霉酚酸酯对糖尿病大鼠足细胞肥大及凋亡的影响及机制研究
|
摘要
研究目的
糖尿病肾病(diabetic nephropathy, DN)以早期肾脏体积增大、晚期肾小球硬化为主要特征,肾脏细胞的增殖、肥大与凋亡参与了DN的发生、发展。本研究采用体内动物实验和体外细胞培养相结合的方法,通过观察糖尿病大鼠及高糖刺激下的肾小球足细胞肥大、凋亡状态,测定细胞周期蛋白激酶抑制剂(cyclin-kinase inhibitors, CKIs)p27kip1、p21cipt及凋亡相关基因bcl-2、bax、cleaved caspase-3的表达,观察霉酚酸酯(mycophenolate mofetil, MMF)对足细胞肥大、凋亡的影响,为治疗DN提供科学的理论依据。
研究方法
体内动物实验：32只雄性健康Wistar大鼠,腹腔内注射链脲佐菌素(STZ),另外8只作为对照组(NC)。注射STZ72小时后剪尾采血,测血糖>16.7mmol/L,尿糖阳性,表明糖尿病模型成功建立。将糖尿病造模成功的32只大鼠,随机分为糖尿病组(DM)、霉酚酸酯组(DM+M)、缬沙坦组(DM+V)及霉酚酸酯+缬沙坦联合治疗组(DM+M.V),每组8只。成模2天后,治疗组分别给予MMF15mg.kg-1.d-1,缬沙坦40mg.kg-1.-1,联合治疗给予MMF15mg.kg-1.d-1+缬沙坦40mg.kg-1.d-1,1mg/d灌胃；而NC组和DM组每日予等量溶媒灌胃,共16周(w)。分别检测五组大鼠4w、8w及16w的左肾重/体重、血糖、血压、内生肌酐清除率及24小时尿蛋白排泄量；光学显微镜和电子显微镜观察糖尿病大鼠肾组织形态学的变化；免疫组织化学法检测肾皮质nephrin.WT1蛋白的表达；TUNEL法检测足细胞的凋亡率；Western-blot半定量及荧光实时定量PCR的分析方法,检测肾组织中p27kip1、p21cipt及bcl-2.bax.cleaved caspase-3的表达。
体外细胞实验：订购的小鼠肾足细胞,按培养液中葡萄糖、霉酚酸(mycophenolic acid,MPA).缬沙坦的含量不同分为：①正常对照组NG(葡萄糖浓度5.6mmol/L+2.5%FBS的DMEM);②高糖组HG(葡萄糖浓度25mmol/L);③HG+M组：HG(葡萄糖浓度25mmol/L)+MPA2.5×10-7mol/L;④HG+V组:HG(葡萄糖浓度25mmol/L)+缬沙坦10-7mol/L;作用72小时(h)。采用流式细胞仪测定12h、24h、48h、72h各时间点足细胞的肥大指数及凋亡率,运用Western-blot半定量及荧光实时定量PCR的分析方法,检测肾小球足细胞中p27kip1、p21cip1及bcl-2、bax、cleaved caspase-3的表达。
结果
1.临床参数
与NC组比较,DM大鼠血糖明显升高、体重减轻、肥大指数增加,24小时尿蛋白排泄量及肌酐清除率均明显增高(P<0.01)。16周末,DM大鼠肾重/体重指数较NC组明显升高(8.49±1.21vs4.57±0.82,P<0.01)；而霉酚酸酯和/或缬沙坦治疗16w后,肾重/体重指数明显降低,差异有显著性(P0.05)。16w末,DM大鼠尿蛋白量较NC组明显升高(55.37±2.33vs10.52±0.66,P<0.05);同样药物治疗16w后,24小时尿蛋白排泄量明显降低,差异有显著性(P<0.01)；各给药组间无统计学差异(P>0.05)。
2.肾脏组织形态学观察
光镜：4周前糖尿病大鼠无明显变化。此后,随着实验周期的延长,糖尿病大鼠肾小球的体积逐渐增大、系膜区逐渐增宽,且基底膜增厚、毛细血管腔受压、变窄；与NC组比较,16周末DM组肾小球的硬化指数(1.15±0.12vs0.15±0.05,P<0.01)及间质纤维化积分(0.58±0.08vs0.12±0.04,P<0.01)明显增加,其差异有显著性；各药物治疗组较DM组均有不同程度的改善。
电镜：糖尿病大鼠肾小球毛细血管基底膜均质性增厚和系膜基质增多,伴足细胞足突的广泛融合,并可见裸露的基底膜及脱落的足细胞。MMF和/或缬沙坦治疗后,足突的融合减轻,系膜基质及足细胞的脱落均明显减少。
3.肾组织中nephrin、WT-1蛋白及mRNA的表达
免疫组织化学染色显示：正常大鼠nephrin、WT1沿肾小球基底膜呈棕黑色线形分布,而糖尿病大鼠的线样沉积变淡、变细、甚至消失,经MMF和/或缬沙坦治疗后,其线样沉积又恢复至粗线状。
从第4周开始糖尿病组大鼠肾组织中nephrin蛋白(2.08±0.42vs2.63±0.51,P<0.05)及WT1蛋白(1.26±0.2vs1.59±0.26,P<0.05)的表达减少,且随着时间的推移nephrin、WT1蛋白的表达进一步降低。各治疗组与DM组比较,nephrin、WT1的表达均有不同程度的升高(P<0.05)；且16w末nephrin蛋白的表达DM+M组(1.83±0.43)及DM+M. V组(1.75±0.31)疗效优于DM+V组(1.39±0.28),有显著性差异(P<0.05)；而各药物治疗组间WT1蛋白的表达无明显差异(P>0.05)。
从第4周开始DM组大鼠肾组织中nephrin mRNA (0.87±0.13vs0.95±0.091, P<0.05)、WT1mRNA (0.92±0.095vs1.04±0.079, P<0.05)的表达下降,且随着时间的推移其mRNA的表达进一步降低。而经霉酚酸酯和/或缬沙坦治疗后,nephrin、WT1mRNA的表达均有不同程度的上调(P<0.05),16周末WT1mRNA的表达霉酚酸酯联合治疗组(1.30±0.25)优于缬沙坦组(1.04±0.23),(P<0.05),而nephrin mRNA的表达三治疗组间比较无明显差异(P>0.05)。
4.足细胞肥大指数
高糖组足细胞肥大从24小时开始(0.828±0.23vs0.678±0.16,P<0.05),且随着时间的推移足细胞进一步肥大。各治疗组与高糖组比较,霉酚酸治疗组足细胞肥大从24小时开始减轻,而缬沙坦治疗组从48小时开始明显减轻,且随着时间推移,疗效逐渐增强(P<0.05)。
5. p27kip1、p21cip1蛋白及mRNA的表达
体内动物实验：从第4周开始DM组大鼠肾组织中p27kip1、p21cipl蛋白及mRNA的表达增强,与正常对照组相比有显著性差异(P<0.05),16周末达高峰。各治疗组与DM组比较,p27kip1、p21cipl蛋白及mRNA的表达从第8周开始降低(P<0.05),且随着实验周期的延长,p27kip1、p21cip1蛋白及mRNA的表达进一步降低；16w末p27kipl蛋白、mRNA及p21cip1mRNA的表达,霉酚酸酯+缬沙坦联合治疗组优于缬沙坦组(P<0.05)。
体外足细胞培养：p27kip1、p21cipl蛋白及mRNA的表达：从24小时开始,HG组小鼠足细胞p27kip1、p21cip1蛋白及mRNA水平开始升高,与NG组相比差异有显著性(P<0.05)。且随着时间的推移,p27kipl和p21cipl蛋白及mRNA的表达进一步升高。霉酚酸及缬沙坦可明显降低p27kip1和p21cip1蛋白的表达,且72小时末p27kip1蛋白的表达霉酚酸的疗效优于缬沙坦(P<0.05)；霉酚酸和缬沙坦亦可明显降低p27kip1mRNA的表达,而p21cip1mRNA的表达仅霉酚酸起到明显的抑制作用。
6.足细胞凋亡率
体内动物实验：实验第8周糖尿病大鼠足细胞凋亡率开始增高(2.63±0.31vs0.84±0.26,P<0.01),且随着实验的延长,足细胞凋亡率进一步增高。各治疗组与DM组相比,均有不同程度的下降；16周末霉酚酸酯单一治疗组(1.81±0.27)及联合治疗组(1.24±0.19)优于缬沙坦组(2.25±0.33),差异有显著性(P<0.05)。
体外足细胞实验：高糖培养48小时后,足细胞凋亡率明显高于NG组(4.97±0.58vs2.77±0.31,P<0.05)。两治疗组,仅霉酚酸治疗72小时后足细胞凋亡率降低(P<0.05),而缬沙坦治疗组无变化(P>0.05),且72小时末霉酚酸的疗效优于缬沙坦,差异有统计学意义(P<0.05)。
7. bax、bcl-2、cleaved caspase-3蛋白及mRNA的表达
体内动物实验：糖尿病大鼠的bax、cleaved caspase-3蛋白及mRNA的表达从实验第8周起明显升高,而bcl-2的表达下降,差异有显著性(P<0.05)。各治疗组与DM组比较,其表达均有不同程度的改善；且16周末bax/bcl-2蛋白的表达,DM+M组(1.42±0.2)、DM+M. V组(1.57±0.26)与DM+V(1.95±0.24)比较差异有显著性,(P<0.01)。
体外足细胞培养：高糖培养下,足细胞的bax、cleaved caspase-3蛋白及mRNA的表达从实验第48小时起升高,而bcl-2的表达下降,差异有显著性(P<0.05),且随着时间的推移比值进一步升高。经霉酚酸或缬沙坦治疗后,其比值较HG组下降,差异有显著性(P<0.01)；且72小时末bax/bcl-2、cleaved caspase-3蛋白的表达,HG+M组较HG+V组明显降低,差异有显著性(P<0.05)。
结论：
1.足细胞的肥大与凋亡参与了糖尿病肾病的发生、发展。糖尿病早期,足细胞的肥大伴随着细胞周期蛋白激酶抑制剂p27kip1、p2cip1表达的增高。随着糖尿病病程的进展,足细胞的凋亡率增高,晚期的肾小球硬化与其过度凋亡有关,并伴随着凋亡相关基因bax、cleaved caspase-3表达的升高及bc1-2表达的降低。
2.霉酚酸酯和缬沙坦可有效的抑制肾小球足细胞的肥大与凋亡,调节细胞周期相关蛋白(p27kip1、p21cip1)和凋亡相关基因(bax、bcl-2、cleaved caspase-3)的表达,起到一定的保护作用。因此,推测霉酚酸酯可能通过调节细胞周期负性调控蛋白p27kip1、p21cip及凋亡相关基因bax、bcl-2及cleaved caspase-3的表达,抑制和阻断肾小球足细胞的肥大与凋亡,从而起到降低蛋白尿、延缓糖尿病肾病进展的作用。
Objective
Diabetic nephropathy (DN), one of the most serious microvascular complications of diabetes mellitus(DM), is a major cause of end-stage renal disease. Renal hypertrophy in the early stage and glomerulosclerosis in the end stage are the characteristics of DN. Both hypertrophy and apoptosis of renal cells are thought to be involved in the pathogenesis of DN. The present study aimed to examine the effect of mycophenolate mofetil (MMF), a new immunosuppressive agent, on the hypertrophy and apoptosis of podocyte, and investigate the underlying mechanisms.
