摘要
目的:糖尿病肾病(diabetic nephropathy,DN)是糖尿病(diabetic melliutus,DM)常见而严重的微血管并发症,是西方国家终末期肾病(end stage renal disease,ESRD)及进行肾替代治疗的最主要原因。在我国DN在ESRD中的比例也逐渐上升,预计在未来的20年中DN将成为终末期肾衰竭最重要的原因。因此探讨DN的发病机制,寻求延缓DN进展的治疗方法具有重要意义。由于众多生物活性物质参与了糖尿病肾病发病过程,具有多途径、多环节相互作用的复杂病理生理机制,其防治一直是临床难点。西医在治疗DN上主要是控制高血糖、控制高血压、低蛋白饮食及纠正脂代谢紊乱等对症治疗,至今尚没有一个能完全阻断DN进展的药物。而中药复方治病的特点就是通过多途径、多靶点实现治疗目的,故应用中药复方防治DN具有很大的潜力。在前期临床实践中,我们根据中医学病机理论,结合糖尿病肾病的临床特征、发病机制及其病理特点,逐步认识到该病的病情虽然复杂,但在早期和中期大多以气阴两虚、瘀血阻络、癥瘕积聚为其基本病机,治疗当以益气养阴、化瘀软坚、消癥通络为主,为此我们结合多年的临床用药经验,拟定了以益气养阴、消癥通络为主的中药复方制剂,主要药物有黄芪、玄参、鳖甲、地龙等。用该方在临床上治疗早中期的糖尿病肾病,取得了良好的临床疗效。为了进一步证实该方的治疗作用,探讨其可能的机制,进行了本项研究。即借助糖尿病肾病动物模型,观察其对糖尿病肾病大鼠干预作用及对TGFβ/Smad信号传导系统的影响。
方法:
1益气养阴消癥通络中药对糖尿病肾病大鼠干预作用
健康雄性SD大鼠46只,适用性饲养一周。按体重随机分为正常组(Normal)10只,造模组36只。除正常组外,造模组大鼠一次性腹腔注射链脲佐菌素(STZ) 60 mg/kg,正常组注射相应量生理盐水,72小时后测血糖,以≥16.7mmol/L为DM模型成模标准。造模组在DM模型成模后,随机分为模型组(Model)、贝那普利组(Benazepril)、中药(ZY)组各12只。成模一周后开始给药,正常组、模型组每天每只大鼠2毫升生理盐水灌胃,治疗各组按成人剂量的20倍给药,即贝那普利组予贝那普利10mg/kg.d灌胃,中药组予中药煎剂26g/kg.d灌胃,每日一次,连续给药23周。于治疗开始后23周末收集24小时尿液,杀检动物,留取血、肾组织,测定实验动物的24小时尿蛋白定量、糖化血红蛋白、血脂、肾功能、观察肾组织超微结构及肾小球硬化指数的变化。
2益气养阴消癥通络中药对糖尿病肾病大鼠TGFβ及其受体的影响
同上方法复制糖尿病肾病大鼠模型,于成功复制糖尿病肾病模型24周末留取肾组织标本,观察益气养阴消癥通络中药对DN大鼠Ⅳ型胶原(Col-Ⅳ)、TGFβ1及TGFβΙ型受体(Transforming growth factor-beta TypeⅠReceptor, TβR-Ⅰ)、Ⅱ型受体(Transforming growth factor-beta TypeⅡReceptor, TβR-Ⅱ)的影响。
3益气养阴消癥通络中药对糖尿病肾病大鼠TGFβ/Smad信号传导系统的影响
同上方法复制糖尿病肾病大鼠模型,于成功复制糖尿病肾病模型24周末留取肾组织标本,观察益气养阴消癥通络中药对DN大鼠smad2、smad3和smad7的影响,进一步探讨其对糖尿病肾病可能的作用机制,
结果:
1益气养阴消癥通络中药治疗糖尿病肾病大鼠的干预作用
1.1各组大鼠24h尿蛋白定量的比较
24h尿蛋白定量,模型组、贝那普利组、中药组与正常组比较均明显升高(P<0.01),贝那普利组、中药组与模型组比较明显降低(P<0.01),而贝那普利组与中药组之间无显著差异(P>0.05)。
1.2各组大鼠糖化血红蛋白(HbA1C)的比较
模型组、贝那普利组、中药组大鼠糖化血红蛋白与正常组相比明显升高(P<0.01),而模型组、贝那普利组、中药组之间无显著性差异(P>0.05)。
1.3各组大鼠血脂的比较
模型组、贝那普利组、中药组血清总胆固醇(TC)明显高于正常组(P<0.01),中药组则比模型组明显偏低(P<0.01),模型组与贝那普利组之间比较无显著性差异(P>0.05)。甘油三酯(TG),两治疗组与正常组均明显低于模型组(P<0.01),而两治疗组与正常组之间无显著性差异(P>0.05)。
1.4各组大鼠肾功能的比较
两个治疗组与正常组的血肌酐(Scr)明显低于模型组(P<0.01),贝那普利组与正常组相比明显升高(P<0.05),而中药组与正常组之间无显著性差异(P>0.05);尿素氮(BUN),两治疗组与正常组均明显低于模型组(P<0.01),而与正常组比较,贝那普利组和中药组的BUN则明显升高(P<0.05),两治疗组之间则无显著性差异(P>0.05)。
1.5肾脏病理形态学观察
光镜:正常组肾小球结构完整,未见肾小球肥大,肾小球毛细血管基底膜、系膜、基质未见明显异常改变;模型组可见肾小球肥大,肾小球毛细血管基底膜增厚,肾小球系膜细胞增生、系膜基质增多,系膜区增宽;部分肾小管发生空泡变性,肾小管萎缩,透明管型形成;贝那普利组与中药组也均有不同程度病理改变,但与模型组相比病变减轻。
电镜:正常组肾脏组织结构正常,肾小球基底膜均匀无增厚,上皮足突分布均匀,模型组大鼠肾小球毛细血管基底膜高度增厚,大部分呈匀质样增厚,部分增生呈丘状隆起;足细胞足突广泛融合;内皮细胞高度融合,窗口大部分消失。贝那普利组与中药组大鼠肾小球毛细血管基底膜较正常呈匀质样增厚,足细胞局部融合,内皮细胞部分融合,窗口部分消失。
各组肾小球硬化指数比较:与正常组相比,模型组和两个治疗组的肾小球硬化指数要明显升高(P<0.05),与模型组比较,贝那普利组和中药组要明显降低(P<0.05),两治疗组比较,贝那普利组高于中药组(P<0.05)。
2益气养阴消癥通络中药对糖尿病肾病大鼠TGFβ及其受体的影响
2.1 Col-Ⅳ免疫组化检测结果
正常组Col-Ⅳ在系膜基质及基底膜及包曼氏囊有基础表达。与正常组相比,模型组Col-Ⅳ表达的范围和强度明显扩大和增强(P<0.01);而中药组、贝那普利组与模型组相比,其表达显著下降(P<0.01)中药组与贝那普利组比较无显著性差异(P>0.05)。
2.2对大鼠肾组织TGF-β1蛋白及其基因表达的影响
免疫组化染色显示:TGF-β1在肾小球上皮细胞及系膜区,毛细血管袢,肾小囊壁层上皮细胞,肾小管上皮细胞胞浆以及小管间质均呈弱阳性表达,半定量分析结果显示:与正常组相比,模型组和两个治疗组的表达要明显加强(P<0.05),与模型组比较,贝那普利组和中药组的表达要明显降低(P<0.05),两治疗组比较,贝那普利组高于中药组(P<0.05)。RT-PCR结果显示:与正常组相比,模型组和各治疗组表达明显升高(P<0.01),而与模型组比较,贝那普利组和中药组大鼠肾组织TGF-β1表达则明显降低(P<0.01);两治疗组比较,贝那普利组高于中药组(P<0.05)。
