摘要
目的:
本实验研究观察雷至胶囊治疗肾病综合征的疗效及减毒增效作用;并探讨雷至胶囊对蛋白尿、肾小球系膜、肾小管的影响及其细胞分子机制。
方法:
将健康雌性SD大鼠60只,随机分为正常组(10只)、阿霉素肾病模型组(10只)、雷公藤多甙组(10只)、雷至胶囊低剂量组(10只)、雷至胶囊中剂量组(10只)、雷至胶囊高剂量组(10只),尾静脉注射法(第一天4mg/kg体重,第七天3mg/kg体重)制作肾病综合征模型。用药剂量及方法:正常组和模型组:生理盐水1ml.100g-1.d-1;雷公藤多甙组:2mg.100g-1.d-1体重;雷至胶囊低剂量组:0.4g.100g-1.d-1;雷至胶囊中剂量组:0.8g.100g-1.d-1;雷至胶囊高剂量组:1.2g/100g-1.d-1,上述用药均为第一次造模2周后开始,每日一次,疗程3周。①观察一般情况及体重,造模前、第一次造模后第14天、给药第7、14、21天,测24HUPQ、尿NAG酶②末次治疗给药后24h后,眼眶取血检测Scr、Bun、ALB、TG和CHO、ALT和AST。③末次给药24h后,West-Blot法检测大鼠肾组织nephrin、podocin、podocalyxin、TGF-β、MMP-9的表达;RT-PCR法检测大鼠肾组织nephrin. podocin、podocalyxin;实时定量PCR法检测大鼠肾组织Dicer酶的表达;免疫荧光法检测大鼠肾组织podocalyxin的表达。④末次给药24h后,将摘除右肾组织,在Jem-1011透射电镜下观察;其余部分肾组织及部分肝脏组织经PAS染色,光镜下观察。(注:分子生物学指标及病理仅检测了正常组、模型组、雷公藤多甙组及雷至胶囊中剂量组)。
结果:
①雷至胶囊对阿霉素肾病大鼠血液生化学及尿蛋白的影响:给药后第7天、给药后第14天、给药后第21天,各治疗组24HUPQ均显著低于模型组(P<0.01);给药后第14天、给药后第21天,雷至胶囊低、中、高剂量组明显低于雷公藤多甙组(P均<0.01)。给药三周后各治疗组血清白蛋白均显著高于模型组(P<0.01);雷至胶囊低、中、高剂量组血清白蛋白明显高于雷公藤多甙组(P<0.01)。血脂结果显示:各治疗组血清TG, CHO含量显著低于模型组(P<0.01);雷至胶囊低、中、高剂量组血清TG、CHO显著低于雷公藤多甙组(P<0.01);雷至胶囊低、中、高剂量组间无统计意义。肝功结果:雷公藤多甙组血清ALT、AST显著高于其它各组(P<0.01)。其它各组间BUN、Scr水平比较无显著性差异(P>0.05)。
②雷至胶囊对阿霉素肾病大鼠病理学的影响:光镜下观察,模型组大鼠可见肾组织:弥漫性肾小球系膜区增宽,系膜细胞、系膜基质增生,肝组织:雷公藤多甙组肝细胞排列紊乱,基质明显增多,其他各组组肝脏结构无明显变化。透射电镜检测结果,模型组可见肾小球足细胞足突的广泛融合,系膜基质增多,肾小球基底膜增厚等;各治疗组病变程度明显较模型组轻,仅见足突部分融合。
③雷至胶囊对阿霉素肾病大鼠肾小球系膜基质的影响:TGF-β在治疗组显著低于模型组(P<0.01),雷至胶囊组与雷公藤多甙组间无显著性差异;MMP-9在治疗组显著高于模型组(P<0.01),在雷至胶囊组也显著高于雷公藤多甙组(P<0.01)。另外相关性研究显不,TGF-β、24HUPQ、TG、CHO与系膜基质指数呈正相关(P<0.01), MMP-9与系膜基质指数呈负相关(P<0.01)。
④雷至胶囊对阿霉素肾病大鼠肾小管的影响:给药后第7天、给药后第14天、给药后第21天,治疗组NAG酶量均显著低于模型组(P均<0.01),雷至胶囊组NAG酶量也明显低于雷公藤多甙组(P<0.05)。另外相关性研究发现,TGF-β、24HUPQ与NAG呈正相关(P<0.01), MMP-9与NAG呈负相关(P<0.01)。
⑤雷至胶囊对阿霉素肾病大鼠肾脏组织SD分子nephrin、podocin的影响:模型组nephrin、podocin蛋白表达降低,而基因表达nephrin下降、podocin上升,与正常组相比均有明显差异(P均<0.01);与模型组相比,各治疗组明显抑制nephrin、podocin模型组改变(P均<0.01);相关性分析显示,nephrin蛋白和基因、podocin蛋白与24HUPQ呈负相关(P均<0.01),podocin基因与24HUPQ呈正相关(P<0.01)。
⑥雷至胶囊对阿霉素肾病大鼠肾脏组织足细胞分子podocalyxin的影响:模型组podocalyxin蛋白表达明显降低,基因表达明显上升,与正常组相比均有明显差异(P均<0.01);与模型组相比,各治疗组明显抑制podocalyxin蛋白和基因模型组改变(P均<0.05)。相关性分析显示,Podocalyxin蛋白与24HUPQ呈负相关,而基因与24HUPQ呈正相关。
⑦雷至胶囊对早期阿霉素肾病大鼠肾脏组织足细胞Dicer酶的影响:模型组Dicer酶mRNA表达显著低于正常组(P<0.01);与模型组相比,雷至胶囊组Dicer酶mRNA表达显著高于模型组(P<0.05);雷至胶囊组与雷公藤多甙组间比较无统计学意义。相关性分析显示Dicer酶mRNA与pococin mRNA、podocalyxin mRNA、24HUPQ呈负相关,Dicer酶mRNA与nephrin mRNA呈正相关。
结论:
1本研究证明雷至胶囊能有效降低阿霉素肾病大鼠蛋白尿,且在改善阿霉素肾病大鼠营养状况、调节蛋白质代谢及脂质代谢方面的良好作用,与雷公藤多甙相比,功效明确,无明显肝脏等毒副作用,体现了减毒增效的效果。
2雷至胶囊治疗后大鼠病理改变明显减轻,为雷至胶囊治疗阿霉素肾病大鼠、减轻蛋白尿提供了组织病理学上的依据。
3雷至胶囊可通过①降低尿蛋白②降低高脂血症③抑制TGF-β和提高MMP-9表达④保护足细胞作用,抑制肾小球系膜基质增生。
4雷至胶囊可通过降低TGF-β蛋白表达、24HUPQ,提高MMP-9蛋白表达,保护阿霉素肾病大鼠肾小管免受损伤。
