来氟米特对糖尿病肾病大鼠肾脏VEGF表达的研究
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摘要
目的：通过制备实验性糖尿病大鼠模型,观察糖尿病大鼠肾损害情况和VEGF的表达以及来氟米特干预后的变化情况,探讨治疗DN可能的作用机制,从而为临床上防治DN提供新的思路和手段。
方法：将50只大鼠行右肾摘除术,两周后按随机数字表法将50只大鼠随机分为正常对照组(A组)12只及DN模型组38只。最后成DN模型36只,死亡两只。成模7d后,将成模糖尿病大鼠随机分成3组：模型对照组(B组)、来氟米特干预组(C组)和氯沙坦干预组(D组)各12只。来氟米特干预组以来氟米特5mg/kg/d灌胃给药,氯沙坦干预组以氯沙坦按20mg/kg/d灌胃给药,正常对照组和模型对照组大鼠用等量蒸馏水灌胃。成模后每两周测1次体重,测1次血糖。于实验第8周、12周末各组分别取6只大鼠用代谢笼收集24h尿,测尿蛋白定量；心内采血测肌酐(Scr)尿素氮(BUN)；解剖肾脏去除包膜、称重、取肾组织做蜡块,切片行PAS染色进行光镜观察病理改变,应用RT-PCR方法检测VEGF的mRNA,应用免疫组化的方法测定VEGF的蛋白表达。
结果：
(1)糖尿病大鼠肾损伤模型死亡两只,成模率96%,成模后无一只死亡。
(2)本实验发现DN大鼠肾脏VEGF mRNA呈持续表达,VEGF蛋白主要分布于肾小球上皮细胞,与既往报道一致。本研究还发现在糖尿病模型组VEGF分布范围较正常大鼠组明显增大,8、12周在肾小球的表达明显增强。均说明VEGF在DN发生、发展过程中可能起到一定的致病作用。
(3)来氟米特组及氯沙坦干预组通过来氟米特及氯沙坦的干预,肾重/体重、Scr、BUN及24hU-TP等生化指标均较糖尿病组有所减少,肾脏病理结构损伤减轻,VEGF蛋白表达及VEGF mRNA表达也均较糖尿病模型组减少。来氟米特干预组与氯沙坦干预组间各项指标无统计学差异,故也可证明来氟米特可能具有减少尿蛋白、保护肾脏结构与功能的作用。
结论：DN时存在VEGF表达的增加,通过来氟米特干预后VEGF表达的减少,提示来氟米特具有保护DN大鼠肾脏的作用。
Objective:To observe the kidney damaged conditions of diabetic rats, the expression of VEGF and the changes after intervention of leflunomide by preparing an experimental diabetic rat model so as to explore the possible mechanism of treating DN and provide new ideas and means for clinical prevention and treatment of DN.
Methods:Fifty rats were given removal of right kidney. Two weeks later,50 rats were randomly divided into a normal control group (group A) with 12 rats and a DN model group with 38 rats according to the random number table. Thirty-six rats formed into the DN model and 2 rats died among them finally. After 7 days of forming model, the diabetic rats were randomly divided into 3 groups again. They were a model control group (group B), a Leflunomide intervention group (group C) and a Losartan intervention group (group D).Each group with 12 rats. The rats of the Leflunomide intervention group(group C) were given Leflunomide of 5 mg/kg/d by intragastric gavage, the rats of the losartan intervention group(group D) were administered losartan of 20mg/kg/d by intragastric gavage and the rats of the normal control group(group A) and the model control group (group B) were administered with equal volume of distilled water. The body weight and the blood glucose were measured once every two weeks. The 24h urine was collected from six rats of each group in metabolic cages in the experiment of the first 8 weeks and 12 weekends so as to measure the urinary protein quantity. Intracardial blood sampling collecting detected the Scr and BUN. Dissecting kidneys removed capsule. Weighing and taking kidney tissues made the paraffin,and then the paraffin sections were stained by PAS to observe the pathological changes by light microscopy. mRNA of VEGF was detected by RT-PCR and the VEGF protein expression was determined by immunohistochemical assay.
Results:
(1). There were 2 rats died in the kidney damaged model of diabetic rats and the model success rate was 96%. no one died after model formed finally.