2.3对大鼠肾组织TGF-βΙ型受体蛋白表达及基因表达的影响
免疫组化结果显示:与正常组相比,模型组和各治疗组表达明显升高(P<0.01),而与模型组比较,贝那普利组和中药组大鼠肾组织TGF-βΙ型受体表达则明显降低(P<0.01);两治疗组比较,贝那普利组高于中药组(P<0.05)。
RT-PCR结果显示:与正常组相比,模型组和各治疗组表达明显升高(P<0.01),而与模型组比较,贝那普利组和中药组大鼠肾组织TGF-βΙ型受体表达则明显降低(P<0.01);两治疗组比较,贝那普利组高于中药组(P<0.05)。
2.4对大鼠肾组织TGF-βⅡ型受体蛋白表达及基因表达的影响
免疫组化染色显示:与正常组相比,模型组和各治疗组表达明显升高(P<0.01),而与模型组比较,贝那普利组和中药组大鼠肾组织TGF-βⅡ型受体表达则明显降低(P<0.01);贝那普利组和中药组比较则无明显差异(P>0.05)。
RT-PCR结果显示:与正常组相比,模型组和各治疗组表达明显升高(P<0.01),而与模型组比较,贝那普利组和中药组大鼠肾组织TGF-βⅡ型受体表达则明显降低(P<0.01);贝那普利组和中药组比较则无明显差异(P>0.05)。
3益气养阴消癥通络中药对糖尿病肾病大鼠TGFβ/Smad信号传导系统的影响
3.1免疫组化检测大鼠肾组织p-Smad2/3蛋白表达的结果与正常组相比,模型组和各治疗组阳性增强的核占小管细胞的百分比显著增加(P<0.05),与模型组比较,贝那普利组和中药组的表达明显降低(P<0.05),贝那普利组和中药组之间的比较则无明显差异(P>0.05)。
3.2 Western印迹检测大鼠肾组织Smad7蛋白表达的结果:
与正常组相比,模型组和各治疗组的表达要明显降低(P<0.05),与模型组比较,贝那普利组和中药组的表达要明显上升(P<0.01),贝那普利组和中药组之间的比较则无明显差异(P>0.05)。
3.3 RT-PCR检测肾组织Smad2mRNA和Smad7mRNA表达结果
3.3.1Smad2mRNA表达结果:
与正常组相比,模型组和各治疗组Smad2mRNA显著增加(P<0.05),与模型组比较,贝那普利组和中药组的表达明显降低(P<0.05),贝那普利组和中药组之间的比较则无明显差异(P>0.05)。
3.3.2Smad7mRNA表达结果:
与正常组相比,模型组和各治疗组的表达要明显降低(P<0.05),与模型组比较,贝那普利组和中药组的表达要明显上升(P<0.01),贝那普利组和中药组之间的比较则无明显差异(P>0.05)。
结论:
1.益气养阴消癥通络中药能改善DN大鼠一般状况,减少尿蛋白的排泄,降低血脂,改善大鼠的肾功能。
2.益气养阴消癥通络中药可减轻减轻肾组织病理损害,从而延缓DN的进展。
3.益气养阴消癥通络中药可降低糖尿病肾病大鼠IV型胶原合成,从而阻止肾小球硬化的发生和发展。
4.益气养阴消癥通络中药能明显降低DN大鼠肾组织中TGF-β1及TGF-βⅠ、Ⅱ型受体蛋白及基因的表达,说明该药可能通过降低肾组织TGF-β1及TGF-βⅠ、Ⅱ型受体蛋白及基因的表达而达到抗纤维化、保护肾功能的作用。
5.益气养阴消癥通络中药能够抑制受体活化性蛋白smad2和smad3的表达,促进抑制性蛋白Smad7的表达,从而抑制DN大鼠肾脏中TGF-β/smad信号传导通路的激活,减少大鼠肾脏ECM沉积,从而延缓DN的进程。
Objectives: Diabetic nephropathy(DN)is a part of micovasclor complication and the most important reason of end stage renal disease. The researchers keep working on the reason and treatment of DN. It is difficult to prevent and cure DN because numerous biotic activator participate in the process of DN. The medical doctor give the DN patients only with symptomatic treatment such as control hyperglycosemia and hypertension, low-protein diet and so on. The traditional Chinese herbs play an important role in preventing DN because of its advantage. We invented an effective traditional medicine, according to the pathogenesis which is at the early stage of DN and clinical experience for treating DN. This medicine has the functions of promoting circulation of qi and yin, dispersing blood stasis and dredging collateral which was composed of astragali, figwort root, carapax amydae, angle worm, ect. The clinical data showed that the prescription had good effect on the early stage of DN. This study aim to observe the curative effect of herbs of supplement qi and nourish yin resolve masses and dredge meridians(ZY) and investigate the mechanism in TGFβ/Smad signal transduction system in DN rats. All the above that have been done was to provide the foundation in order to generalization and exploitation.
