Megsin在糖尿病肾病发病机制中的作用
|
摘要
目的:糖尿病肾病(diabetic nephropathy, DN)是糖尿病最常见、最严重的慢性微血管并发症。随着全球范围内糖尿病(diabetes mellitus,DM)发病率逐年升高,糖尿病肾病逐渐成为导致终末期肾衰竭(end-stage renal failure,ESRF)的首位或主要病因,患者最终只能依靠透析或肾移植维持生命,严重影响了生存质量,给家庭和社会带来沉重的经济负担和精神压力。如何早期有效防治糖尿病肾病是当今国内外学者热切关注的课题。
糖尿病肾病的病理特征是肾小球系膜细胞增生、肥大,细胞外基质积聚,最终发生肾小球硬化。肾小球系膜细胞(glomerular mesangial cell,GMC)是肾脏重要的固有细胞(inherent cell)之一,在肾脏的组织结构和生理功能方面发挥着重要作用,具有产生细胞外基质、分泌细胞因子、吞噬和清除异物等多种功能。肾小球系膜细胞也是多种致病因子作用的主要靶细胞,在病变进展中扮演着重要角色。Megsin是一种系膜细胞优势表达基因,定位于18q21.3,编码蛋白N末端可变反应活性环(reactive site loop,RSL)与丝氨酸蛋白酶结合,发挥丝氨酸蛋白酶抑制剂(serine protease inhibitor,serpin)活性。Serpin家族成员众多,功能涉及凝血、纤溶、炎症、细胞增殖、凋亡、信号转导、消化等多个系统,serpin与其作用底物丝氨酸蛋白酶之间的动态平衡与机体各项生理活动的正常进行息息相关,推测megsin作为serpin的成员之一,势必参与系膜细胞的某些生理活动,探讨megsin在肾小球系膜细胞中的作用对于深入揭示糖尿病肾病的发生机制具有重要意义。
本研究拟通过临床和基础研究两方面,观察megsin在糖尿病状态下肾组织中的表达变化;分别构建megsin表达质粒和megsin siRNA表达质粒,转染CD-1小鼠和小鼠肾脏系膜细胞,从整体、细胞和分子水平探讨megsin与系膜细胞增殖程度、细胞外基质代谢关键酶基质金属蛋白酶-2(matrix metalloproteinase,MMP-2)/组织型抑制剂-2(tissue inhibitor of metalloproteinase,TIMP-2)以及细胞周期抑制蛋白P27水平之间的关系,探讨megsin在糖尿病肾病早期的致病机制;通过Sprague-dawlay(SD)大鼠糖尿病模型干预实验,观察羟甲基戊二酰辅酶A(Ahydroxy-methyl-glutaryl coenzyme A,HMG-CoA)还原酶抑制剂(HMG-CoA reductase inhibitor , HCRI)对肾组织megsin表达的影响,最终来阐明megsin参与早期糖尿病肾病发生的作用,筛选从基因或蛋白水平阻断megsin致病作用的有效途径,为进一步阐明糖尿病肾病发病的分子机制及其防治提供一条新思路。方法:
第一部分:从我院肾内科2006年6月至2007年6月住院患者中选择符合1999年WHO糖尿病诊断标准的2型糖尿病患者60例和原发性肾病综合征患者38例,经肾活检病理检查并结合临床资料确诊糖尿病肾病18例和微小病变性肾小球病,确定为研究对象,另外5例正常肾组织(选自我院泌尿外科肾癌患者手术切除的肾脏病灶远周部分)作为正常对照组,进行免疫组织化学染色,并半定量分析各组肾组织标本中megsin的表达。
第二部分:建立单侧肾切除+链脲佐菌素诱导的CD-1小鼠糖尿病模型,构建megsin表达质粒并经尾静脉注射转染小鼠(C组,n=15),每周1次,同时设立单纯单侧肾切除组(A组,n=15)和单侧肾切除+糖尿病+空质粒转染组(B组,n=15),实验共12周,分别于第1、2和12周末收集各组小鼠血、尿和肾组织标本,测定血糖(blood glucose,BG)、血肌酐(serum creatinine,Scr)、肾重/体重比值(Kidney weight/body weight ratio,KW/BW)、尿蛋白(Urinary protein,UP),分别应用免疫组织化学染色和western blot测定肾组织中megsin、MMP-2、TIMP-2、P27和IV型胶原的表达;建立稳定表达megsin的小鼠系膜细胞株(D组),高糖环境中培养48小时,同时设立野生型小鼠系膜细胞正常糖培养组(A组)、高糖培养组(B组)和megsin质粒对照+高糖培养组(C组),分别于12、24和48小时末收集各组细胞及上清,提取蛋白,应用western blot测定各组总蛋白中megsin、MMP-2、TIMP-2和P27的表达,放免法测定细胞上清液中IV型胶原浓度(结果用总蛋白校正)。
第三部分:动物模型制备同第二部分,构建megsin siRNA表达质粒并经尾静脉注射转染糖尿病小鼠(C组,n=15),每周1次,同时设立单纯单侧肾切除组(A组,n=15)和单侧肾切除+糖尿病+空质粒转染组(B组,n=15),实验共12周,分别于第1、2和12周末收集各组小鼠血、尿和肾组织标本,测定血糖(blood glucose,BG)、血肌酐(serumcreatinine,Scr)、肾重/体重比值(Kidney weight/body weight ratio,KW/BW)、尿蛋白(Urinary protein,UP),分别应用免疫组织化学染色和western blot测定肾组织中megsin、MMP-2、TIMP-2、P27和IV型胶原的表达;体外培养小鼠系膜细胞,应用脂质体2000瞬时转染megsin siRNA质粒,高糖环境中培养48小时,同时设立小鼠系膜细胞正常糖培养组、高糖培养组和空质粒+高糖培养组,分别于12、24和48小时末收集各组细胞及上清,提取蛋白,应用western blot测定各组总蛋白中megsin、MMP-2、TIMP-2和P27的表达,放免法测定细胞上清液中IV型胶原浓度(结果用总蛋白校正)。
第四部分:建立链脲佐菌素诱导的SD大鼠糖尿病模型,给予氟伐他汀干预(2mg·kg-1·d-1灌胃,DF组,n=6),同时设立糖尿病对照组(DC组,n=6)和正常对照组(NC组,n=6),于实验第6周末用代谢笼收集各组大鼠血、尿及肾组织标本,测定BG、血清总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、血肌酐(serum creatinine,Scr)和尿肌酐(urine creatinine,Ucr),并计算肌酐清除率(creatinine clearance rate,Ccr),结果用体重校正,放免法测定24h尿白蛋白排泄率(urine albumin excretion rate,UAER),免疫组织化学染色半定量分析各组大鼠肾组织megsin、IV型胶原和层黏连蛋白(laminin,LN)的表达,并行肾组织过碘酸-希夫(periodic acid-schiff,PAS)染色,普通光镜下观察肾小球病理学改变。
结果:
第一部分:正常肾组织和微小病变性肾小球病患者肾组织中megsin在肾小球系膜区无表达或仅微弱表达,两组间差异无显著性(p>0.05),而在糖尿病肾病患者肾组织肾小球表达增强,与其他两组比较差异有显著性(p<0.01)。
第二部分:
1.动物实验糖尿病小鼠成模4～5天后出现多饮、多食、多尿表现;第1周末B组小鼠肾小球固有细胞数目增多,免疫组化和western blot结果显示肾小球megsin、TIMP-2、P27和IV型胶原表达增强,MMP-2表达减弱,C组变化更显著,三组间差异有显著性意义(p<0.05);第2周末上述各指标变化更为明显,三组间比较有显著性差异(p<0.05);第12周末B组小鼠肾小球固有细胞数目减少,与A组无差异(p>0.05),P27表达水平回落,但仍高于A组(p<0.05),与C组无差异(p>0.05),其他指标如megsin、TIMP-2和IV型胶原进一步增强,但仍弱于C组,MMP-2表达水平进一步降低,但仍高于C组(p<0.05)。第12周末UP、Scr和KW/BW比值在A组最低,在C组最高,B组次之,三组间差异有显著性意义(P<0.05)。
2.细胞培养从12h开始,B组较A组小鼠系膜细胞megsin、P27和TIMP-2蛋白水平开始升高,48h达到高峰,48h时MMP-2水平降至最低(P<0.05),C组和B组相比差异无显著性(P>0.05),但D组变化趋势更明显,与B组和C组相比差异有显著性意义(P<0.05);从12h开始,B组小鼠系膜细胞上清液中IV型胶原浓度(细胞总蛋白校正)较A组开始升高,48h达到高峰(P<0.05),C组与B组相比差异无显著性(P>0.05),但D组系膜细胞上清液中IV型胶原浓度升高最明显,与其他组相比差异有显著性意义(P<0.05)。
第三部分:
1.动物实验糖尿病小鼠成模4～5天后出现与第二部分类似的糖尿病症状;第1周末B组小鼠与A组相比,肾小球固有细胞数目增多,免疫组化和western blot结果显示肾小球megsin、TIMP-2、P27和IV型胶原表达增强,MMP-2表达减弱,C组上述各指标介于A组和B组之间,三组间差异有显著性意义(p<0.05);第2周末B组小鼠肾小球固有细胞数目、组织中megsin、MMP-2、TIMP-2、P27和IV型胶原表达变化更明显,C组上述各指标介于A组和B组之间,三组间比较有显著性差异(p<0.05);第12周末B组小鼠肾小球固有细胞数目减少,P27表达水平下降,但二者仍高于A组或C组(p<0.05),其他指标如megsin、TIMP-2和IV型胶原进一步增强,MMP-2表达水平进一步降低,C组上述各指标介于A组和B组之间,三组间差异有显著性意义(p<0.05)。第12周末UP、Scr和KW/BW比值在A组最低,在B组最高,C组次之,三组间差异有显著性意义(P<0.05)。
2.细胞培养从12h开始,B组较A组小鼠系膜细胞megsin、P27和TIMP-2蛋白水平开始升高,48h达到高峰,48h时MMP-2水平降至最低(P<0.05),C组和B组相比差异无显著性(P>0.05);D组与B组或C组同期相比,megsin、P27和TIMP-2蛋白水平降低,MMP-2升高,差异有显著性意义(P<0.05);从12h开始,B组小鼠系膜细胞上清液中IV型胶原浓度(细胞总蛋白浓度校正)较A组开始升高,48h达到高峰(P<0.05),C组与B组相比差异无显著性(P>0.05);D组与C组相比,系膜细胞上清液中IV型胶原浓度降低,差异有显著性意义(P<0.05)。第四部分:实验第6周末DF组和DC组大鼠血糖无差异,但均高于NC组(P<0.01);DC组大鼠肾小球嗜PAS阳性物轻度增多,固有细胞数目增多,血清中TC、TG、Ccr、UAER、肾脏/体重比值以及免疫组化结果显示组织中megsin、IV型胶原和层黏连蛋白水平高于NC组,DF组上述指标低于DC组,但高于NC组,三组间差异有显著性意义(P<0.05)。
结论:
1.临床和实验研究表明,糖尿病状态下megsin在肾小球表达增强,与肾小球系膜增殖、细胞外基质积聚相一致,提示megsin在糖尿病肾病发生机制中起一定作用。
2.体内和体外实验证实,过表达megsin可上调肾组织或系膜细胞TIMP-2、P27的表达,下调MMP-2的表达,提示megsin促发糖尿病肾病发生的机制与MMP-2/TIMPs和P27有关,但具体作用途径有待进一步研究阐明。
3. megsin siRNA质粒和HMG-CoA还原酶抑制剂可分别从基因水平和蛋白水平下调megsin的表达,进而下调TIMP-2和P27、上调MMP-2在肾组织中的表达来延缓系膜增殖、细胞外基质积聚病理过程,为人类糖尿病肾病的防治提供了新思路。
Objective: Diabetic nephropathy (DN) is the most common and serious chronic microvascular complications of Diabetes mellitus (DM). With the increased incidence of DM in the whole world, DN gradually becomes the major or first cause of end-stage renal failure (ESRF). Patients have to rely on dialysis or kidney transplantation to live, which seriously affected the quality of life and brought about a heavy financial burden and stress to the family and the community. How to effectively prevent and treat early diabetic nephropathy is currently a very hot subject concerned by scholars at home and abroad.