(2) The experiment showed VEGF mRNA in DN rat kidneys was continuous expression. The VEGF protein mainly distributed in glomerular epithelial cells, which accorded with past reports. The study also found that the distribution of VEGF in the diabetic model group was significantly wider than that of the normal rat group and the expression in kidney tubular of 8 and 12 weeks was markedly increased. So it suggests that the VEGF play a pathogenic role in the happening and development of DN.
(3) The kidney weight/body weight, Scr, BUN,24hU-TP and other biochemical criteria in the Leflunomide group and the losartan group intervened by leflunomide and losartan decreased than those in the diabetic group. And the injuries of kidney pathology structure were milder. The expression of VEGF protein and VEGF mRNA also reduced than those in the diabetic model group. There was no significant statistical difference between the Leflunomide intervention group and the losartan intervention group. Therefore, it can also be proved that leflunomide may be of the role of reducing the urine protein to protect the structure and function of kidney.
Conclusion:The expression of VEGF may increase when DN exists. While the expression of VEGF decreases when intervened by leflunomide. It indicates that leflunomide be of protective role for DN rat kidneys.
方法：将50只大鼠行右肾摘除术,两周后按随机数字表法将50只大鼠随机分为正常对照组(A组)12只及DN模型组38只。最后成DN模型36只,死亡两只。成模7d后,将成模糖尿病大鼠随机分成3组：模型对照组(B组)、来氟米特干预组(C组)和氯沙坦干预组(D组)各12只。来氟米特干预组以来氟米特5mg/kg/d灌胃给药,氯沙坦干预组以氯沙坦按20mg/kg/d灌胃给药,正常对照组和模型对照组大鼠用等量蒸馏水灌胃。成模后每两周测1次体重,测1次血糖。于实验第8周、12周末各组分别取6只大鼠用代谢笼收集24h尿,测尿蛋白定量；心内采血测肌酐(Scr)尿素氮(BUN)；解剖肾脏去除包膜、称重、取肾组织做蜡块,切片行PAS染色进行光镜观察病理改变,应用RT-PCR方法检测VEGF的mRNA,应用免疫组化的方法测定VEGF的蛋白表达。
结果：
(1)糖尿病大鼠肾损伤模型死亡两只,成模率96%,成模后无一只死亡。
(2)本实验发现DN大鼠肾脏VEGF mRNA呈持续表达,VEGF蛋白主要分布于肾小球上皮细胞,与既往报道一致。本研究还发现在糖尿病模型组VEGF分布范围较正常大鼠组明显增大,8、12周在肾小球的表达明显增强。均说明VEGF在DN发生、发展过程中可能起到一定的致病作用。
(3)来氟米特组及氯沙坦干预组通过来氟米特及氯沙坦的干预,肾重/体重、Scr、BUN及24hU-TP等生化指标均较糖尿病组有所减少,肾脏病理结构损伤减轻,VEGF蛋白表达及VEGF mRNA表达也均较糖尿病模型组减少。来氟米特干预组与氯沙坦干预组间各项指标无统计学差异,故也可证明来氟米特可能具有减少尿蛋白、保护肾脏结构与功能的作用。
结论：DN时存在VEGF表达的增加,通过来氟米特干预后VEGF表达的减少,提示来氟米特具有保护DN大鼠肾脏的作用。
Objective:To observe the kidney damaged conditions of diabetic rats, the expression of VEGF and the changes after intervention of leflunomide by preparing an experimental diabetic rat model so as to explore the possible mechanism of treating DN and provide new ideas and means for clinical prevention and treatment of DN.
Methods:Fifty rats were given removal of right kidney. Two weeks later,50 rats were randomly divided into a normal control group (group A) with 12 rats and a DN model group with 38 rats according to the random number table. Thirty-six rats formed into the DN model and 2 rats died among them finally. After 7 days of forming model, the diabetic rats were randomly divided into 3 groups again. They were a model control group (group B), a Leflunomide intervention group (group C) and a Losartan intervention group (group D).Each group with 12 rats. The rats of the Leflunomide intervention group(group C) were given Leflunomide of 5 mg/kg/d by intragastric gavage, the rats of the losartan intervention group(group D) were administered losartan of 20mg/kg/d by intragastric gavage and the rats of the normal control group(group A) and the model control group (group B) were administered with equal volume of distilled water. The body weight and the blood glucose were measured once every two weeks. The 24h urine was collected from six rats of each group in metabolic cages in the experiment of the first 8 weeks and 12 weekends so as to measure the urinary protein quantity. Intracardial blood sampling collecting detected the Scr and BUN. Dissecting kidneys removed capsule. Weighing and taking kidney tissues made the paraffin,and then the paraffin sections were stained by PAS to observe the pathological changes by light microscopy. mRNA of VEGF was detected by RT-PCR and the VEGF protein expression was determined by immunohistochemical assay.