Motheds:
1 Effect of ZY on the treatment in DN rats:
Forty-six male Sprague-Dauley rats were selected in this experiment. According to the weight, the animals were randomly divided into normal group(10 rats)and DM model group(36 rats). Besides the normal group, all the rats were injected with streptozotocin (STZ) in the dose of 60 mg/kg, the normal group were given the corresponding quantity’s physiological saline. After 72 hours, measured their blood glucose, took“≥16.7mmol/L”as the DM model standard. According to the blood glucose level, the rats of DM model were randomly divided into three groups: 12 in the model group, 12 in benazepril group and 12 in ZY group. After one week of DM models were made successfully, the drugs started be given to the rats according to adult dosage 20 times, once daily. The rats of ZY group were given the ZY by gavage, 26g/kg, the benazepril group were administrated with benazepril by gavage, 10mg/kg, the normal group and the model group were given physiological saline by gavage. Each group was given the medicine for 23 weeks. After 24 week of DM models made successfully, all rats were put into metabolism cage to obtain 24 hour urine. All animals were killed and the blood and the renal tissue were obtained. 24 hour urinary protein quantitative, glycohemoglobin(HbA1C), total cholesterol(TC), triglyceride (TG), serum creatinine (Scr), urea nitrogen (BUN) were assayed with the automaticbiochemistry analyzer. Pathomorphology change and the glomerulosclerosis index were observed.
2 Effect of ZY on TGFβand receptor in DN rats:
DM model was duplicated the same as the above.After 24 week of DM models made successfully, the renal tissue were obtained. The Col-Ⅳ、transforming growth factor-beta , transforming growth factor-beta typeⅠreceptor, transforming growth factor-beta typeⅡreceptor were measured by immunohistochemical method and RT-PCR.
3 Effect of ZY on TGFβ/Smad signal transduction system in DN rats:
DM model was duplicated the same as the above.After 24 week of DM models made successfully, the renal tissue were obtained. The smad2、smad3 and smad7 were measured by immunohistochemical method and RT-PCR.
Results:
1 Effect of ZY on the treatment in DN rats:
1.1 The 24 hour urinary protein quantitative of each group
The 24 hour urinary protein quantitative of the rats in model group, benazepril group and ZY group was significantly higher than that of normal group (P <0.01), compared with model group, the 24 hour urinary protein quantitative of benazepril group and ZY group was obviously lower (P <0.01). There was no difference between benazepril group and ZY group (P >0.05).
1.2 The HbA1C of each group
Compared with normal group, the HbA1C of the rats in model group, benazepril group and ZY group was significantly higher (P <0.01), but there was no difference among model group, benazepril group and ZY group (P >0.05).
1.3 The TC and TG of each group
The TC of the rats in model group, benazepril group and ZY group was significantly higher than that of normal group (P <0.01), compared with model group, the TC of ZY group was obviously lower (P <0.01), there was no difference between benazepril group and model group (P >0.05). The TG of the rats in normal group, benazepril group and ZY group was significantly lower than that of model group (P <0.01), there was no difference among normal group, benazepril group and ZY group (P>0.05).
1.4 The Scr and BUN of each group
Compared with model group, the Scr of the rats in normal group, benazepril group and ZY group was significantly lower (P<0.01), and benazepril group was obviously higher than that of normal group (P<0.05). There was no difference between normal group and ZY group (P>0.05). The BUN of the rats in normal group, benazepril group and ZY group was significantly lower than that of model group (P<0.01). Compared with normal group, the BUN of benazepril group and ZY group was significantly higher (P<0.01), and there was no difference between benazepril group and ZY group (P>0.05).
1.5 The pathomorphology change of kidney
Observations under light microscope:In the normal group: the structure of renal glomerulus was integrated, glomerular capillary basement membrane, mesenterium and base material were normal. In the model group: glomerular hypertrophy, thickening of glomerular capillary basement membrane, mesangial cell proliferation, mesangial matrix increased, mesangial area broadening and tubulointerstitial fibrosis were seen. vacuolar degeneration, analosis and hyaline cast were seen in some renal tubule. The benazepril group and ZY group also showed different degree of pathological changes, but the pathological degree was obviously lighter than the changes of the model group.
Observations under transmission electron microscopy: In normal group: the structure of glomerular capillary basement membrane was clear and complete, microcirculatary endothelial cell, foot processes was normal. In the model group: most of the glomerular capillary basement membrane showed significant homogeneous thicking, partial basement membrane showed elevation in the shape of hills; microcirculatary endothelial cell significantly confluence and the most of window structure disappeared; the fusion of the foot processes was extensive. The benazepril group and ZY group also showed the similar pathological changes, but the pathological degree was lighter than that in model group.
The glomerulosclerosis index in each group: The glomerulosclerosis index in model and treatment groups were higher than that in normal group (P<0.05);Compared with model group, the glomerulosclerosis index in benazepril group and ZY group was significantly lower (P<0.05), and the glomerulosclerosis index in benazepril group was higher than that in ZY group(P<0.05).