Pathological features of DN are the glomerular mesangial cell proliferation, hypertrophy, extracellular matrix accumulation, and ultimately occurring glomerular sclerosis. Glomerular mesangial cel(lGMC)is one of the important inherent cells in kidney. GMCs play an important role in maintaining the structure and function of the glomerulus, by producing extracellular matrix, secreting cytokines, swallowing and removing foreign bodies and so on. GMCs are the major target cells to a variety of pathogenic factors and play an important role in the lesions progress. Megsin is a mesangium-predominant gene, located in 18q21.3, which encodes protein N-terminal variable reactive site loop (RSL) binding with the serine protease, and plays a serine protease inhibitor (serpin) activity. Serpin family is composed of a large number of members, their functions relate to blood coagulation, fibrinolysis, inflammation, cell proliferation, apoptosis, signal transduction, digestive and other systems. The dynamic balance between serpin and their substrates is closely related to the normal physiological activities in the body. We speculate that megsin, as a member of the serpin family, participates in certain physical activities of mesangial cells. To discuss megsin’s important roles in glomerular mesangial cells was greatly significant to reveal the mechanism of diabetic nephropathy.
In this study we observed the megsin expression in clinical renal biopsy sections,renal tissues of diabetic animals and GMCs in vitro. We Constructed megsin siRNA expression plasmid and megsin cDNA expression plasmid, transfected them to STZ-induced diabetic mice and cultured mesangial cells, then investigated the relationship between megsin and mesangial cell proliferation, matrix metalloproteinase (MMP-2)/tissue inhibitor of metalloproteinase (TIMP-2), and Cell cycle inhibitor P27, to discuss the possible roles of megsin in the pathogenesis of early DN. We also observated the effect of hydroxymethyl-Coenzyme A(Ahydroxy-methyl-glutaryl coenzyme A, HMG-CoA) reductase inhibitors (HMG-CoA reductase inhibitor, HCRI) on megsin expression in STZ-induced diabetic rats. We finally explained the roles of megsin in early DN, which may provide an effective way in blocking pathogenic role of megsin in the levels of gene or protein, and may also provide a new idea to further elucidate the molecular pathogenesis of DN and prevent or treat DN.
Methods:
Part 1: 60 diabetes patients in line with the diagnostic criteria for type 2 diabetes of American Diabetes Academy in 1998 and 38 cases of primary nephrotic syndrome were screened out from the department of nephrology in 3rd hospital of Hebei Medical Universty(HBMU) from June 2006 to June 2007.18 cases in diabetic nephropathy and 29 cases in minimal change glomerulopathy were confirmed by renal biopsy and clinical data as research subjects.In addition, five control cases were received from the normal tissues around the resected renal tumors of hospitalized patients in 3rd hospital of HBMU.The expression of megsin in renal tissue of every group was semi-quantitatively analysed by immunohistochemical staining.
Part 2: In vivo experiment:after 2 weeks of uninephrectomy, 12-week-old male CD-1 mice were divided into three groups: a non-diabetic control group (A group,n=15), a diabetic group administered a pCMVsport6.1 control plasmid (B group,n=15), and a diabetic group administered a pCMVsport6.1 megsin cDNA plasmid (C group,n=15).The mice received a single intravenous dose of 150 mg/kg streptozotocin in citrate buffer to induce diabetes. A group received the same dose of citrate buffer. Hyperglycemia (>15 mmol/L) was confirmed 3 days after STZ administration. Plasmids were then delivered weekly by tail vein injection using the TransIT-EE Hydrodynamic Delivery System each week. Animals were sacrificed at week-1, week-2 and week-12. At each time point, urine and blood samples were collected, and kidney tissues were harvested. Blood glucose (blood glucose, BG), serum creatinine (serum creatinine, Scr), kidney weight / body weight ratio (Kidney weight / body weight ratio, KW / BW) , urinary protein (Urinary protein, UP) were measured.Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy and immunohistochemistry. The paraformaldehyde-fixed and paraffin-embedded kidney tissues were cut into sections of 2-4μm thick. 2μm sections were stained with Hematoxylin and Eosin (HE). 4μm sections were used for immunohistochemistry staining. HE staining were used to observe the pathological changes. Immunohistochemistry was used to analyze the renal expression of megsin, MMP-2, TIMP-2, P27 and collagen IV. The expression of megsin, MMP-2, TIMP-2, P27 and collagen IV were also detected by Western blotting. In vitro experiments:Mouse mesangial cells were grown in DMEM F12 containing 5% FBS, penicillin (100 U/ml), streptomycin (100μg/ml) at 37°C and 5% CO2. The cells were divided into 4 groups: a normal glucose group (A), a high glucose group (B), high glucose transfected a pBAsi mU6 Neo megain siRNA control plasmid group (C), and high glucose transfected a pCMVsport6.1 megsin cDNA plasmid group (D). When the cells grown to 80% confluence,transplanted the cells to 6-well culture plates (2×106 cells per well). After 24 hours, B group, C group and D goup cells were further cultured in DMEM F12 containing high glucose and A group cells were cultured in normal glucose for up to 48 hours. Cells in 6-well culture plates were collected for protein extraction and the culture medium were collected for Collagen IV measurement at hour-12,hour-24 and hour-48.The levels of megsin,MMP-2,TIMP-2 and P27 in GMCs were measured by Western blot and supernatant Collagen IV were measured by RIA.All of the data was analysed by SPSS15.0 statistics software, P value<0.05 was considered to have a statistical significance.
Part 3:The preparation of animal models is the same as the second part. 12-week-old male CD-1 mice were divided into three groups: a non-diabetic control group (A group,n=15), a diabetic group administered a pBAsi mU6 Neo control plasmid (B group,n=15), and a diabetic group administered a pBAsi mU6 Neo megsin siRNA plasmid (C group,n=15).The mice received a single intravenous dose of 150 mg/kg streptozotocin in citrate buffer to induce diabetes. A group received the same dose of citrate buffer. Hyperglycemia (>15 mmol/L) was confirmed 3 days after STZ administration. Plasmids were then delivered weekly by tail vein injection using the TransIT-EE Hydrodynamic Delivery System each week. Animals were sacrificed at week-1, week-2 and week-12. At each time point, urine and blood samples were collected, and kidney tissues were harvested. Blood glucose (blood glucose, BG), serum creatinine (serum creatinine, Scr), kidney weight / body weight ratio (Kidney weight / body weight ratio, KW / BW) , urinary protein (Urinary protein, UP) were measured.Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy and immunohistochemistry. The paraformaldehyde-fixed and paraffin-embedded kidney tissues were cut into sections of 2-4μm thick. 2μm sections were stained with Hematoxylin and Eosin (HE). 4μm sections were used for immunohistochemistry staining. HE staining were used to observe the pathological changes. Immunohistochemistry was used to analyze the renal expression of megsin, MMP-2, TIMP-2, P27 and collagen IV. The expression of megsin, MMP-2, TIMP-2, P27 and collagen IV were also detected by Western blotting. In vitro experiments: Mouse mesangial cells were grown in DMEM F12 containing 5% FBS, penicillin (100 U/ml), streptomycin (100μg/ml) at 37°C and 5% CO2. The cells were divided into 4 groups: a normal glucose group (A), a high glucose group (B), high glucose transfected a pBAsi mU6 Neo control plasmid group (C), and high glucose transfected a pBAsi mU6 Neo megsin siRNA plasmid group (D). When the cells grown to 80% confluence,transplanted the cells to 6-well culture plates (2×106 cells per well).Cells were cultured without antibiotics for 24 hours , then were transfected with pBAsi mU6 Neo megsin siRNA plasmid or pBAsi mU6 Neo control plasmid using lipofectamine 2000 reagent.After 24 hours, B group, C group and D goup cells were further cultured in DMEM F12 containing high glucose and A group cells were cultured in normal glucose for up to 48 hours. Cells in 6-well culture plates were collected for protein extraction and the culture medium were collected for Collagen IV measurement at hour-12,hour-24 and hour-48.The levels of megsin,MMP-2,TIMP-2 and P27 in GMCs were measured by Western blot and supernatant Collagen IV were measured by RIA.All of the data was analysed by SPSS15.0 statistics software, P value<0.05 was considered to have a statistical significance.
Part4: The male SD rats(weight about 200+/-10g) were divided into three groups: a non-diabetic control group (NC,n=6), a diabetic group(DC,n=6), and a diabetic group treated with fluvastatin(DF,n=6,2mg·kg-1·d-1,ig). The animals of DC and DF groups received a single intravenous dose of 65 mg/kg streptozotocin in citrate buffer to induce diabetes. NC group received the same dose of citrate buffer.Hyperglycemia (>15 mmol/L) was confirmed 3 days after STZ administration. The animals were sacrificed at 6 week. The urine and blood samples were collected, and kidney tissues were harvested. Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy and immunohistochemistry. The paraformaldehyde-fixed and paraffin-embedded kidney tissues were cut into sections of 2-4μm thick. 2μm sections were stained with periodic acid-Schiff (PAS). 4μm sections were used for immunohistochemistry and immune-double staining. HE and PAS staining were used to observe the pathological changes. Immunohistochemistry was used to analyze the renal expression of megsin,collagen IV and laminin(LN). BG, serum total cholesterol (TC) , triglyceride (TG), serum creatinine (Scr) and urine creatinine (Ucr), creatinine clearance rate (Ccr) and hypertrophy index(the ratio of kidney weight :body weight) were evaluated in every group. All the data were analysed by SPSS15.0 statistics software, P value<0.05 was considered to have statistical significance.
Results:
Part1:megsin expressed none or only weakly in mesangial area of renal tissue of A group and B group.There wasn’t a significant difference between two groups (p> 0.05).But megsin was strengthened significantly in the renal tissues of diabetic nephropathy patients compared with the other two groups(p<0.01).
Part2:In vivo experiment:diabetic mice occurred polydipsia,polyuria and polyphagia at 4 or 5 days after being injected to STZ.At week-1,the number of glomerular inherent cells in B group mice was more than in A group(p<0.05) but less than in C group(p<0.05).The results of immunohistochemistry and Western blot showed that the levels of megsin,TIMP-2,P27 and collagen IV were higher than A group(p<0.05),lower than C group(p<0.05) and MMP-2 lower than A group(p<0.05) but higher than C group(p<0.05).At week-2 all of the results were changed more markedly and there was the significant difference among three groups(p<0.05).The number of glomerular inherent cells of group B decreased at week-12, there is no difference compared with A group (p> 0.05), the expression level of P27 dropped, but still higher than the A group (p <0.05), and no difference compared with group C(p> 0.05), the other parameters such as megsin,TIMP-2 and type IV collagen further enhanced,but still weaker than C group,MMP-2 expression level of further lower, but still higher than C group (p<0.05). At week-12 UP, Scr and KW / BW ratio are the lowest in the A group,the highest in the C group,followed by B group,there are significant differences among the three groups(P<0.05).In vitro experiment:Since hour-12,the protein levels of megsin, P27, and TIMP-2 of mesangial cells in groupB began to increase,peaked at hour-48,the level of MMP-2 was the most lowest at hour-48(P<0.05),there was no significant difference between C group and B group (P>0.05),but the trend in D group is more obvious,the difference was significant compared with the B group and C group(P<0.05);since hour-12,the concentration of supernatant collagen IV (cell total protein correction) became higher in B group than in A group,peaked at hour-48(P<0.05),there is no significant difference between C group and B group (P> 0.05),but the concentration of type IV collagen in D group increased most obviously,there is a significant difference compared with the other groups(P <0.05).
