泛素在大鼠慢性同种移植肾病中的表达及作用

目的:观察泛素在慢性移植肾病(CAN)大鼠肾组织中的表达及其与肾功能改变、病理学改变及肾组织正常活化T细胞表达及分泌趋化因子(RANTES)之间的关系,探讨泛素在CAN中的作用.方法:实验组采用近交系大鼠的同种异基因动物间的左肾原位移植,雄性F344大鼠40只为供体,雄性LEW大鼠40只为受体,移植手术后第10 d行右肾切除术,对照组采用单肾切除术的雄性F344和LEW大鼠各25只.分别于4周、8周、12周、16周及24周处死大鼠,做肾功能、移植肾组织学检测,并应用免疫组织化学与免疫印迹方法检测肾组织中泛素的表达,应用免疫组织化学方法检测肾组织RANTES的表达.结果:移植组大鼠尿蛋白定量、血清肌酐水平于移植后16周时显著增高,24周时更为显著.肾脏病理显示:移植后12周之前,主要表现为肾间质单个核细胞浸润,血管平滑肌细胞增殖及血管内膜的断裂;移植后16周时可观察到肾小球硬化、肾间质纤维化及血管硬化等慢性病理改变.免疫组织化学及Western-blot结果显示泛素在CAN病程中的表达呈逐渐升高后又降低的过程,移植12周为其表达高峰,后逐渐降低,24周时降至最低.免疫组织化学结果显示RANTES表达部位,表达时相与泛素表达是一致的.相关分析显示,肾移植大鼠泛素表达水平与24 h尿蛋白定量、血清肌酐水平在CAN早期呈正相关,在CAN晚期呈负相关.与肾间质纤维化程度呈负相关.移植12周组泛素表达水平与RANTES呈显著正相关.结论:泛素在CAN早晚期病变中起连接作用,泛素对上调增殖起基本作用,并可能通过对RANTES的调节,参与了CAN的全过程.     

大鼠慢性同种移植肾病肾组织中Smad7的表达及意义

目的研究大鼠慢性同种移植肾病肾组织中Smad7的表达及意义。方法制作原位肾移植大鼠模型，分别于4周、8周、12周、16周及24周处死大鼠，检测24h尿蛋白定量、肾功能；移植肾组织标本切片行光镜检测；免疫组织化学与免疫印迹、逆转录-多聚酶链反应方法检测肾组织中Smad7蛋白、Smad7mRNA的表达；免疫印迹方法检测肾组织p-smad2／3蛋白的表达水平，逆转录-多聚酶链反应方法检测肾组织转化生长因子-81mRNA的表达。结果移植组大鼠尿蛋白定量、血清肌酐水平于移植后16周时显著增高，24周时更为显著。Smad7蛋白在移植组逐渐降低，24周时降至最低，为对照组的15％。Smad7mRNA在移植后4周即升高，随着移植时间逐渐延长，24周时为对照组的2．73倍（P〈0．01）；p-smad2／3在移植后随疾病进展逐渐升高，24周时达高峰。免疫组织化学结果显示Smad7主要表达在小管间质。相关分析显示，肾移植大鼠Smad7蛋白表达水平与24h尿蛋白定量、血清肌酐水平、肾间质纤维化程度呈负相关（P〈0．05，P〈O．01）。结论Smad7蛋白表达下调在慢性同种移植肾病发病机制中起重要作用。     

整合素连接激酶在慢性移植肾肾病患者肾组织中的表达及意义

目的：探讨移植肾组织中整合素连接激酶（ILK）及Ⅳ型胶原的表达,及与转化生长因子-β1（TGF-β1）表达和慢性移植肾肾病（CAN）的关系。方法：用免疫组织化学技术和计算机真彩色图像分析系统半定量检测30例CAN患者移植肾组织中ILK、Ⅳ型胶原和TGF-β1的表达情况,分析三者间及与CAN病理分级之间的关系。10例正常肾组织作为对照。结果：CAN移植肾组织中ILK、Ⅳ型胶原、TGF-β1的表达比正常肾组织明显增加（P〈0.001）,并随CAN病理分级呈逐渐递增的趋势。移植肾组织中ILK的表达与TGF-β1、Ⅳ型胶原呈正相关（r分别为0.822、0.715,P〈0.001）。结论：研究结果显示ILK可能介导TGF-β1促进CAN细胞外基质（ECM）异常沉积发病机制,Ⅳ型胶原的异常沉积是CAN患者移植肾纤维化的重要表现,且ILK在CAN移植肾纤维化进程中起重要作用。     

