摘要
目的
1、探讨阿霉素肾病模型的改良方法,建立稳定的肾病大鼠模型。
2、通过动物实验,观察药物对阿霉素肾病大鼠模型电荷屏障、nephrin的影响,探讨肾特灵加泼尼松治疗肾病综合征的作用机制。
方法
1、采用大鼠尾静脉一次性注射阿霉素7.5mg/kg的方法建立大鼠肾病模型。检测24h尿蛋白定量、血清总蛋白、白蛋白、胆固醇、尿素氮及肌酐;剖腹取左肾,留取肾组织标本,用于电镜、光镜检查。
2、将大鼠随机分为对照组(10只)和造模组(30只)。大鼠尾静脉一次性注射阿霉素7.5mg/kg建立大鼠肾病模型。造模后第14d,将造模组大鼠按尿蛋白量先分层再随机分为模型组、泼尼松组、肾特灵加泼尼松组,每组10只。给予药物干预,检测24h尿蛋白定量、血清总蛋白、白蛋白、胆固醇、尿素氮及肌酐;采用PEI、AB染色法检测电荷屏障;采用反转录—聚合酶链反应(RT—PCR)方法检测nephrin。
结果
1、造模组尿蛋白量逐渐增多,胆固醇、尿素氮、肌酐升高,均于第28d达到高峰;血总蛋白、白蛋白下降,第28d天达到最低值;肾小球光镜下无明显改变,电镜下改变明显。对照组未见病变。
2、给药后,模型组24h尿蛋白量逐渐升高,在第28d达到高峰,在同一时间点泼尼松组、肾特灵加泼尼松组均明显低于模型组。肾特灵加泼尼松组与泼尼松组相比,尿蛋白定量、血清白蛋白、胆固醇、尿素氮、肌酐有统计学意义。电镜下可见模型组大鼠肾小球电荷屏障明显改变;肾特灵加泼尼松组与泼尼松组比较,肾小球AB深染色及电荷屏障改变有差异。与对照组相比,模型组nephrin下调70%、泼尼松组nephrin下调38%、肾特灵加泼尼松组nephrin下调22%。
结论
1、采用一次性大鼠尾静脉注射7.5mg/kg阿霉素的方法可成功的建立阿霉素肾病大鼠模型,且具有缩短实验时间、其表现与人类肾病综合征表现更接近的优点。
2、阿霉素肾病大鼠肾小球足细胞足突的结构改变早于尿蛋白的发生,其严重程度与尿蛋白排出量相一致。
3、电荷屏障的损伤、nephrin的表达减少是大量蛋白尿的主要原因。
4、肾特灵加泼尼松治疗肾病综合征的作用机制之一是修复电荷屏障的损伤、提高肾小球细胞nephrin的表达,其治疗作用优于单纯使用泼尼松。
Objective:1.To study a method of improving the model of adriamycin nephropathy and establish a stable rats' model of adriamycin nephropathy.2.To observe the effect to barrier and nephrine on rats' model founded by adriamycin and discuss the mechanism for treating NS by animal's experiments.
Methods:1.To found rat' s nephropathy models by being received a single tail-vein infection of 7.5mg/kg adriamycin.24 hours urinary protein,ALB,BUN,Scr,Chol Were detected.Kidney tissue were detected with microscope and electronmicroscope.
2.Rats were divided randomly into control group(10 rats)and making models group(30 rats).Then,nephropathy rat' s models were founded with a single tail-vein infection of 7.5mg/kg adriamycin.Making models group were divided randomly into model group,Prednisone group and Shenteling adding Prednisone group 14 days after making models,10 rats each group.Drugs were given to rats of Prednisone group and Shenteling adding Prednisone group,then,all rats were detected 24 hours urinary protein,ALB,BUN, Scr,Chol;barrier with PEI,AB and nephrine with RT-PCR.
Results:1.The level of urinary protein and Chol increased gradually,urinary protein and Chol set at peak,on the other hand,ALB declined,the level of ALB was set at the lowest peak in experimental group in 28~(th).At the same time,the level of BUN and Scr elevated not too obvious.Glomerular form had no obvious alterations with microscope and obvious alterations with electronmicroscope.There had no alterations in control group.