Methods
In vivo studies:Forty male rats were randomly divided into two groups:healthy control group (NC, n=8) and diabetes mellitus group (DM, n=32). Diabetic rat models were induced by streptozotocin(STZ) injected intraperitoneally. After the DM model was established successfully, DM group was subdivided into four groups:group treated with the dissolvent (DM), group treated with mycophenolate mofetil (DM+M), group treated with valsartan (DM+V) and group treated with mycophenolate mofetil and valsartan (DM+M.V). After16weeks of treatment, the weight of kidney(Kw) and body(Bw), serum glucose, blood pressure, serum creatinine and24hours urinary protein excretion(UP) was detected. Histomorphology of renal tissue was observed by an optical microscope and electron microscope. The expressions of nephrin and Wilm's tumor suppressor gene (WT1) were detected by immunohistochemistry. Apoptosis of podocytes were determined by transferase-mediated dUTP nick-end labeling (TUNEL) test. The protein and mRNA expressions of p27kip1,p21cip1, bax and bcl-2were examined by Western blot and competitive reverse transcription-polymerase chain reaction (RT-PCR).
In vitro studies:Cultured rat podocyte were exposed to5.6mmol/L normal glucose (NG),25mmol/L high glucose (HG) with mycophenolic acid(MPA)(HG+M) or Valsartan (HG+V) for72hours. Hypertrophy and apoptosis of podocytes was determined by flow cytometry. The protein and mRNA expressions of p27kip1, p21cip1, bax and bcl-2were examined by Western blot and RT-PCR.
Results
1. Animal data
After16weeks, the serum glucose in DM group was higher than that in NC group, but it couldn't be reduced by MMF or/and Valsartan. The systolic blood pressure and serum creatinine in DM group were not different from those in NC group.The ratio of kidney to body weight and24hours urinary protein excretion were significantly higher in DM (Kw/Bw:8.49±1.21vs4.57±0.82,P<0.01),(UP:55.37±2.33vs10.52±0.66, P<0.05), these increase were rescued by treatmeat with MMF or/and valsartan.
2. Renal morphological changes of rats
Light microscope:After16weeks, rat glomerular volume increased, the glomerular basement membrane thickened, mesangial area expanded and glomerular capillary lumen narrowed by compression in DM group. The glomerulosclerosis and interstitial fibrotic lesion in DM group increased dramatically as compared with those in NC group (1.15±0.12vs0.15±0.05, P<0.01),(0.58±0.08vs0.12±0.04, P<0.01),but they could be reduced in different degrees by treatment with MMF or/and valsartan.
Electron microscope:In DM group, the foot process of podocytes fused and flattened. Glomerular basement membrane exposed and some podocytes ablated. The glomerular basement membrane thickened and the mesangial matrix increased. However, in the three treatment groups, glomerular foot process fusion was reduced, the ablated podocytes decreased and the extracellular matrix was also reduced.
3. The expressions of nephrin and WT1in the renal cortex of rats
Nephrin and WT1expressed at the glomerular basement membrane in normal renal tissue. But in the DM renal tissue, the intensity and the positive cells number decreased or even disappeared. However, in the treatment groups, it was restored significantly.
The protein expressions of nephrin and WT1were reduced significantly in the DM group at4weeks (nephrin:2.08±0.42vs2.63±0.51, P<0.05),(WT1:1.26±0.2vs1.59±0.26, P<0.05), and continued to decrease with the prolongation of time. This decrease was also inhibited by MMF or/and valsartan. In addition, the effect of MMF(1.83±0.43) and MMF+valsartan (1.75±0.31) on the protein expressions of nephrin better than valsartan(1.39±0.28) after16weeks (P<0.05).
The mRNA expressions of nephrin and WT1were also markedly decreased in the DM group at4weeks (nephrin:0.87±0.13vs0.95±0.091, P<0.05),(WT1:0.92±0.095vs1.04±0.079, P<0.05), and with a gradual decrease thereafter. But in the treatment groups, it was restored significantly (P<0.05). In addition, the effect of MMF+valsartan (1.30±0.25) on the mRNA expressions of WT1better than valsartan(1.04±0.23) after16weeks (P<0.05).
4. Celluar hypertrophy
In vitro results:The ratio of proterin/cell numbers as a well-established hypertrophy index revealed a significant increase in podocytes exposed to high glucose (0.828±0.23vs0.678±0.16, P<0.05) for24hours, and the increment in the ratio of protein/cell numbers by high glucose was inhibited with MPA or Valsartan in podocytes(P<0.05).
5. The protein and mRNA expressions of p27kip1, p21cip1
In vivo results:P27kip1and p21cip1protein expression were significantly increased in DM compared to NC rats at4weeks (p27kip1:1.83±0.25vs1.0±0.08, P<0.05; p21cipl:1.62±0.24vs0.96±0.05, P<0.05), and continued to increase with the prolongation of time. In additoin, the mRNA exprossion of these cyclin-kinase inhibitors showed similar patterns to their protein expression (P<0.05). This increase was also inhibited by MMF or/and valsartan. Moreover, Combination of MMF with Valsartan had more significantly inhibitory effect on the protein and mRNA expressions of P27kip1than valsartan after16weeks (P<0.05).
In vitro results:P27kipl and p21cipl protein expression were significantly increased in podocytes exposed to HG compared to NG for24hours (p27kip1:1.24±0.13vs0.97±0.09, P O.05; p21cipl:1.27±0.140vs0.97±0.09, P<0.05), and with a gradual increase thereafter. In additoin, the mRNA exprossion of these cyclin-kinase inhibitors showed similar patterns to their protein expression (P<0.05). These changes induced by HG were significantly abrogated by MPA.
6. Apoptosis of podocytes
In vivo results:Then number of apoptotic podocytes was significantly increased in DM (2.63±0.31) compared to NC(0.84±0.26)(P<0.01) at8weeks, and continued to increase with the prolongation of time. It could be inhibited by MMF and Valsartan. MMF with/or Valsartan had more significantly inhibitory effect on the apoptosis of podocytes in the glomerulus of rats with diabetic nephropathy (P<0.05). In addition, the effect of MMF(1.81±0.27) and MMF+valsartan (1.24±0.19) on the apoptosis of podocytes better than valsartan(2.25±0.33) after16weeks (P<0.05).
In vitro results:Exposure to HG for48hours resulted in a remarkable increase in apoptotic podocytes(4.97±0.58vs2.77±0.31, P<0.05), and these increments in apoptosis were markedly ameliorated by the administreation of MPA after72hours. There were no differences in the apoptotic podocytes between HG and HG+V groups.
7. The protein and mRNA expressions of of bax, bcl-2and cleaved caspase-3
In vivo results:Bax and cleaved caspase-3protein and mRNA expressions were significantly increased,while bcl-2protein and mRNA expressions were markedly decreased in the DM compared to NC at8weeks (P<0.05). Administration of MMF or/and valsartan significantly ameliorated the increases in the radio of bax/bcl-2(P<0.05) and cleaved caspase-3protein and mRNA expressions in DM glomeruli(.P<0.05). In addition, MMF with/or Valsartan had more significantly inhibitory effect on the radio of bax/bcl-2protein expressions in DM glomeruli after16weeks (P<0.05).
In vitro results:HG significantly increased bax and cleaved caspase-3protein and mRNA expressions and significantly reduced bcl-2protein and mRNA expressions in cultured podocytes for48h(P<0.05). These changes induced by HG was significantly abrogated by MPA or valsartan. Moreover, MPA had more significantly inhibitory effect than valsartan on the radio of bax/bcl-2and cleaved caspase-3protein expressions in HG after72h (P<0.05).
Conclusion
1. These results demonstrated hypertrophy and apoptosis of podocytes may play important roles in pathogenesis of diabetic nephropathy. It was proved that the expression of cyclin-kinase inhibitors such as p27kip1, p21cip1was significantly increased followed by the hypertrophy of podocyte. With the progression of diabetic nephropathy, the number of apoptotic podocytes increased gradually and the balance between proliferation and apoptosis was broken in diabetic kidney. Apoposis which was regulated by apoptosis related genes such as bax, bcl-2and cleaved caspase-3.
2. MMF and valsartan can inhibit abnormal hypertrophy and apoptosis of podocytes in diabetes, partly by regulating the expression of cell cycle related protein p27kip1, p21cip1and apoptosis related genes,sueh as bax, bcl-2and cleaved caspase-3. These suggest that the protective effects of MMF on renal function maybe partly through inhibiting abnormal renal cell growth by regulating cell cycle related protein or apoptosis related genes.