Part3:In vivo experiment:A similar symptoms of diabetes with the second part was appeared.The number of glomerular inherent cells in B group increased,comparing with A group at week-1,the results of immunohistochemistry and Western blot showed that the expression of megsin,TIMP-2,P27and type IV collagen strengthened, MMP-2’s expression decreased.The above-mentioned indicators in C group expressed between A group and B group,there was a significant difference among three groups(p <0.05).The number of glomerular inherent cells, the expression levels of megsin,MMP-2,TIMP-2,P27 and type IV collagen at week-2 were more significantly different,the above indicators in C group expressed between A group and B group,there were significantly difference among the three groups(p<0.05).The number of glomerular inherent cells decreased in B group at week-12,the expression levels of P27 decreased,but still higher than in A group and C group (p<0.05),the expression of other indicators such as megsin, TIMP-2 and type IV collagen further enhanced,the expression level of MMP-2 further reduced,the expression of above-mentioned indicators in C group was between A group B group,there was significant difference among the three groups(p<0.05).UP,Scr and KW/BW ratio were in the lowest in the A group,in the highest in B group,followed by C group at week-12,the differences among the three groups was significant(P<0.05).In vitro experiment:Since hour-12,the protein levels of megsin, P27, and TIMP-2 in mice mesangial cells began to increase in B group than A goup, peaked at hour-48,the level of MMP-2 dropped to the lowest at hour-48(P<0.05),there was no significant difference between C group and B group (P>0.05);the protein level of megsin,P27 and TIMP-2 reduced in D group,comparing with B group or C group,MMP-2 increased, there was a significant difference(P<0.05);from the start of hour-12,the concentrations of type IV collagen (cell total protein concentration correction)started to rise in B group than in A group,peaked at hour-48(P<0.05),there was no significant difference between B group and C group (P>0.05); comparing with C group,the concentration of collagen type IV in D group was lower,the difference was significant(P<0.05). Part4:There was no difference about blood glucose between DC group and DF group at week-6,but higher than NC group (P<0.01);the PAS-positive material on rats glomerular increased mildly at DC group,the number of inherent cells incresed,serum TC,TG,Ccr,UAER,kidney/body weight ratio increased,as well as immunohistochemical results showed that the expression levels of megsin, IV collagen and laminin was higher than at NC group,the above-mentioned indicators in DF group was lower than at DC group, but higher than at NC group,the difference among three groups was significant(P<0.05).
Conclusions:
I.Clinical and experimental studies have shown that the expression of megsin in the glomerular strengthen at diabetic state,consisting with mesangial proliferation,extracellular matrix accumulation and suggesting that megsin plays an important role in the mechanism of the occurrence of diabetic nephropathy.
II.The experiments in vivo and in vitro have confirmed that over-expressed megsin may increase the expression levels of TIMP-2, P27 in renal mesangial cells,but decrease the expression of MMP-2,suggesting that megsin promotes the mechanism of early diabetic nephropathy and concerned with MMP-2/TIMPs and P27.However the specific ways have to be further studied.
III.Megsin siRNA plasmid and HMG-CoA reductase inhibitor can decrease the expression level of megsin in the gene level and protein level, reduce the expression of TIMP-2 and P27 and increase the expression of MMP-2 in renal tissue,then retard the pathological process of mesangial proliferation and extracellular matrix accumulation,which provides a new idea for prevention and treatment of diabetic nephropathy.
糖尿病肾病的病理特征是肾小球系膜细胞增生、肥大,细胞外基质积聚,最终发生肾小球硬化。肾小球系膜细胞(glomerular mesangial cell,GMC)是肾脏重要的固有细胞(inherent cell)之一,在肾脏的组织结构和生理功能方面发挥着重要作用,具有产生细胞外基质、分泌细胞因子、吞噬和清除异物等多种功能。肾小球系膜细胞也是多种致病因子作用的主要靶细胞,在病变进展中扮演着重要角色。Megsin是一种系膜细胞优势表达基因,定位于18q21.3,编码蛋白N末端可变反应活性环(reactive site loop,RSL)与丝氨酸蛋白酶结合,发挥丝氨酸蛋白酶抑制剂(serine protease inhibitor,serpin)活性。Serpin家族成员众多,功能涉及凝血、纤溶、炎症、细胞增殖、凋亡、信号转导、消化等多个系统,serpin与其作用底物丝氨酸蛋白酶之间的动态平衡与机体各项生理活动的正常进行息息相关,推测megsin作为serpin的成员之一,势必参与系膜细胞的某些生理活动,探讨megsin在肾小球系膜细胞中的作用对于深入揭示糖尿病肾病的发生机制具有重要意义。
本研究拟通过临床和基础研究两方面,观察megsin在糖尿病状态下肾组织中的表达变化;分别构建megsin表达质粒和megsin siRNA表达质粒,转染CD-1小鼠和小鼠肾脏系膜细胞,从整体、细胞和分子水平探讨megsin与系膜细胞增殖程度、细胞外基质代谢关键酶基质金属蛋白酶-2(matrix metalloproteinase,MMP-2)/组织型抑制剂-2(tissue inhibitor of metalloproteinase,TIMP-2)以及细胞周期抑制蛋白P27水平之间的关系,探讨megsin在糖尿病肾病早期的致病机制;通过Sprague-dawlay(SD)大鼠糖尿病模型干预实验,观察羟甲基戊二酰辅酶A(Ahydroxy-methyl-glutaryl coenzyme A,HMG-CoA)还原酶抑制剂(HMG-CoA reductase inhibitor , HCRI)对肾组织megsin表达的影响,最终来阐明megsin参与早期糖尿病肾病发生的作用,筛选从基因或蛋白水平阻断megsin致病作用的有效途径,为进一步阐明糖尿病肾病发病的分子机制及其防治提供一条新思路。方法:
第一部分:从我院肾内科2006年6月至2007年6月住院患者中选择符合1999年WHO糖尿病诊断标准的2型糖尿病患者60例和原发性肾病综合征患者38例,经肾活检病理检查并结合临床资料确诊糖尿病肾病18例和微小病变性肾小球病,确定为研究对象,另外5例正常肾组织(选自我院泌尿外科肾癌患者手术切除的肾脏病灶远周部分)作为正常对照组,进行免疫组织化学染色,并半定量分析各组肾组织标本中megsin的表达。
第二部分:建立单侧肾切除+链脲佐菌素诱导的CD-1小鼠糖尿病模型,构建megsin表达质粒并经尾静脉注射转染小鼠(C组,n=15),每周1次,同时设立单纯单侧肾切除组(A组,n=15)和单侧肾切除+糖尿病+空质粒转染组(B组,n=15),实验共12周,分别于第1、2和12周末收集各组小鼠血、尿和肾组织标本,测定血糖(blood glucose,BG)、血肌酐(serum creatinine,Scr)、肾重/体重比值(Kidney weight/body weight ratio,KW/BW)、尿蛋白(Urinary protein,UP),分别应用免疫组织化学染色和western blot测定肾组织中megsin、MMP-2、TIMP-2、P27和IV型胶原的表达;建立稳定表达megsin的小鼠系膜细胞株(D组),高糖环境中培养48小时,同时设立野生型小鼠系膜细胞正常糖培养组(A组)、高糖培养组(B组)和megsin质粒对照+高糖培养组(C组),分别于12、24和48小时末收集各组细胞及上清,提取蛋白,应用western blot测定各组总蛋白中megsin、MMP-2、TIMP-2和P27的表达,放免法测定细胞上清液中IV型胶原浓度(结果用总蛋白校正)。
第三部分:动物模型制备同第二部分,构建megsin siRNA表达质粒并经尾静脉注射转染糖尿病小鼠(C组,n=15),每周1次,同时设立单纯单侧肾切除组(A组,n=15)和单侧肾切除+糖尿病+空质粒转染组(B组,n=15),实验共12周,分别于第1、2和12周末收集各组小鼠血、尿和肾组织标本,测定血糖(blood glucose,BG)、血肌酐(serumcreatinine,Scr)、肾重/体重比值(Kidney weight/body weight ratio,KW/BW)、尿蛋白(Urinary protein,UP),分别应用免疫组织化学染色和western blot测定肾组织中megsin、MMP-2、TIMP-2、P27和IV型胶原的表达;体外培养小鼠系膜细胞,应用脂质体2000瞬时转染megsin siRNA质粒,高糖环境中培养48小时,同时设立小鼠系膜细胞正常糖培养组、高糖培养组和空质粒+高糖培养组,分别于12、24和48小时末收集各组细胞及上清,提取蛋白,应用western blot测定各组总蛋白中megsin、MMP-2、TIMP-2和P27的表达,放免法测定细胞上清液中IV型胶原浓度(结果用总蛋白校正)。
第四部分:建立链脲佐菌素诱导的SD大鼠糖尿病模型,给予氟伐他汀干预(2mg·kg-1·d-1灌胃,DF组,n=6),同时设立糖尿病对照组(DC组,n=6)和正常对照组(NC组,n=6),于实验第6周末用代谢笼收集各组大鼠血、尿及肾组织标本,测定BG、血清总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、血肌酐(serum creatinine,Scr)和尿肌酐(urine creatinine,Ucr),并计算肌酐清除率(creatinine clearance rate,Ccr),结果用体重校正,放免法测定24h尿白蛋白排泄率(urine albumin excretion rate,UAER),免疫组织化学染色半定量分析各组大鼠肾组织megsin、IV型胶原和层黏连蛋白(laminin,LN)的表达,并行肾组织过碘酸-希夫(periodic acid-schiff,PAS)染色,普通光镜下观察肾小球病理学改变。
结果:
第一部分:正常肾组织和微小病变性肾小球病患者肾组织中megsin在肾小球系膜区无表达或仅微弱表达,两组间差异无显著性(p>0.05),而在糖尿病肾病患者肾组织肾小球表达增强,与其他两组比较差异有显著性(p<0.01)。
第二部分:
1.动物实验糖尿病小鼠成模4～5天后出现多饮、多食、多尿表现;第1周末B组小鼠肾小球固有细胞数目增多,免疫组化和western blot结果显示肾小球megsin、TIMP-2、P27和IV型胶原表达增强,MMP-2表达减弱,C组变化更显著,三组间差异有显著性意义(p<0.05);第2周末上述各指标变化更为明显,三组间比较有显著性差异(p<0.05);第12周末B组小鼠肾小球固有细胞数目减少,与A组无差异(p>0.05),P27表达水平回落,但仍高于A组(p<0.05),与C组无差异(p>0.05),其他指标如megsin、TIMP-2和IV型胶原进一步增强,但仍弱于C组,MMP-2表达水平进一步降低,但仍高于C组(p<0.05)。第12周末UP、Scr和KW/BW比值在A组最低,在C组最高,B组次之,三组间差异有显著性意义(P<0.05)。
2.细胞培养从12h开始,B组较A组小鼠系膜细胞megsin、P27和TIMP-2蛋白水平开始升高,48h达到高峰,48h时MMP-2水平降至最低(P<0.05),C组和B组相比差异无显著性(P>0.05),但D组变化趋势更明显,与B组和C组相比差异有显著性意义(P<0.05);从12h开始,B组小鼠系膜细胞上清液中IV型胶原浓度(细胞总蛋白校正)较A组开始升高,48h达到高峰(P<0.05),C组与B组相比差异无显著性(P>0.05),但D组系膜细胞上清液中IV型胶原浓度升高最明显,与其他组相比差异有显著性意义(P<0.05)。
第三部分:
1.动物实验糖尿病小鼠成模4～5天后出现与第二部分类似的糖尿病症状;第1周末B组小鼠与A组相比,肾小球固有细胞数目增多,免疫组化和western blot结果显示肾小球megsin、TIMP-2、P27和IV型胶原表达增强,MMP-2表达减弱,C组上述各指标介于A组和B组之间,三组间差异有显著性意义(p<0.05);第2周末B组小鼠肾小球固有细胞数目、组织中megsin、MMP-2、TIMP-2、P27和IV型胶原表达变化更明显,C组上述各指标介于A组和B组之间,三组间比较有显著性差异(p<0.05);第12周末B组小鼠肾小球固有细胞数目减少,P27表达水平下降,但二者仍高于A组或C组(p<0.05),其他指标如megsin、TIMP-2和IV型胶原进一步增强,MMP-2表达水平进一步降低,C组上述各指标介于A组和B组之间,三组间差异有显著性意义(p<0.05)。第12周末UP、Scr和KW/BW比值在A组最低,在B组最高,C组次之,三组间差异有显著性意义(P<0.05)。
2.细胞培养从12h开始,B组较A组小鼠系膜细胞megsin、P27和TIMP-2蛋白水平开始升高,48h达到高峰,48h时MMP-2水平降至最低(P<0.05),C组和B组相比差异无显著性(P>0.05);D组与B组或C组同期相比,megsin、P27和TIMP-2蛋白水平降低,MMP-2升高,差异有显著性意义(P<0.05);从12h开始,B组小鼠系膜细胞上清液中IV型胶原浓度(细胞总蛋白浓度校正)较A组开始升高,48h达到高峰(P<0.05),C组与B组相比差异无显著性(P>0.05);D组与C组相比,系膜细胞上清液中IV型胶原浓度降低,差异有显著性意义(P<0.05)。第四部分:实验第6周末DF组和DC组大鼠血糖无差异,但均高于NC组(P<0.01);DC组大鼠肾小球嗜PAS阳性物轻度增多,固有细胞数目增多,血清中TC、TG、Ccr、UAER、肾脏/体重比值以及免疫组化结果显示组织中megsin、IV型胶原和层黏连蛋白水平高于NC组,DF组上述指标低于DC组,但高于NC组,三组间差异有显著性意义(P<0.05)。
结论:
1.临床和实验研究表明,糖尿病状态下megsin在肾小球表达增强,与肾小球系膜增殖、细胞外基质积聚相一致,提示megsin在糖尿病肾病发生机制中起一定作用。
2.体内和体外实验证实,过表达megsin可上调肾组织或系膜细胞TIMP-2、P27的表达,下调MMP-2的表达,提示megsin促发糖尿病肾病发生的机制与MMP-2/TIMPs和P27有关,但具体作用途径有待进一步研究阐明。
3. megsin siRNA质粒和HMG-CoA还原酶抑制剂可分别从基因水平和蛋白水平下调megsin的表达,进而下调TIMP-2和P27、上调MMP-2在肾组织中的表达来延缓系膜增殖、细胞外基质积聚病理过程,为人类糖尿病肾病的防治提供了新思路。
Objective: Diabetic nephropathy (DN) is the most common and serious chronic microvascular complications of Diabetes mellitus (DM). With the increased incidence of DM in the whole world, DN gradually becomes the major or first cause of end-stage renal failure (ESRF). Patients have to rely on dialysis or kidney transplantation to live, which seriously affected the quality of life and brought about a heavy financial burden and stress to the family and the community. How to effectively prevent and treat early diabetic nephropathy is currently a very hot subject concerned by scholars at home and abroad.