肾小管上皮细胞转分化在慢性移植肾肾病中的作用

目的探讨肾小管上皮细胞-肌成纤维细胞转分化在慢性移植肾肾病(CAN)的发生、发展中的作用。方法36例移植肾穿刺标本分为正常组(N组),CAN组(C组),依Banff 97标准将C组按CANⅠ、Ⅱ、Ⅲ级分为C_1、C_2、C_3组,每组9例。免疫组化染色半定量分析比较α-平滑肌肌动蛋白(α-SMA)、波形蛋白(VIM)、角质蛋白(CK_(AEI/AE3))和转化生长因子β1(TGF-β1)在各组中的表达,线性相关分析TGF—β1和α-SMA、VIM、CK_(AE1/AE3)表达的相关性。结果C_1、C_2、C_3组α-SMA表达积分分别为0．95±0．07、1．78±0．12、2．42±0．31,VIM表达积分分别为0．74±0．05、1．31±0．18、2．34±0．25,组间呈递增趋势,均明显高于N组α-SMA表达积分0．07±0．02、VIM表达积分0．09±0．02(P＜0．05)；移植肾组织中TGF-β1表达与α-SMA、VIM呈正相关(r分别为0．73、0．68,P＜0．05),而与CKAE1/AE3表达呈负相关(r=-0．71,P＜0．05)。结论肾小管上皮细胞-肌成纤维细胞转分化参与了CAN的发生、发展,而局部肾组织中TGF-β1的表达上调可能是介导肾小管上皮细胞转分化的重要原因。     

大豆异黄酮对大鼠慢性移植肾肾病的防治作用

目的通过对肾移植大鼠的饮食营养干预，观察大豆异黄酮对慢性移植肾肾病的防治作用。方法选择近交系雄性Fisher（F344）大鼠作为供者，雄性Lewis（Lew）大鼠作为受者，采用显微外科技术制作肾移植模型。将受者随机分为三组，分别给予高异黄酮大豆蛋白饲料（HIS组）、低异黄酮大豆蛋白饲料（LIS组）或酪蛋白饲料（CAS组）。移植前和移植后第4、12和24周时检测血压，并收集受者的血和尿样，检测尿蛋白和血肌酐含量。24周时处死大鼠获取移植肾，行组织学和免疫组织化学检查。结果在移植后4周时，HIS组受者的尾动脉收缩压、24h尿蛋白含量和血肌酐浓度即低于LIS组和CAS组，但差异无统计学意义（P〉0．05）；移植后12周和24周时，HIS组的受者尾动脉收缩压、24h尿蛋白含量和血肌酐浓度均较LIS组和CAS组显著降低（P〈0．05）；移植后24周时，HIS组移植肾组织的间质纤维化和炎症、血管硬化、肾小球硬化和肾小管萎缩等慢性病变均较LIS组和CAS组为轻（P〈0．05）；HIS组移植肾组织中转化生长因子-β1（TGF-β1）的表达和分泌均较LIS组和CAS组为少（P〈0．05）。结论大豆异黄酮对移植肾功能和结构有保护作用，可作为一种防治慢性移植肾肾病的新方法。     

铁调素在大鼠慢性移植肾肾病中的表达及其意义

目的 探讨铁调素(Hepcidin)在大鼠慢性移植肾肾病(CAN)动物模型中的表达变化及临床意义.方法 以F344近交系大鼠为供体、Lewis近交系大鼠为受体,原位肾移植,建立20只CAN大鼠模型;于模型建立术前、术后1、2及4个月分别收集大鼠的静脉血和尿样,检测肾功能;酶联免疫吸附法(ELISA)检测血清、尿Hepcidin、血清白细胞介素-6(IL-6)和促红细胞生成素(EPO)的表达;并于移植后2个月及4个月分别处死大鼠10只,观察各组大鼠移植肾组织病理学变化.结果 血清Hepcidin、IL-6表达随着移植术的时间逐渐增高,术前、术后1、2及4个月分别为(8.18±1.60)、(10.94±2.10)、(12.71 ±2.10)、(15.81±2.40) μg/L; (2.52±0.41)、(15.58 ±6.94)、(18.16±7.08)、(25.71 ±5.71) ng/L.尿Hepcidin排出逐渐减少,术前、术后l、2及4个月分别为(17.52±2.60)、(13.65 ±2.80)、(12.02±1.30)、(10.13 ±1.80) μg/L,EPO表达逐渐减少术前、术后1、2及4个月分别为(15.51 ±0.76)、(10.29 ±0.58)、(6.37 ±0.82)、(1.23±0.15) U/L,术前与术后比较差异有统计学意义(P＜0.05);移植术后大鼠血肌酐(Cr)、尿素氮(BUN)逐渐升高,并且Cr与血清Hepcidin呈正相关;移植术后血清Hepcidin的表达与IL-6呈正相关,与EPO呈负相关;肾脏病理形态、HE染色符合CAN的病理改变.结论 随着移植肾功能的减退,大鼠体内微炎性反应增加,Hepcidin在大鼠CAN模型表达变化与肾功能有关.Hepcidin随着时间和炎症状态表达的变化可反映肾功能损伤.     