Results:
(1). There were 2 rats died in the kidney damaged model of diabetic rats and the model success rate was 96%. no one died after model formed finally.
(2) The experiment showed VEGF mRNA in DN rat kidneys was continuous expression. The VEGF protein mainly distributed in glomerular epithelial cells, which accorded with past reports. The study also found that the distribution of VEGF in the diabetic model group was significantly wider than that of the normal rat group and the expression in kidney tubular of 8 and 12 weeks was markedly increased. So it suggests that the VEGF play a pathogenic role in the happening and development of DN.
(3) The kidney weight/body weight, Scr, BUN,24hU-TP and other biochemical criteria in the Leflunomide group and the losartan group intervened by leflunomide and losartan decreased than those in the diabetic group. And the injuries of kidney pathology structure were milder. The expression of VEGF protein and VEGF mRNA also reduced than those in the diabetic model group. There was no significant statistical difference between the Leflunomide intervention group and the losartan intervention group. Therefore, it can also be proved that leflunomide may be of the role of reducing the urine protein to protect the structure and function of kidney.
Conclusion:The expression of VEGF may increase when DN exists. While the expression of VEGF decreases when intervened by leflunomide. It indicates that leflunomide be of protective role for DN rat kidneys.
引文
[1]Bates DO,Harper SJ.Regulation of vascular permeability by vascular endothelial growth factors[J]. Vascular Pharmacol,2002,39(4-5):225-237.
[2]Amemiya T,Sasamura H,Mifune M,et al.Vascular endothelial growth factor activates MAP kinase and enhances collagen synthesis in human mesangial cells[J].kidney Int,1999,56(6):2055-2063.
[3]WU GJ, WANG H, YU L, et al. Immunosuppressive therapy for renal transplant recipients with cellcept, cyclosporine A and Prednisone [J]. China Journal of Modern Medicine,2006,16(17):2640-2642.
[4]IoH,H am ada C, Ro Y, et a 1. M o rp ho logy changes of peritoneum and exp ression of V EGF in encapsulated peritoneal sclerosisrat models. K idney In t,2004; 65 (5):1927-1936.
[5]Tsilibary E C.Microvascular basement membranes in diabetes mellitus [J]. J Pathol,2003,200 (4):537-546.
[6]Gudehithlu KP, Singh AK Vascular endothelial growth factor and diabetic nephropathy Int J A rtif O rgans,1999; 22(5):297-299
[7]Flyvbjerb A. Putative pathophysiological role of growth factors and cytokines in experim ental diabetic kidney disease. D iabetologia,2000; 43 (10):1205-1223
[8]De Vriese AS,Tilton RG,Elger M,Stephan CC,Kriz W,Lameire NH.Antibodies Against vascular endothelial growthfactor improve early renal dysfunction in experimental diabetes.J Am Soc Nephrol 2001;12(5):993-1000.
[9]DEVRIESE AS, TILTON RG, ELGER M, et al. Antibodies against vascular endothelial growth factorimprove early renal dysfunction in experimental diabetes [J]. J Am Soc Nephrol, 2001,12:993-1000.
[10]SENTHIL D, CHOUDHURY GG, MCLAURIN C, et al. Vascular endothelial growth factor induces protein synthesis in renal epithelial cells:A potential role in diabetic nephropathy[J]. Kidney Int,2003,64:468-479.
[11]AMEMIYA T, SASAMURA H, MIFUNE M, et al. Vascular endothelial growth factor activates MAP kinase and enhances collagensynthesis in human mesangial cells[J]. Kidney Int, 1999,56:2055-2063.
[12]Si on M, Grone HJ, Joh ren O, et al. Exp ression of vascu lar mendo thelial g row th facto r and its receptors in hum an renal on togenesis and in adu It kidney. Am J Physio 1,1995; 268 (2 P t2):F 240-F 250.