Pathological features of DN are the glomerular mesangial cell proliferation, hypertrophy, extracellular matrix accumulation, and ultimately occurring glomerular sclerosis. Glomerular mesangial cel(lGMC)is one of the important inherent cells in kidney. GMCs play an important role in maintaining the structure and function of the glomerulus, by producing extracellular matrix, secreting cytokines, swallowing and removing foreign bodies and so on. GMCs are the major target cells to a variety of pathogenic factors and play an important role in the lesions progress. Megsin is a mesangium-predominant gene, located in 18q21.3, which encodes protein N-terminal variable reactive site loop (RSL) binding with the serine protease, and plays a serine protease inhibitor (serpin) activity. Serpin family is composed of a large number of members, their functions relate to blood coagulation, fibrinolysis, inflammation, cell proliferation, apoptosis, signal transduction, digestive and other systems. The dynamic balance between serpin and their substrates is closely related to the normal physiological activities in the body. We speculate that megsin, as a member of the serpin family, participates in certain physical activities of mesangial cells. To discuss megsin’s important roles in glomerular mesangial cells was greatly significant to reveal the mechanism of diabetic nephropathy.
In this study we observed the megsin expression in clinical renal biopsy sections,renal tissues of diabetic animals and GMCs in vitro. We Constructed megsin siRNA expression plasmid and megsin cDNA expression plasmid, transfected them to STZ-induced diabetic mice and cultured mesangial cells, then investigated the relationship between megsin and mesangial cell proliferation, matrix metalloproteinase (MMP-2)/tissue inhibitor of metalloproteinase (TIMP-2), and Cell cycle inhibitor P27, to discuss the possible roles of megsin in the pathogenesis of early DN. We also observated the effect of hydroxymethyl-Coenzyme A(Ahydroxy-methyl-glutaryl coenzyme A, HMG-CoA) reductase inhibitors (HMG-CoA reductase inhibitor, HCRI) on megsin expression in STZ-induced diabetic rats. We finally explained the roles of megsin in early DN, which may provide an effective way in blocking pathogenic role of megsin in the levels of gene or protein, and may also provide a new idea to further elucidate the molecular pathogenesis of DN and prevent or treat DN.
Methods:
Part 1: 60 diabetes patients in line with the diagnostic criteria for type 2 diabetes of American Diabetes Academy in 1998 and 38 cases of primary nephrotic syndrome were screened out from the department of nephrology in 3rd hospital of Hebei Medical Universty(HBMU) from June 2006 to June 2007.18 cases in diabetic nephropathy and 29 cases in minimal change glomerulopathy were confirmed by renal biopsy and clinical data as research subjects.In addition, five control cases were received from the normal tissues around the resected renal tumors of hospitalized patients in 3rd hospital of HBMU.The expression of megsin in renal tissue of every group was semi-quantitatively analysed by immunohistochemical staining.
Part 2: In vivo experiment:after 2 weeks of uninephrectomy, 12-week-old male CD-1 mice were divided into three groups: a non-diabetic control group (A group,n=15), a diabetic group administered a pCMVsport6.1 control plasmid (B group,n=15), and a diabetic group administered a pCMVsport6.1 megsin cDNA plasmid (C group,n=15).The mice received a single intravenous dose of 150 mg/kg streptozotocin in citrate buffer to induce diabetes. A group received the same dose of citrate buffer. Hyperglycemia (>15 mmol/L) was confirmed 3 days after STZ administration. Plasmids were then delivered weekly by tail vein injection using the TransIT-EE Hydrodynamic Delivery System each week. Animals were sacrificed at week-1, week-2 and week-12. At each time point, urine and blood samples were collected, and kidney tissues were harvested. Blood glucose (blood glucose, BG), serum creatinine (serum creatinine, Scr), kidney weight / body weight ratio (Kidney weight / body weight ratio, KW / BW) , urinary protein (Urinary protein, UP) were measured.Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy and immunohistochemistry. The paraformaldehyde-fixed and paraffin-embedded kidney tissues were cut into sections of 2-4μm thick. 2μm sections were stained with Hematoxylin and Eosin (HE). 4μm sections were used for immunohistochemistry staining. HE staining were used to observe the pathological changes. Immunohistochemistry was used to analyze the renal expression of megsin, MMP-2, TIMP-2, P27 and collagen IV. The expression of megsin, MMP-2, TIMP-2, P27 and collagen IV were also detected by Western blotting. In vitro experiments:Mouse mesangial cells were grown in DMEM F12 containing 5% FBS, penicillin (100 U/ml), streptomycin (100μg/ml) at 37°C and 5% CO2. The cells were divided into 4 groups: a normal glucose group (A), a high glucose group (B), high glucose transfected a pBAsi mU6 Neo megain siRNA control plasmid group (C), and high glucose transfected a pCMVsport6.1 megsin cDNA plasmid group (D). When the cells grown to 80% confluence,transplanted the cells to 6-well culture plates (2×106 cells per well). After 24 hours, B group, C group and D goup cells were further cultured in DMEM F12 containing high glucose and A group cells were cultured in normal glucose for up to 48 hours. Cells in 6-well culture plates were collected for protein extraction and the culture medium were collected for Collagen IV measurement at hour-12,hour-24 and hour-48.The levels of megsin,MMP-2,TIMP-2 and P27 in GMCs were measured by Western blot and supernatant Collagen IV were measured by RIA.All of the data was analysed by SPSS15.0 statistics software, P value<0.05 was considered to have a statistical significance.
Part 3:The preparation of animal models is the same as the second part. 12-week-old male CD-1 mice were divided into three groups: a non-diabetic control group (A group,n=15), a diabetic group administered a pBAsi mU6 Neo control plasmid (B group,n=15), and a diabetic group administered a pBAsi mU6 Neo megsin siRNA plasmid (C group,n=15).The mice received a single intravenous dose of 150 mg/kg streptozotocin in citrate buffer to induce diabetes. A group received the same dose of citrate buffer. Hyperglycemia (>15 mmol/L) was confirmed 3 days after STZ administration. Plasmids were then delivered weekly by tail vein injection using the TransIT-EE Hydrodynamic Delivery System each week. Animals were sacrificed at week-1, week-2 and week-12. At each time point, urine and blood samples were collected, and kidney tissues were harvested. Blood glucose (blood glucose, BG), serum creatinine (serum creatinine, Scr), kidney weight / body weight ratio (Kidney weight / body weight ratio, KW / BW) , urinary protein (Urinary protein, UP) were measured.Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy and immunohistochemistry. The paraformaldehyde-fixed and paraffin-embedded kidney tissues were cut into sections of 2-4μm thick. 2μm sections were stained with Hematoxylin and Eosin (HE). 4μm sections were used for immunohistochemistry staining. HE staining were used to observe the pathological changes. Immunohistochemistry was used to analyze the renal expression of megsin, MMP-2, TIMP-2, P27 and collagen IV. The expression of megsin, MMP-2, TIMP-2, P27 and collagen IV were also detected by Western blotting. In vitro experiments: Mouse mesangial cells were grown in DMEM F12 containing 5% FBS, penicillin (100 U/ml), streptomycin (100μg/ml) at 37°C and 5% CO2. The cells were divided into 4 groups: a normal glucose group (A), a high glucose group (B), high glucose transfected a pBAsi mU6 Neo control plasmid group (C), and high glucose transfected a pBAsi mU6 Neo megsin siRNA plasmid group (D). When the cells grown to 80% confluence,transplanted the cells to 6-well culture plates (2×106 cells per well).Cells were cultured without antibiotics for 24 hours , then were transfected with pBAsi mU6 Neo megsin siRNA plasmid or pBAsi mU6 Neo control plasmid using lipofectamine 2000 reagent.After 24 hours, B group, C group and D goup cells were further cultured in DMEM F12 containing high glucose and A group cells were cultured in normal glucose for up to 48 hours. Cells in 6-well culture plates were collected for protein extraction and the culture medium were collected for Collagen IV measurement at hour-12,hour-24 and hour-48.The levels of megsin,MMP-2,TIMP-2 and P27 in GMCs were measured by Western blot and supernatant Collagen IV were measured by RIA.All of the data was analysed by SPSS15.0 statistics software, P value<0.05 was considered to have a statistical significance.