慢性移植肾肾病研究进展

本文主要阐述了慢性移植肾肾病的发病机制、诊断和治疗方面研究现状及进展。     

磷酸化肌球蛋白轻链在大鼠慢性移植肾肾病早期病变中的作用及其机制

目的 探讨磷酸化肌球蛋白轻链(pMLC)在慢性移植肾肾病(CAN)中的作用与机制.方法 依照标准的CAN大鼠模型进行左肾原位移植,受者为LEW大鼠,供者为F344大鼠,另取雄性F344大鼠和LEW大鼠仅行单肾切除术,分别作为对照.分别于术后4、8及12周时,收集各组大鼠的24 h尿量,并检测其尿肌酐水平,计算肌酐清除率.然后取各组大鼠血液标本,测定血清肌酐水平.采用Banff分级标准评定各组肾小球肾病、肾小管萎缩、间质纤维化及血管内膜增厚程度.采用免疫组织化学法和采用蛋白质印迹法检测肾组织中磷酸化MLC(pMLC)和整合素连接激酶(ILK)的表达部位及表达水平.结果 移植组大鼠术后4周时肾间质可见单个核细胞浸润,12周时可见血管平滑肌细胞的移行与增殖.移植组大鼠各时相肾组织pMLC和ILK表达水平显著高于Lewis对照组及F344对照组,且随着移植时间的延长有逐渐增高趋势.大鼠移植肾组织中pMLC表达水平与24 h尿蛋白定量、血清肌酐水平、肾间质单个核细胞浸润、肾小动脉血管平滑肌细胞数量、Banff评分等呈显著正相关,相关系数(r)分别为0.273(P＜0.05)、0.434(P＜0.01)、0.525(P＜0.01)、0.676(P＜0.01)、0.570(P＜0.01),在移植后4周时,pMLC表达水平与肾小管间质ILK表达水平呈显著正相关,r=0.778(P＜0.01).结论 pMLC在慢性移植肾病早期病理变化中发挥重要作用,移植肾肾小管间质及肾小动脉中pMLC表达上调与ILK的作用机制相关.

Abstract：

Objective To investigate the role and mechanism of phosphate myosin light chain (pMLC) in the rat kidney of chronic allograft nephropathy (CAN) model. Methods The left donor kidneys from Fisher (F344) rats were orthotopically transplanted into Lewis recipients. Meanwhile, the F344 rats and LEW rats with resection of the right kidney served as control groups. Animals were harvested respectively at the 4th, 8th and 12th week after transplantation. The creatinine clearance rate (CCr) was calculated by urine creatinine of 24-h urine. Blood samples were collected from rats for determination of serum creatinine. The expression of pMLC was detected by using Western blotting and immunohistochernistry, and that of integrin-linked kinase (ILK) by using immunohistochemistry. Results Mononuclear cells infiltration of allografts was markedly aggravated as compared to the controls. Allografts got severe interstitial fibrosis and tubular atrophy at 12th week after transplantation. The expression of pMILC and ILK was up-regulated in the kidney of CAN rats after transplantation, and increased more significantly as the time went on. The expression of pMILC was significantly correlated with 24-h urine protein excretion (r= 0. 273, P＜0. 05), serum creatinine levels (r = 0. 434, P＜0. 01 ), the number of tubulointerstitial infiltrated mononuclear cells (r = 0. 525, P＜0. 01 ), the number of smooth muscle cells (SMC) in vascular wall (r= 0. 676, P＜0. 01 ) and the extent of interstitial fibrosis (r= 0. 570, P＜0. 01 ).There was a significantly positive correlation between ILK and pMLC in CAN rats at the 4th week after transplantation (r= 0. 778, P＜0. 01 ). Conclusion pMLC might play an key role in CAN, and the over-expression of ILK might be involve in the pathogenesis of CAN.     

