吡格列酮对糖尿病肾病大鼠肾脏血管内皮生长因子及podocin表达的影响实验研究
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摘要
目的:
本研究拟观察糖尿病肾病(diabetic nephropathy,DN)大鼠尿蛋白、肾功能及肾脏表达的血管内皮生长因子(Vascular endothelial growth factor,VEGF)、podocin表达的变化,以及吡格列酮对其干预的影响,探讨血管内皮生长因子、podocin在DN发病中的作用及吡格列酮治疗糖尿病肾病的机制,为DN的防治提供依据。
方法:
1、雄性SD大鼠经单次腹腔注射链脲佐菌素(STZ,55mg/Kg),建立糖尿病模型,将糖尿病大鼠随机分为糖尿病组(DM组)、糖尿病吡格列酮(10mg.kg~(-1)d~(-1))干预组(DP组),并以正常大鼠(NC组)作对照。
2、干预8周后,检测各组大鼠尿蛋白、肾功能、血糖、血脂,用光镜观察各组大鼠肾脏病理改变,用透射电镜观察肾小球足细胞病理改变,RT-PCR测定VEGF、podocin mRNA表达,用免疫组织化学测定VEGF蛋白表达。
结果:
1、DM组大鼠24h尿蛋白、BUN、SCr、肾皮质VEGF mRNA及蛋白表达均较NC组明显升高;DP组大鼠24h尿蛋白、BUN、SCr、肾皮质VEGF mRNA及蛋白表达较DM组均有降低。
2、DM组大鼠肾皮质podocin mRNA表达较NC组明显降低;DP组大鼠肾皮质podocin mRNA表达较DM组显着增加。
3、DM组血糖、血脂与NC组相比,差异有统计学意义,与DP组比较差异无统计学意义。
4、光镜及电镜结果NC组肾小球体积正常,基底膜均匀一致,无足突细胞融合。DM组肾小球体积增大,系膜细胞显着增多,基质增生,肾小球毛细血管壁狭窄,足突增粗、破坏、融合、消失。经吡格列酮干预治疗后,DP组与DM组比较肾小球基质及系膜细胞数减少,毛细血管袢开放良好,系膜基质轻微增生,足突结构基本正常,仅小部分发生融合,较DM组明显改善。
5、VEGF蛋白及mRNA表达与24小时蛋白尿均呈正相关关系(r=0.717,P<0.01)r=0.697,P<0.01),podocin mRNA表达与24小时蛋白尿呈负相关关系(r=-0.603,P<0.01);VEGF蛋白及mRNA表达与血肌酐(SCr)均呈正相关关系(r=0.448,P<0.05;r=0.611,P<0.01),podocin mRNA表达与血肌酐(SCr)呈负相关关系(r=-0.635,P<0.01)。
结论:
吡格列酮可以降低DN大鼠的尿蛋白,改善肾功能,减轻肾脏病理改变,纠正肾脏VEGF、podocin表达的异常,改善肾脏滤过屏障功能障碍及血流动力学异常,但是对血糖、血脂并没有明显影响。因此我们认为吡格列酮可通过抑制糖尿病大鼠肾脏VEGF的表达及上调podocin的表达,延缓糖尿病肾病的发生、发展,发挥直接肾保护作用。
Objective:To investigate the changes about excretion of urine protein,renal function and the expressions of VEGF and podocin in rats with diabetic nephropathy (DN),and the relationship among these changes,study the effects of pioglitazone on the expressions of vascular endothelial growth factor and podocin in the kidney of rats with diabetic nephropathy
Methods:
1、Diabetic rats model were established by injection of streptozotocin(55mg/kg). The model rats were divided randomly into 2 groups:pioglitazone-treated group (DP group),and model control group(DM group),nomal rats group(NC group) served as control.
2、Eight weeks later,blood glucose,serum lipid profile,excretion of urine protein and renal function were detected.The expression of VEGF protein was detected by immunohistochemistry and the expressions of VEGF and podocin mRNA were detected by Reverse transcription-PCR.Histomorphologic changes were observed by optics microscope and electron microscope.
Results:
1、Eigh weeks later,compared with normal rats,blood glucose,excretion of urine protein,blood urea nitrogen(BUN)and serum creatinine(SCr)were significantly increased in DM group,while excretion of urine protein,blood urea nitrogen(BUN) and serum creatinine(SCr)were descreased after treated by pioglitazone.