2.Following drugs given,the level of urinary protein increased gradually and set at peak in model group in 28~(th),at the same time,the level of urinary protein in Prednisone group and Shenteling adding Prednisone group was belower than model group.Comparing with Prednisone group,the level of urinary protein,ALB,Chol,AB dyeing rate of glomerular and barrier had statistically significant in Shenteling adding Prednisone group.Barrier altered with electronmicroscope in model group.
Conclusions.1.It can found the rats' model about adriamycin nephropathy successfully with a single tail-vein infection of 7.5mg/kg adriamycin.It also can shorten experimental times and it' s character is agreement with human NS.
2.Foot processes' uhrastructure changes of adriamycin nephropathy rats is ealier than urinary protein and it' s severity is agreement with urinary protein excretion.
3.Both barrier damage and nephrine expression decrease are the main etiology of a lot of proteinuria.
4.One of the effective mechanism of Shenteling adding Prednisone is repairing barrier damage and increasing nephrine expression,it' s therapentical effect is better than Prednisone onely.
引文
1.徐叔云,卞如濂,陈修.药理实验方法学[M].北京:人民卫生出版社,2003,1232
2.王新颖,王新良,牟兆新,等.阿霉素肾病大鼠肾小球nephrin、podocin 蛋白表达[J].中华儿科杂志,2005,43(4):297
3.许涛,吕磊,钱琛,等.大鼠阿霉素肾病的病理学观察[J].中国临床药理学与治疗学杂志,2001,6(1):43-45
4.郑镇,贾玉汉,王玉学。血管紧张素Ⅱ受体拮抗剂对阿霉素肾病模型骨调素表达的影响[J].华中科技大学学报(医学版),2003,32(1):51-54
5.彭征屏,李荣辉,冯伟.调激宁配合强的松对大鼠阿霉素肾病模型的作用[J].中国医学杂志,2004,2(2):56-57
6.毕丽娟,司富春,王小琴。肾舒Ⅱ号对肾病综合征大鼠模型影响的实验研究[J].中医研究杂志,2004,17(2):16-20
7.王彩云,邓虹珠,李辉。胡枝子总黄酮治疗大鼠微笑病变型肾病综合征的实验研究[J].中国中医杂志,2005,3 0(8):614-617
8.张丽芬,黄文政,朱小棣,等.阿霉素肾病肾小球硬化动物模型的研究[J].中国中西医结合肾病杂志,2005,6(4):195-199
9.孔令媛,留莉,梅其炳.低蛋白血症大鼠模型的建立[J].西北国防医学杂志,2006,27(4):282-284
10.关小玲,张霞,陈欣欣.蛋白消合剂对肾病综合征大鼠SOD、MDA和-OH 影响的实验研究[J].河南中医学院学报,2006,21(3):24-28
11.Okuda S,et al.Adriamycin-induced nephropathy as a model of chronic progressive glomerular disease.Kidney International,1986,29:502
12.Egido J.Involvenent of lipid mediators in the pathogenesis of experimental nephrosis in rats:its pharmacological modulation.Ren Fail.1991,13:95-101
13.Haidle CW,McKinney SH.Adriamycin-mediated introduction of a limited number of single-strand breaks into supercoiled DNA.Cancer Biochem Biophys,1986,8(4):327-330
14.Pedrycz A,Wieczorski M,Czerny K.Features of proteinuria in rat kidney in experimental nephritic syndrome.Ann Univ Mariae Curie Sklodowska,2000,55(2):275-279
15.Whiteside C,Prutis K,Cameron R,et al.Glomerular epithelial detachment,not reduced charge density,correlates with proteinuria in adriamycin and puromycin nephrosis.Lab Invest,1989,61(6):650-655
16.王新颖,牟兆新,王新良,等.阿霉素肾病大鼠肾脏nephrin、podocinmRNA表达[J].河北医科大学学报,2005,26(6):418-422
17.毕丽娟,司富春,王小琴,等.肾舒Ⅱ号对肾病综合征模型大鼠的疗效观察[J].上海中医药杂志,2005,39(2):49-52
18.陈钺,丁国华,尤燕舞.苯那普利对阿霉素肾病大鼠足细胞损伤的保 护作用[J].中华肾脏病杂志,2004,20(3):215-216
19.邢燕,丁洁,范青锋.足细胞分子高表达致阿霉素肾病大鼠发生蛋白尿[J].中华肾脏病杂志,2006,22(1):27-32
20.刘丽华,朱春芳,欧周罗.单次注射嘌呤霉素氨基核苷所致大鼠肾病模型的优化及评价[J].复旦学报(医学版),2005,32(4):488-492
21.于为民,李荣山,张佩珍,等.肾康对嘌呤霉素肾病大鼠血液流变学的影响[J].中国中西医结合肾病杂志,2001,2(8):453-455
22.李荣山.现代肾脏病实验技术[M].北京:军事医学出版社,2006,33
23.陈重义,姜新献.研究肾小球电荷屏障方法的概况[J].国外医学泌尿系统分册,1990,6:257-260
24.Huaiping Y,Emiko T,David J.Podocyte slit-diaphragm protein nephrin is linked to the actin cytoskeleton.Am J Physio Renal physio,2002,282(4):F585-591.