慢性移植肾肾病的治疗方法探讨

目的：探讨治疗慢性移植肾肾病(CAN)的有效途径。方法：按照不同治疗方案将CAN234例患者分为4组，比较各组的治疗效果。结果：234例的总有效率为34．2％，4组的有效率分别为19．3％、32．6％、46．9％、42．9％结论：随着免疫抑制方案的不同，临床上所见慢性排斥发生机制也有差别，治疗上宜根据患者具体情况，采取相应的综合措施，抗病毒、降血脂、降血压、治疗糖尿病等，可取得一定效果；结合传统中药治疗，可明显提高有效率。     

Smad核转录共抑因子在人近端小管上皮细胞转分化中的表达及作用

目的：观察Smad核转录共抑因子（SnoN）在TGF-β1致人近端小管上皮细胞转分化过程中的表达,探讨SnoN蛋白对TGF-β1所致的小管上皮细胞转分化的作用。方法：体外培养的人近端小管上皮细胞（HK-2）,随机分为正常对照组、TGF-β1（5ng/ml）组和TGF-β1（5ng/ml）＋MG-132（5μmol/ml）组。Western-blot检测细胞中SnoN蛋白水平的改变和Smad2/3磷酸化水平的改变;半定量RT-PCR方法观察细胞中SnoN mRNA表达的改变。免疫荧光检测间充质细胞标记物α-SMA的表达。结果：Western-blot的结果显示：（1）TGF-β1作用于细胞,SnoN蛋白表达迅速减少,10min为对照组的38%,并呈时间依赖进行性减少,60min较0min降低89%（P〈0.01）,24h有所恢复但仍较0min明显降低（P〈0.01）;TGF-β1＋MG-132组中SnoN蛋白表达与对照组差异无统计学意义,与TGF-β1组相比,SnoN蛋白水平明显上调。（2）半定量RT-PCR显示：TGF-β1作用于细胞后,与SnoN蛋白表达不同,SnoN mRNA迅速升高,60min其表达增高近1倍（与0min比较,P〈0.01）;TGF-β1＋MG-132组中SnoNmRNA的表达与TGF-β1组差异无统计学意义。（3）TGF-β1诱导细胞10min即可引发Smad2/3磷酸化（与0min比较,P〈0.01）,随着作用时间的延长,细胞中磷酸化的Smad2/3蛋白表达水平逐渐升高,TGF-β1＋MG-132组中,Smad2/3磷酸化蛋白的表达减少80%（P〈0.01）。（4）TGF-β1作用于细胞24h后免疫荧光检测到重新表达的α-SMA,TGF-β1＋MG-132组α-SMA的表达被抑制。结论：TGF-β1可诱导SnoNmRNA表达上调,但SnoN蛋白的表达遭遇泛素系统的降解显著减少,SnoN蛋白水平的下调可能是TGF-β1导致小管上皮细胞纤维化作用的必要条件。     

慢性移植肾病与整合素连接激酶

慢性移植肾病(cAN)已成为移植肾远期失功的一个主要原因,其发生原因和发病机制目前尚不清楚.CAN多发生于肾移植数月后,其分子生物学发病机制研究已广泛展开.本文就CAN发病机制研究现状予以综述,有利于更好理解本病发病机制以及开展临床CAN防治研究.     

Role of Peritubular Capillaries and Vascular Endothelial Growth Factor in Chronic Allograft Nephropathy

Objective

To investigate the role of peritubular capillary damage and vascular endothelial growth factor (VEGF) in chronic allograft injury and to evaluate their correlation with clinical factors.

Patients and Methods

The study included 56 patients who underwent transplantation between 1987 and 2004 and experienced chronic graft dysfunction. CD34 (peritubular capillaries) and VEGF were evaluated at histologic analysis. Patients were classified into 3 groups: 47 with chronic allograft injury, 9 with pure cyclosporine toxicity, and 26 who served as the control group (time 0 biopsy).

Results

Compared with the control group, CD34 total expression in chronic nephropathy was indirectly proportional to Banff stage (P < .05), and VEGF was increased in chronic allograft injury grade I or II or nephrotoxicity (P < .05). CD34 expression was correlated with age (P < .007) and number of acute rejection episodes (P = .005). A negative correlation was observed between expression of CD34 and of VEGF (P < .001). Low expression of CD34 was associated with risk of graft loss of 1.45 (95% confidence interval, 1.15-7.24; P = .04).

Conclusion

Peritubular capillaries decreased progressively with development of chronic allograft injury. The VEGF demonstrated a bimodal behavior, increasing at the onset of nephropathy and decreasing in the final stages. Loss of peritubular capillaries was associated with worse graft survival and overexpression of VEGF.     