5雷至胶囊可通过调控炎症因子(TGF-β、MMP-9)、Dicer酶、足细胞分子nephrin、podocin、podocalyxin等多靶点治疗阿霉素肾病大鼠蛋白尿。
Objective:
Based on the theories of Traditional Chinese Medicine and modern medicine. This experimental research is designed to study the impact of Leizhi capsule on Adrianmycin-inducded nephritic syndrome in rats and part of the mechanism of action from the perspectives of Hemo-Biochemistry,Histopathology and Molecular Biology.
Methods:
60 female SD rats were divided at random into the following groups:the normal group(n=10), the model group(n=10), the high-dosed Leizhi capsule group(n=10), the middle-dosed Leizhi capsule group(n=10), the low-dosed Leizhi capsule group(n=10), the tripterygium glycosides group (n=10). The nephritic syndrome mode induced by etail intravenous injection of adriamycin in day 1 and day 7. The administrations of medicin were as follows:the normal and model groups, saline lml.100g-1.d-1; the tripterygium glycosides 2mg.100g-1.d-1; the high-dosed Leizhi capsule group 1.2mg.100g-1.d-1; the middle-dosed Leizhi capsule group 0.8g.100g-1.d-1; the low-dosed Leizhi capsule group 0.4g.100g-1.d-1. All the administrations metioned above began two week after the models had been made, each medicine was administered once a day. The period of treatment was three weeks.①Observe the general conditions and weight of body;check 24 hour urea protein quantity(24HUPQ), before and 14 days after the model had been made first, and on the 7th,14th,21th day after theadministation of related medieine.②24 hours after the last administration of medieine, check Scr, Bun, ALB, TG,CHO.③24 hours after the last administration of medicine, the expression of nephrin,podocin, podocalyxin mRNA and protein of the kidney tissues of rats were examined through RT-PCR and West-Blot method. The expression of TGF-βand MMP-9 protein of the kidney tissues of rats were examined through West-Blot method.The expression of Dicer mRNA of the kidney tissues of rats were examined through real-time PCR method. The expression of podocalyxin of the kidney tissues of rats were examined through indirect immunofluorescence stainin method.④24 hours after the last administration of medicine, the removed kidney tissues were observed under Jem-1011 penetrating electric microscope; The part of kidney tissues and hepatic tissues, which were dyed with PAS after being dealt with, were observed under light microscope. (Note:The indicators of molecular biology and pathology detected only in the middle-dosed Leizhi capsule group).