[13]Miceli-Richard C, Dougados M. Leflunomide for the treatment for the treatment of rheumatoid arthritis [J]. Expert Opin Pharmacother,2003,4 (6):987-997.
[14]张永,罗昌霞,张建鄂,等.来氟米特活性代谢产物对肿瘤坏死因子-α诱导肾小管 上皮细胞内核因子-κB活性的影响[J].微循环学杂志,2006,16(2)：18-20.
[15]付宇,王丹,石鹏,等.来氟米特治疗DN大量蛋白尿20例临床分析[J].临床内科杂志,2007,24(7)：470-471.
[1]Senger S, Mirzolt H, CNner Ce, Lorenz AF, et al. VEGF receptor antagonist inhibits the gugmentation of metastasis induced by VEGF of lipopolysaccharide in a human elnom/nude mouse system. Int J. Cancer,1998,75:780-785.
[2]Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun,1989; 161(2):851.
[3]Mantovani G, Maccio A, Pisano M, et al. Tumor associated lympho monocytes from neoplatic effusions are immunogically defectiove in comparison with patient autogous PBNCS but are capable of releasing high amounts of various cytokines. Int J Cancer; 1997,7(1)724-731.
[4]Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial through alternative exon splicing [J]. J Biol Chem,1991; 266(2):11947.
[5]Mantovani G, Maccio A, Pisano M, et al. Hypoxia induces endothelin in cultured human endothelium J Clin Invest 1991,88(9),1054-1065.
[6]Wellmann S, Taube T, Paal K, et al. Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology. Clin Chem,2001;47(4):654.
[7]Ferrara N, Carver Moore K, Chen H, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene[J]. Nature,1996,380(2):439-442
[8]Tomas KA, vascular endothelial growth factor, a potent and selective angiogenic agent[J]. J Biol Chem,1996,271(2):603.
[9]McTigue MA, Wickersham, JA, Pinko C, et al. Crystal structure of the kinase domain of human vascular endothelial growth factor receptor 2:a key enzyme in angiogenesis. Structure Fold Des,1999; 7(3):319.
[10]Brock TA, Dvorak HF, Senger DR. Tumor-secreted vascular perme abilityfactor increases cytosolic Ca 2+and von Willebrand factor release in human endothelial cells. Am J Pathol, 1991;138(1):213
[11]Shifren J, Dildi N, Ferrara N, et al. In the human fetus, vascular endothelial growth factor is expressed in epithelial cells and myocytes, but not vascular endothelium:implications for mode of action [J].J Clin Endocrinol Metal,1994,79(1):316.
[12]Shweiki D, Itin A, Soffer D, et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis [J]. Nature,1992,359(3):843.
[13]Anand IS. Hypoxia and the pulmonary circulation. Throax,994,49:19-28.
[14]Siegel Q Malmsten M. The role of the VEGF in inflammation and tumor metastasis. Int J Microcirc Clin Exp,1997,17:257-262.
[15]Simon M, Rockl W Hornig C, et al. Receptors of vascular endothelial growth factor/vascular permeability factor(VEGF/VPF) in fetal and adult human kidney; localization and VEGF binding dites [J], J Am Soc Nephrol,1998 9(10):1032-1044.
[16]Simon M, Grone growth factor and Johren 0, et al. Expression of vascular endothelialreceptons in hukan renal ontogenesis and in adult kidney[J]. Am J Physiol,1995, 268(1):F240-F250.
[17]Brown L F, Berse B, Winer J et al. Vascular permeability factor mRNA and protein expression in human kidney. Kidney Int,1992,42:1457.
[18]Zachary 1:Vascular endothelial growth factor:How it transmits its signal.Exp Nephrol, 1998,6:480-487.
[19]Stehouwer CDA, Schaper NC:The pathogenesis of vascular complications of diabetes mellitus:One voice or Many? Eur J Clin Invest,1996,26:535-543.
[20]Copper M E, Vranes D, Youssef S, et al. Increase renal expression of vascular endothelial growth factor(VEGF) and its receptor VEGFR-2 in experimental diabetes[J]. Diabetes,1999, 48:2229-2239.
[21]Tolentino MJ, Miller JW, Gragoudas ES, Jakobiec FA, Flynn E Chatzistefanou K, Ferrara N, Adams BV:Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate. Ophthalmology,1996 103:1820-1828.