糖尿病肾病(diabetic nephropathy, DN)以早期肾脏体积增大、晚期肾小球硬化为主要特征,肾脏细胞的增殖、肥大与凋亡参与了DN的发生、发展。本研究采用体内动物实验和体外细胞培养相结合的方法,通过观察糖尿病大鼠及高糖刺激下的肾小球足细胞肥大、凋亡状态,测定细胞周期蛋白激酶抑制剂(cyclin-kinase inhibitors, CKIs)p27kip1、p21cipt及凋亡相关基因bcl-2、bax、cleaved caspase-3的表达,观察霉酚酸酯(mycophenolate mofetil, MMF)对足细胞肥大、凋亡的影响,为治疗DN提供科学的理论依据。
研究方法
体内动物实验：32只雄性健康Wistar大鼠,腹腔内注射链脲佐菌素(STZ),另外8只作为对照组(NC)。注射STZ72小时后剪尾采血,测血糖>16.7mmol/L,尿糖阳性,表明糖尿病模型成功建立。将糖尿病造模成功的32只大鼠,随机分为糖尿病组(DM)、霉酚酸酯组(DM+M)、缬沙坦组(DM+V)及霉酚酸酯+缬沙坦联合治疗组(DM+M.V),每组8只。成模2天后,治疗组分别给予MMF15mg.kg-1.d-1,缬沙坦40mg.kg-1.-1,联合治疗给予MMF15mg.kg-1.d-1+缬沙坦40mg.kg-1.d-1,1mg/d灌胃；而NC组和DM组每日予等量溶媒灌胃,共16周(w)。分别检测五组大鼠4w、8w及16w的左肾重/体重、血糖、血压、内生肌酐清除率及24小时尿蛋白排泄量；光学显微镜和电子显微镜观察糖尿病大鼠肾组织形态学的变化；免疫组织化学法检测肾皮质nephrin.WT1蛋白的表达；TUNEL法检测足细胞的凋亡率；Western-blot半定量及荧光实时定量PCR的分析方法,检测肾组织中p27kip1、p21cipt及bcl-2.bax.cleaved caspase-3的表达。
体外细胞实验：订购的小鼠肾足细胞,按培养液中葡萄糖、霉酚酸(mycophenolic acid,MPA).缬沙坦的含量不同分为：①正常对照组NG(葡萄糖浓度5.6mmol/L+2.5%FBS的DMEM);②高糖组HG(葡萄糖浓度25mmol/L);③HG+M组：HG(葡萄糖浓度25mmol/L)+MPA2.5×10-7mol/L;④HG+V组:HG(葡萄糖浓度25mmol/L)+缬沙坦10-7mol/L;作用72小时(h)。采用流式细胞仪测定12h、24h、48h、72h各时间点足细胞的肥大指数及凋亡率,运用Western-blot半定量及荧光实时定量PCR的分析方法,检测肾小球足细胞中p27kip1、p21cip1及bcl-2、bax、cleaved caspase-3的表达。
结果
1.临床参数
与NC组比较,DM大鼠血糖明显升高、体重减轻、肥大指数增加,24小时尿蛋白排泄量及肌酐清除率均明显增高(P<0.01)。16周末,DM大鼠肾重/体重指数较NC组明显升高(8.49±1.21vs4.57±0.82,P<0.01)；而霉酚酸酯和/或缬沙坦治疗16w后,肾重/体重指数明显降低,差异有显著性(P
2.肾脏组织形态学观察
光镜：4周前糖尿病大鼠无明显变化。此后,随着实验周期的延长,糖尿病大鼠肾小球的体积逐渐增大、系膜区逐渐增宽,且基底膜增厚、毛细血管腔受压、变窄；与NC组比较,16周末DM组肾小球的硬化指数(1.15±0.12vs0.15±0.05,P<0.01)及间质纤维化积分(0.58±0.08vs0.12±0.04,P<0.01)明显增加,其差异有显著性；各药物治疗组较DM组均有不同程度的改善。
电镜：糖尿病大鼠肾小球毛细血管基底膜均质性增厚和系膜基质增多,伴足细胞足突的广泛融合,并可见裸露的基底膜及脱落的足细胞。MMF和/或缬沙坦治疗后,足突的融合减轻,系膜基质及足细胞的脱落均明显减少。
3.肾组织中nephrin、WT-1蛋白及mRNA的表达
免疫组织化学染色显示：正常大鼠nephrin、WT1沿肾小球基底膜呈棕黑色线形分布,而糖尿病大鼠的线样沉积变淡、变细、甚至消失,经MMF和/或缬沙坦治疗后,其线样沉积又恢复至粗线状。
从第4周开始糖尿病组大鼠肾组织中nephrin蛋白(2.08±0.42vs2.63±0.51,P<0.05)及WT1蛋白(1.26±0.2vs1.59±0.26,P<0.05)的表达减少,且随着时间的推移nephrin、WT1蛋白的表达进一步降低。各治疗组与DM组比较,nephrin、WT1的表达均有不同程度的升高(P<0.05)；且16w末nephrin蛋白的表达DM+M组(1.83±0.43)及DM+M. V组(1.75±0.31)疗效优于DM+V组(1.39±0.28),有显著性差异(P<0.05)；而各药物治疗组间WT1蛋白的表达无明显差异(P>0.05)。
从第4周开始DM组大鼠肾组织中nephrin mRNA (0.87±0.13vs0.95±0.091, P<0.05)、WT1mRNA (0.92±0.095vs1.04±0.079, P<0.05)的表达下降,且随着时间的推移其mRNA的表达进一步降低。而经霉酚酸酯和/或缬沙坦治疗后,nephrin、WT1mRNA的表达均有不同程度的上调(P<0.05),16周末WT1mRNA的表达霉酚酸酯联合治疗组(1.30±0.25)优于缬沙坦组(1.04±0.23),(P<0.05),而nephrin mRNA的表达三治疗组间比较无明显差异(P>0.05)。
4.足细胞肥大指数
高糖组足细胞肥大从24小时开始(0.828±0.23vs0.678±0.16,P<0.05),且随着时间的推移足细胞进一步肥大。各治疗组与高糖组比较,霉酚酸治疗组足细胞肥大从24小时开始减轻,而缬沙坦治疗组从48小时开始明显减轻,且随着时间推移,疗效逐渐增强(P<0.05)。
5. p27kip1、p21cip1蛋白及mRNA的表达
体内动物实验：从第4周开始DM组大鼠肾组织中p27kip1、p21cipl蛋白及mRNA的表达增强,与正常对照组相比有显著性差异(P<0.05),16周末达高峰。各治疗组与DM组比较,p27kip1、p21cipl蛋白及mRNA的表达从第8周开始降低(P<0.05),且随着实验周期的延长,p27kip1、p21cip1蛋白及mRNA的表达进一步降低；16w末p27kipl蛋白、mRNA及p21cip1mRNA的表达,霉酚酸酯+缬沙坦联合治疗组优于缬沙坦组(P<0.05)。
体外足细胞培养：p27kip1、p21cipl蛋白及mRNA的表达：从24小时开始,HG组小鼠足细胞p27kip1、p21cip1蛋白及mRNA水平开始升高,与NG组相比差异有显著性(P<0.05)。且随着时间的推移,p27kipl和p21cipl蛋白及mRNA的表达进一步升高。霉酚酸及缬沙坦可明显降低p27kip1和p21cip1蛋白的表达,且72小时末p27kip1蛋白的表达霉酚酸的疗效优于缬沙坦(P<0.05)；霉酚酸和缬沙坦亦可明显降低p27kip1mRNA的表达,而p21cip1mRNA的表达仅霉酚酸起到明显的抑制作用。
6.足细胞凋亡率
体内动物实验：实验第8周糖尿病大鼠足细胞凋亡率开始增高(2.63±0.31vs0.84±0.26,P<0.01),且随着实验的延长,足细胞凋亡率进一步增高。各治疗组与DM组相比,均有不同程度的下降；16周末霉酚酸酯单一治疗组(1.81±0.27)及联合治疗组(1.24±0.19)优于缬沙坦组(2.25±0.33),差异有显著性(P<0.05)。
体外足细胞实验：高糖培养48小时后,足细胞凋亡率明显高于NG组(4.97±0.58vs2.77±0.31,P<0.05)。两治疗组,仅霉酚酸治疗72小时后足细胞凋亡率降低(P<0.05),而缬沙坦治疗组无变化(P>0.05),且72小时末霉酚酸的疗效优于缬沙坦,差异有统计学意义(P<0.05)。
7. bax、bcl-2、cleaved caspase-3蛋白及mRNA的表达
体内动物实验：糖尿病大鼠的bax、cleaved caspase-3蛋白及mRNA的表达从实验第8周起明显升高,而bcl-2的表达下降,差异有显著性(P<0.05)。各治疗组与DM组比较,其表达均有不同程度的改善；且16周末bax/bcl-2蛋白的表达,DM+M组(1.42±0.2)、DM+M. V组(1.57±0.26)与DM+V(1.95±0.24)比较差异有显著性,(P<0.01)。
体外足细胞培养：高糖培养下,足细胞的bax、cleaved caspase-3蛋白及mRNA的表达从实验第48小时起升高,而bcl-2的表达下降,差异有显著性(P<0.05),且随着时间的推移比值进一步升高。经霉酚酸或缬沙坦治疗后,其比值较HG组下降,差异有显著性(P<0.01)；且72小时末bax/bcl-2、cleaved caspase-3蛋白的表达,HG+M组较HG+V组明显降低,差异有显著性(P<0.05)。
结论：
1.足细胞的肥大与凋亡参与了糖尿病肾病的发生、发展。糖尿病早期,足细胞的肥大伴随着细胞周期蛋白激酶抑制剂p27kip1、p2cip1表达的增高。随着糖尿病病程的进展,足细胞的凋亡率增高,晚期的肾小球硬化与其过度凋亡有关,并伴随着凋亡相关基因bax、cleaved caspase-3表达的升高及bc1-2表达的降低。
2.霉酚酸酯和缬沙坦可有效的抑制肾小球足细胞的肥大与凋亡,调节细胞周期相关蛋白(p27kip1、p21cip1)和凋亡相关基因(bax、bcl-2、cleaved caspase-3)的表达,起到一定的保护作用。因此,推测霉酚酸酯可能通过调节细胞周期负性调控蛋白p27kip1、p21cip及凋亡相关基因bax、bcl-2及cleaved caspase-3的表达,抑制和阻断肾小球足细胞的肥大与凋亡,从而起到降低蛋白尿、延缓糖尿病肾病进展的作用。
Objective
Diabetic nephropathy (DN), one of the most serious microvascular complications of diabetes mellitus(DM), is a major cause of end-stage renal disease. Renal hypertrophy in the early stage and glomerulosclerosis in the end stage are the characteristics of DN. Both hypertrophy and apoptosis of renal cells are thought to be involved in the pathogenesis of DN. The present study aimed to examine the effect of mycophenolate mofetil (MMF), a new immunosuppressive agent, on the hypertrophy and apoptosis of podocyte, and investigate the underlying mechanisms.