2 Effect of ZY on TGFβ1 and receptor in DN rats
2.1 Expression of CollegeⅣexamined by immunohistochemistry:
In normal group, Col-Ⅳhas the basal expression. Compared with normal group, the expression of Col-Ⅳin model group was stronger(P<0.01). Compared with model group, the expression of Col-Ⅳin benazepril group and ZY group was significantly lighter (P<0.01), and there was no difference between benazepril group and ZY group (P >0.05).
2.2 Expression of TGFβ1 in renal tissue
The results detected by immunohistochemistry showed: The expression of TGFβ1 in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβ1 in benazepril group and ZY group was significantly decreased (P <0.01), and the TGFβ1 in benazepril group was higher than that in ZY group(P<0.05).
The results detected by RT-PCR showed: The expression of TGFβ1 in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβ1 in benazepril group and ZY group was significantly decreased (P<0.01), and the TGFβ1 in benazepril group was higher than that in ZY group(P<0.05).
2.3 Expression of TGFβtypeⅠreceptor in renal tissue
The results detected by immunohistochemistry showed: The expression of TGFβtypeⅠreceptor in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβtypeⅠreceptor in benazepril group and ZY group was significantly decreased (P <0.01), and the TGFβtypeⅠreceptor in benazepril group was higher than that in ZY group(P<0.05).
The results detected by RT-PCR showed: The expression of TGFβtypeⅠreceptor in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβtypeⅠreceptor in benazepril group and ZY group was significantly decreased (P <0.01), and the TGFβtypeⅠreceptor in benazepril group was higher than that in ZY group(P<0.05).
2.4 Expression of TGFβtypeⅡreceptor in renal tissue
The results detected by immunohistochemistry showed: The expression of TGFβtypeⅡreceptor in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβtypeⅡreceptor in benazepril group and ZY group was significantly decreased (P<0.01), and there was no difference between benazepril group and ZY group (P>0.05).
The results detected by RT-PCR showed: The expression of TGFβtypeⅡreceptor in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβtypeⅡreceptor in benazepril group and ZY group was significantly decreased (P <0.01), and there was no difference between benazepril group and ZY group (P >0.05).
3 Effect of ZY on TGFβ/Smad signal transduction system in DN rats
3.1 Expression of p-Smad2/3 protein in renal tissue
Compared with normal group, the expression of p-Smad2/3 in model group was stronge(rP<0.01). Compared with model group, the expression of p-Smad2/3 in benazepril group and ZY group was significantly lighter (P <0.01), and there was no difference between benazepril group and ZY group (P >0.05).
3.2 Expression of Smad7 protein in renal tissue by Westernblotting
Compared with normal group, the expression of Smad7 in model group was lower(P<0.01). Compared with model group, the expression of Smad7 in benazepril group and ZY group was significantly stronger (P<0.01), and there was no difference between benazepril group and ZY group (P >0.05).
3.3 Expression of Smad2 and Smad7mRNA by
3.3.1 Smad2mRNA
Compared with normal group, the expression of Smad2 in model group was stronger(P<0.01). Compared with model group, the expression of Smad2 in benazepril group and ZY group was significantly lighter (P<0.01), and there was no difference between benazepril group and ZY group (P >0.05).
3.3.2 Smad7mRNA
Compared with normal group, the expression of Smad7 in model group was lower(P<0.01). Compared with model group, the expression of Smad7 in benazepril group and ZY group was significantly stronger (P<0.01), and there was no difference between benazepril group and ZY group (P >0.05).
Conclusions:
1. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could decreased the DN rat’s proteinuria, improve the lipid metabolism, decrease Scr and BUN
2. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could lessen the damage of nephridial tissue.
3. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could decrease the ColIV deposition in order to slow down the progress of glomerulosclerosis.
4. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could decreased TGF-β1and receptorⅠ、Ⅱwhich could resist renal fibrosis and protect renal founction.
5. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could inhibit the expression of smad2, smad3 and promote the expression of smad7which could delay the process of DN.
引文
1 Gamille AJ, Andrzej SK, John R, et al. Epidemic of end-atage renal Disease In people with diabetes In the United States population : Do we know the cause ? Kidney Int, 2005,67(5):1684-1691
2 Paul CW, Van D, Kitty J, et al. Renal replacement therapy for diabetic end-stage disease, Data fromto registries Europe(1991-2000). Kidney Int, 2005,67(4):1489-1499
3 Gorss LJ,de Azevedo MJ,Silveiro SP,et al. Diabetic nephropathy: diangosis,Prevention,and teratment[J].Diabetes care,2005,28(l):164-176
4郭登洲、王月华、陈志强等。活血化瘀消癥通络中药治疗糖尿病肾病76例临床研究,中国全科医学,2007, 10(20): 1692-1693
5王月华、郭登洲、陈志强等。活血化瘀消癥通络中药对糖尿病肾病患者Ⅳ型胶原及层黏连蛋白的影响,临床荟萃,2008,23(21),1573-1574
6雷作熹,罗仁,董晓蕾等. STZ诱导糖尿病肾病大鼠模型的建立,中国实验动物学报,2005,13(3):163-165
7徐叔云,卞如濂,陈修,主编.药理实验方法学,人民卫生出版社,2003, 9:1202-1203
8 Raij L ,Azar S,Keane W. Mesangial immune injury, hypertension and progressive glomerular damage in Dahl rats. Kidney Int ,1984 ,26 2 :137 - 140
9向红丁.糖尿病肾病的预防措施[J].临床内科杂志, 2005, 22 (3): 147-149
10 Adler SG, Pahl M, Seldin MF. Deciphering diabetic nephropathy: progressusing strategies [J ]. Curr Opin Nephrol Hypertens, 2000, 9: 99-106
11林善锬.糖尿病肾病[J ].中华内科杂志, 2005 ,44 (3):229-231
12周建辉,陈香美,谢院生等.糖尿病肾病不同程度蛋白尿的相关因素分析及随访.中华肾脏病杂志, 2005, 21(5): 251-255
13付海霞.糖尿病肾病的研究进展.中国实验诊断学, 2005,9(1):31-33
14赵进喜,邓德强,李靖.糖尿病肾病相关中医病名考辨[J].南京中医药大学学报(自然科学版),2005,21(5):288-289
15董振华,季元,等.祝谌予经验集.北京:人民卫生出版社,1999,40～42.