[22]Sone H, Kawakami Y, Okuda Y, Sekine Y, Honmura S, Matsuo K, Segawa T, Suzuki H, Yamashita K:Ocular vascular endothelial growth factor levels in diabetic rats are elevated before observable retinal proliferative changes. Diabetologia,1997,40:726-730.
[23]Cha D R, Kim N H, Yoon J W, et al. Role of vascular endothelial growth factor in diabetic nephropathy[J].Kidney Int,2000,58(77):104-112.
[24]刘志红,胡可斌,周虹等。2型DN患者肾组织中血管内皮细胞生长因子及其受体的变化。肾脏病与透析肾移植杂志,2001,10(5)：401.
[25]Kim NH, Jung HH, Cha DR, et al. Expression of vascular endothelial growth factor in response to high glucose in rat mesangial cells[J]. J Endocrinol,2000,165(3):617-624.
[26]Esser S, Wolburg K, Volburg H, et al. Vascular endothelial growth factor induces endothelial fenestration in vivo[J]. J Cell Biol,1998,140:947-959.
[27]Dvorak AM, Feng D. The vesiculo-vacuolar organelle(VVO). A new endothelial cell permeability organelle. J Histochem Cytochem,2001;49(4):419.
[28]Amemiya T, Sasamura H, Mifune M, et al. Vascular endothelial growth factor activates MAP kinase and enhances collagen synthesis in human mesangial cells[J]. Kidney Int,1999,56: 2055-2063.
[2]Amemiya T,Sasamura H,Mifune M,et al.Vascular endothelial growth factor activates MAP kinase and enhances collagen synthesis in human mesangial cells[J].kidney Int,1999,56(6):2055-2063.
[3]WU GJ, WANG H, YU L, et al. Immunosuppressive therapy for renal transplant recipients with cellcept, cyclosporine A and Prednisone [J]. China Journal of Modern Medicine,2006,16(17):2640-2642.
[4]IoH,H am ada C, Ro Y, et a 1. M o rp ho logy changes of peritoneum and exp ression of V EGF in encapsulated peritoneal sclerosisrat models. K idney In t,2004; 65 (5):1927-1936.
[5]Tsilibary E C.Microvascular basement membranes in diabetes mellitus [J]. J Pathol,2003,200 (4):537-546.
[6]Gudehithlu KP, Singh AK Vascular endothelial growth factor and diabetic nephropathy Int J A rtif O rgans,1999; 22(5):297-299
[7]Flyvbjerb A. Putative pathophysiological role of growth factors and cytokines in experim ental diabetic kidney disease. D iabetologia,2000; 43 (10):1205-1223
[8]De Vriese AS,Tilton RG,Elger M,Stephan CC,Kriz W,Lameire NH.Antibodies Against vascular endothelial growthfactor improve early renal dysfunction in experimental diabetes.J Am Soc Nephrol 2001;12(5):993-1000.
[9]DEVRIESE AS, TILTON RG, ELGER M, et al. Antibodies against vascular endothelial growth factorimprove early renal dysfunction in experimental diabetes [J]. J Am Soc Nephrol, 2001,12:993-1000.
[10]SENTHIL D, CHOUDHURY GG, MCLAURIN C, et al. Vascular endothelial growth factor induces protein synthesis in renal epithelial cells:A potential role in diabetic nephropathy[J]. Kidney Int,2003,64:468-479.
[11]AMEMIYA T, SASAMURA H, MIFUNE M, et al. Vascular endothelial growth factor activates MAP kinase and enhances collagensynthesis in human mesangial cells[J]. Kidney Int, 1999,56:2055-2063.
[12]Si on M, Grone HJ, Joh ren O, et al. Exp ression of vascu lar mendo thelial g row th facto r and its receptors in hum an renal on togenesis and in adu It kidney. Am J Physio 1,1995; 268 (2 P t2):F 240-F 250.
[13]Miceli-Richard C, Dougados M. Leflunomide for the treatment for the treatment of rheumatoid arthritis [J]. Expert Opin Pharmacother,2003,4 (6):987-997.
[14]张永,罗昌霞,张建鄂,等.来氟米特活性代谢产物对肿瘤坏死因子-α诱导肾小管 上皮细胞内核因子-κB活性的影响[J].微循环学杂志,2006,16(2)：18-20.