Methods
In vivo studies:Forty male rats were randomly divided into two groups:healthy control group (NC, n=8) and diabetes mellitus group (DM, n=32). Diabetic rat models were induced by streptozotocin(STZ) injected intraperitoneally. After the DM model was established successfully, DM group was subdivided into four groups:group treated with the dissolvent (DM), group treated with mycophenolate mofetil (DM+M), group treated with valsartan (DM+V) and group treated with mycophenolate mofetil and valsartan (DM+M.V). After16weeks of treatment, the weight of kidney(Kw) and body(Bw), serum glucose, blood pressure, serum creatinine and24hours urinary protein excretion(UP) was detected. Histomorphology of renal tissue was observed by an optical microscope and electron microscope. The expressions of nephrin and Wilm's tumor suppressor gene (WT1) were detected by immunohistochemistry. Apoptosis of podocytes were determined by transferase-mediated dUTP nick-end labeling (TUNEL) test. The protein and mRNA expressions of p27kip1,p21cip1, bax and bcl-2were examined by Western blot and competitive reverse transcription-polymerase chain reaction (RT-PCR).
In vitro studies:Cultured rat podocyte were exposed to5.6mmol/L normal glucose (NG),25mmol/L high glucose (HG) with mycophenolic acid(MPA)(HG+M) or Valsartan (HG+V) for72hours. Hypertrophy and apoptosis of podocytes was determined by flow cytometry. The protein and mRNA expressions of p27kip1, p21cip1, bax and bcl-2were examined by Western blot and RT-PCR.
Results
1. Animal data
After16weeks, the serum glucose in DM group was higher than that in NC group, but it couldn't be reduced by MMF or/and Valsartan. The systolic blood pressure and serum creatinine in DM group were not different from those in NC group.The ratio of kidney to body weight and24hours urinary protein excretion were significantly higher in DM (Kw/Bw:8.49±1.21vs4.57±0.82,P<0.01),(UP:55.37±2.33vs10.52±0.66, P<0.05), these increase were rescued by treatmeat with MMF or/and valsartan.
2. Renal morphological changes of rats
Light microscope:After16weeks, rat glomerular volume increased, the glomerular basement membrane thickened, mesangial area expanded and glomerular capillary lumen narrowed by compression in DM group. The glomerulosclerosis and interstitial fibrotic lesion in DM group increased dramatically as compared with those in NC group (1.15±0.12vs0.15±0.05, P<0.01),(0.58±0.08vs0.12±0.04, P<0.01),but they could be reduced in different degrees by treatment with MMF or/and valsartan.
Electron microscope:In DM group, the foot process of podocytes fused and flattened. Glomerular basement membrane exposed and some podocytes ablated. The glomerular basement membrane thickened and the mesangial matrix increased. However, in the three treatment groups, glomerular foot process fusion was reduced, the ablated podocytes decreased and the extracellular matrix was also reduced.
3. The expressions of nephrin and WT1in the renal cortex of rats
Nephrin and WT1expressed at the glomerular basement membrane in normal renal tissue. But in the DM renal tissue, the intensity and the positive cells number decreased or even disappeared. However, in the treatment groups, it was restored significantly.
The protein expressions of nephrin and WT1were reduced significantly in the DM group at4weeks (nephrin:2.08±0.42vs2.63±0.51, P<0.05),(WT1:1.26±0.2vs1.59±0.26, P<0.05), and continued to decrease with the prolongation of time. This decrease was also inhibited by MMF or/and valsartan. In addition, the effect of MMF(1.83±0.43) and MMF+valsartan (1.75±0.31) on the protein expressions of nephrin better than valsartan(1.39±0.28) after16weeks (P<0.05).
The mRNA expressions of nephrin and WT1were also markedly decreased in the DM group at4weeks (nephrin:0.87±0.13vs0.95±0.091, P<0.05),(WT1:0.92±0.095vs1.04±0.079, P<0.05), and with a gradual decrease thereafter. But in the treatment groups, it was restored significantly (P<0.05). In addition, the effect of MMF+valsartan (1.30±0.25) on the mRNA expressions of WT1better than valsartan(1.04±0.23) after16weeks (P<0.05).
4. Celluar hypertrophy
In vitro results:The ratio of proterin/cell numbers as a well-established hypertrophy index revealed a significant increase in podocytes exposed to high glucose (0.828±0.23vs0.678±0.16, P<0.05) for24hours, and the increment in the ratio of protein/cell numbers by high glucose was inhibited with MPA or Valsartan in podocytes(P<0.05).
5. The protein and mRNA expressions of p27kip1, p21cip1
In vivo results:P27kip1and p21cip1protein expression were significantly increased in DM compared to NC rats at4weeks (p27kip1:1.83±0.25vs1.0±0.08, P<0.05; p21cipl:1.62±0.24vs0.96±0.05, P<0.05), and continued to increase with the prolongation of time. In additoin, the mRNA exprossion of these cyclin-kinase inhibitors showed similar patterns to their protein expression (P<0.05). This increase was also inhibited by MMF or/and valsartan. Moreover, Combination of MMF with Valsartan had more significantly inhibitory effect on the protein and mRNA expressions of P27kip1than valsartan after16weeks (P<0.05).
In vitro results:P27kipl and p21cipl protein expression were significantly increased in podocytes exposed to HG compared to NG for24hours (p27kip1:1.24±0.13vs0.97±0.09, P O.05; p21cipl:1.27±0.140vs0.97±0.09, P<0.05), and with a gradual increase thereafter. In additoin, the mRNA exprossion of these cyclin-kinase inhibitors showed similar patterns to their protein expression (P<0.05). These changes induced by HG were significantly abrogated by MPA.
6. Apoptosis of podocytes
In vivo results:Then number of apoptotic podocytes was significantly increased in DM (2.63±0.31) compared to NC(0.84±0.26)(P<0.01) at8weeks, and continued to increase with the prolongation of time. It could be inhibited by MMF and Valsartan. MMF with/or Valsartan had more significantly inhibitory effect on the apoptosis of podocytes in the glomerulus of rats with diabetic nephropathy (P<0.05). In addition, the effect of MMF(1.81±0.27) and MMF+valsartan (1.24±0.19) on the apoptosis of podocytes better than valsartan(2.25±0.33) after16weeks (P<0.05).
In vitro results:Exposure to HG for48hours resulted in a remarkable increase in apoptotic podocytes(4.97±0.58vs2.77±0.31, P<0.05), and these increments in apoptosis were markedly ameliorated by the administreation of MPA after72hours. There were no differences in the apoptotic podocytes between HG and HG+V groups.
7. The protein and mRNA expressions of of bax, bcl-2and cleaved caspase-3
In vivo results:Bax and cleaved caspase-3protein and mRNA expressions were significantly increased,while bcl-2protein and mRNA expressions were markedly decreased in the DM compared to NC at8weeks (P<0.05). Administration of MMF or/and valsartan significantly ameliorated the increases in the radio of bax/bcl-2(P<0.05) and cleaved caspase-3protein and mRNA expressions in DM glomeruli(.P<0.05). In addition, MMF with/or Valsartan had more significantly inhibitory effect on the radio of bax/bcl-2protein expressions in DM glomeruli after16weeks (P<0.05).
In vitro results:HG significantly increased bax and cleaved caspase-3protein and mRNA expressions and significantly reduced bcl-2protein and mRNA expressions in cultured podocytes for48h(P<0.05). These changes induced by HG was significantly abrogated by MPA or valsartan. Moreover, MPA had more significantly inhibitory effect than valsartan on the radio of bax/bcl-2and cleaved caspase-3protein expressions in HG after72h (P<0.05).
Conclusion
1. These results demonstrated hypertrophy and apoptosis of podocytes may play important roles in pathogenesis of diabetic nephropathy. It was proved that the expression of cyclin-kinase inhibitors such as p27kip1, p21cip1was significantly increased followed by the hypertrophy of podocyte. With the progression of diabetic nephropathy, the number of apoptotic podocytes increased gradually and the balance between proliferation and apoptosis was broken in diabetic kidney. Apoposis which was regulated by apoptosis related genes such as bax, bcl-2and cleaved caspase-3.
2. MMF and valsartan can inhibit abnormal hypertrophy and apoptosis of podocytes in diabetes, partly by regulating the expression of cell cycle related protein p27kip1, p21cip1and apoptosis related genes,sueh as bax, bcl-2and cleaved caspase-3. These suggest that the protective effects of MMF on renal function maybe partly through inhibiting abnormal renal cell growth by regulating cell cycle related protein or apoptosis related genes.
引文
1.贾伟平.加强糖尿病慢性并发症的筛查,是提高防治效果的关键.上海医学,2009,32(5)：367-368.
2. Wild S,Roglic G,Green A, et al.Global Prevalence of diabetes estimates for the year 2000 and Projeetions for 2030[J].Diabetes Care,2004,27(5):1047-1053.
3. Barntt A. Prevention of loss of renal funetion over time in Patients with diabetie nephr-opathy [J].Am J Med,2006,119(5 Supp 11):S40-S47.