16江苏新医学院主编.中药大辞典.上海科学技术出版社,1992:2038
17石君华,章如虹.黄芪对实验性糖尿病大鼠肾脏保护作用的实验研究.中国中医药科技,1999,40(5):314-316
18郁杰,张庆怡,陆敏,等.黄芪对糖尿病大鼠肾皮质TGF-β表达的影响.中华内分泌代谢杂志,1998,14(5):312-314
19祁忠华,林善锬,黄字峰.黄芪改善糖尿病早期肾血流动力学异常的研究.中华糖尿病杂志,1999,7(3):147-249
20张卫平.浅谈活血化瘀法在糖尿病肾病治疗中的运用.实用中医药杂志,2003,19(5):268
21志云,马琼英.丹参注射液对肾小球系膜细胞增殖及产生IL-6、Ⅳ型胶原影响的实验研究.中国中西医结合肾病杂志,2001;2(7):421
22韩彩芝,余素清,李季芳等.蚯蚓提取液纤溶及抗凝作用的实验研究.中国生化药物杂志,1997;18(5):252
23叶任高.中西医结合肾脏病学,北京:人民卫生出版社,2003;第一版:624
24 Remuzzi G,Ruggenenti P,Benigni A. Understnading the natuer of renal. Disease Progression[J].Kidney Int,1997,51:2-10
25 Jafar TH,Stark PC,Schmid CH,et al. Proteinnuai as modifiable risk factor for the Progression of non diabetic renal disease[J].kidney Int,2001,60:1131-1137
26 Burton C, Harper SJ, Bailey E, et al. Turnover of human tubular cells exposed to proteins in vivo and in vitro [J].kidney Int,2001,59:507-511
27 Zatz R,Meyer TW,Rennke HG,et al.Predominance of hemodynamic rather than metabolic factor in the pathogenesis of diabetice glomerulopathy,ProcNatl Acad Sci USA,1985,82:59-63
28 En SF, Goetz FC, Keen H, et al. Effect of lowprotein diet on experimental diabetic nephropathy in the rats [J], Lab Clin Med, 1985,106:589
29王秀问.脂代谢异常与糖尿病肾病,国外医学.内分泌学分册,1991,3:147-148
30 Marshall SM. Recent advances in diabetic nephropathy. Clin Med 2004 May-Jun; 4(3): 277-282
31 Witch WE. Insights into the abnormalities of chronic renal disease attributed to mainutrition. J Am Soc nephrol , 2002, 13: 22-24
32 Doyonnas R, Kershaw DB, Duhme C Anuria, Omphalocele, and Perinatal Lethality in Mice Lacking the CD34-related Protein Podocalyxin. J. Exp. Med, Jun 2001; 194: 13-28
33 Asanuma K and Mundel P. The role of podocytes in glomerular pathobiology. Clin Exp Nephrol, 2003; 7: 255-259
34 Matsumae T, Jimi S, Uesugi N, et al. Clinical and morphometrical interrelationships in patients with overt nephropathy induced by non-insulin dependent diabetes mellitus,Nephron,1999, 81(1): 41-48
35 Hayashi H, Karasawa R, Inn H,et a1. An electron microscopic study of glomeruli in Japanese patients with non-insulin dependent diabetes mellitus, Kidney Int, 1992,41(4): 749-757
36 Ota K, Shikata K, Makino H,et al.Ultrastructural changes of the glomerular basement membrane in diabetic nephropathy revealed by newly devised tissue negative staining method,Acta Med Okayama, 1993,47(4): 267-272
37 Wolf G, Ziyadeh FN·Cellular and molecular mechanisms of proteinuria in diabetic nephropathy·Nephron Physiol, 2007,106(2): p26-p31
38 Shankland SJ·The podocytes' response to injury: role in proteinuria and glomerulosclerosis·Kidney Int, 2006, 69(12):2131-2147
39 Iglesias-de la Cruz MC, Ziyadeh FN, Isono M, et al·Effects of high glucose and TGF-beta on the expression of collagenⅣand vascular endothelial growth factor in mouse podocytes·Kidney Int, 2004, 62(3): 901-913
1 Rapits AE ,Viberti G. Pathogenesis of diabetic nephropathy. Exp Clin Endocrinol Diabetes , 2001 ,109 (2) :S424-437
2 Ziyadeh FN. Mediators of diabetic renal disease: the case of tgf-Beta as the major mediator[J]. Joumal of the American of Society of NePhorlogy,2004,15SuPPl 1:S55-7
3 Schena PF, Gesualdo L. Pathogenetic mechanism of diabetic nephropathy. J Am Soc Nephrol,2005,16(suppl 1):S30-S33
4 Fernandes L , Fortes ZB , Nigro D , et al . potentiation of bradykinin by angiotensin-(1-7) on arterioles of spontaneously hypertensive rat s studies in vivo. Hypertension , 2001 , 37 :703
5 Shonklacd ST ,Scholey TW. Expression of transforming growth factor-β1 during diabetic renal hypertrophy. Kidney Int ,1994 ,46 (2) :430-442
6 Jong Hoon OH ,Hunjoo Ha ,Mi Ra Yu ,et al . Sequential effects of high glucose on mesangial cell transforming growth factor -β1 and fibronectin synthesis. Kidney Int ,1998 ,54 (4) :1872-1878
7 Fraser O ,Wakefield L ,Phillips A. Independent regulation of transforming growth factor - betal transcription and translation by glucose and platelet growth factors. AmJ Pathol ,2002 ,161 (3) :1039-1049
8 Inak , Kitamnra H , Tatsukawa S ,et al . Transformation of interstitial fibroblasts and tubulointerstitial fibrosis in diabetic nephropathy. Med Electron Microsc ,2002 ,35 (2) :87-95