[15]付宇,王丹,石鹏,等.来氟米特治疗DN大量蛋白尿20例临床分析[J].临床内科杂志,2007,24(7)：470-471.
[1]Senger S, Mirzolt H, CNner Ce, Lorenz AF, et al. VEGF receptor antagonist inhibits the gugmentation of metastasis induced by VEGF of lipopolysaccharide in a human elnom/nude mouse system. Int J. Cancer,1998,75:780-785.
[2]Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun,1989; 161(2):851.
[3]Mantovani G, Maccio A, Pisano M, et al. Tumor associated lympho monocytes from neoplatic effusions are immunogically defectiove in comparison with patient autogous PBNCS but are capable of releasing high amounts of various cytokines. Int J Cancer; 1997,7(1)724-731.
[4]Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial through alternative exon splicing [J]. J Biol Chem,1991; 266(2):11947.
[5]Mantovani G, Maccio A, Pisano M, et al. Hypoxia induces endothelin in cultured human endothelium J Clin Invest 1991,88(9),1054-1065.
[6]Wellmann S, Taube T, Paal K, et al. Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology. Clin Chem,2001;47(4):654.
[7]Ferrara N, Carver Moore K, Chen H, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene[J]. Nature,1996,380(2):439-442
[8]Tomas KA, vascular endothelial growth factor, a potent and selective angiogenic agent[J]. J Biol Chem,1996,271(2):603.
[9]McTigue MA, Wickersham, JA, Pinko C, et al. Crystal structure of the kinase domain of human vascular endothelial growth factor receptor 2:a key enzyme in angiogenesis. Structure Fold Des,1999; 7(3):319.
[10]Brock TA, Dvorak HF, Senger DR. Tumor-secreted vascular perme abilityfactor increases cytosolic Ca 2+and von Willebrand factor release in human endothelial cells. Am J Pathol, 1991;138(1):213
[11]Shifren J, Dildi N, Ferrara N, et al. In the human fetus, vascular endothelial growth factor is expressed in epithelial cells and myocytes, but not vascular endothelium:implications for mode of action [J].J Clin Endocrinol Metal,1994,79(1):316.
[12]Shweiki D, Itin A, Soffer D, et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis [J]. Nature,1992,359(3):843.
[13]Anand IS. Hypoxia and the pulmonary circulation. Throax,994,49:19-28.
[14]Siegel Q Malmsten M. The role of the VEGF in inflammation and tumor metastasis. Int J Microcirc Clin Exp,1997,17:257-262.
[15]Simon M, Rockl W Hornig C, et al. Receptors of vascular endothelial growth factor/vascular permeability factor(VEGF/VPF) in fetal and adult human kidney; localization and VEGF binding dites [J], J Am Soc Nephrol,1998 9(10):1032-1044.
[16]Simon M, Grone growth factor and Johren 0, et al. Expression of vascular endothelialreceptons in hukan renal ontogenesis and in adult kidney[J]. Am J Physiol,1995, 268(1):F240-F250.
[17]Brown L F, Berse B, Winer J et al. Vascular permeability factor mRNA and protein expression in human kidney. Kidney Int,1992,42:1457.
[18]Zachary 1:Vascular endothelial growth factor:How it transmits its signal.Exp Nephrol, 1998,6:480-487.
[19]Stehouwer CDA, Schaper NC:The pathogenesis of vascular complications of diabetes mellitus:One voice or Many? Eur J Clin Invest,1996,26:535-543.
[20]Copper M E, Vranes D, Youssef S, et al. Increase renal expression of vascular endothelial growth factor(VEGF) and its receptor VEGFR-2 in experimental diabetes[J]. Diabetes,1999, 48:2229-2239.
[21]Tolentino MJ, Miller JW, Gragoudas ES, Jakobiec FA, Flynn E Chatzistefanou K, Ferrara N, Adams BV:Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate. Ophthalmology,1996 103:1820-1828.
[22]Sone H, Kawakami Y, Okuda Y, Sekine Y, Honmura S, Matsuo K, Segawa T, Suzuki H, Yamashita K:Ocular vascular endothelial growth factor levels in diabetic rats are elevated before observable retinal proliferative changes. Diabetologia,1997,40:726-730.
[23]Cha D R, Kim N H, Yoon J W, et al. Role of vascular endothelial growth factor in diabetic nephropathy[J].Kidney Int,2000,58(77):104-112.
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