25.叶任高,陆再英.内科学[M].北京:人民卫生出版社,2004,508
26.叶任高,杨念生,郑智华.肾病综合征[M].北京:人民卫生出版社,2005,3
27.王海燕,李惊子,潘缉圣,等.中药黄芪当归合剂对肾病综合征肾损伤的保护作用及对代谢紊乱的影响[J].北京大学学报(医学版),2002,34(5):542-552
28.蒋小芸,陈述枚,林穗珍,等.芪归合剂促进肾病综合征鼠肝IGF-Ⅰ、IGFBP-3表达的研究[J].中华儿科杂志,2001,39(9):550-553
29.鲁艳芳,周桂元,黄琼霞,等.温阳活血方对大鼠阿霉素肾病模型 的影响[J].湖北中医学院学报,2001,3(1):20-22
30.张淑平,李旺,季增荣,等.复肾Ⅵ服液对肾病综合征治疗作用的实验研究[J].陕西中医,2002,23(12):1142-1143
31.韩贵清,张淑平,李旺,等.僵蚕粉胶囊加服龙蜂保肾汤消除蛋白尿的实验研究[J].河北中医,2003,25(1):74-76
32.张秋林,陈思源.复方雷公藤片治疗阿霉素肾病大鼠的实验研究[J].湖北中医学院学报,2000,2(4):20-21
33.章文平,宋国维,谢瑞珍.益气活血剂对阿霉素肾病模型的影响[J].广州中医药大学学报,2000,17(2):155-157
34.肖旭腾,江英能,邓国安,等.活血利水汤对大鼠肾病模型的作用[J].广州中医药大学学报,2001,13(4):342-345
35.赵军宁,王晓东,彭龙玲,等.加味实脾饮治疗肾病综合征的实验研究[J].中国实验方剂学杂志,1996,2(6):12-15
36.陈志强,黄文政.肾炎3号方治疗微小病变型肾病的实验研究[J].天津中医,2000,17(6):30-31
37.谭海荣,刘恩棋,潘竞锵,等.益肾Ⅰ方对阿霉素诱导大鼠肾病综合征的治疗作用[J].中国新药杂志,2003,12(4):264-267
38.董兴刚,安增梅,杨海春.汉防己甲素对肾病综合征大鼠肾皮质Ⅳ型胶原基因表达的影响[J].上海中医药大学学报,2001,15(3):49-50
39.李燕,蔡东联,陈小莉,等.大豆异黄酮对实验性肾病综合征疗效观察[J].中华肾脏病杂志,2002,18(3):207-210
40.徐志良,王长松.肾病Ⅳ号对阿霉素大鼠红细胞免疫功能的影响 [J].南京中医药大学学报(自然科学版),2001,17(6):370-371
41.樊忠民,刘光陵,夏正坤,等.黄芪对阿霉素肾病大鼠白细胞介素-2及其受体的影响[J].医学研究生报,2003,26(10):740-742
42.王海云,石君杰,盛丽先.肾乐冲剂对阿霉素大鼠血清TNF、IL-8水平的干预作用[J].中国中医药信息杂志,2003,10(5):18-19
43.郑健,吴群励,曾章超,等.肾康灵干预阿霉素肾病大鼠的实验研究[J].中医药学刊,2004,22(3):441-45
44.Johnson RJ.Am J Kid Dis.2000,36(1):214-219
45.李玉林,唐建武.病理学[M].北京:人民卫生出版社,2004,266-267
46.牛建英,胡新伟.蛋白尿损害肾脏的机制及其干预措施[J].肾脏病与透析肾移植杂志,2003,12(1):66-69
47.董兴刚,安增梅.雷公藤治疗蛋白尿的机理研究进展[J].现代中西医结合杂志,2001,10(3):202-203
48.王丽.低密度脂蛋白对系膜细胞TGF和FNmRAN表达的影响[J]第二军医大学学报,2000,21(3):219-221
49.Arovin BH.LDL stimulates messangial fibronectin production-and chemoattactant expression.Kidney Int,1993,43:218
50.程叙扬,李晓枚,唐嘉薇,等.微小病变患者尿蛋白对肾小球上皮细胞的损伤作用及其机制研究[J].中华肾脏病杂志,2002,18(6):398-402
51.Burton CJ,Harper SJ,Bailey E et al.Turnover of human tubular cells exposed to proteins in vivo and in vitro.Kidney Int, 2001,59:507
52.Abbate M,Zoja C,Coma D et al.In progressive nephropathies,overload of tubular cells with filtered proteins translates glomerular permeability dysfunction into cellular signals of interstitial inflammation.J AM So Nephrol,1998,9:1213
53.崔世维,邵亚男,钱桐荪,等.糖皮质激素对肾小球滤过膜通透性的影响[J].天津医药,1997,25(3):136-138
54.Damico G,Ferrario F,Rastaldi MP.Tubulointerstitial damage in glomerular disease:it role in the progression of renal damage.Am J kidney Dis,1995,1:124