Results:
①The research of the impact of the high-dosed Leizhi capsule group on the Hemo-biochemistry and urea protein of the Adrianmycin-induced rats with kidney disease. On the 7 th, 14th.,21th day after the administration of medicine,24HUPQ of the treatment groups was significantly lower than that of the model group(P<0.01),24HUPQ of the high-dosed,the middle-dosed, the low-dosed group was significantly lower than that of the the tripterygium glycosides group(P<0.01)。
3 weeks after the administration of medicine, the serum ALB of the treatment groups were examined to be significantly higher than that of model group(P<0.01);the serum level of ALB the high-dosed,the middle-dosed, the low-dosed groups was examined to be higher than that of the tripterygium glycosides group(P<0.01). The content of TG and CHO in the treatment groups was significantly lower than that in the model group(P<0.01); The level of TG and CHO in the high-dosed,the middle-dosed,the low-dosed groups was significantly lower than that in the tripterygium glycosides group(P<0.01); There was no significant difference between the high-dosed,the middle-dosed,the low-dosed groups (p>0.05). There was no significant difference when the levels of Bun and Scr in different groups were compared with each other(p>0.05).
②The pathological research of the effect of Leizhi capsule on the Adrianmycin-induced rats with kidney disease.Under the light microscope, the diffuse hyperplasia of mesangium was seen in the model group. It could be seen under penetrating electric microscope that the main pathological changes of Adrianmycin induced rats with kidney disease were the widespread fusion of foot protrusion in the glomerulus of kidney and hyperplasia of mesanguim matrix, thickening of glomerular basement membrane;In the treated groups the degree of pathological changes in rats was obviously slighter than that in the model group, and only part of the fusion of foot protrusion could be seen.
③The research of the impact of Leizhi capsule on the glomerular mesangial matrix of Adrianmycin-induced rats with kidney disease.The TGF-βin the treated groups were significantly lower than that in the model group(P<0.01).There was no significant difference when the levels of TGF-βin different groups were compared with each other(p>0.05). The MMP-9 in the treated groups were significantly higher than that in the model group(P<0.01);The MMP-9 in the Leizhi capsule groups were significantly higher than that in the the tripterygium glycosides group(P<0.01). Correlation studies have shown that TGF-β,24HUPQ, TG, CHO and mesangial matrix index were positively correlated (P<0.01), MMP-9 and mesangial matrix index was negatively correlated (P<0.01.
④The research of the impact of Leizhi capsule on the tubular cells of Adrianmycin-induced rats with kidney disease. On the 7 th,14th.,21th day after the administration of medicine, the NAG in the treated groups were significantly lower than that in the model group(P<0.01), the NAG in the Leizhi capsule groups were significantly lower than that in the tripterygium glycosides group(P<0.05). Correlation studies have shown that TGF-β,24HUPQ and NAG were positively correlated (P<0.01), MMP-9 and NAG was negatively correlated (P <0 .01).
④The research of the impact of Leizhi capsule on nephrin,podocin of Adrianmycin-induced rats with kidney disease. The protein expression of nephrin, podocin was reduced, while gene expression of nephrin was decreased, gene expression of podocin was increased in the model group. There was significant difference when the levels of nephrin, podocin in model and nomal glycosides groups were compared with each other(p<0.01). Leizhi capsule and tripterygium was significantly inhibited changes of nephrin, podocin in the model group(p<0.01). Correlation studies have shown that protein and mRNA of nephrin,protein of podocin and 24HUPQ were negatively correlated (P<0.01), mRNA of podocin and 24HUPQ was positively correlated (P <0 .01).
⑥The research of the impact of Leizhi capsule on podocalyxin of Adrianmycin-induced rats with kidney disease. The protein expression of podocalyxin was reduced, while gene expression of podocalyxin was increased in the model group. There was significant difference when the levels of podocalyxin in model and nomal glycosides groups were compared with each other(p<0.01). Leizhi capsule and tripterygium was significantly inhibited changes of podocalyxin in the model group(p<0.01). Correlation studies have shown that protein of podocalyxin,protein of podocin and 24HUPQ were negatively correlated (P<0.01), mRNA of podocalyxin and 24HUPQ was positively correlated (P<0.01).
⑦The research of the impact of Leizhi capsule on Dicer of Adrianmycin-induced rats with kidney disease. The mRNA of Dicer in the model group were significantly higher than that in the nomal group(P<0.01); The mRNA of Dicer in the Leizhi capsule groups were significantly higher than that in the model group(P<0.01); There was no significant difference in dicer expression between Leizhi capsule group tripterygium glycosides group. Correlation studies have shown that mRNA of dicer and pococin mRNA、podocalyxin mRNA,24HUPQ were negatively correlated (P<0.01), mRNA of dicer and nephrin mRNA was positively correlated (P <0 .01).