4. Marshall SM. Recent advances in diabetic nephropat hy [J].Clin Med,2004,4 (3):277-282.
5. Osterby R. Glomerular structural changes in type 1 (insulin-dependent) diabetes mellitus:causes, consequences, and prevention. Diabetologia,1992,35:803-812.
6. Adler S. Structure-function relationships associated with extracellular matrix alterations in diabetic glomerulopathy. J Am Soc Nephrol,1994,5:1165-1172.
7. Baumgartl HJ, Sigl G, Banholzer P, et al. On the prognosis of IDDM patients with large kidneys. Nephrol Dial Transplant,1998,13:630-634.
8. Young BA, Johnson RJ, Alpers CE, et al. Cellular events in the evolution of experimen-tal diabetic nephropathy. Kidney Int,1995,47:935-944.
9. Hoffman BB, Sharma K, Ziyadeh FN. Potential role of TGF-β in diabetic nephropathy. Miner Electrolyte Metab,1998,24:190-196.
10. Leehey DJ, Singh AK, Alavi N, et al. Role of angiotensin Ⅱ in diabeticn nephropathy.Kidney Int,2000,77(Suppl 77):S93-S98.
11. Saikumar P, Dong Z, Mikhailov V, et al. Apoptosis:definition, mechanisms, and relevance to disease. Am J Med,1999,107:489-506.
12. Dalla Vestra M, Saller A, MauerMet, et al. Role of mesangial expansion in the pathogenesis of diabetic nephropathy. J Nephrol,2001,14(Suppl 4):S51-S57.
13. Susztak K, Raff AC, Schiffer M, et al. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes,2006,55:225-233.
14. Verzola D, Gandolfo MT, Ferrario F, et al. Apoptosis in the kidneys of patients with type II diabetic nephropathy. Kidney Int,2007,72:1262-1272.
15. Mar shall SM.The podocyte:a major player in the development of diabetic nephropathy. Hormmetab Res,2005,37(Supp 11):9-16.
16. Doyonnas R,Kershaw DB,Duhme C, et al.Anuria,omphalocele,and perinatal lethality in mice laeking the CD34-related protein podoealyxin [J].J EXP Med,2001,194 (1):13-27.
17. Toyoda M,Naiafian B,Kim Y ET, et al.Podocyte detachment and reduced glomerular capillary endothelial fenestration in human type 1 diabetic nephropathy. Diabetes, 2007,56(8):2155-2160.
18. Tryggvason K,Wartiovaara J.How does the kidncy filter Plasma? [J]. Physiology (Bethesda),2005,20(1):96-101.
19. Zou J,Yaoita E,Watanabe Y, et al.Upregulation of nestin,vmieniin,and desmin in rat podocytes in response to injury[J].Virchows Areh,2006,448 (4):485-492.
20. Wharram BL, GoyalM,Wiggings JE, et al. Podocyte depletion causes glomeruloscler-osis:diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene. J Am Soc Nephrol,2005,16:2941-2952.
21. Brian Siu,Jharna Saha,William E Smoyer, et al.Reduction in podocyte density as a pathologic feature in early diabetic nephropathy in rodents:Prevention by lipoic acid treament.BMC Nephrology,2006,7:6.
22. Menini C.Iacobini G.Oddi C, et al.Increased glomerular cell (podocyte) apoptosis in rats with streptozotocin-induced diabeties mellitus:role in the development of diabetic glo-merular disease. Diabetologia,2007,DOI 10.1007/s00125-007-0821.
23. Haikong H, Zhihong L, Xuejin Z, et al. Mycophenolate mofetil inhibits endothelial cell NF-KB activity [J]. Acta Pharmacol Sin,2002,2.
1. Norbury C, Nurse P. Animal cell cycles and their control.AnnuRev. Bioehem,1989, 61:441-447.
2. Maekay K,Striker LJ,Stauffer JW, etal.Relationship of glomerular. Hypertrophy and sclerosis:Studies in SV40 transgenic mice.Kidney Int,1990,37:741-748.
3. Wolf G.Angiotensin as a renal growth promotting factor.Adv Exp. Med.Biol,1995, 377:225-236.
4. Fujihara CK, De Lourdes Noronha I, Malheiros, et al. Combined mycophen-olate mofetil and losartan therapy arrests established injury in the remnant kidney[J]. Am Soc Nephrol,2000,11 (2):283-290.
5. Rudnicki M, Mayer G. Significance of genetic polymorphisms of the Reninangiotensin-aldosterone system in cardiovascular and renal disease. Pharmaco-enomics,2009,10 (3):463-476.
6. Berrou J, Tostivint I, Verrecchia F, et a 1. Advanced glycation end products regulate extracellular matrix protein and protease expression by human glome-rular mesangial cells. Int J Mol Med,2009,23(4):513-520.
7. Fukami K, Ueda S,Yamagishi S, et al. AGEs Activate Mesangial TGF-beta-Smad Signaling Via an Angiotensin Ⅱ Type I Receptor Interaction. Kidney Int,2004,66 (6):2137-2147.
8. 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:S42-45.
9. Balakumar P, Chakkarwar VA, Krishan P, et al. Vascular endothelial dysfunction: a tug of war in diabetic nephropathy? Biomed Pharmacother,2009,63(3):171-179.
10. Maeda S. Genetics of diabetic nephropathy. Ther Adv Cardiovasc Dis,2008,2(5):363-371.
11. Rogus JJ, Poznik GD, Pezzolesi MG, et al. High-density single nucleotide polymorph-ism genome-wide linkage scan for susceptibility genes for diabetic nephropathy in type 1 diabetes:discordant sibpair approach. Diabetes,2008,57(9):2519-2526.
12. 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):141-150.
13. Makni K, Jarraya F, Rebai M, et al. Risk genotypes and haplotypes of the GLUT 1 gene for type 2 diabetic nephropathy in the Tunisian population. Ann Hum Biol,2008, 35 (5):490-498.
14. 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):308-313.
15. Maekay K,Striker LJ,Stauffer JW, et al.Relationship of glomerular hypertrophy and selerosis:Studies in SV40 transgenic mice.Kdney Int,1990,37:741-748.
16. Shankland SJ.Cell-cycle control and renal disease[J].Kdney lnt,1997,52(2):294-308.
17. Obaya AJ,Sedivy JM. Regulation of cyclin-Cdk aetivity in mammalian cells. Cell Mol Life Sci,2002,59(1):126-142.
18. Wolf G. Molecular mechanisms of renal hypertrophy:role of P27Kip1.[J]. Kidney Int, 1999,56(4):1262-1265.
19. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 Cyclin-dependent kinases. Genes Dev,1995,9:1149-1163.
20. Pangno M,et al. Role of the ubiquilin-proteosome pathway in regulating abundance of the cyclin-dependent kinase inhibitor P27 Science,1995,269:682-686.
21.陈世金,冯胜刚.周期素激酶抑制剂p21和糖尿病肾病[J].西部医学,2007,19(1)：133-135.
22. Umar A, Buermeyer AB, Simon JA, et al. Reouirement for PCNA mismatch repair at a step preceding DNA resynthesis[J]. Cell,1996,87(1):65-73.
23. Shankland SJ. Cell cycle regulatory proteins in glomerular disease[J]. Kidney Int, 1999,56(4):1208-1205.
24. Kuan CJ, AI-Douahji M, Shankland SJ. The cyclin kinase inhibitor p21 Wafl/Cipl is increased in experimental diabetic nephropathy:potential role in glomerular hypertrophy[J]. J Am Soc Nephrol.1998,9(6):986-993.
25. AI-Douahji M, Brugarolas J, Brown PAJ, et al. The cyclin kinase inhibitor p21 Wafl /Cipl is required for glomerular hypertrophy in experimental diabetic nephropathy [J]. Kidney Int,1999,56:1691-1699.
26. Brizzi MF, Dentelli P, Rosso A, et al. RAGE and TGF-β receptor-mediated signals converge on STAT5 and p21waf to control cell-cycle progression of mesangial cells:a possible role in the development and progression of diabetic nephropathy [J]. Faseb j,2004,18:1249-1251.
27. Kim YG, Alpers CE, Brugarolas J, et al. The cyclin kinase inhibitor p21 CIP1/WAF1 limits glomerular epithelial cell proliferation in experimental glomeruto nephritis[J]. Kidney Int,1999,55(6):2349-2361.
28. Petermann AT, Hiromura K, Blonski M, et al. Mechanical stress reduces podocyte proliferation in vitro [J]. Kidney Int,2002,61(1):40-50.
29. David J,Leehey,Ashok K,Singh,Nahid Alavi,Rekha Singh. Role of angiote-nsin II in diabeic nephropathy.Kindey Int,2001,58(Suppl 77):93-98.
30. Tete Hamken,Regine Schroeder,Rolf AK,Stahl,Gunter wolf. Angiotensin II mediated expression of P27 and induction of celluar hypertrophy in renal tubular cells depend on the generation of oxygen redicals. Kidney Int,1998,54:1923-1933.
31. Allison AC,Eugui EM.The design and development of animmunosuppressive drug,mycophenolate mofetil.Springer Semini mmunopathol,1993,14 (4):353-380.
32. Wu YG,Lin H,Qi X M,et al.Prevention of early renal injury by mycopheno-late mofetil and its mechanism in experimental diabetes [J].Int Immunopharmacol,2006,6 (3):445-453.
33. Xu ZG,Yoo TH,Ryu DR,et al.Angiotensin Ⅱ receptor blocker inhibits p27 Kip1 expre-ssion in glucose-stimulated podocytes and in diabetic glomeruli. Kidney Int,2005, 67:944-952.
1. Savill J.Apoptosis:a mechanism for regulation of the cell complement of inflamed glomeruli.Kidney Int,1992,47:606-612.
2. Savill J.Apoptosis and the kidney.J Am Soc Nephrol,1994,5:12-21.
3. Shimizu A,Masuda Y,Kitamura H,et al.Appotosis in progressive crescentic glomeru-lonephritis.Lab Invest,1996,74(5):941-951.