55.崔世维,潘征平,邵亚男,等.卡托普利不能抑制激素对大鼠阿霉素肾病肾小球硬化的恶化作用[J].中国中西医结合肾病杂志,2001,2(12):692-693
56.Kerjaschki D,Sharkey DJ.Identification and characterization of podocalyxin the major sialoprotein of the renal glomerular epithelial cell.Cell Biol,1984,98:1596
57.Deen WM,Satvat B.Theoretical model for glomerular filtration of charged solutes.Am J Physiol,1980,238:F126
58.叶任高,杨念生,郑智华.肾病综合征[M].北京:人民卫生出版社,2005,3
59.Kanwar YS,Farquhar MG.Detachment of endothelium and epithelium from the glomerular basement membrane produced by perfusion with neuraminidase.Lab Invest,1980,42:378-379
60.陈重义,刘利平.蛋白尿大鼠肾小球基底膜阴离子位点变化[J].中华病理学杂志,1993,22(6):350-352
61.C.J.ILSE RAATS,JACOB VAN DEN BORN,JO H.M.BERDEN.Glomerular heparan sulfate alterations:Mechanisms and relevance for proteinuria.Kidney Int,2000,57:385-388
62.陈重义,郭仁寿.肾小球电荷屏障的实质[[J].国外医学·泌尿系统分册,1991,3:65-68
63.陈重义,姜新献.研究肾小球电荷屏障方法的概况[[J].国外医学·泌尿系统分册,1990,6:257-260
64.M.D.AL-NAWAB,D.R.DAVIES.Alterations in theglomerularcharge barrier in human lupus nephritis.J pathol,1994,173(1):45-52
65.M.D.AL-NAWAB,D.R.DAVTES.Alterations in the glomerular charge barrier in human lupus nephritis.J patho,1994,173(1):45-52
66.李丽光,佐藤茂,相原熏,等.碱性铁蛋白对肾小球基膜及系膜区通透性的影响[J].中华病理学杂志,1997,26(5):301-302
67.Y KITANO,N.YOSHIKAWA,H.NAKAMURA.Glomerularanionic sites in minimal change nephritic syndrome and focal segmental glomerulosclerosis.Clin Nephrol,1993,40(4):199-202
68.D.Zhang,S.Seno,M.Akita,etal.Histochemical studies on the mechanism of macromolecule leakage across the glomerular capillary wall.Histochemistry,1991,96:115-117
69.Niaudel P.Nephrotic syndrome in children.Current Opin Pediatr,1993,5:174-179
70.陈重义,姜新献.研究肾小球电荷屏障方法的概况[J].国外医学泌尿系统分册,1990(6):257-260
71.Wang SX,Mene P,Holthofer H.Nephrin mRNA regulation by protein kinase C.J Nephrol,2001:14(2):98-103
72.Ahola H,Wang S,Luimula P,et al.Cloning and expression of the rat nephrin homolog.Am J Pathol,1999,155(3):907-913
73.Kestila M,Lenkkeri U,Mannikk0 M,et al.Positionally cloned gene for a novel glomerular protein-nephrin is mutated in congenital nephritic syndrome.Mon Cell,1998,1(4):575-582
74.Hamano Y,Grunkemeyer JA,Sudhakar A,et al.Determinants of vascular permeability in the kidney glomerulus.J Biol Chem,2002,27(34):31154-31158
75.Fatness PN,Hall LL,Shaw JA,et al.Glomerular expression of nephrin is decreased in acquired human nephritic syndrome.Nephrol Dial Transplant,1999,14(5):1234-1237