Part4: The male SD rats(weight about 200+/-10g) were divided into three groups: a non-diabetic control group (NC,n=6), a diabetic group(DC,n=6), and a diabetic group treated with fluvastatin(DF,n=6,2mg·kg-1·d-1,ig). The animals of DC and DF groups received a single intravenous dose of 65 mg/kg streptozotocin in citrate buffer to induce diabetes. NC group received the same dose of citrate buffer.Hyperglycemia (>15 mmol/L) was confirmed 3 days after STZ administration. The animals were sacrificed at 6 week. The urine and blood samples were collected, and kidney tissues were harvested. Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy and immunohistochemistry. The paraformaldehyde-fixed and paraffin-embedded kidney tissues were cut into sections of 2-4μm thick. 2μm sections were stained with periodic acid-Schiff (PAS). 4μm sections were used for immunohistochemistry and immune-double staining. HE and PAS staining were used to observe the pathological changes. Immunohistochemistry was used to analyze the renal expression of megsin,collagen IV and laminin(LN). BG, serum total cholesterol (TC) , triglyceride (TG), serum creatinine (Scr) and urine creatinine (Ucr), creatinine clearance rate (Ccr) and hypertrophy index(the ratio of kidney weight :body weight) were evaluated in every group. All the data were analysed by SPSS15.0 statistics software, P value<0.05 was considered to have statistical significance.
Results:
Part1:megsin expressed none or only weakly in mesangial area of renal tissue of A group and B group.There wasn’t a significant difference between two groups (p> 0.05).But megsin was strengthened significantly in the renal tissues of diabetic nephropathy patients compared with the other two groups(p<0.01).
Part2:In vivo experiment:diabetic mice occurred polydipsia,polyuria and polyphagia at 4 or 5 days after being injected to STZ.At week-1,the number of glomerular inherent cells in B group mice was more than in A group(p<0.05) but less than in C group(p<0.05).The results of immunohistochemistry and Western blot showed that the levels of megsin,TIMP-2,P27 and collagen IV were higher than A group(p<0.05),lower than C group(p<0.05) and MMP-2 lower than A group(p<0.05) but higher than C group(p<0.05).At week-2 all of the results were changed more markedly and there was the significant difference among three groups(p<0.05).The number of glomerular inherent cells of group B decreased at week-12, there is no difference compared with A group (p> 0.05), the expression level of P27 dropped, but still higher than the A group (p <0.05), and no difference compared with group C(p> 0.05), the other parameters such as megsin,TIMP-2 and type IV collagen further enhanced,but still weaker than C group,MMP-2 expression level of further lower, but still higher than C group (p<0.05). At week-12 UP, Scr and KW / BW ratio are the lowest in the A group,the highest in the C group,followed by B group,there are significant differences among the three groups(P<0.05).In vitro experiment:Since hour-12,the protein levels of megsin, P27, and TIMP-2 of mesangial cells in groupB began to increase,peaked at hour-48,the level of MMP-2 was the most lowest at hour-48(P<0.05),there was no significant difference between C group and B group (P>0.05),but the trend in D group is more obvious,the difference was significant compared with the B group and C group(P<0.05);since hour-12,the concentration of supernatant collagen IV (cell total protein correction) became higher in B group than in A group,peaked at hour-48(P<0.05),there is no significant difference between C group and B group (P> 0.05),but the concentration of type IV collagen in D group increased most obviously,there is a significant difference compared with the other groups(P <0.05).
Part3:In vivo experiment:A similar symptoms of diabetes with the second part was appeared.The number of glomerular inherent cells in B group increased,comparing with A group at week-1,the results of immunohistochemistry and Western blot showed that the expression of megsin,TIMP-2,P27and type IV collagen strengthened, MMP-2’s expression decreased.The above-mentioned indicators in C group expressed between A group and B group,there was a significant difference among three groups(p <0.05).The number of glomerular inherent cells, the expression levels of megsin,MMP-2,TIMP-2,P27 and type IV collagen at week-2 were more significantly different,the above indicators in C group expressed between A group and B group,there were significantly difference among the three groups(p<0.05).The number of glomerular inherent cells decreased in B group at week-12,the expression levels of P27 decreased,but still higher than in A group and C group (p<0.05),the expression of other indicators such as megsin, TIMP-2 and type IV collagen further enhanced,the expression level of MMP-2 further reduced,the expression of above-mentioned indicators in C group was between A group B group,there was significant difference among the three groups(p<0.05).UP,Scr and KW/BW ratio were in the lowest in the A group,in the highest in B group,followed by C group at week-12,the differences among the three groups was significant(P<0.05).In vitro experiment:Since hour-12,the protein levels of megsin, P27, and TIMP-2 in mice mesangial cells began to increase in B group than A goup, peaked at hour-48,the level of MMP-2 dropped to the lowest at hour-48(P<0.05),there was no significant difference between C group and B group (P>0.05);the protein level of megsin,P27 and TIMP-2 reduced in D group,comparing with B group or C group,MMP-2 increased, there was a significant difference(P<0.05);from the start of hour-12,the concentrations of type IV collagen (cell total protein concentration correction)started to rise in B group than in A group,peaked at hour-48(P<0.05),there was no significant difference between B group and C group (P>0.05); comparing with C group,the concentration of collagen type IV in D group was lower,the difference was significant(P<0.05). Part4:There was no difference about blood glucose between DC group and DF group at week-6,but higher than NC group (P<0.01);the PAS-positive material on rats glomerular increased mildly at DC group,the number of inherent cells incresed,serum TC,TG,Ccr,UAER,kidney/body weight ratio increased,as well as immunohistochemical results showed that the expression levels of megsin, IV collagen and laminin was higher than at NC group,the above-mentioned indicators in DF group was lower than at DC group, but higher than at NC group,the difference among three groups was significant(P<0.05).
Conclusions:
I.Clinical and experimental studies have shown that the expression of megsin in the glomerular strengthen at diabetic state,consisting with mesangial proliferation,extracellular matrix accumulation and suggesting that megsin plays an important role in the mechanism of the occurrence of diabetic nephropathy.
II.The experiments in vivo and in vitro have confirmed that over-expressed megsin may increase the expression levels of TIMP-2, P27 in renal mesangial cells,but decrease the expression of MMP-2,suggesting that megsin promotes the mechanism of early diabetic nephropathy and concerned with MMP-2/TIMPs and P27.However the specific ways have to be further studied.
III.Megsin siRNA plasmid and HMG-CoA reductase inhibitor can decrease the expression level of megsin in the gene level and protein level, reduce the expression of TIMP-2 and P27 and increase the expression of MMP-2 in renal tissue,then retard the pathological process of mesangial proliferation and extracellular matrix accumulation,which provides a new idea for prevention and treatment of diabetic nephropathy.
引文
1 Rychlík I.Epidemiology of diabetic nephropathy.Vnitr Lek,2008,54(5):488-93
2 Iseki K.Epidemiology of CKD in Japan.Nippon Rinsho,2008 ,66(9):1650-6
3 Miyata T,Nangaku M, Suzuki D,et al.A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy.J Clin Invest,1998, 1209(4):828-836
4 Miyata T,Ming Li,Xueqing Yu,et al. Megsin gene: Its genomic analysis, pathobiological functions, and therapeutic perspectives.Current Genomics, 2007,8(3):203-208
5 Miyata T,Inagi R,Nangaku M,et al.Overexpression of the serpin megsin induces progressive mesangial cell proliferation and expansion.J Clin Invest,2002,109(5):585–593
6 Giunti S, Barit D, Cooper ME.Mechanisms of diabetic nephropathy: role of hypertension.Hypertension.2006,48(4):519-26
7 Miyata T,Ming Li,Xueqing Yu,et al. Megsin gene: Its genomic analysis, pathobiological functions, and therapeutic perspectives.Current Genomics, 2007;8(3):203-208
8陈楠,王伟铭,俞海瑾,等.肾小球肾炎肾组织megsin的表达及其意义.肾脏病与透析肾移植杂志,2003;12(5):412-415
1 Rudnicki M, Mayer G.Significance of genetic polymorphisms of the renin-angiotensin-aldosterone system in cardiovascular and renal disease. Pharmacogenomics,2009,10(3):463-76
2 Berrou J, Tostivint I, Verrecchia F,et al. Advanced glycation end products regulate extracellular matrix protein and protease expression by human glomerular mesangial cells. Int J Mol Med,2009,23(4):513-20
3 Fukami K,Ueda S,Yamagishi S,et al.AGEs Activate Mesangial TGF-beta- Smad Signaling Via an AngiotensinⅡTypeⅠReceptor Interaction. Kidney Int,2004,66(6):2137-2147
4 Ha H,Hwang IA,Park JH,et al.Role of reactive oxygen species in the pathogenesis of diabetic nephropathy.Diabetes Res Clin Pract,2008,82 Suppl 1:S42-5
5 Balakumar P,Chakkarwar VA,Krishan P,et al.Vascular endothelial dysfunction:a tug of war in diabetic nephropathy? Biomed Pharmacother, 2009,63(3):171-9
6 Maeda S.Genetics of diabetic nephropathy. Ther Adv Cardiovasc Dis,2008,2(5):363-71
7 Rogus JJ, Poznik GD, Pezzolesi MG,et al. High-density single nucleotide polymorphism genome-wide linkage scan for susceptibility genes for diabetic nephropathy in type 1 diabetes: discordant sibpair approach. Diabetes,2008,57(9):2519-26
8 Ahluwalia TS, Ahuja M, Rai TS,et al. ACE Variants Interact with the RAS Pathway to Confer Risk and Protection against Type 2 Diabetic Nephropathy. DNA Cell Biol,2009,28(3):141-50