Conclusion:
1.This research proved that Leizhi capsule could effeetively bring down the protein uria of the Adrianmycin-induced rats with kidney disease, and play a wholesome role in improving the nutrient conditions of Adrianmycin-induced rats and regulating the metabolism of protein and lipid. Compared with tripterygium glycosides, Leizhi capsule has the obviouse anti-proteinuria effectiveness and no obvious side effects such as the liver which reflects the attenuated, synergistic effect.
2.After the treatment with Leizhi capsule, the pathological change were significantly relieved, this provided a histo-pathological evidence for the suggestion that Leizhi capsule could be used to treat adrianmycin-induced rats with kidney disease and reduce the proteinuria.
3.Leizhi capsule inhibit proliferation of glomerular mesangial matrix probably through①reducing urinary protein②reducing hyperlipidemia③inhibitting TGF-βand enhance MMP-9 expression④protecting podocyte.
4.Leizhi capsule protect renal tubular cells probably through reducing urinary protein,inhibitting TGF-βand enhancing MMP-9 expression.
5. Leizhi capsule could bring down the proteinuria of the Adrianmycin-induced rats through the multi-target regulation of inflammatory factors (TGF-P, MMP-9), Dicer enzyme, nephrin, podocin, podocalyxin etc.
引文
[1]Levey AS, Eckardt KU, Tsukamoto Y, et al. Definition and classification of chronic kidney disease:a position statement from Kidney Disease:Improving Global Outcomes (KDIGO). Kidney Int,2005,67: 2089-100.
[2]Palmer BF. Proteinuria as a therapeutic target in patients with chronic kidney disease. Am J Nephrol, 2007,27:287-93.
[3]Doublier S, Salvidio G, Lupia E, et al. Nephrin expression is reduced in humandiabetic nephropathy: evidence for a distinct role for glycated albumin and angiotensin II. Diabetes,2003,52:1023-1030.
[4]Peter V,Hauser,Federica Collino, et al. Nephrin and endothelial injury.Nephrology and Hypertension,2009, 18:3-8.
[5]Philippe A, Weber S, E squivel EL,et al. A missense mutation in podocin leads to early and severe renal disease in mice. Kidney Int,2008,73:1038-1047.
[6]Tryggvason K, Patrakka J, Wartiovaara J. Hereditary proteinuria syndromes and mechanisms of proteinuria. N Engl J Med,2006,354:1387-1401.
[7]朱吉莉,丁国华.蛋白尿形成相关足细胞的结构蛋白[J].国际泌尿系统杂志,2006,27(7):540-544.
[8]Kriz W.Podocyte is the major culprit accounting for the progression of chronic renal disease[J].Microsc Res Tech,2002,57(4):189-195.
[9]Durvasula RV,Shankland SJ,et al Podocyte injury and targeting therapy:an update. Curr Opin Nephrol Hypertens,2006,15(1):1-7.
[10]Wartiovaara J, OfverstedtLG, Khoshnoodi J, etal Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography. J Clin Invest,2004,114:1475-1483.
[11]Doyonnas R,Kershaw DB,Duhme C,et al.Anuria,omphalocele and perinatal lethality in mice lacking the CD34-related protein podocalyxin[J].J Exp Med,2001,194(1):13-27.
[12]王惠,何方方,张春.足细胞损伤机制研究进展[J].中国中西医结合肾病杂志,2006,9(11):1018-1020.
[13]Koop K, Eikmans M, Baelde HJ, et al. Expression of podocyte-associated molecules in acquired human kidney diseases[J]. J Am Soc Nephrol,2003,14(8):2063-2071.
[14]杜鹏,刘蕊,朱广博.尿足细胞标志蛋白检测对肾脏疾病诊断临床意义的探讨[J].现代检验医学杂志,2007,33(4):87-88.
[15]Nakamura T, Ushiyama C, Suzuki S, et al·Effects of angiotensin-converting enzyme inhibitor, angiotensin Ⅱ receptor antagonist and calcium antagonist on urinary podocytes in patients with IgA nephropathy. Am J Nephrol,2000,20:373-379·
[16]Nakamura T, Ushiyama C, Hirokawa K, et al.Effect of cerivastatin on proteinuria and urinary podocytes in patients with chronic glomerulonephritis.Nephrol Dial Transplant,2002,17:798-802·
[17]Asanuma K,Mundel P.The role sf podocytes in glomerular pathobiology.Clin Exp Nephrol,2003, 7 (4):255-259.
[18]KestilaM,LenkkeriU,MannikkoM, et al. Positionally cloned gene for a novel glomerular protein-nephrin is mutated in congenital nephrotic syndrome.MolCell,1998,1(4):575-582.
[19]贾俊亚,丁国华.Nephrin信号转导机制研究进展[J].生理科学进展杂志,2006,37(3):276-279.
[20]Kawachi H,Koikc H,Kurihara H,et al.Cloning of rat nephrin:expressin in developing glomeruli and in proteinuric states[J].Kidney Int,2000,57 (5):1949-1961.
