏｒ，ＧＬＵＴ１）在肾小球系膜细胞上的表达与功能，探讨共在糖尿病肾病发病机制中的作用。方法：ＧＬＵＴ１ｍＲＮＡ蛋白质表达及其功能的检测分别采用ＲＴ－ＰＣＲ，细胞免疫荧光染色，流式细胞仪分析技术２－Ｄｅｏｘｙ－（＾３Ｈ）－Ｄ－Ｇｌｕｃｏｓｅ摄入法与根皮素竞争性抑制实验。结果：证实小鼠肾小球系膜细胞ＧＬＵＴ１ｍＲＮＡ和蛋白质的表达并表肯定了系