甘草酸二胺对输尿管梗阻大鼠肾间质纤维化的治疗作用及机制

目的：探讨甘草酸二胺对肾间质纤维化的作用及其机制。方法：以Wistar大鼠单侧输尿管梗阻（UUO）为模型，在不同的时间点（7d、14d、28d）观察梗阻侧肾间质纤维化指数；致纤维化的转化生长因子β1（TGF-β1）的表达情况；肾皮质中与肾脏纤维化相关的Smurf2、Smad7信号蛋白等的mRNA及蛋白表达。结果：（1）随着梗阻时间的延长，肾间质纤维化程度逐渐加重，Smurf2基因和蛋白质明显上调，呈时间依赖性（P〈0．01）；Smad7蛋白呈时间依赖性下调（P〈0．01），Smad7基因在各个时间点无明显变化；TGF-β1基因在UUO后第7d达高峰，此后逐渐下降，但仍然高于假手术组（P〈0．01）。（2）甘草酸能改善UUO所致的肾间质纤维化程度（P〈0．01），下调肾脏组织TGF-β1基因的表达（尸〈0．01）；减少Smurf2基因和蛋白质的表达，同时上调TGF-β1信号传导中抑制性因子Smad7的表达（P〈0．01）。结论：甘草酸二胺能保护UUO所致的肾间质纤维化损伤。其可能的作用机制为减少Smurf2核酸和蛋白质的表达，增加抗纤维化作用的Smad7蛋白表达；减少TGF-β1表达，阻止TGF-β1信号传导，从而阻断肾间质的纤维化。     

Prediction of Chronic Allograft Nephropathy Using Classification Trees

Introduction

For its intrinsic potential to mine causal relations, machine learning techniques are useful to identify new risk indicators. In this work, we have shown two classification trees to predict chronic allograft nephropathy (CAN), through an evaluation of routine blood and urine tests.

Methods

We retrospectively analyzed 80 renal transplant patients with 60-month follow-up (mean = 55.20 ± 12.74) including 52 males and 28 females of overall average age of 41.65 ± 12.52 years. The primary endpoint was biopsy-proven CAN within 5 years from transplantation (n = 16). Exclusion criteria were multiorgan transplantations, patients aged less than 18 years, graft failure, or patient death in the first 6 months posttransplantation. Classification trees based on the C 4.8 algorithm were used to predict CAN development starting from patient features at transplantation and biochemical test at 6-month follow-up. Model performance was showed as sensitivity (S), false-positive rate (FPR), and area under the receiver operating characteristic curve (AUC).

Results

The two class of patients (no CAN versus CAN) showed significant differences in serum creatinine, estimated Glomerular Filtration Rate with Modification of Diet in Renal Disease study formula (MDRD), serum hemoglobin, hematocrit, blood urea nitrogen, and 24-hour urine protein excretion. Among the 23 evaluated variables, the first model selected six predictors of CAN, showing S = 62.5%, TFP = 7.2%, and AUC = 0.847 (confidence interval [CI] 0.749-0.945). The second model selected four variables, showing S = 81.3%, TFP = 25%, and AUC = 0.824 (CI 0.713-0.934).

Conclusions

Identification models have predicted the onset of multifactorial, complex pathology, like CAN. The use of classification trees represent a valid alternative to traditional statistical models, especially for the evaluation of interactions of risk factors.     

Epithelial-to-Mesenchymal Transition and Oxidative Stress in Chronic Allograft Nephropathy

Epithelial-to-mesenchymal transition (EMT) and oxidative stress contribute to kidney tissue fibrosis in various forms of native kidney disease. However, their role in chronic allograft nephropathy (CAN) remains somewhat uncertain. To address this question, kidney transplants were performed in 3-month-old rats, using the Fisher 344 --> Lewis model of CAN. Six-month posttransplant, kidney allografts displayed significant tubular atrophy, interstitial fibrosis and vascular wall thickening. Allograft recipients had significantly higher levels of serum creatinine (4.7 +/- 1.3 versus 0.59 +/- 0.08 mg/dL, p = 0.03) and proteinuria (380 +/- 102 versus 30.2 +/- 8 mg/dL, p = 0.04) compared to syngeneic grafts. Semiquantitative PCR, immunoblot and immunohistochemical analyses demonstrated increased alpha-smooth muscle actin (alpha-SMA) mRNA and protein levels coupled with reduced E-cadherin mRNA and protein immunoreactivity, confirming the presence of CAN-associated EMT. Allograft alpha-SMA levels were increased as early as 1-2 weeks posttransplant. Immunohistochemical studies for collagen type I and III, superoxide anion (O(2) (-)), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) confirmed that tubular O(2) (-), eNOS and iNOS, and interstitial collagen I, III and O(2) (-) levels were significantly increased in CAN-associated EMT. In conclusion, these observations suggest that CAN-associated EMT may be a link between oxidative stress and allograft fibrosis.