[21]Mundel P,Shanldand SJ.Podocyte biology and response to injury[J].J Am Soc Nephrol,2002,13:3005-3015.
[22]Schwarz K, Simons M, Reiser J, et al. Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin[J]. Clin Invest,2001,108(11):1621-1629.
[23]Boute N, Gribouval O,Roselli S, et al.NPHS2,eneoding the glomerular protein podocin,is mutated in autosonmal recessive steoid-resistant nephritic syndrome.Nat Genet,2000,24(4):349-354.
[24]Kaplan JM, Kim SH, North KN,et al. Mutations in ACTN4,encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis[J]. Nature Genet,2000,24:251-256.
[25]Goto H,Wakui H, Komatsuda A,et al. Renal alpha-actinin-4:purification and puromycin aminonucleoside-binding property[J].Nephron,2003,93:27-35.
[26]邢燕,丁洁,范青锋,等.三种不同药物作用下足细胞分子的变化[J].中华肾脏病杂志,2006,22(5):275-281.
[27]郑春霞,刘志红,孙吉平,等.雷公藤甲素对嘌呤霉素模型足细胞病变的影响.肾脏病与透析肾移植杂志2007,16(2):110-118.
[28]秦卫松,刘志红,曾彩虹,等.雷公藤甲素对Heymann肾炎模型足细胞病变的影响.肾脏病与透析肾移植杂志,2007,16(2):101-109.
[29]邢燕,丁洁,范青锋,等.足细胞分子高表达致阿霉素肾病大鼠发生蛋白尿.中华肾脏病杂志,2006,22(1):27-32.
[30]刘航,陈香美.足细胞表面标志及尿中足细胞的检测[J].国外医学泌尿系统分册,2003,23(4):45.
[31]Tetsuro T.podocyte cytoskeleton is connected to the integral membrane protein podocalyxin through Na+/H+-exchanger regulatory factor 2 and ezrin[J].Clin Exp Nephrol,2003,7:260-269.
[32]Kloosterman WP, Plasterk RH. The diverse functions of microRNAs in animal development and disease. Dev Cell.2006,11:441-50.
[33]Rajewsky N. microRNA target predictions in animals. Nat Genet.2006,38[Suppl]:S8-13.
[34]Ho J, Ng KH, Rosen S, et al. Podocyte-specific loss of functional microRNAs leads to rapid glomerular and tubular injury. J Am Soc Nephrol.2008,19:2043-2046.
[35]Harvey SJ, Jaral G, Cunningham J, et al. Podocyte-specific deletion of dicer alters cytoskeletal dynamics and causes glomerular disease. J Am Soc Nephrol.2008,19:2150-2158.
[36]Shi S, Yu L, Chin C, et al. Podocyte-selective deletion of dicer induces proteinuria and glomerulosclerosis. J Am Soc Nephrol 2008,19:2159-2169.
[37]Remuzzi G,Bertani T.Pathophysilogy of progressive nephropathies [J] N Engl J Me d,1998,339:1448.
[38]Bohle A, Strutz F, Muller GA. On the pathogenesis of Chronic Renal Failure in Primary glomernlopathies [J].Exp Nephrol,1994,2:205-208.
[39]Benigni A, Zoja C, Remuzzi G. The renal toxicity of sustained glomerular protein traffic[J].Lab Invest, 1995,73(4):461-468.
[40]Wheeler DC, Chana RS, ToPley N, et al. Oxidation of low density lipoprotein by mesangial cells may promote glomerular injury.Kidney Int,1994,45:1628-1636.
[41]Michel LH, Cornelia K, Valerie E, et al. Podocyte bridges between the Tuft and Bowman's capsule:An early event in experimental crescentic glomerulonephritis [J]. J Am SocNephrol,2001,12(10):2060-2071.
[42]张秋业.蛋白尿在肾小球疾病进展中的作用.青岛大学医学院学报,2002,38(2):185.
[43]顾连方.蛋白尿与肾损伤的关系.临床荟萃,1999,14(12):544.
[44]刘志红,唐政.蛋白尿与慢性肾脏病变的进展.肾脏病与透析肾移植杂志,1997,6(4):359-36.
[45]Knopp RH, Frohich J, Jokubaitis LA, et al, Efficacy and safety of fluvastatin in patients with non-insulin dependent diabetes mellitus and hyperlipidemia.Am J Med,1994,96(Suppl6A):69-78.
[46]Oda H,Kenne W.Lipids in progression of renal disease.Kid Int,1997,52(Suppl 62):36-38.
[47]田庆印,吴葆杰.氧化型低密度脂蛋白与血栓形成[J].中国动脉硬化杂志,2000,8(3):279-282
[48]Liu Y. Renal fibrosis:new insights into the pathogenesis and therapeutics. Kidney Int,2006,69:213-217.