9 Yang CW,Hsueh S,Wu MS,et al. Glomerular transoforming growth factor-betal mRNA as a markerof glomeruloscleorsis-application in renalbiopsies[J].Nephron,1997,77(3):290-297
10 Chen S, Hong SW, Lglesias de la cruz MC, et al, The key role of the transforming growth factor-beta system in the pathogenesis of diabetic nephropathy. Ren Fail, 2001,23(4):471-481
11 Leehey DJ, Singh AK, Alavi N, et al. Role of AngiotensionⅡin diabetic nephropathy. Kidney Int,2000,58(suppl77):s93-s98
12 Singh R, Song RH, Alavi N, et al. High glucose decrease matrix metalloproteinase-2 activity in rat mesangial cells via transforming growth factor-beta. Exp Nephrol, 2001,9(4)249-257
13 Chen S, Jim B, Ziyadeh FN. Diabetic nephropathy and TGF- beta: transforming our view of glomerulosclerosis and fibrosis buildup. Semin Nephrol, 2003,23(6)532-543
14 Nichosas SB, Advances in pathogenetic mechanisms of diabetic nephropathy.Cell Mol Biol,2003,49(8)1319-1325
15 Ziyadeh FN,Hoffman BB,Han DC, et a.l Long-term prevention of renal insufficiency, excessmatrix gene expression, and glomerular mesangial matrix expansion by treatmentwith monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice. Poc Natl Acad Sci USA, 2000,97:8015-8020
16 Attisnao L , Wrnaa LJ. Singal transduction by theTGF-βsuper family[J].Seience,2002,296(5573):1646-1647
17 Massague J, Chen YG, Controlling TGF-βsignaling Genes Dev, 2000,14(6): 627-644
18 Hong SW, Iono M, Chen S , et al . Increased glomerular expression of transforming growth factor - beta1 ,its type II recepor and activation of the Smad signaling pathway in the db/db mouse. AmJ Pathol , 2001, 158(5) : 1653-1663
19 Kang MJ ,Ingram A ,Ly H ,et al . Effects of diabetes and hypertension on glomerular transforming growth factor - beta receptor expression. Kidney Int ,2000 ,58(4) :1677-1685
20 Wolf G, Ziyadeh FN·Cellular and molecular mechanisms of proteinuria indiabetic nephropathy·Nephron Physio l, 2007,106(2): p26-p31
21 Shankland SJ·The podocytes' response to injury: role in proteinuria and glomerulosclerosis·Kidney Int, 2006, 69(12):2131-2147
22 Iglesias-de la Cruz MC, Ziyadeh FN, Isono M, et al·Effects of high glucose and TGF-beta on the expression of collagenⅣand vascular endothelial growth factor in mouse podocytes·Kidney Int, 2004, 62(3): 901-913
1 Derynck R ,Zhang YE. Smad-dependent and Smad- independent pathways in TGF - beta family signaling. Nature ,2003 ,425 (6958) : 577 -584
2 WolfG, Chen S, Ziyadeh FN·From the periphery of the glomerular capillarywall toward the center of disease: podocyte injury comes of age in diabetic nephropathy·Diabetes,2005, 54(6): 1626-1634
3 Yu HT·Progression of chronic renal failure·Arch Intem Med, 2003, 163(12): 1417-1429
4 Luukko K, YlikorkalaA,MakelaTP·Developmentally regulated expression of Smad3, Smad4, Smad6, and Smad7 involved in TGF-beta signaling·Mech Dev, 2001, 101(1-2):209-212
5 Itoh S, Itoh F,DaiC, eta.l Signaling of transforming growth factor-beta familymembers through Smad proteins J. Eur J Biochem, 2000, 267(24):6954-6967
6 Vrljicak P,MyburghD,RyanAK, eta.l Smad expression during kindey development. Am J Physiol Renal Physiol ,2004,286:625-633
7 Liliana Attisano. Signal transduction by the TGF - beta superfamily. Science ,2002 ,296(5573) :1646-1647
8 Schnaper HW,Hayashida T,Poncelet AC.It’s a smad word: Regulation of TGF-βsignaling in the kidney[J].J Am Soc Nephrol,2002,13(4):1126-1128
9 Runyan CE,Schnaper HW,Poncelet AC.Smad3 and PKC deltamediate TGF-beta1-induced collagenⅠexpression in human mesangial cells[J].Am J Physiol Renal Physiol,2003,285(3):F413-422
10 Hayashi H, Abdollah S, Qiu Y, et al·The MAD-related protein Smad7 associates with the TGF-βreceptor and functions as an antagonist of TGF-beta signaling·Cell, 1997, 89(7): 1165-1173
11 Huang Y,Border W A,Noble N A Perspectives on blockade of TGF-βoverexpression[J]. Kidney Int,2006,69(10):1713
12 Kim HW,Kim BC,Song CY,et al. Heteorzygous mice for TGF-betaⅡR gene are rersistant to the Porgression of streptozotocin-induced dibaetic-nephropathy[JlKidney Int,2004,66(5):1859-1865
13 IsonoM,Chen S,HongSW, et al. Smad pathway isactivated in the diabetic mouse kindey and Smad3 mediated TGF-beta-induced fibronection in mesangial cells. Biochem Biophys Res Commun, 2002, 296:1356-1365