4. Kumar D,Zimpelmann J,Robertson S,Tubular and interstitial cell apoptosis in the streptozotocin-diabetic rat kidney [J].Nephron Exp Nephrol,2004,96(3):e77-e88.
5. Susztak K,Raff AC,Schiffer M,Bottinger EP,et al.Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy.Diabetes,2006,55:225-233.
6. MundelP,Shankland Stuart J.Podoeyte biology and response to injury J Am Socne-phrol,2002,13:3005-3015.
7. Ruotsalainen V,Ljungberg P,Wartiovaara J,et al.Nephrin is specifically located at the slit diaphragm of glomerular podocytes.Proe Natl Acad Sei USA,1999,96 (14):7962-7967.
8. Holzman LB,St John PL,Kovari IA,et al.Nephrin localizes to the slit pore of the glomerular epithelial cell.Kidney Int,1999,56(4):1481-1491.
9. Wagner N,Wagner KD, XingY,et al.The major podoeyte protein nephrin is transcrip tionally activated by the Wilms'tumor suppressor WT1[J].J Am Soe Nephrol,2004, 15(12):3044-3050.
10. Aaltonen P,Luimula P,Astrom E,et al.Changes in the expression of nephrin gene and protein in experimental diabetic nephropathy.Lab Invest,2001,81 (9):1185-1190.
11. Doublier S,Salvidio G,Lupia E,et al.Nephrin expression is reduced in human diabetic nephropathy:evidence for a distinct role for glycated albumin and angiotensin II.Diabetes,2003,Apr:52(4):1023-1030.
12. Nishikawa T,Edelstein D,Du XL,et al.Normalizing mitochondrial superoxide produc -tion blocks three pathways of hyperglycemic damage. Nature,2000,404 (677 9):787-790.
13. Takano Y,Yamauchi K,Hayakawa K,et al.Transcriptional suppression of nephrin in podocytes by macrophages:roles of inflammatory cytokines and involvement of The PI3K/Akt Pathway.FEBS Letters,2007,581(3):421-426.
14. Lee S B,Haber D A,et al.Wilms'tumor and the WT1 gene[J].Exp cell Res,2001, 264(1):74-99.
15. Smoyer W E.Podocyte alpha-actinin induction precedes foot process Effa-cement in expermiental nephrotic syndrome[J].Anl J Physiol Renal Physiol,1997,273:150-157.
16. Pollak MR. Inherited podocytopathies:FSGS and nephrotic syndrome from a genetic viewpoint.J Am Soc Nephrol,2002,13:3016-3023.
17. Narita I,Goto S,Saito N,et al.Genetic polymorphism of NPHS1 modifies the clinical manifestations of IgA nephropathy.Lab Invest,2003,83:1193-1200.
18. Guo J K, Menke AL, Gubler MC, et al. WT1 is a key regulator of podocyte function: reduced expression levelscause crescentic glomerulonephritis and mesangials clerosis.Hum Mol Genet,2002,11:651-659.
19. Palmer R. E., Kotsianti A., Cadman B. et al, WT1 regulates the expressionof the major glomerular podocyte membrane protein Podocalyxin,Curr.Biol,2001,11(22):1805-1809.
20. Wagner N, Wagner KD, Xing Y, et al. The major podocyte protein nephrin is transcriptionally activated by the Wilms'tumor suppressor WT1. J Am Soc Nephrol, 2004,15:3044-3051.
21. Srichai MB,Konieczkowski M, Padiyar A,et al.A WT1 co-regulator controls podoeyte phenotype by shuttling between adhesion structures and nucleus[J].J Biol Chem,2004,279(14):14398-14408.
22. Jung D S, Lee S H, Kwak S J, Li J J, Kim do H, Nam BY, et al. Apoptosis occurs differentially according to glomerular size in diabetic kidney disease[J]. Nephrol Dial Transplant,2012,27:259-266.
23. Seung J K, Jisun P, Do H K, Sun H L, Bo Y Nam, Hye Y K, et al. Local kallikrein-kinin system is involved in podocyte apoptosis under diabetic conditions [J]. Apoptosis,2011,16:478-490.
24. Susztak K, Raff AC, Schiffer M et al. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes,2006;55:225-233.
25. Verzola D, Gandolfo MT, Ferrario F et al. Apoptosis in the kidneys of patients with type Ⅱ diabetic nephropathy. Kidney Int,2007;72:1262-1272.
26. Daina NN, Korsmeyer SJ. Cell death:critical control points. Cell,2004,116 (2):205-219.
27. Aziz MH,Sundling KE,Dreckschmidt NE.Protein kinsase cepsilon inhibits UVR indu-ced expression of FADD,an adaptor protein,linked to both fas and TNFR1-mediated apoptosis[J].J Invest Dermatol,2009,129(8):2011-2021.
28. Caraccio N,Cuccato S,Pratesi F,et al.Effect of type I interferon(s) on cell viability and apoptosis in primary human thyrocyte cultures[J]. Thyroid,2009,19(2):149-155.
29. Gentilini A,Lottnin B,Brogi M,et al.Evaluation of intracellular signalling pathways in response to insulin-like growth factor I in apoptotic-resistant activated human hepatic stellate cell[J]. Fibrogenesis Tissue Repair,2009,2(l):1.
30. Genovese T,Esposito E,Mazzon E,et al.Absence of endogenous Interleukin-enhances secondary inflammatory process after spinal cord compression injury in mice[J].J Neurochem,2009,108(6):1360-1372.
31. Altuwaijri S,Chuang KH,Lai KP,et al.Susceptibility to autoimmunity and B cell resis-tance to apoptosis in mice lacking androgen receptor in B cells [J].Mol Endocrinol,2009,23(4):444-453.
32. Sasaki M,Uehara S,Ohta,et al.Losartan ameliorates progression of glomerular Structural changes in diabetic KKAy mice.Life Seienees,2004,75(7):869-880.
33. Andersen S,Jacobsen P,Tarnow L,et al.Time course of the antiproteinuric and antihy-pertensive effect of losartan in diabetic nephropathy.Nephrol Dial Transplant, 2003,18:293-297.
34. Brenner BM,Cooper ME,Zeeuw DD,et al.Effect of losartan on renal cardiovascular outcomes in patients with type 2 diabetes and nephropathy.New England J Med,2001,345(12):861-869.
1. Molitch ME,DeFronzo RA, Franz MJ, et al. Nephropathy in diabetes. American Diabetes Association. Diabetes Care,2004,27(suppl 1):S79-S83.
2. Asanuma K,Mundel P.The role of podocytes in glomerular pathobiology.Clin Exp Nephrol,2003,7(4):255-259.
3. Akhtar M,Al Mana H. Molecular basis of proteinuria.Adv Anat Pathol,2004,11 (6):304-309.
4. White KE,Bilous RW,Diabiopsies Study Group.Structural alterations to the podocyte are realated to proteinuriain type 2 diabetic patients.Nephrol Dial Transplant, 2004,19(6):1437-1440.
5. Benigni A,Gagliardini E,Tomasoni S,et al.Selective impairment of gene expression and assembly of nephrin in human diabetic nephropathy.Kidney Int,2004,65 (6):2193-2200.
6. Pavenstadt H, Kriz W, KretzlerM.Cell biology of the glomerular podocyte[J]. Physiol Rev,2003,83(1):253-307.
7. Khoshnoodi J,Sigmundsson K,Ofverstedt LG,et al.Nephrin promotes cell-cell adhesion through homophilic interactions[J]. Am J Pathol,2003,163(6):2337-2346.
8. Huber TB,Hartleben B,Kim J,et al.Nephrin and CD2AP associate with phosphoinosi-tide 3-OH kinase and stimulate AKT2dependent signaling [J]. Mol Cell Biol, 2003,23(14):4917-4928.
9. Susztak K, RaffAC, SchifferM, et al. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy [J].Diabetes,2006,55(1):225-233.
10. KumarD,Zimpelmann J,Robertson S,et al.Tubular and interstitial cell apoptosis in the streptozotocin-diabetic rat kidney[J]. Nephron Exp Nephrol,2004,96(3):77-88.
11. Lerco MM,Macedo CS,Silva RJ,et al.The number of podocyte and slit diaphragm is decreased in experimental diabetic nephropathy[J].Acta Cir Bras,2006,21(2):87-91.
12. Dalla VM,Masiero A,RoiterAM,et al.Is podocyte injury relevant in diabetic neph-ropathy? Studies in patients with type 2 diabetes[J].Diabetes,2003,52(4):1031-1035.
13. White KE,Bilous RW,Marshall SM,et al.Podocyte number in normotensive type 1 diabetic patientswith albuminuria[J].Diabetes,2002,51(10):3083-3089.
14. Tejada T, Catanuto P, Ijaz A, et,al. Failure to phosphorylate AKT in podocy-tes from mice with early diabetic nephropathy promotes cell death. Kidney Int,2008,73, 1385-1393.
15. Isermann B,Vinnikov IA,Madhusudhan T,et al.Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis.Nat Med, 2007,13(11):1349-1358.
16. Wendt TM,Tanji N,Guo J, et al.RAGE drives the development of glomerulo-sclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy[J].Am J Pathol,2003,162(4):1123-1137.
17. Ruster C,Bondeva T,Franke S,et al.Advanced glycation end-products induce cell cycle arrest and hypertrophy in podocytes.Nephrol Dial Transp-lant,2008,23:2179-2191.
18. Ding G,Reddy K,Kapasi AA, et al.Angiotensin Ⅱ induces apoptosis in rat glomerular epithelial cells[J].Am J Physiol Renal Physiol,2002,283 (1):F173-180.
19. Lee EY,Chung CH,KimJH,et al.Antioxidants ameliorate the expression of vascular endothelial factor mediated by protein kinase C in diabetic podocytes[J].Nephrol Dial Transplant,2006,21 (6):1496-1503.
2. Wild S,Roglic G,Green A, et al.Global Prevalence of diabetes estimates for the year 2000 and Projeetions for 2030[J].Diabetes Care,2004,27(5):1047-1053.