[49]Leask A, Abraham DJ. TGF-beta signaling and the Wbrotic response. FASEB J,2004,18:816-827.
[50]Chiharu Ito,Tetsu AkimoIto,Takashi Ioka, et al. TGF-β Inhibits Vascular Sprouting through TGF-β type Ⅰ Receptor in the Mouse Embryonic Aorta[J]. Tohoku J. Exp. Med,2009,218(1),63-71.
[51]李雄,柳飞,付平.TGF-β/CTGF在肾脏纤维化机制中的作用及中药治疗影响[J].中国中西医结合肾病杂志,2006,7(6):370-372.
[52]Yih-Hsin Chang, I-Ling Lin, Gregory J,et al. The Canadian Society of Clinical Chemists [J]. Clinical Biochemistry,2008,41:955-959.
[53]Taylor JL, Hattle JM, Dreitz SA, et al. Role formatrix metallo-proteinase 9 in granuloma formation during pulmonary Mycobacterium tuberculosis infection. Infect Immun,2006,74:6135-6144.
[54]卢祖礼.浅析肾纤维化的中医病机[J].湖北中医杂志,2004,26(11):18-19.
[55]王继明,陈继红,孙伟.孙伟治疗慢性肾脏病用药特色拾偶[J].辽宁中医杂志,2006,33(12):1543-1544.
[56]孙伟,曾安平,严志林,等.湿热病邪与慢性肾病关系探讨:附152例临床资料分析.中医研究2000,13(1):36.
[57]孙伟.湿热之邪在慢性肾炎进展中的作用[J].江苏中医药,2006,27(6):6-7.
[58]卢玲,梁冰.中医药抗肾纤维化的研究进展[J].广西中医药2001,24(6):5.
[59]安辉.肾病综合征难治因素的分析.湖北中医学院学报.2004,6(2),42-44.
[60]潘振帮,刘俊,黄蓝君.肾性蛋白尿中医病机及其方剂现代药理探索.中国中医药信息杂志,1995, 2(4),14-15.
[61]孙伟,高坤,周栋,等.益肾清利活血综合疗法治疗局灶节段性肾小球硬化及lgA肾病患者64例临床观察.中医杂志,2005,16(10),753-755.
[62]黎磊石,刘志红.应用雷公藤治疗肾炎二十五载的体会.肾脏病与透析肾移植杂志,2003;12(3):246-247.
[63]胡大伟,刘沛霖.雷公藤与强的松药理作用的互补性.中国中西医结合杂志,1997,17(2):94-96.
[64]黎磊石,刘志红.雷公藤在肾脏病领域应用的前景.肾脏病与透析肾移植杂志,1997;6(3):203-204.
[65]王海燕,李惊子,于宏,等.雷公藤及黄芪、当归对微小病变肾病鼠的肾小球滤过膜通透性的影响.中华医学杂志,1988,68(9):513-516.
[66]刘喆,邹万忠.肿瘤坏死因子受体在肾小球系膜细胞中的基因表达及蛋白合成,肾脏病与透析肾移植杂志,1997,6(4):110-112.
[67]杨俊伟,黎磊石.雷公藤的药理和临床研究.中国药理学年鉴,1993,40.
[68]孙伟,曾安平,王钢,等.肾炎灵颗粒治疗难治性肾病综合征的疗效观察.中国中西医结合肾病杂志,2002,3(2):98-100.
[69]赵军宁,王晓东,彭龙玲,等.雷公藤多甙预防大鼠肾病的体视学研究.中国药理学通报,1995,11:468.
[70]韦登明,黄光照.雷公藤及其单体的药理和毒理病理学研究进展.中药材,2003,26(12):894-897.
[71]和西云.雷公藤制剂毒副反应的观察和护理,泰山卫生,2002,29(1):35.
[72]杨小红,张伟程.雷公藤制剂的应用及其副作用.中医药临床杂志,2004,16(5):511-512.
[73]张骁,束梅英.女贞子药理研究进展.中国医药报,2004,36期,A7版.
[74]旱莲草乙醇提取物对实验性大鼠和小鼠肝损伤的肝保护作用[J]国外医学(中医中药分册),1994,(02).
[75]冯明功,才迎,张巍峨.旱莲草对老龄小鼠脾淋巴细胞功能的影响[J].中国煤炭工业医学杂志,1998,02:174-175.
[76]康文红,王庆伟,高翔,等.旱莲草乙酸乙酯总提物对小鼠外周血T淋巴细胞亚群影响[J].安徽中医学院学报,2000(6):53-54.
[77]操红缨,谈博.二至丸对小鼠巨噬细胞产生IL-1的影响[J].中药新药与临床药理,2000,11(3):154-156.