9 Makni K, Jarraya F, Reba? M,et al. Risk genotypes and haplotypes of the GLUT1 gene for type 2 diabetic nephropathy in the Tunisian population. Ann Hum Biol,2008,35(5):490-8
10 Rudofsky G Jr, Schlotterer A, Reismann P,et al.The -174G>C IL-6 gene promoter polymorphism and diabetic microvascular complications.Horm Metab Res,2009,41(4):308-13
11 Miyata T,Ming Li,Xueqing Yu,et al.Megsin gene:Its genomic analysis,pathobiological functions,and therapeutic perspectives.Current Genomics, 2007,8(3):203-208
12 Miyata T,Nangaku M, Suzuki D,et al.A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy.J Clin Invest,1998,1209(4):828-836
13 Ohtomo S,Nangaku M,Izuhara Y,et al.The role of megsin, a serine protease inhibitor,in diabetic mesangial matrix accumulation.Kidney Int,2008,74(6):768-774
14 Wolf G,Schanze A,Stahl RA,et al.p27(Kip1) Knockout mice are protected from diabetic nephropathy: evidence for p27(Kip1) haplotype insufficiency. Kidney Int,2005,68(4):1583-9
15 Rysz J,Banach M,Stolarek RA,et al.Serum matrix metalloproteinases MMP-2 and MMP-9 and metalloproteinase tissue inhibitors TIMP-1 and TIMP-2 in diabetic nephropathy.J Nephrol,2007,20(4):444-52
16 Thrailkill KM, Bunn RC, Moreau CS,et al.Matrix metalloproteinase-2 dysregulation in type 1 diabetes.Diabetes Care,2007,30(9):2321-6
17 Han SY, Jee YH, Han KH,et al.An imbalance between matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 contributes to the development of early diabetic nephropathy.Nephrol Dial Transplant,2006,21(9):2406-16
18 Liang J,Shao SH,Xu ZX,et al.The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis.Nat Cell Biol,2007,9(2):218-24
19 Ding Z,Liang J,Lu Y,et al.A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners. Proc Natl Acad Sci USA,2006,103(41):15014-9
1 Rychlík I.Epidemiology of diabetic nephropathy.Vnitr Lek,2008,54(5):488-93
2 Iseki K.Epidemiology of CKD in Japan.Nippon Rinsho,2008 ,66(9):1650-6
3林善锬.糖尿病肾病.中华内科杂志,2005,44(3):229-231
4 Asci?-Buturovi? B, Kacila M, Kuli? M.Effects of aggressive approach to the multiple risk factors for diabetic nephro-pathy on proteinuria reduction in diabetes type 2 patients.Bosn J Basic Med Sci,2009,9(1):44-8
5 Volker V, Roland CB, Scott T, et al. Glomerular hyperfiltration and the salt paradox in rarly type 1 diabetes mellitus: a tubulo-centric view. J Am Soc Nephrol, 2003, 14(2): 530-537
6 Batlle D. Clinical and cellular markers of diabetic nephropathy.Kidney Int, 2003, 63(6): 2319-2330
7 Doi T, Mima A, Matsubara T,et al.The current clinical problems for early phase of diabetic nephropathy and approach for pathogenesis of diabetic nephropathy.Diabetes Res Clin Pract,2008,82 Suppl 1:S21-4
8 Pantaleo V, Szittya G, Burgyán J.Molecular bases of viral RNA targeting by viral small interfering RNA-programmed RISC.J Virol,2007,81(8):3797-806
9 Culver KW, Osborne WR, Miller AD, et al.Correction of ADA deficiency in human T lymphocytes using retroviral-mediated gene transfer.Transplant Proc,1991,23(1 Pt 1):170-1
10 Blaese RM.Progress toward gene therapy.Clin Immunol Immunopathol,1991,61(2 Pt 2):S47-55
11 Glorioso JC, Fink DJ.Gene therapy for pain: introduction to the special issue.Gene Ther,2009,16(4):453-4
12 Brown PA,Bodles-Brakhop AM,Pope MA,et al.Gene therapy by electroporation for the treatment of chronic renal failure in companion animals.BGMC Biotechnol,2009,16(9):4
13 Martínez Castelao A.Advances in diabetes mellitus, diabetic nephropathy, metabolic syndrome and cardio-vascular-renal risk.Nefrologia,2008,28(Suppl 5):79-84
14 Kawasaki H, Taira K.Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Nature,2004, 431 (7005) : 211-217
15 Wynter CV.The dialectics of cancer:A theory of the initiation and development of cancer through errors in RNAi.Med Hypotheses, 2006, 66 (3) : 612-635
16 Hammond SM,Bernstein E,Beach D, et al.An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.Nature, 2000,404(6775):293-296
17 Bernstein E,Caudy AA,Hammond SM, et al.Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature,2001, 409 (6818) :363-366
18 Ohtomo S,Nangaku M,Izuhara Y,et al.The role of megsin, a serine protease inhibitor,in diabetic mesangial matrix accumulation.Kidney Int,2008,74(6):768-774
1 Miyata T,Ming Li,Xueqing Yu,et al.Megsin gene:Its genomic analysis,pathobiological functions,and therapeutic perspectives.Current Genomics, 2007,8(3):203-208
2 Miyata T,Nangaku M, Suzuki D,et al.A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy.J Clin Invest,1998,1209(4):828-836
3 Ohtomo S,Nangaku M,Izuhara Y,et al.The role of megsin, a serine protease inhibitor,in diabetic mesangial matrix accumulation.Kidney Int,2008,74(6):768-774
4 Zachary T.Diabetic Nephropathy.Diabetes Care,2005,28(3):745-751
5卫生部疾病控制司,中华医学会糖尿病分会.中国糖尿病防治指南[M].北京:北京大学出版社,2004:3-4
6 Radbill B,Murphy B,LeRoith D.Rationale and strategies for early detection and management of diabetic kidney disease.Mayo Clin Proc,2008,83(12):1373-81
7 Cortinovis M, Cattaneo D, Perico N,et al.Investigational drugs for diabetic nephropathy.Expert Opin Investig Drugs,2008,17(10):1487-500
8 G?rtner V, Eigentler TK.Pathogenesis of diabetic macro- and microangiopathy.Clin Nephrol,2008,70(1):1-9
9 Miyata T,Nangaku M,Suzuki D,et al.A mesangium-predominantgene,megsin,is a new serpin upregulated in IgA nephropathy.J Clin Invest,1998,1209(4):828-836
10陈楠,王伟铭,俞海瑾,等.肾小球肾炎肾组织megsin的表达及其意义.肾脏病与透析肾移植杂志,2003,12(5):412-415
11 Ohtomo S,Nangaku M,Izuhara Y,et al.The role of megsin, a serine protease inhibitor,in diabetic mesangial matrix accumulation.Kidney Int,2008,74(6):768-74
12 Ceska R,Tesar V.Diabetes,dyslipidaemia and kidney diseases.Vnitr Lek,2008,54(5):511-7
13 Li H,Li X,Duan L,et al.Inhibition of lovastatin on proliferation and expression of proinflammatory cytokines in cultured human glomerular mesangial cells.Chin Med J(Engl),2003,116:1366-1369
14刘茂东,李英,迟彦青,等.2型糖尿病肾病患者血清脂蛋白(a)质量浓度与纤溶功能的相关性探讨.中国实用内科杂志,2004,10(4):237-8
15 Peng F,Wu D,Gao B,et al.RhoA/Rho-kinase contribute to the pathogenesis of diabetic renal disease.Diabetes,2008,57(6):1683-92
16 Kolavennu V,Zeng L,Peng H,et al.Targeting of RhoA/ROCK signaling ameliorates progression of diabetic nephropathy independent of glucose control.Diabetes,2008,57(3):714-23
17 Fujii M,Inoguchi T,Maeda Y,et al.Pitavastatin ameliorates albuminuria and renal mesangial expansion by downregulating NOX4 in db/db mice. Kidney Int,2007,72(4):473-80
18 Parsons RB,Price GC,Farrant JK,et al.Statins inhibit the dimerization of beta-secretase via both isoprenoid- and cholesterol-mediated mechanisms.Biochem J,2006,399(2):205-14
1 Caroline B. Marshall Stuart J. Cell Cycle and Glomerular Disease:A Minireview. Nephron Exp Nephrol 2006;102:e39–e48
2 Kawamura K, Kobayashi Y, Tanaka T, et al. Intracellular localization of proliferating cell nuclear antigen during the cell cycle in renal cell carcinoma.Anal Quant Cytol Histol,2000,20(2):107-113
3 Schocklmann H Q, Lang S, Stelzel R B. Regulation of mesangial cell proliferation. Kidney Int, 1999,66(4):1199~1207
4 Pan DA, Lillioja S, Milner MR, et al. Skeletal muscle membrane lipid composition is related to adiposity and in-sulin action. J Clin Invest, 1995,96(6):2802
5 Kurogi Y. Mesangial cell proliferation inhibitors for the treatment of proliferative glomerular disease. Med Res Rev, 2003,23(1):15
6丁炜.转化生长因子-β与肾脏疾病进展的关系.国外医学.泌尿系统分册,1997,17(6):273
7 Gressner A M, Weiskirchen R, Breitkopf K, et al. Roles of TGF-beta in hepatic fibrosis. Front Biosci,2002,7:793-807
8史伟,梁馨苓,刘双信,等.转化生长因子β反义RNA对肾小球GMC基质合成及分泌的影响.中华肾脏病杂志,2002,18(2):103-106
9 Frazier K, Williams S, Kothapalli D, et al. Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor, nvest Dematol, 1996,107(3):404-411
10 ItoY, Goldschmeding R, Bende R.et al. Kinetics of connective tissue growth factor expression during experimental proliferative glomerulonephritis. J Am Soc Nephrol, 2001,12(3):472-484
11 Yokoi H,Mukoyama M, Sugawara A, et al.Role of Connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis. Am J Physiol Renal Physiol,2002,282(5):933-942
12史伟.何朝生.刘双信.等.转化生长因子β1及Smad信号转导通路在肾小球硬化中的作用.中华肾脏病杂志,2005,21(5):270-273
13 Chen R, Huang C, Morinelli TA, et al. Blockade of the effects of TGF-beta 1 on mesangial cells by overexpression of Smad7. J Am Soc Nephrol, 2002,13(4):887-893
14王凯旋.洪小苏.转化生长因子β与Smad7在肾脏疾病中的作用.国外医学.内科学分册.2004,31(12):535-537
15 Inagi R,Nangaku M,Miyata T,et al.Mesangial cell-predominant functional gene, megsin. Clin Exp Nephrol,2003,7:87–92
16 Inagi R, Miyata T, Suzuki D, et al.Specific tissue distribution of megsin, a novel serpin, in the glomerulus and its up-regulation in IgA nephropathy. Biochem Biophys Res Commun 2001;286:1098–106
17 Nangaku M, Miyata T, Suzuki D, et al. Cloning of rodent megsin revealed its up-regulation in mesangioproliferative nephritis. Kidney Int 2001;60:641–52
18 Miyata T, Inagi R, Nangaku M,et al.Overexpression of the serpin megsin induces progressive mesangial cell proliferation and expansion. J Clin Invest 2002;109:585–93
19 Narayanan Parameswaran*, Wojciech Nowak, Carolyn S. Hall,et al. Cellular and molecular actions of adrenomedullin in glomerular mesangial cells. Peptides, 2001,22:1919–1924
20 Wang Pi,Takezawa T.Reconstruction of renal glomerular tissue using collagen vitrigel scaffold.Journal of bioscience and bioengineering.2005,99(6):529-40
1 Cove-Smith A, Hendry BM.The regulation of mesangial cell proliferation.Nephron Exp Nephrol,2008;108(4):e74-9
2 Miyata T,Nangaku M, Suzuki D,et al.A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy.J Clin Invest,1998, 1209(4):828-836
3 Miyata T,Li M,Yu X,et al.Megsin gene:its genomic analysis, pathobiological functions, and therapeutic perspectives.Current Genomics, 2007, 8(3):203-8
4 Lomas DA, Carrell RW.Serpinopathies and the conformational dementias. Nat Genet Rev, 2002,3: 759–767
5 Lomas DA, Mahadeva R.Alpha1-antitrypsin polymerization and the serpinopathies: Pathobiology and prospects for therapy.J Clin Invest,2002, 110:1585–1590