76.Luimula P,Sandstrom N,Novikov D,et al.Podocyte-associated molecules in puromycin aminonucleoside nephrosis of the rat.Lab Invest,2002,82(6):713-718
77.Ruotsalainen V,Ljunglxerg P.Proc Natl Acad Sci USA,1999, 96(14):7962-7967
78.Kawachi H,Kurihara H,Topham PS,etal.Am J Physiol,1997,27(36):F985-993
79.Ahala H,Wang SX,Luimula P,etal.Am J Pathol,1999,15(53):907-913
80.Wang SX,Ahola H,Palment,et al.Exp.Nephrol,2001,9(5):327-331
81.Topham PS,Kanachi H,Haydar SA,et al.J Clin Invent,1999,104(11):1559-1566
82.CooperME,Mun delP,BonerG.Roleofnephrinin renal diseasein.cluding diabetic nephmpathy.Semin Nephml,2002,22(5):393
83.Kestila M,Lenkkeri U,Mannikka M,etal.ETAL.Mol Cell,1998,1(4):575-582
84.Holzman LB,John PLT.Kidney Int,1999,56(4):1481-1485
85.河内裕.肾病综合征的发病机理-蛋白尿发病机制的最新认识[J].日本医学介绍,2005,26(9):385-387
86.袁伟杰,崔若英.现代肾脏病药物治疗学[M].北京:人民军医出版社,2001,255
1.叶任高,陆再英.内科学[M].北京:人民卫生出版社,2004,508
2.叶任高,杨念生,郑智华.肾病综合征[M].北京:人民卫生出版社,2005,3
3.李玉林,唐建武.病理学[M].北京:人民卫生出版社,2004,266-267
4.袁伟杰,崔若英.现代肾脏病药物治疗学[M]。北京:人民军医出版社,2001,255
5.Fogo A.Nephrotic syndrome:molecular and genetic basis.Nephron,2000,85(1):8-13
6.Kerjaschki D,Sharkey DJ.Identification and characterization of podocalyxin the major sialoprotein of the renal glomerular epithelial cell.Cell Biol,1984,98:1596
7.Deen WM,Satvat B.Theoretical model for glomerular filtration of charged solutes.Am J Physiol,1980,238:F126
8.Kanwar YS,Farquhar MG.Detachment of endothelium and epithelium from the glomerular basement membrane produced by perfusion with neuraminidase.Lab Invest,1980,42:378-379 1998,1(4):575-582
18.Holzman LB,John PLT.Kidney Int,1999,56(4):1481-1491
19.河内裕.肾病综合征的发病机理-蛋白尿发病机制的最新认识[J].日本医学介绍,2005,26(9):385-387
20.管娜,丁洁,张敬京,等.嘌呤霉素肾病大鼠肾小球中nephrin,podocin 和α-actinin与蛋白尿的关系[J].中华肾脏病杂志,2004,20(1):26-32
21.Stochand JD,Sansom SC.Glomerular mesangial cells:electrophysiology and regulation of contraction.Physiol Rev,1998,78(3):723-744.
22.Yoshioka T,Nitarai T,Kon V,etal.Role of angiotensin Ⅱin aggressive proteinuria.Kidney Int,1986,30:538-545
23.Yoshioka T,Ichikawa I,Fogo A.Reactive oxygen metabolites cause massive,reversible proteinuria and glomerular sieving defect without apparent ultrastructural abnormality.J Am Soc Nephrol,1991,2:902-912