2、The expressions of VEGF protein and mRNA in the kidney of DM group were both significantly higher than NC group,whereas the expression of podocin mRNA in the kidney of DM group was significantly lower than NC group;the expressions of VEGF protein and mRNA in the kidney of DP group were both significantly descreased after treated by pioglitazone,and the expression of podocin mRNA in the kidney of DP group was significantly increased after treated by pioglitazone.
3、Respectively compared with DM group,blood glucose,triglyceride,total cholesterol,high density lipoprotein cholesterol and low density lipoprotein cholesterol in DP group did not differ significantly.
4、Renal histopathological changes in DM gruop were similar to those of diabetic nephropathy.Glomerular hypertrophy、mesangial cell proliferation、extracellular matrix accumulation were observed by optics microscope in DM group.The result of electron microscope showed that podocytes were tumefied、foot processes were fusioned into a band and adhered to the basement membrane in DM group.Above abnormal histomorphologic changes were improved in DP group.
5、The levels of VEGF protein and mRNA were positive correlated with 24h urine protein excretion(r=0.717,P<0.01;r=0.697,P<0.01) and the level of podocin mRNA was significantly negative correlated with 24h urine protein excretion(r=—0.603,P<0.01);the levels of VEGF protein and mRNA were positive correlated with serum creatinine(SCr)(r=0.448,P<0.05;r=0.611,P<0.01) and the level of podocin mRNA was significantly negative correlated with serum creatinine(SCr)(r=—0.635, P<0.01).
Conclusion:Pioglitazone may decrease proteinuria,improve renal function, alleviate renal histopathological damage and retrieve the changes of podocin and VEGF expressions to alleviate renal filtration barrier function in DN rats,but it do not significantly change blood glucose and serum lipid profile in DP group.Pioglitazone may up-regulate podocin expression and down-regulate VEGF expression to produce direct nephroprotective effect independent of glucose and lipid metabolism.
本研究拟观察糖尿病肾病(diabetic nephropathy,DN)大鼠尿蛋白、肾功能及肾脏表达的血管内皮生长因子(Vascular endothelial growth factor,VEGF)、podocin表达的变化,以及吡格列酮对其干预的影响,探讨血管内皮生长因子、podocin在DN发病中的作用及吡格列酮治疗糖尿病肾病的机制,为DN的防治提供依据。
方法:
1、雄性SD大鼠经单次腹腔注射链脲佐菌素(STZ,55mg/Kg),建立糖尿病模型,将糖尿病大鼠随机分为糖尿病组(DM组)、糖尿病吡格列酮(10mg.kg~(-1)d~(-1))干预组(DP组),并以正常大鼠(NC组)作对照。
2、干预8周后,检测各组大鼠尿蛋白、肾功能、血糖、血脂,用光镜观察各组大鼠肾脏病理改变,用透射电镜观察肾小球足细胞病理改变,RT-PCR测定VEGF、podocin mRNA表达,用免疫组织化学测定VEGF蛋白表达。
结果:
1、DM组大鼠24h尿蛋白、BUN、SCr、肾皮质VEGF mRNA及蛋白表达均较NC组明显升高;DP组大鼠24h尿蛋白、BUN、SCr、肾皮质VEGF mRNA及蛋白表达较DM组均有降低。
2、DM组大鼠肾皮质podocin mRNA表达较NC组明显降低;DP组大鼠肾皮质podocin mRNA表达较DM组显着增加。
3、DM组血糖、血脂与NC组相比,差异有统计学意义,与DP组比较差异无统计学意义。
4、光镜及电镜结果NC组肾小球体积正常,基底膜均匀一致,无足突细胞融合。DM组肾小球体积增大,系膜细胞显着增多,基质增生,肾小球毛细血管壁狭窄,足突增粗、破坏、融合、消失。经吡格列酮干预治疗后,DP组与DM组比较肾小球基质及系膜细胞数减少,毛细血管袢开放良好,系膜基质轻微增生,足突结构基本正常,仅小部分发生融合,较DM组明显改善。
5、VEGF蛋白及mRNA表达与24小时蛋白尿均呈正相关关系(r=0.717,P<0.01)r=0.697,P<0.01),podocin mRNA表达与24小时蛋白尿呈负相关关系(r=-0.603,P<0.01);VEGF蛋白及mRNA表达与血肌酐(SCr)均呈正相关关系(r=0.448,P<0.05;r=0.611,P<0.01),podocin mRNA表达与血肌酐(SCr)呈负相关关系(r=-0.635,P<0.01)。
结论:
吡格列酮可以降低DN大鼠的尿蛋白,改善肾功能,减轻肾脏病理改变,纠正肾脏VEGF、podocin表达的异常,改善肾脏滤过屏障功能障碍及血流动力学异常,但是对血糖、血脂并没有明显影响。因此我们认为吡格列酮可通过抑制糖尿病大鼠肾脏VEGF的表达及上调podocin的表达,延缓糖尿病肾病的发生、发展,发挥直接肾保护作用。
Objective:To investigate the changes about excretion of urine protein,renal function and the expressions of VEGF and podocin in rats with diabetic nephropathy (DN),and the relationship among these changes,study the effects of pioglitazone on the expressions of vascular endothelial growth factor and podocin in the kidney of rats with diabetic nephropathy
Methods:
1、Diabetic rats model were established by injection of streptozotocin(55mg/kg). The model rats were divided randomly into 2 groups:pioglitazone-treated group (DP group),and model control group(DM group),nomal rats group(NC group) served as control.