1 Ziyadeh FN. Mediators of diabetic renal disease: the case of tgf-Beta as the major mediator[J]. Joumal of the American of Society of NePhorlogy,2004,15SuPPl 1:S55-7
2刘志红,黎磊石.糖尿病肾病发病机理[J].中华肾脏病杂志,1999,15(2:)120-123
3 Deryncrk R, Zhnag YE, Smad-depended and Smad-independed Pathway in TGF-beta family singalling[J]. Nature,2003,425(6958):577-584
4 Chen S, Hong SW, Lglesias de la cruz MC, et al, The key role of the transforming growth factor-beta system in the pathogenesis of diabetic nephropathy. Ren Fail, 2001,23(4):471-481
5 Leehey DJ, Singh AK, Alavi N, et al. Role of AngiotensionⅡin diabetic nephropathy. Kidney Int,2000,58(suppl77):s93-s98
6 Singh R, Song RH, Alavi N, et al. High glucose decrease matrix metalloproteinase-2 activity in rat mesangial cells via transforming growth factor-beta. Exp Nephrol, 2001,9(4)249-257
7 Chen S, Jim B, Ziyadeh FN. Diabetic nephropathy and TGF- beta: transforming our view of glomerulosclerosis and fibrosis buildup. Semin Nephrol, 2003,23(6)532-543
8 Nichosas SB, Advances in pathogenetic mechanisms of diabetic nephropathy.Cell Mol Biol,2003,49(8)1319-1325
9 Ziyadeh FN,Hoffman BB,Han DC, et a.l Long-term prevention of renal insufficiency, excessmatrix gene expression, and glomerular mesangial matrix expansion by treatmentwith monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice. Poc Natl Acad Sci USA, 2000,97:8015-8020
10 Luukko K, YlikorkalaA,MakelaTP·Developmentally regulated expression of Smad3, Smad4, Smad6, and Smad7 involved in TGF-beta signaling·Mech Dev, 2001, 101(1-2):209-212
11 Itoh S, Itoh F,DaiC, eta.l Signaling of transforming growth factor-beta familymembers through Smad proteins J. Eur J Biochem, 2000, 267(24):6954-6967
12 Vrljicak P,MyburghD,RyanAK, eta.l Smad expression during kindey development. Am J Physiol Renal Physiol ,2004,286:625-633
13 Liliana Attisano. Signal transduction by the TGF - beta superfamily. Science ,2002 ,296(5573) :1646-1647
14 Schnaper HW,Hayashida T,Poncelet AC.It’s a smad word: Regulation of TGF-βsignaling in the kidney[J].J Am Soc Nephrol,2002,13(4):1126-1128
15 Runyan CE,Schnaper HW,Poncelet AC.Smad3 and PKC deltamediateTGF-beta1-induced collagenⅠexpression in human mesangial cells[J].Am J Physiol Renal Physiol,2003,285(3):F413-422
16 Hayashi H, Abdollah S, Qiu Y, et al·The MAD-related protein Smad7 associates with the TGF-βreceptor and functions as an antagonist of TGF-beta signaling·Cell, 1997, 89(7): 1165-1173
17 Kim HW,KimBC ,Song CY,et al . Heterozygous mice for TGF - beta IIR gene are resistant to the progression of streptozotocin-induced diabetic nephropathy. Kidney Int ,2004 ,66 (5) :1859-1865
18 Ellis LR ,Warner DR ,Greene RM,et al . Interaction of Smads with collagen types I、III and V. Biochem Biophys Res Commun ,2003 ,310 (4) :1117-1123
19 Poncelet AC ,Schnaper HW. Sp1 and smad proteins cooperate to mediate transforming growth factor-β1- inducedα2 (Ⅰ) collagen expressiom in human glomerular mesangial cell . J Bio Chem,2001 ,276 (10) :6983 -6992
20 Kanamaru Y,Nakao A ,Tanaka Y,et al . Involvement of p300 in TGF-beta/ Smad pathway mediated alpha2 ( I) collagen expression in mouse mesangial cells. Nephron Exp Nephrol ,2003 ,95(1) :36-42
21 Singh R ,Song RH ,Alavi N ,et al . High glucose decreases matrix metalloproteinase-2 activity in rat mesangial cells via transforming growth factor - beta (1) . Exp Nephrol ,2001 ,9(4) :249-257
22 Hall MC ,Young DA ,Waters JG,et al . The comparative role of activator protein 1 and Smad factors in the regulation of TIMP - 1 and MMP-1 gene expression by transforming growth factor - beta 1. J Biol Chem, 2003 ,278 (12) :10304 - 10313
23 Rerolle JP , Hertig A ,Nguyen G, et al . Plasminogen activator inhibitor type 1 is a potential target in renal fibrogenesis. Kidney Int, 2000, 58(5): 1841-1850
24 Li JH ,Zhu HJ ,Huang XR ,et al . Smad 7 inhibits fibrotic effect of TGF -beta on renal tubular epithelial cells by blocking smad 2 activaiton. J Am Soc Nephrol ,2002 ,13 (6) :1464 - 1472
25 Terada Y,Hanada S ,Nakao A ,et al . Gene transfer of Smad7 usingelectroporation of adenovirus prevents renal fibrosis in post - obstructed kidney. Kidney Int ,2002 ,1 (Supll) :94-98