6 Davis RL, Shrimpton AE, Holohan PD, et al.Familial dementia caused by polymerization of mutant neuroserpin. Nature,1999,401(6751): 376–379
7 Benarafa C, Cooley J, Zeng W,et al.Characterization of four murine homologs of the human ov-serpin monocyte neutrophil elastase inhibitor MNEI (SERPINB1).J Biol Chem,2002,277(44):42028-33
8 Lee W, Haslinger A, Karin M,et al.Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40.Nature,1987,325: 368–372
9 Foletta VC, Segal DH, Cohen DR.Transcriptional regulation in the immune system: all roads lead to AP-1. J Leuk Biol,1998,63:139–152
10 Gewirtz AM, Anfossi G, Venturelli D, et al.G1/S transition in normal human T-lymphocyte requires the nuclear protein encoded by c-myb. Science,1989,245:180–183
11 Mucenski ML, McLain K, Kier AB, et al.A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis.Cell,1991,65: 677–689
12 Rosenfeld MG.POU-domain transcription factors: Powerful developmental regulators. Genes Dev,1991,5: 897–907
13 Guijarro C, Egido J.Transcription factor-kappa B (NF-κB) and renal disease. Kidney Int,2001,59: 415–424
14 Morrissey JJ, Klahr S.Transcription factor NF-κB regulation of renal fibrosis during ureteral obstruction.Semin Nephrol,1998,18:603–611
15 Inagi R,Miyata T,Nangaku M,et al.Transcriptional regulation of a mesangium-predominant gene, megsin.J Am Soc Nephrol,2002,13: 2715–2722
16 Evdokimova V, Ovchinnikov LP, Sorensen PH.Y-box binding protein 1: providing a new angle on translational regulation.Cell Cycle,2006,5(11):1143-7
17 Mertens PR, Harendza S, Pollock AS,et al.Glomerular mesangial cells-specific transactivation of matrix metalloproteinase-2 transcription is mediated by YB-1. J Biol Chem, 1997,272:22905–22912
18 Inagi R,Miyata T, Imasawa T,et al.Mesangial cell-predominant gene, megsin.Nephrol Dial Transplant,2002,17(Suppl 9):32–33
19 Nangaku M,Miyata T,Suzuki D,et al.Cloning of rodent megsin revealed its up-regulation in mesangioproliferative nephritis.Kidney International,2001,60 (pp):641–652
20 Miyata T,Inagi R,Nangaku M,et al.Overexpression of the serpin megsin induces progressive mesangial cell proliferation and expansion.J Clin Invest,2002,109(5):585–593
21 Inagi R,Yamamoto Y,Nangaku M,et al. A severe diabetic nephropathy model with early development of nodule-like lesions induced by megsin overexpression in RAGE/iNOS transgenic mice.Diabetes,2006,55:356-365
22 Schipper R, Loof A, de Groot J,et al.Salivary protein/peptide profiling with SELDI-TOF-MS.Ann N Y Acad Sci,2007,1098:498-503
23 Schipper R, Loof A, de Groot J,et al.SELDI-TOF-MS of saliva: methodology and pre-treatment effects.J Chromatogr B Analyt Technol Biomed Life Sci,2007,847(1):45-53
24 Ohtomo S,Nangaku M,Izuhara Y,et al.The role of megsin,a serine protease inhibitor,in diabetic mesangial matrix accumulation.Kidney Int,2008,74(6):768-74
25 Jewett MC, Calhoun KA, Voloshin A,et al.An integrated cell-free metabolic platform for protein production and synthetic biology.Mol Syst Biol,2008;4:220
26 Inagi R,Nangaku M,Usuda N,et al.Novel serpinopathy in rat kidney and pancreas induced by overexpression of megsin.J Am Soc Nephrol,2005,16: 1339–1349
27 Szelestei T, B?hring S, Kovács T,et al.Association of a uteroglobin polymorphism with rate of progression in patients with IgA nephropathy.Am J Kidney Dis,2000,36(3):468-73
28 Lim CS, Kim SM, Oh YK, et al.Megsin 2093T-2180C haplotype at the 3' untranslated region is associated with poor renal survival in Korean IgA nephropathy patients.Clin Nephrol,2008,70(2):101-9
29 MaixnerováD, Merta M, ReiterováJ, et al.The influence of two megsin polymorphisms on the progression of IgA nephropathy.Folia Biol (Praha),2008,54(2):40-5
30王朝晖,陈楠,潘晓霞等.Megsin基因单核苷酸多态性与IgA肾病相关性研究.中华医学杂志,2006,86(19):1337-1441
31 Xia YF, Huang S, Li X,et al.A family-based association study of megsin A23167G polymorphism with susceptibility and progression of IgA nephropathy in a Chinese population.Clin Nephrol,2006,65(3):153-9
2 Iseki K.Epidemiology of CKD in Japan.Nippon Rinsho,2008 ,66(9):1650-6
3 Miyata T,Nangaku M, Suzuki D,et al.A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy.J Clin Invest,1998, 1209(4):828-836
4 Miyata T,Ming Li,Xueqing Yu,et al. Megsin gene: Its genomic analysis, pathobiological functions, and therapeutic perspectives.Current Genomics, 2007,8(3):203-208
5 Miyata T,Inagi R,Nangaku M,et al.Overexpression of the serpin megsin induces progressive mesangial cell proliferation and expansion.J Clin Invest,2002,109(5):585–593
6 Giunti S, Barit D, Cooper ME.Mechanisms of diabetic nephropathy: role of hypertension.Hypertension.2006,48(4):519-26
7 Miyata T,Ming Li,Xueqing Yu,et al. Megsin gene: Its genomic analysis, pathobiological functions, and therapeutic perspectives.Current Genomics, 2007;8(3):203-208
8陈楠,王伟铭,俞海瑾,等.肾小球肾炎肾组织megsin的表达及其意义.肾脏病与透析肾移植杂志,2003;12(5):412-415
1 Rudnicki M, Mayer G.Significance of genetic polymorphisms of the renin-angiotensin-aldosterone system in cardiovascular and renal disease. Pharmacogenomics,2009,10(3):463-76
2 Berrou J, Tostivint I, Verrecchia F,et al. Advanced glycation end products regulate extracellular matrix protein and protease expression by human glomerular mesangial cells. Int J Mol Med,2009,23(4):513-20
3 Fukami K,Ueda S,Yamagishi S,et al.AGEs Activate Mesangial TGF-beta- Smad Signaling Via an AngiotensinⅡTypeⅠReceptor Interaction. Kidney Int,2004,66(6):2137-2147
4 Ha H,Hwang IA,Park JH,et al.Role of reactive oxygen species in the pathogenesis of diabetic nephropathy.Diabetes Res Clin Pract,2008,82 Suppl 1:S42-5
5 Balakumar P,Chakkarwar VA,Krishan P,et al.Vascular endothelial dysfunction:a tug of war in diabetic nephropathy? Biomed Pharmacother, 2009,63(3):171-9
6 Maeda S.Genetics of diabetic nephropathy. Ther Adv Cardiovasc Dis,2008,2(5):363-71
7 Rogus JJ, Poznik GD, Pezzolesi MG,et al. High-density single nucleotide polymorphism genome-wide linkage scan for susceptibility genes for diabetic nephropathy in type 1 diabetes: discordant sibpair approach. Diabetes,2008,57(9):2519-26
8 Ahluwalia TS, Ahuja M, Rai TS,et al. ACE Variants Interact with the RAS Pathway to Confer Risk and Protection against Type 2 Diabetic Nephropathy. DNA Cell Biol,2009,28(3):141-50
9 Makni K, Jarraya F, Reba? M,et al. Risk genotypes and haplotypes of the GLUT1 gene for type 2 diabetic nephropathy in the Tunisian population. Ann Hum Biol,2008,35(5):490-8
10 Rudofsky G Jr, Schlotterer A, Reismann P,et al.The -174G>C IL-6 gene promoter polymorphism and diabetic microvascular complications.Horm Metab Res,2009,41(4):308-13
11 Miyata T,Ming Li,Xueqing Yu,et al.Megsin gene:Its genomic analysis,pathobiological functions,and therapeutic perspectives.Current Genomics, 2007,8(3):203-208
12 Miyata T,Nangaku M, Suzuki D,et al.A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy.J Clin Invest,1998,1209(4):828-836
13 Ohtomo S,Nangaku M,Izuhara Y,et al.The role of megsin, a serine protease inhibitor,in diabetic mesangial matrix accumulation.Kidney Int,2008,74(6):768-774
14 Wolf G,Schanze A,Stahl RA,et al.p27(Kip1) Knockout mice are protected from diabetic nephropathy: evidence for p27(Kip1) haplotype insufficiency. Kidney Int,2005,68(4):1583-9
15 Rysz J,Banach M,Stolarek RA,et al.Serum matrix metalloproteinases MMP-2 and MMP-9 and metalloproteinase tissue inhibitors TIMP-1 and TIMP-2 in diabetic nephropathy.J Nephrol,2007,20(4):444-52
16 Thrailkill KM, Bunn RC, Moreau CS,et al.Matrix metalloproteinase-2 dysregulation in type 1 diabetes.Diabetes Care,2007,30(9):2321-6
17 Han SY, Jee YH, Han KH,et al.An imbalance between matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 contributes to the development of early diabetic nephropathy.Nephrol Dial Transplant,2006,21(9):2406-16
18 Liang J,Shao SH,Xu ZX,et al.The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis.Nat Cell Biol,2007,9(2):218-24
19 Ding Z,Liang J,Lu Y,et al.A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners. Proc Natl Acad Sci USA,2006,103(41):15014-9
1 Rychlík I.Epidemiology of diabetic nephropathy.Vnitr Lek,2008,54(5):488-93
2 Iseki K.Epidemiology of CKD in Japan.Nippon Rinsho,2008 ,66(9):1650-6
3林善锬.糖尿病肾病.中华内科杂志,2005,44(3):229-231
4 Asci?-Buturovi? B, Kacila M, Kuli? M.Effects of aggressive approach to the multiple risk factors for diabetic nephro-pathy on proteinuria reduction in diabetes type 2 patients.Bosn J Basic Med Sci,2009,9(1):44-8
5 Volker V, Roland CB, Scott T, et al. Glomerular hyperfiltration and the salt paradox in rarly type 1 diabetes mellitus: a tubulo-centric view. J Am Soc Nephrol, 2003, 14(2): 530-537
6 Batlle D. Clinical and cellular markers of diabetic nephropathy.Kidney Int, 2003, 63(6): 2319-2330
7 Doi T, Mima A, Matsubara T,et al.The current clinical problems for early phase of diabetic nephropathy and approach for pathogenesis of diabetic nephropathy.Diabetes Res Clin Pract,2008,82 Suppl 1:S21-4
8 Pantaleo V, Szittya G, Burgyán J.Molecular bases of viral RNA targeting by viral small interfering RNA-programmed RISC.J Virol,2007,81(8):3797-806
9 Culver KW, Osborne WR, Miller AD, et al.Correction of ADA deficiency in human T lymphocytes using retroviral-mediated gene transfer.Transplant Proc,1991,23(1 Pt 1):170-1
10 Blaese RM.Progress toward gene therapy.Clin Immunol Immunopathol,1991,61(2 Pt 2):S47-55
11 Glorioso JC, Fink DJ.Gene therapy for pain: introduction to the special issue.Gene Ther,2009,16(4):453-4
12 Brown PA,Bodles-Brakhop AM,Pope MA,et al.Gene therapy by electroporation for the treatment of chronic renal failure in companion animals.BGMC Biotechnol,2009,16(9):4
13 Martínez Castelao A.Advances in diabetes mellitus, diabetic nephropathy, metabolic syndrome and cardio-vascular-renal risk.Nefrologia,2008,28(Suppl 5):79-84
14 Kawasaki H, Taira K.Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Nature,2004, 431 (7005) : 211-217
15 Wynter CV.The dialectics of cancer:A theory of the initiation and development of cancer through errors in RNAi.Med Hypotheses, 2006, 66 (3) : 612-635
16 Hammond SM,Bernstein E,Beach D, et al.An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.Nature, 2000,404(6775):293-296