2、Eight weeks later,blood glucose,serum lipid profile,excretion of urine protein and renal function were detected.The expression of VEGF protein was detected by immunohistochemistry and the expressions of VEGF and podocin mRNA were detected by Reverse transcription-PCR.Histomorphologic changes were observed by optics microscope and electron microscope.
Results:
1、Eigh weeks later,compared with normal rats,blood glucose,excretion of urine protein,blood urea nitrogen(BUN)and serum creatinine(SCr)were significantly increased in DM group,while excretion of urine protein,blood urea nitrogen(BUN) and serum creatinine(SCr)were descreased after treated by pioglitazone.
2、The expressions of VEGF protein and mRNA in the kidney of DM group were both significantly higher than NC group,whereas the expression of podocin mRNA in the kidney of DM group was significantly lower than NC group;the expressions of VEGF protein and mRNA in the kidney of DP group were both significantly descreased after treated by pioglitazone,and the expression of podocin mRNA in the kidney of DP group was significantly increased after treated by pioglitazone.
3、Respectively compared with DM group,blood glucose,triglyceride,total cholesterol,high density lipoprotein cholesterol and low density lipoprotein cholesterol in DP group did not differ significantly.
4、Renal histopathological changes in DM gruop were similar to those of diabetic nephropathy.Glomerular hypertrophy、mesangial cell proliferation、extracellular matrix accumulation were observed by optics microscope in DM group.The result of electron microscope showed that podocytes were tumefied、foot processes were fusioned into a band and adhered to the basement membrane in DM group.Above abnormal histomorphologic changes were improved in DP group.
5、The levels of VEGF protein and mRNA were positive correlated with 24h urine protein excretion(r=0.717,P<0.01;r=0.697,P<0.01) and the level of podocin mRNA was significantly negative correlated with 24h urine protein excretion(r=—0.603,P<0.01);the levels of VEGF protein and mRNA were positive correlated with serum creatinine(SCr)(r=0.448,P<0.05;r=0.611,P<0.01) and the level of podocin mRNA was significantly negative correlated with serum creatinine(SCr)(r=—0.635, P<0.01).
Conclusion:Pioglitazone may decrease proteinuria,improve renal function, alleviate renal histopathological damage and retrieve the changes of podocin and VEGF expressions to alleviate renal filtration barrier function in DN rats,but it do not significantly change blood glucose and serum lipid profile in DP group.Pioglitazone may up-regulate podocin expression and down-regulate VEGF expression to produce direct nephroprotective effect independent of glucose and lipid metabolism.
引文
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2.Sharma K,Ziyadeh FN.The case for transforming growth factor-beta as a key mediator.Diabetes,1995;44:1139-1146.
3.Ritz E.RAS blockade in diabetic nephropathy.Early intervention can dramatically decrease loss of function.MMW Fortschr Med.2004,146(38):12.
4.Yamagishi S,Inagaki Y,Okamoto T,et al.Advanced glycation end productinduced apoptosis and overexpression of vascular endothelial growth factor and monocyte chemoattractant protein-1 in human-cultured mesangial cells.J Biol Chem,2002,277(23):20309-20315.
5.Cha DR,Kang YS,Han SY,et al.Vascular endothelial growth factor is increased during early stage of diabetic nephropathy in type 2 diabetic rats.Endocrinol,2004,183(1):183-194.
6.Mannhalter C,Koizar D,Mitterbauer G.Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA.Clin Chen Lab Med,2000,38(2):171-177.
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