26 Guh JY,Chuang TD ,Chen HC ,et al . Beta - hydroxybutyrate– induced growth inhibition and collagen production in HK- 2 cells are dependent on TGF - beta and Smad 3. Kidney Int ,2003 ,64 (6) :2041-2051
27 Hu B ,Wu Z ,Phan SH. Smad3 mediates transforming growth factor - beta- induced alpha - smooth muscle actin expression. AmJ Respir Cell Mol Biol ,2003 ,29 (3 Pt 1) :397-404
28 Strutz F ,Muller GA. Interstitial pathomechanisms underlying progressive tubulointerstitial damage. Kidney Blood Press Res ,1999 ,22 :71-80
1 Keane WF,Zhang Z,Lyle PA,et al.Risk scores for predicting outcomes in patients with type 2 diabetes and nephropathy;the RENAAL study[J].Clin J Am Soc Nephrol,2006 (1);761-767
2向红丁,糖尿病肾脏病变.国外医学·内分泌学分册,2004 24 (2);125
3 Dikow R,Browatzeki M,Adamczak M,et al.Handbook of Nutrition and the Kidney[M].4rd ed.Philadelphia;lippincott Williama&Wilkins,2002.157
4 CollinsAJ,Kasiske B,HerzogC, et al United States renal data system 2006 annual data report abstract Am J Kidney Dis, 2007 49;6
5 F.Blicklé, J.Doucet, T.Krummel,et al Diabetes in the elderly Part 2 [J]Diabetes & Metabolism, 200733(4); S40-S55
6叶山东,朱禧星.临床糖尿病学.合肥;安徽科学技术出版社,2005.194
7 R.D.Toto, M.Tian, K.Fakouhi, A.Champion, Effects of calcium channel blockers on proteinuria in patients with diabetic nephropathy, [J] Clin Hypertens (Greenwich) 2008 (10); 761-769
8 Baba S,The J-Mind Study Group. Nifedipine and enalapril equaly reduce the progression of nephropathy in hypertensive type 2 diabetes[J].Diabetes Res Clin Pract,2001,54;191-201
9 Danesh FR,Sadeghi M M,Amro N,et a1.3-Hydroxy-3-methyl-glutaryl CoAreductase inhibitors prevent high glucose-induced proliferation of mesangial cells via modulation of Rho GTPase/p21 signaling pathway;Implications for diabetic nephropathy [J]. Proc NatAcad Sci, 2002(11);8301-8305
10 Yokota N,ODonnellM,DanMsF,ct a1.Protective effect of HMG~ CoAreductase inhibitor on experimental renal ischemia-reperfusion injury [J]. Am J Nephrol, 2003 (23);13-17
11谌贻璞.糖尿病肾病的诊断与治疗.西藏医药杂志, 2004, 25(1): 24-27
12 Body-White J,Williams JC.Effect of cross-linking on matrix permeability;a model for AGE-modified basement membranes.Kidney Int,1996 46 (3);348-353
13 G. Remmuzzi, D. Cattaneo and N. Perico, The aggravating mechanisms of aldosterone on kidney fibrosis,[J]Am Soc Nephrol 2008 (29);1459-1462
14 S.Y. Han, C.H. Kim, H.S. Kim, Y.H. Jee, H.K. Song and M.H. Lee et al., Spironolactone prevents diabetic nephropathy through an anti-inflammatory mechanism in type 2 diabetic rats, [J] Am Soc Nephrol 2006 (17);1362-1372
15 Y.S. Kang, G.J. Ko, M.H. Lee, H.K. Song, S.Y. Han and K.H. Han et al., Effect of eplerenone, enalapril and their combination treatment on diabetic nephropathy in type II diabetic rats, Nephrol Dial Transplant 2009 (24);73-84
16 Galkina E, Ley K.Leukocyte recruitment and vascular injury in diabetic nephropathy. J Am Soc Nephrol, 2006 17 (2);368-377
17 Richard E, Gilbert, MarkE.Cooper. The tubulo interstitium in progressive diabetic kidney disease; More than an aftermath of glomerular injury Kidney Int 1999 56 (5); 1627-1637
18亢秋云,张晓东,方敬爱等霉酚酸酯对糖尿病肾病肾组织Nephrin表达的影响[J]中国中西医结合肾病杂志2008 9(8);700-701
19叶任高.中西医结合肾脏病学[M].北京;人民卫生出版社,2003; 305-307
20张珍先.糖尿病肾病的辨证论治[J].光明中医,2006, 21 (1);16
21南征.消渴肾病(糖尿病肾病)研究[M].吉林;吉林科学技术出版社, 2001; 3
22仝小林,张志远.糖尿病肾病的中医治疗[J].中国医药学报,1998,13(4);50-51
23林兰,等.糖尿病中西医结合论治.第1版.北京科学技术出版社1992; 229
24吕仁和.糖尿病及其并发症中西医诊治学.第1版.北京;人民卫生出版社,1997.328
25周保林,柏喜林,庞莉.贺永清主任医师治疗糖尿病肾病经验[J].陕西中医学院学报, 2004, 27(3): 14-15
26张建伟.糖尿病肾病中医分期辨证论治体会[J].陕西中医.2007, 28(11): 1571-1572
27陈延强,史伟,黄玉茵糖尿病肾病中医分期辨治的探讨[J].新中医2009 41(3) 3-4
28石咏军,王川,刘冠贤.雷公藤多苷治疗早中期糖尿病肾病的临床研究[J].现代中西医结合杂志,2006,4,15(8):987-988.
29樊瑞芬.黄芪改善肾病综合征患者血液高凝状态的临床观察[J].中国中西医结合肾病杂志,2001,2(11):673-675
30王光浩,张敬芳,杨雪琴.黄芪注射液治疗糖尿病肾病的实验研究[J].微循环学杂志,2007,17(1):20-22
31杜鹃,王琼书,贾汝汉.大黄对肾硬化大鼠肾小球细胞增殖和p27蛋白表达的影响[J].医药导报,2005,4(4):278-281
32陈湘东.川芎嗪治疗糖尿病微量白蛋白尿疗效观察[J]右江医学.2005,33(5):498-499
33林柳如,丹参注射液对血液流变学的影响[J].中医药研究,1998, 14(5):13
34徐东波,张军平,张文志.丹参注射液对血管内皮细胞氧化损伤保护作用的实验研究[J].天津医科大学学报, 2001, 7(1):21-23
35圈启芳.银杏达莫注射液治疗早期糖尿病肾病临床观察[J].临床荟萃, 2003, 18(18): 1059-1060
36郭登洲,王月华,陈志强.红花黄色素粉针与贝那普利联合治疗糖尿病肾病39例疗效观察[J]中国中西医结合杂志2008 28(4)360-363