3. Barntt A. Prevention of loss of renal funetion over time in Patients with diabetie nephr-opathy [J].Am J Med,2006,119(5 Supp 11):S40-S47.
4. Marshall SM. Recent advances in diabetic nephropat hy [J].Clin Med,2004,4 (3):277-282.
5. Osterby R. Glomerular structural changes in type 1 (insulin-dependent) diabetes mellitus:causes, consequences, and prevention. Diabetologia,1992,35:803-812.
6. Adler S. Structure-function relationships associated with extracellular matrix alterations in diabetic glomerulopathy. J Am Soc Nephrol,1994,5:1165-1172.
7. Baumgartl HJ, Sigl G, Banholzer P, et al. On the prognosis of IDDM patients with large kidneys. Nephrol Dial Transplant,1998,13:630-634.
8. Young BA, Johnson RJ, Alpers CE, et al. Cellular events in the evolution of experimen-tal diabetic nephropathy. Kidney Int,1995,47:935-944.
9. Hoffman BB, Sharma K, Ziyadeh FN. Potential role of TGF-β in diabetic nephropathy. Miner Electrolyte Metab,1998,24:190-196.
10. Leehey DJ, Singh AK, Alavi N, et al. Role of angiotensin Ⅱ in diabeticn nephropathy.Kidney Int,2000,77(Suppl 77):S93-S98.
11. Saikumar P, Dong Z, Mikhailov V, et al. Apoptosis:definition, mechanisms, and relevance to disease. Am J Med,1999,107:489-506.
12. Dalla Vestra M, Saller A, MauerMet, et al. Role of mesangial expansion in the pathogenesis of diabetic nephropathy. J Nephrol,2001,14(Suppl 4):S51-S57.
13. Susztak K, Raff AC, Schiffer M, et al. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes,2006,55:225-233.
14. Verzola D, Gandolfo MT, Ferrario F, et al. Apoptosis in the kidneys of patients with type II diabetic nephropathy. Kidney Int,2007,72:1262-1272.
15. Mar shall SM.The podocyte:a major player in the development of diabetic nephropathy. Hormmetab Res,2005,37(Supp 11):9-16.
16. Doyonnas R,Kershaw DB,Duhme C, et al.Anuria,omphalocele,and perinatal lethality in mice laeking the CD34-related protein podoealyxin [J].J EXP Med,2001,194 (1):13-27.
17. Toyoda M,Naiafian B,Kim Y ET, et al.Podocyte detachment and reduced glomerular capillary endothelial fenestration in human type 1 diabetic nephropathy. Diabetes, 2007,56(8):2155-2160.
18. Tryggvason K,Wartiovaara J.How does the kidncy filter Plasma? [J]. Physiology (Bethesda),2005,20(1):96-101.
19. Zou J,Yaoita E,Watanabe Y, et al.Upregulation of nestin,vmieniin,and desmin in rat podocytes in response to injury[J].Virchows Areh,2006,448 (4):485-492.
20. Wharram BL, GoyalM,Wiggings JE, et al. Podocyte depletion causes glomeruloscler-osis:diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene. J Am Soc Nephrol,2005,16:2941-2952.
21. Brian Siu,Jharna Saha,William E Smoyer, et al.Reduction in podocyte density as a pathologic feature in early diabetic nephropathy in rodents:Prevention by lipoic acid treament.BMC Nephrology,2006,7:6.
22. Menini C.Iacobini G.Oddi C, et al.Increased glomerular cell (podocyte) apoptosis in rats with streptozotocin-induced diabeties mellitus:role in the development of diabetic glo-merular disease. Diabetologia,2007,DOI 10.1007/s00125-007-0821.
23. Haikong H, Zhihong L, Xuejin Z, et al. Mycophenolate mofetil inhibits endothelial cell NF-KB activity [J]. Acta Pharmacol Sin,2002,2.
1. Norbury C, Nurse P. Animal cell cycles and their control.AnnuRev. Bioehem,1989, 61:441-447.
2. Maekay K,Striker LJ,Stauffer JW, etal.Relationship of glomerular. Hypertrophy and sclerosis:Studies in SV40 transgenic mice.Kidney Int,1990,37:741-748.
3. Wolf G.Angiotensin as a renal growth promotting factor.Adv Exp. Med.Biol,1995, 377:225-236.
4. Fujihara CK, De Lourdes Noronha I, Malheiros, et al. Combined mycophen-olate mofetil and losartan therapy arrests established injury in the remnant kidney[J]. Am Soc Nephrol,2000,11 (2):283-290.
5. Rudnicki M, Mayer G. Significance of genetic polymorphisms of the Reninangiotensin-aldosterone system in cardiovascular and renal disease. Pharmaco-enomics,2009,10 (3):463-476.
6. Berrou J, Tostivint I, Verrecchia F, et a 1. Advanced glycation end products regulate extracellular matrix protein and protease expression by human glome-rular mesangial cells. Int J Mol Med,2009,23(4):513-520.
7. Fukami K, Ueda S,Yamagishi S, et al. AGEs Activate Mesangial TGF-beta-Smad Signaling Via an Angiotensin Ⅱ Type I Receptor Interaction. Kidney Int,2004,66 (6):2137-2147.
8. 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:S42-45.
9. Balakumar P, Chakkarwar VA, Krishan P, et al. Vascular endothelial dysfunction: a tug of war in diabetic nephropathy? Biomed Pharmacother,2009,63(3):171-179.
10. Maeda S. Genetics of diabetic nephropathy. Ther Adv Cardiovasc Dis,2008,2(5):363-371.
11. Rogus JJ, Poznik GD, Pezzolesi MG, et al. High-density single nucleotide polymorph-ism genome-wide linkage scan for susceptibility genes for diabetic nephropathy in type 1 diabetes:discordant sibpair approach. Diabetes,2008,57(9):2519-2526.
12. 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):141-150.
13. Makni K, Jarraya F, Rebai M, et al. Risk genotypes and haplotypes of the GLUT 1 gene for type 2 diabetic nephropathy in the Tunisian population. Ann Hum Biol,2008, 35 (5):490-498.
14. 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):308-313.
15. Maekay K,Striker LJ,Stauffer JW, et al.Relationship of glomerular hypertrophy and selerosis:Studies in SV40 transgenic mice.Kdney Int,1990,37:741-748.
16. Shankland SJ.Cell-cycle control and renal disease[J].Kdney lnt,1997,52(2):294-308.
17. Obaya AJ,Sedivy JM. Regulation of cyclin-Cdk aetivity in mammalian cells. Cell Mol Life Sci,2002,59(1):126-142.
18. Wolf G. Molecular mechanisms of renal hypertrophy:role of P27Kip1.[J]. Kidney Int, 1999,56(4):1262-1265.
19. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 Cyclin-dependent kinases. Genes Dev,1995,9:1149-1163.
20. Pangno M,et al. Role of the ubiquilin-proteosome pathway in regulating abundance of the cyclin-dependent kinase inhibitor P27 Science,1995,269:682-686.
21.陈世金,冯胜刚.周期素激酶抑制剂p21和糖尿病肾病[J].西部医学,2007,19(1)：133-135.
22. Umar A, Buermeyer AB, Simon JA, et al. Reouirement for PCNA mismatch repair at a step preceding DNA resynthesis[J]. Cell,1996,87(1):65-73.
23. Shankland SJ. Cell cycle regulatory proteins in glomerular disease[J]. Kidney Int, 1999,56(4):1208-1205.
24. Kuan CJ, AI-Douahji M, Shankland SJ. The cyclin kinase inhibitor p21 Wafl/Cipl is increased in experimental diabetic nephropathy:potential role in glomerular hypertrophy[J]. J Am Soc Nephrol.1998,9(6):986-993.
25. AI-Douahji M, Brugarolas J, Brown PAJ, et al. The cyclin kinase inhibitor p21 Wafl /Cipl is required for glomerular hypertrophy in experimental diabetic nephropathy [J]. Kidney Int,1999,56:1691-1699.
26. Brizzi MF, Dentelli P, Rosso A, et al. RAGE and TGF-β receptor-mediated signals converge on STAT5 and p21waf to control cell-cycle progression of mesangial cells:a possible role in the development and progression of diabetic nephropathy [J]. Faseb j,2004,18:1249-1251.
27. Kim YG, Alpers CE, Brugarolas J, et al. The cyclin kinase inhibitor p21 CIP1/WAF1 limits glomerular epithelial cell proliferation in experimental glomeruto nephritis[J]. Kidney Int,1999,55(6):2349-2361.
28. Petermann AT, Hiromura K, Blonski M, et al. Mechanical stress reduces podocyte proliferation in vitro [J]. Kidney Int,2002,61(1):40-50.
29. David J,Leehey,Ashok K,Singh,Nahid Alavi,Rekha Singh. Role of angiote-nsin II in diabeic nephropathy.Kindey Int,2001,58(Suppl 77):93-98.
30. Tete Hamken,Regine Schroeder,Rolf AK,Stahl,Gunter wolf. Angiotensin II mediated expression of P27 and induction of celluar hypertrophy in renal tubular cells depend on the generation of oxygen redicals. Kidney Int,1998,54:1923-1933.
31. Allison AC,Eugui EM.The design and development of animmunosuppressive drug,mycophenolate mofetil.Springer Semini mmunopathol,1993,14 (4):353-380.
32. Wu YG,Lin H,Qi X M,et al.Prevention of early renal injury by mycopheno-late mofetil and its mechanism in experimental diabetes [J].Int Immunopharmacol,2006,6 (3):445-453.
33. Xu ZG,Yoo TH,Ryu DR,et al.Angiotensin Ⅱ receptor blocker inhibits p27 Kip1 expre-ssion in glucose-stimulated podocytes and in diabetic glomeruli. Kidney Int,2005, 67:944-952.
1. Savill J.Apoptosis:a mechanism for regulation of the cell complement of inflamed glomeruli.Kidney Int,1992,47:606-612.