[78]刘大基,刘解生,徐继勋,等.二至丸对小鼠淋巴细胞免疫功能的调节作用[J].湖北中医杂志,2002,24(2):48-49.
[79]操红缨,梁颂名,荣向路,等.二至丸对阴虚模型的影响.神经内分泌免疫网.
[80]丁玉琴,马凤巧,尚喜雨.二至丸对D-半乳糖所致衰老小鼠皮肤组织的抗氧化作用[J].中国临床康复,2006,10(19):141-143.
[81]王淑梅,王灿岭.二至丸对亚急性衰老模型小鼠抗衰老作用的实验研究[J].中医研究,2006,19(5):21-22.
[82]李胜志,赵雪莹,李冀.二至丸对D-半乳糖致衰老模型大鼠SOD、MDA影响的实验研究[J].中医药 信息,2008,25(3):37-38.
[83]孙洪涛.二至丸对衰老小鼠模型脑细胞Na+-K+-ATP酶、Ca2+-ATP酶活性影响的研究[J].中外医疗,2008(13):78.
[84]王莉英.二至丸的保肝降酶作用研究[J].海峡药学,2002,11(1):14-15.
[85]姚干,蔡应繁,江怀仲,等.二至丸有效部位群组方对小鼠实验性肝损伤的保护作用[J].中成药,2009,31(3):461-463.
[86]赵雪莹,李胜志,李冀.二至丸对衰老大鼠血液流影响的实验研究[J].辽宁中医杂志,2008,35(6):945-946.
[87]Zhang H,Xing W,Li YS,et al.Effects of a traditional Chinese herbal reparation on osteoblasts and osteoclasts[J]. aturitas 2008,61(4):334-339.
[88]郑里翔,朱卫丰.二至丸对阴虚荷瘤小鼠抑瘤作用的研究[J].江西中医学院学报,2007,19(6):58-59.
[89]胡慧娟,杭秉茜.二至丸的抗炎作用[J].江西中医学院学报,1993,5(2):37-38.
[90]鲁斌,李新民,马融.对改造的阿霉素肾病模型的评价.实验动物科学与管理,1999,3:5-9.
[91]毕黎琦,刘晓敏.肾病综合征大鼠模型的建立及酶酚酸酷对其作用的实验研究.中国免疫学杂志,2002,16(12):681-683.
[92]Bertani T, Poggi A, Pozzoni R, et al. Adriamycin-induced nephrotic syndrome in rats.Laboratory Investigation,1982,46:16.
[93]Okuda S.Adriamycin-induced nephropathy as a model of chronic progressive glomerular disease.Kidney International,1986,29:502.
[94]李保春,崔茗兰,张晓英,等.大鼠阿霉素肾病模型的建立.第二军医大学学报,1992,13(5):48-50.
[95]周铁成,卢宝弼.天门冬氨酸氨基转移酶测定诊断肝病的意义.第四军医大学学报,1993,14(1):77-78.
[96]刘伟,曹勇,王凤娟,等.阿魏酸钠干预雷公藤多甙致小鼠肝损伤.武汉大学学报,2006,27(4):468-470.
[97]吴永贵,林善琰.蛋白尿与慢性肾功能衰竭发病机制新见解[J].中华内科杂志,1998,37(11):772-774.
[98]陈灏珠.实用内科学[M].11版.北京:人民卫生出版社,2002:1930-1932.
[99]Davies DJ, Messina A, Thumwood CM, et al. Glomerular podocyte injury in protein overload proteinuria[J]. Pathology,1985,17(3):412-419.
[100]Mauro A, Carla Z, Marina M, et al. Transforming growth factorβ-1 is up-regulated by podocytes in response to excess intraglomerular passage of proteins[J]. Am J Pathol,2002,161(6):2179-2193.
[101]Michel LH, Cornelia K, Valerie E, et al. Podocyte bridges between the Tuft and Bowman's capsule:An early event in experimental crescentic glomerulonephritis [J]. J Am SocNephrol,2001,12(10):2060-2071.
[102]Katsuhiko A, Isao S,Kazumi I, et al. Selective modulation of the secretion of proteinases and their inhibitors by growth factors in cultured differentiated podocytes[J].Kidney Int,2002,62(3):822-831.
[103]Border WA, Noble NA, Ketteler M. TGF-P:a cytokine mediator of glomerulosclerosis and a target for therapeutic intervention.Kidney Int,1995,47(Suppl 49):59-61.
[104]Mario S, Markus B, Ian SDR, et al. Apoptosis in podocytes induced by TGFβand Smad7[J]. J Clin Invest, 2001,108(6):807-816.
[105]Bottinger EP, Bitzer M. TGF-beta signaling in renal disease. J Am Soc Nephrol,2002,13:2600-2610.