17 Bernstein E,Caudy AA,Hammond SM, et al.Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature,2001, 409 (6818) :363-366
18 Ohtomo S,Nangaku M,Izuhara Y,et al.The role of megsin, a serine protease inhibitor,in diabetic mesangial matrix accumulation.Kidney Int,2008,74(6):768-774
1 Miyata T,Ming Li,Xueqing Yu,et al.Megsin gene:Its genomic analysis,pathobiological functions,and therapeutic perspectives.Current Genomics, 2007,8(3):203-208
2 Miyata T,Nangaku M, Suzuki D,et al.A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy.J Clin Invest,1998,1209(4):828-836
3 Ohtomo S,Nangaku M,Izuhara Y,et al.The role of megsin, a serine protease inhibitor,in diabetic mesangial matrix accumulation.Kidney Int,2008,74(6):768-774
4 Zachary T.Diabetic Nephropathy.Diabetes Care,2005,28(3):745-751
5卫生部疾病控制司,中华医学会糖尿病分会.中国糖尿病防治指南[M].北京:北京大学出版社,2004:3-4
6 Radbill B,Murphy B,LeRoith D.Rationale and strategies for early detection and management of diabetic kidney disease.Mayo Clin Proc,2008,83(12):1373-81
7 Cortinovis M, Cattaneo D, Perico N,et al.Investigational drugs for diabetic nephropathy.Expert Opin Investig Drugs,2008,17(10):1487-500
8 G?rtner V, Eigentler TK.Pathogenesis of diabetic macro- and microangiopathy.Clin Nephrol,2008,70(1):1-9
9 Miyata T,Nangaku M,Suzuki D,et al.A mesangium-predominantgene,megsin,is a new serpin upregulated in IgA nephropathy.J Clin Invest,1998,1209(4):828-836
10陈楠,王伟铭,俞海瑾,等.肾小球肾炎肾组织megsin的表达及其意义.肾脏病与透析肾移植杂志,2003,12(5):412-415
11 Ohtomo S,Nangaku M,Izuhara Y,et al.The role of megsin, a serine protease inhibitor,in diabetic mesangial matrix accumulation.Kidney Int,2008,74(6):768-74
12 Ceska R,Tesar V.Diabetes,dyslipidaemia and kidney diseases.Vnitr Lek,2008,54(5):511-7
13 Li H,Li X,Duan L,et al.Inhibition of lovastatin on proliferation and expression of proinflammatory cytokines in cultured human glomerular mesangial cells.Chin Med J(Engl),2003,116:1366-1369
14刘茂东,李英,迟彦青,等.2型糖尿病肾病患者血清脂蛋白(a)质量浓度与纤溶功能的相关性探讨.中国实用内科杂志,2004,10(4):237-8
15 Peng F,Wu D,Gao B,et al.RhoA/Rho-kinase contribute to the pathogenesis of diabetic renal disease.Diabetes,2008,57(6):1683-92
16 Kolavennu V,Zeng L,Peng H,et al.Targeting of RhoA/ROCK signaling ameliorates progression of diabetic nephropathy independent of glucose control.Diabetes,2008,57(3):714-23
17 Fujii M,Inoguchi T,Maeda Y,et al.Pitavastatin ameliorates albuminuria and renal mesangial expansion by downregulating NOX4 in db/db mice. Kidney Int,2007,72(4):473-80
18 Parsons RB,Price GC,Farrant JK,et al.Statins inhibit the dimerization of beta-secretase via both isoprenoid- and cholesterol-mediated mechanisms.Biochem J,2006,399(2):205-14
1 Caroline B. Marshall Stuart J. Cell Cycle and Glomerular Disease:A Minireview. Nephron Exp Nephrol 2006;102:e39–e48
2 Kawamura K, Kobayashi Y, Tanaka T, et al. Intracellular localization of proliferating cell nuclear antigen during the cell cycle in renal cell carcinoma.Anal Quant Cytol Histol,2000,20(2):107-113
3 Schocklmann H Q, Lang S, Stelzel R B. Regulation of mesangial cell proliferation. Kidney Int, 1999,66(4):1199~1207
4 Pan DA, Lillioja S, Milner MR, et al. Skeletal muscle membrane lipid composition is related to adiposity and in-sulin action. J Clin Invest, 1995,96(6):2802
5 Kurogi Y. Mesangial cell proliferation inhibitors for the treatment of proliferative glomerular disease. Med Res Rev, 2003,23(1):15
6丁炜.转化生长因子-β与肾脏疾病进展的关系.国外医学.泌尿系统分册,1997,17(6):273
7 Gressner A M, Weiskirchen R, Breitkopf K, et al. Roles of TGF-beta in hepatic fibrosis. Front Biosci,2002,7:793-807
8史伟,梁馨苓,刘双信,等.转化生长因子β反义RNA对肾小球GMC基质合成及分泌的影响.中华肾脏病杂志,2002,18(2):103-106
9 Frazier K, Williams S, Kothapalli D, et al. Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor, nvest Dematol, 1996,107(3):404-411
10 ItoY, Goldschmeding R, Bende R.et al. Kinetics of connective tissue growth factor expression during experimental proliferative glomerulonephritis. J Am Soc Nephrol, 2001,12(3):472-484
11 Yokoi H,Mukoyama M, Sugawara A, et al.Role of Connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis. Am J Physiol Renal Physiol,2002,282(5):933-942
12史伟.何朝生.刘双信.等.转化生长因子β1及Smad信号转导通路在肾小球硬化中的作用.中华肾脏病杂志,2005,21(5):270-273
13 Chen R, Huang C, Morinelli TA, et al. Blockade of the effects of TGF-beta 1 on mesangial cells by overexpression of Smad7. J Am Soc Nephrol, 2002,13(4):887-893
14王凯旋.洪小苏.转化生长因子β与Smad7在肾脏疾病中的作用.国外医学.内科学分册.2004,31(12):535-537
15 Inagi R,Nangaku M,Miyata T,et al.Mesangial cell-predominant functional gene, megsin. Clin Exp Nephrol,2003,7:87–92
16 Inagi R, Miyata T, Suzuki D, et al.Specific tissue distribution of megsin, a novel serpin, in the glomerulus and its up-regulation in IgA nephropathy. Biochem Biophys Res Commun 2001;286:1098–106
17 Nangaku M, Miyata T, Suzuki D, et al. Cloning of rodent megsin revealed its up-regulation in mesangioproliferative nephritis. Kidney Int 2001;60:641–52
18 Miyata T, Inagi R, Nangaku M,et al.Overexpression of the serpin megsin induces progressive mesangial cell proliferation and expansion. J Clin Invest 2002;109:585–93
19 Narayanan Parameswaran*, Wojciech Nowak, Carolyn S. Hall,et al. Cellular and molecular actions of adrenomedullin in glomerular mesangial cells. Peptides, 2001,22:1919–1924
20 Wang Pi,Takezawa T.Reconstruction of renal glomerular tissue using collagen vitrigel scaffold.Journal of bioscience and bioengineering.2005,99(6):529-40
1 Cove-Smith A, Hendry BM.The regulation of mesangial cell proliferation.Nephron Exp Nephrol,2008;108(4):e74-9
2 Miyata T,Nangaku M, Suzuki D,et al.A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy.J Clin Invest,1998, 1209(4):828-836
3 Miyata T,Li M,Yu X,et al.Megsin gene:its genomic analysis, pathobiological functions, and therapeutic perspectives.Current Genomics, 2007, 8(3):203-8
4 Lomas DA, Carrell RW.Serpinopathies and the conformational dementias. Nat Genet Rev, 2002,3: 759–767
5 Lomas DA, Mahadeva R.Alpha1-antitrypsin polymerization and the serpinopathies: Pathobiology and prospects for therapy.J Clin Invest,2002, 110:1585–1590
6 Davis RL, Shrimpton AE, Holohan PD, et al.Familial dementia caused by polymerization of mutant neuroserpin. Nature,1999,401(6751): 376–379
7 Benarafa C, Cooley J, Zeng W,et al.Characterization of four murine homologs of the human ov-serpin monocyte neutrophil elastase inhibitor MNEI (SERPINB1).J Biol Chem,2002,277(44):42028-33
8 Lee W, Haslinger A, Karin M,et al.Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40.Nature,1987,325: 368–372
9 Foletta VC, Segal DH, Cohen DR.Transcriptional regulation in the immune system: all roads lead to AP-1. J Leuk Biol,1998,63:139–152
10 Gewirtz AM, Anfossi G, Venturelli D, et al.G1/S transition in normal human T-lymphocyte requires the nuclear protein encoded by c-myb. Science,1989,245:180–183
11 Mucenski ML, McLain K, Kier AB, et al.A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis.Cell,1991,65: 677–689
12 Rosenfeld MG.POU-domain transcription factors: Powerful developmental regulators. Genes Dev,1991,5: 897–907
13 Guijarro C, Egido J.Transcription factor-kappa B (NF-κB) and renal disease. Kidney Int,2001,59: 415–424
14 Morrissey JJ, Klahr S.Transcription factor NF-κB regulation of renal fibrosis during ureteral obstruction.Semin Nephrol,1998,18:603–611
15 Inagi R,Miyata T,Nangaku M,et al.Transcriptional regulation of a mesangium-predominant gene, megsin.J Am Soc Nephrol,2002,13: 2715–2722
16 Evdokimova V, Ovchinnikov LP, Sorensen PH.Y-box binding protein 1: providing a new angle on translational regulation.Cell Cycle,2006,5(11):1143-7
17 Mertens PR, Harendza S, Pollock AS,et al.Glomerular mesangial cells-specific transactivation of matrix metalloproteinase-2 transcription is mediated by YB-1. J Biol Chem, 1997,272:22905–22912
18 Inagi R,Miyata T, Imasawa T,et al.Mesangial cell-predominant gene, megsin.Nephrol Dial Transplant,2002,17(Suppl 9):32–33
19 Nangaku M,Miyata T,Suzuki D,et al.Cloning of rodent megsin revealed its up-regulation in mesangioproliferative nephritis.Kidney International,2001,60 (pp):641–652
20 Miyata T,Inagi R,Nangaku M,et al.Overexpression of the serpin megsin induces progressive mesangial cell proliferation and expansion.J Clin Invest,2002,109(5):585–593
21 Inagi R,Yamamoto Y,Nangaku M,et al. A severe diabetic nephropathy model with early development of nodule-like lesions induced by megsin overexpression in RAGE/iNOS transgenic mice.Diabetes,2006,55:356-365
22 Schipper R, Loof A, de Groot J,et al.Salivary protein/peptide profiling with SELDI-TOF-MS.Ann N Y Acad Sci,2007,1098:498-503
23 Schipper R, Loof A, de Groot J,et al.SELDI-TOF-MS of saliva: methodology and pre-treatment effects.J Chromatogr B Analyt Technol Biomed Life Sci,2007,847(1):45-53
24 Ohtomo S,Nangaku M,Izuhara Y,et al.The role of megsin,a serine protease inhibitor,in diabetic mesangial matrix accumulation.Kidney Int,2008,74(6):768-74
25 Jewett MC, Calhoun KA, Voloshin A,et al.An integrated cell-free metabolic platform for protein production and synthetic biology.Mol Syst Biol,2008;4:220
26 Inagi R,Nangaku M,Usuda N,et al.Novel serpinopathy in rat kidney and pancreas induced by overexpression of megsin.J Am Soc Nephrol,2005,16: 1339–1349
27 Szelestei T, B?hring S, Kovács T,et al.Association of a uteroglobin polymorphism with rate of progression in patients with IgA nephropathy.Am J Kidney Dis,2000,36(3):468-73
28 Lim CS, Kim SM, Oh YK, et al.Megsin 2093T-2180C haplotype at the 3' untranslated region is associated with poor renal survival in Korean IgA nephropathy patients.Clin Nephrol,2008,70(2):101-9
29 MaixnerováD, Merta M, ReiterováJ, et al.The influence of two megsin polymorphisms on the progression of IgA nephropathy.Folia Biol (Praha),2008,54(2):40-5
30王朝晖,陈楠,潘晓霞等.Megsin基因单核苷酸多态性与IgA肾病相关性研究.中华医学杂志,2006,86(19):1337-1441
31 Xia YF, Huang S, Li X,et al.A family-based association study of megsin A23167G polymorphism with susceptibility and progression of IgA nephropathy in a Chinese population.Clin Nephrol,2006,65(3):153-9