2. Savill J.Apoptosis and the kidney.J Am Soc Nephrol,1994,5:12-21.
3. Shimizu A,Masuda Y,Kitamura H,et al.Appotosis in progressive crescentic glomeru-lonephritis.Lab Invest,1996,74(5):941-951.
4. Kumar D,Zimpelmann J,Robertson S,Tubular and interstitial cell apoptosis in the streptozotocin-diabetic rat kidney [J].Nephron Exp Nephrol,2004,96(3):e77-e88.
5. Susztak K,Raff AC,Schiffer M,Bottinger EP,et al.Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy.Diabetes,2006,55:225-233.
6. MundelP,Shankland Stuart J.Podoeyte biology and response to injury J Am Socne-phrol,2002,13:3005-3015.
7. Ruotsalainen V,Ljungberg P,Wartiovaara J,et al.Nephrin is specifically located at the slit diaphragm of glomerular podocytes.Proe Natl Acad Sei USA,1999,96 (14):7962-7967.
8. Holzman LB,St John PL,Kovari IA,et al.Nephrin localizes to the slit pore of the glomerular epithelial cell.Kidney Int,1999,56(4):1481-1491.
9. Wagner N,Wagner KD, XingY,et al.The major podoeyte protein nephrin is transcrip tionally activated by the Wilms'tumor suppressor WT1[J].J Am Soe Nephrol,2004, 15(12):3044-3050.
10. Aaltonen P,Luimula P,Astrom E,et al.Changes in the expression of nephrin gene and protein in experimental diabetic nephropathy.Lab Invest,2001,81 (9):1185-1190.
11. Doublier S,Salvidio G,Lupia E,et al.Nephrin expression is reduced in human diabetic nephropathy:evidence for a distinct role for glycated albumin and angiotensin II.Diabetes,2003,Apr:52(4):1023-1030.
12. Nishikawa T,Edelstein D,Du XL,et al.Normalizing mitochondrial superoxide produc -tion blocks three pathways of hyperglycemic damage. Nature,2000,404 (677 9):787-790.
13. Takano Y,Yamauchi K,Hayakawa K,et al.Transcriptional suppression of nephrin in podocytes by macrophages:roles of inflammatory cytokines and involvement of The PI3K/Akt Pathway.FEBS Letters,2007,581(3):421-426.
14. Lee S B,Haber D A,et al.Wilms'tumor and the WT1 gene[J].Exp cell Res,2001, 264(1):74-99.
15. Smoyer W E.Podocyte alpha-actinin induction precedes foot process Effa-cement in expermiental nephrotic syndrome[J].Anl J Physiol Renal Physiol,1997,273:150-157.
16. Pollak MR. Inherited podocytopathies:FSGS and nephrotic syndrome from a genetic viewpoint.J Am Soc Nephrol,2002,13:3016-3023.
17. Narita I,Goto S,Saito N,et al.Genetic polymorphism of NPHS1 modifies the clinical manifestations of IgA nephropathy.Lab Invest,2003,83:1193-1200.
18. Guo J K, Menke AL, Gubler MC, et al. WT1 is a key regulator of podocyte function: reduced expression levelscause crescentic glomerulonephritis and mesangials clerosis.Hum Mol Genet,2002,11:651-659.
19. Palmer R. E., Kotsianti A., Cadman B. et al, WT1 regulates the expressionof the major glomerular podocyte membrane protein Podocalyxin,Curr.Biol,2001,11(22):1805-1809.
20. Wagner N, Wagner KD, Xing Y, et al. The major podocyte protein nephrin is transcriptionally activated by the Wilms'tumor suppressor WT1. J Am Soc Nephrol, 2004,15:3044-3051.
21. Srichai MB,Konieczkowski M, Padiyar A,et al.A WT1 co-regulator controls podoeyte phenotype by shuttling between adhesion structures and nucleus[J].J Biol Chem,2004,279(14):14398-14408.
22. Jung D S, Lee S H, Kwak S J, Li J J, Kim do H, Nam BY, et al. Apoptosis occurs differentially according to glomerular size in diabetic kidney disease[J]. Nephrol Dial Transplant,2012,27:259-266.
23. Seung J K, Jisun P, Do H K, Sun H L, Bo Y Nam, Hye Y K, et al. Local kallikrein-kinin system is involved in podocyte apoptosis under diabetic conditions [J]. Apoptosis,2011,16:478-490.
24. Susztak K, Raff AC, Schiffer M et al. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes,2006;55:225-233.
25. Verzola D, Gandolfo MT, Ferrario F et al. Apoptosis in the kidneys of patients with type Ⅱ diabetic nephropathy. Kidney Int,2007;72:1262-1272.
26. Daina NN, Korsmeyer SJ. Cell death:critical control points. Cell,2004,116 (2):205-219.
27. Aziz MH,Sundling KE,Dreckschmidt NE.Protein kinsase cepsilon inhibits UVR indu-ced expression of FADD,an adaptor protein,linked to both fas and TNFR1-mediated apoptosis[J].J Invest Dermatol,2009,129(8):2011-2021.
28. Caraccio N,Cuccato S,Pratesi F,et al.Effect of type I interferon(s) on cell viability and apoptosis in primary human thyrocyte cultures[J]. Thyroid,2009,19(2):149-155.
29. Gentilini A,Lottnin B,Brogi M,et al.Evaluation of intracellular signalling pathways in response to insulin-like growth factor I in apoptotic-resistant activated human hepatic stellate cell[J]. Fibrogenesis Tissue Repair,2009,2(l):1.
30. Genovese T,Esposito E,Mazzon E,et al.Absence of endogenous Interleukin-enhances secondary inflammatory process after spinal cord compression injury in mice[J].J Neurochem,2009,108(6):1360-1372.
31. Altuwaijri S,Chuang KH,Lai KP,et al.Susceptibility to autoimmunity and B cell resis-tance to apoptosis in mice lacking androgen receptor in B cells [J].Mol Endocrinol,2009,23(4):444-453.
32. Sasaki M,Uehara S,Ohta,et al.Losartan ameliorates progression of glomerular Structural changes in diabetic KKAy mice.Life Seienees,2004,75(7):869-880.
33. Andersen S,Jacobsen P,Tarnow L,et al.Time course of the antiproteinuric and antihy-pertensive effect of losartan in diabetic nephropathy.Nephrol Dial Transplant, 2003,18:293-297.
34. Brenner BM,Cooper ME,Zeeuw DD,et al.Effect of losartan on renal cardiovascular outcomes in patients with type 2 diabetes and nephropathy.New England J Med,2001,345(12):861-869.
1. Molitch ME,DeFronzo RA, Franz MJ, et al. Nephropathy in diabetes. American Diabetes Association. Diabetes Care,2004,27(suppl 1):S79-S83.
2. Asanuma K,Mundel P.The role of podocytes in glomerular pathobiology.Clin Exp Nephrol,2003,7(4):255-259.
3. Akhtar M,Al Mana H. Molecular basis of proteinuria.Adv Anat Pathol,2004,11 (6):304-309.
4. White KE,Bilous RW,Diabiopsies Study Group.Structural alterations to the podocyte are realated to proteinuriain type 2 diabetic patients.Nephrol Dial Transplant, 2004,19(6):1437-1440.
5. Benigni A,Gagliardini E,Tomasoni S,et al.Selective impairment of gene expression and assembly of nephrin in human diabetic nephropathy.Kidney Int,2004,65 (6):2193-2200.
6. Pavenstadt H, Kriz W, KretzlerM.Cell biology of the glomerular podocyte[J]. Physiol Rev,2003,83(1):253-307.
7. Khoshnoodi J,Sigmundsson K,Ofverstedt LG,et al.Nephrin promotes cell-cell adhesion through homophilic interactions[J]. Am J Pathol,2003,163(6):2337-2346.
8. Huber TB,Hartleben B,Kim J,et al.Nephrin and CD2AP associate with phosphoinosi-tide 3-OH kinase and stimulate AKT2dependent signaling [J]. Mol Cell Biol, 2003,23(14):4917-4928.
9. Susztak K, RaffAC, SchifferM, et al. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy [J].Diabetes,2006,55(1):225-233.
10. KumarD,Zimpelmann J,Robertson S,et al.Tubular and interstitial cell apoptosis in the streptozotocin-diabetic rat kidney[J]. Nephron Exp Nephrol,2004,96(3):77-88.
11. Lerco MM,Macedo CS,Silva RJ,et al.The number of podocyte and slit diaphragm is decreased in experimental diabetic nephropathy[J].Acta Cir Bras,2006,21(2):87-91.
12. Dalla VM,Masiero A,RoiterAM,et al.Is podocyte injury relevant in diabetic neph-ropathy? Studies in patients with type 2 diabetes[J].Diabetes,2003,52(4):1031-1035.
13. White KE,Bilous RW,Marshall SM,et al.Podocyte number in normotensive type 1 diabetic patientswith albuminuria[J].Diabetes,2002,51(10):3083-3089.
14. Tejada T, Catanuto P, Ijaz A, et,al. Failure to phosphorylate AKT in podocy-tes from mice with early diabetic nephropathy promotes cell death. Kidney Int,2008,73, 1385-1393.
15. Isermann B,Vinnikov IA,Madhusudhan T,et al.Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis.Nat Med, 2007,13(11):1349-1358.
16. Wendt TM,Tanji N,Guo J, et al.RAGE drives the development of glomerulo-sclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy[J].Am J Pathol,2003,162(4):1123-1137.
17. Ruster C,Bondeva T,Franke S,et al.Advanced glycation end-products induce cell cycle arrest and hypertrophy in podocytes.Nephrol Dial Transp-lant,2008,23:2179-2191.
18. Ding G,Reddy K,Kapasi AA, et al.Angiotensin Ⅱ induces apoptosis in rat glomerular epithelial cells[J].Am J Physiol Renal Physiol,2002,283 (1):F173-180.
19. Lee EY,Chung CH,KimJH,et al.Antioxidants ameliorate the expression of vascular endothelial factor mediated by protein kinase C in diabetic podocytes[J].Nephrol Dial Transplant,2006,21 (6):1496-1503.