[106]Kriz W, LeHir M. Pathways to nephron loss starting from glomerular diseases:Insights from animal models. Kidney Int,2005,67:404-419.
[107]Wharram BL, Goyal M, Wiggins JE, et al. Podocyte depletion causes glomerulosclerosis:Diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptortransgene. J Am Soc Nephrol,2005,16:2941-2952.
[108]Wiggins R.The spectrum of podocytopathies:A unifying view of glomerular diseases. Kidney Int, 2007,71:1205-1214.
[109]Malleson, Lociitch G, Mackinnon M, et al. Renal disease in chronic arthritis of childhood:A Study of urinary N-acetyl-betaglcos aminidase and beta-2-microglobulin exeretion. Arthritics Rheum,1990,33:156.
[110]Dance N. The excretion of N-acetyl-β-D-glucosa-minidase and β-galactosidase by patients with renal disease. Clin Chim Acta,1970,27:89.
[111]Shi-Nong Wang, Raimund Hirschberg. Tubular epithelial cell activation and interstitial fibrosis,The role of glomerular ultrafiltration of growth factors in the nephrotic syndrome and diabetic-nephropathy. Nephrol Dial Transplant,1999,14:2072-2074.
[112]张少宁.nephrin在肾脏疾病中的研究进展.实用医学杂志,2006,22(6):727-728.
[113]Boute N,Gribouval O,Roselli S,et al.NPHS2,eneoding the glomerular protein podocin,is mutated in autosonmal recessive steoid-resistant nephritic syndrome.Nat Genet,2000,24(4):349-354.
[114]Kawachi H,Koikc H,Kurihara H,et al. Cloning of Rat Homologue of Podocin:Expression in Proteinuric States and in Developing Glomeruli[J]. J Am Soc Nephrol,2003,14(1):46-56.
[115]Kestda M,Lenkkeri U,Mannikko M,et al.Positionally cloned gene for a novel glomerular protein nephrin is mutated in congenital nephritic syndrome.J Mol Cell,1998,1(4):575-582.
[116]Nakatsue T,Koike H,Han GD,et al.Nephrin and podocin dissociate at the onset of proteinuria in experimental membranous nephropathy.Kidney Int,2005,67(6):2239-2253.
[117]Guan N,Ding J,Zhang J,et al.Expression of nephrin,podocin,alpha-actinin,and WT1 in children with nephritic sydrome.Pediatr Nephrol,2003,18(11):1122-1127.
[118]Huber TB,Kottgen M, Schilling B,et al. Interaction with podocin facilitates nephrin signaling[J]. J Biol Chem,2001,276(45):41543-41546.
[119]Liux L,Kilpelainen P,Hellman U,et al.characterization of the interactions of the nephrin intercellular domain:Evidence that the scaffolding protein IQCAP1 associates with nephrin[J].FEBS J,2005,272(1):228-243.
[120]Nakhoul F, Ramadan R, Khankin E,et al. Glomerular abundance of nephrin and podocin in experimental nephritic syndrome:Different effects of antiproteinuric therapies[J].Am J Physiol Renal Physiol,2005,289(4): F880-F890.
[121]Luimula P, Ahola H, Wang SX,et al. Nephrin in experimental glomerular disease[J]. Kidney Int,2000, 58(4):146-1468.
[122]Lih P,Lemay S,Aoudjit I,et al.Src-family kinsase fyn phosphorylates the cytoplasmic domain of nephrin and modulates its interaction with podocin[J].J Am Soc Nephol,2004,15(12):3006-3015.
[123]Matsui I, Ito T,Kurihara H,et al. Snail a transcriptional regulator, represses nephrin expression in glomerular epithelial cells of nephritic rats[J].Lab Invest,2007,87(3):273-283.
[124]Huber TB, Hartleben B, Kim J,et al. Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling[J].Mol Cell Biol,2003,23(14):4917-4928.
[125]Huber T,Simons M,Hartleben B,et al.Molecular basis of the functional podocon-nephrin complex: mutations in the NPHS2 gene disrupt nephrin targeting to lipid raft microdomain[J].Hub Mol Genet,2003,12 (14):3397-3405.
[126]Nasanori Hara,ToshioYana. Immunohistochemical and urinary markers of podocyte injury[J].Pediatr NePhrol,1998,12:43-48.
[127]Orlando RA, Takeda T, Zak B,et al. The glomerular epithelial cell anti-adhesin, podocalyxin, associates with the actin cytoskeleton through interactionswith ezrin. J Am Soc Nephrol,2001,12(8):1589-1598.
[128]Takeda T,Go WY,Orlando RA,et al.Expression of podocalyxin inhibits cell-cell adhesion and modifies junctional properties in Madin-Darby canine kidney cells.Mol Biol Cell,2000,11 (2),3219-3232.