一氧化氮在肾缺血再灌注肾小球损伤中的作用

目的:探讨一氧化氮(NO)对肾缺血再灌注（ischemia-reperfusion injury，I-RI）时大鼠肾小球超微结构及负电荷位点的影响。 方法:SD大鼠15只，建立肾缺血再灌注模型，动物随机分为5组：（1）假手术（sham）组（n=6）；（2）I-RI组（n=6），缺血前20 min舌静脉注入生理盐水0.3 mL；（3）SNP+I-RI组（n=6），缺血前20 min舌静脉注入2.5 μg/kg硝普钠（SNP）；（4）AG+I-RI组（n=6），缺血前20 min舌静脉注入10 mg/kg氨基胍（AG）；（5）L-NNA+I-RI组（n=6），缺血前20 min舌静脉注入10 mg/kg L-硝基精氨酸（L-NNA）。以聚乙烯亚胺（PEI）为阳离子探针标记肾小球滤过膜负电荷位点，透射电镜观察肾I-RI对大鼠肾小球超微结构及负电荷位点的影响。 结果:(1) sham组电镜下见肾小球结构正常，肾小球基底膜（GBM）外透明层负电荷位点（AS）清晰，呈连续的规则点线状排列［（19.3±1.7）个/1 000 nm］。I-RI组肾小球足细胞足突有明显的融合现象；GBM外透明层AS排列稀疏［（16.6±1.0）个/1 000 nm，P<0.05］，PEI颗粒小。（2）与I-RI组相比，给予SNP使肾I-RI大鼠肾小球滤过膜上皮细胞足突融合现象加重，肾小球GBM的AS［（11.7±3.2）个/1 000 nm］显著少于假手术组（P<0.05），且PEI颗粒的电子致密度也明显低于假手术组；而AG的应用使I-RI大鼠肾小球滤过膜损伤减轻，可见清晰的足突间隙；L-NNA+I-RI组大鼠肾小球上皮细胞足突融合也明显加重，但和I-RI组相比，L-NNA+I-RI组大鼠GBM的AS数量［（14.7±0.9）个/1 000 nm］无显著差异（P>0.05）。 结论:肾I-RI时出现肾小球上皮细胞足突融合、肾小球滤过膜的负电荷位点减少等病理性损伤，NO可加重这些损伤；肾I-RI时肾小球滤过膜超微结构的损伤与NO的生成及其作用有关。     

肾组织中perlecan的表达及其与蛋白尿的关系

目的 观察基膜蛋白多糖（perlecan）在不同病理改变儿童原发性肾病综合征及阿霉素肾病大鼠的肾组织中表达变化,探讨其参与蛋白尿发生的机制。方法 应用免疫组化法检测阿霉素肾病大鼠在阿霉素注射后第7、14、28天时肾组织perlecan的表达变化,并与24h尿蛋白进行相关分析。在69例不同病理类型原发性肾病综合征和血尿患儿的肾组织,应用免疫组化法检测perlecan表达,并分别与尿蛋白肌酐比值及电镜下平均足突宽度（FPW）进行相关分析。结果 在阿霉素肾病大鼠中随着蛋白尿的加重,perlecan在肾小球内染色强度明显减低,且与24h尿蛋白呈显著负相关（P〈0．01）。Perlecan在正常肾组织中沿肾小球血管襻及肾小管基膜分布。肾小球perlecan的表达在MCD、FSGS中分别较正常对照或TBMN显著减低,而在MN中沿增宽的GBM分布,表达显著升高。在IgA肾病中,肾小球内可见perlecan沿血管襻及系膜区分布,蛋白尿组的肾小球中perlecan表达量较单纯血尿组显著减低（P〈0．05）。MCD肾小球中perlecan免疫组化指数与尿蛋白肌酐比值呈负相关,与FPW无显著相关。结论 Perlecan在肾小球内表达减低与阿霉素肾病大鼠及不同病理类型的儿童原发性肾病综合征的蛋白尿的发生有关。     

实验性糖尿病肾小球的超微结构及其病变分级

目的:探讨糖尿病肾病的形态学特点及其变化规律.方法: 采用链脲佐菌素制备糖尿病大鼠模型.采用透射电子显微镜,对比观察14、30、60d肾病变.结果: 糖尿病模型组在14d时,肾小球等结构未见明显异常变化;在30d时,可见少数肾小球有系膜基质灶性增多;在60d时,少部分肾小球有系膜细胞增生,系膜基质灶性增多.透射电镜观察可见,糖尿病模型组在14d时,肾小球毛细血管基底膜不均匀,个别足突相互融合;在30d时,肾小球毛细血管基底膜节段性增厚,足突细胞部分足突融合,系膜区出现灶性电子致密物沉积.在60d时,肾小球毛细血管基底膜节段性增厚,部分基底膜的3层结构变得不清晰,部分系膜区增宽,可见灶性电子致密物沉积.结论: 链脲佐菌素诱导的糖尿病大鼠模型可出现肾病变,随着病程进展,可将糖尿病肾病变分为5级(DN0～DN4).     

足细胞损伤与肾小球疾病

足细胞（podocyte），即肾小球脏层上皮细胞，它附着于肾小球基底膜（glomerular basement membrane，GBM）的外侧，连同GBM和毛细血管内皮细胞一起，构成肾小球血液滤过屏障。足细胞这一独特的解剖位置，使得其体内研究较为困难；又由于正常成年机体的肾脏足细胞是一种终末分化细胞，体外培养的原代细胞不能增殖，     

钙调神经蛋白在抗肾小球基底膜病肾小球内的表达增强

 目的 观察抗肾小球基底膜（GBM）病患者肾小球足细胞钙调神经蛋白（CaN）的表达,了解其与临床病理特点及肾脏长期存活的关系。方法 选取临床病理资料完整的抗GBM病患者29例,对照为8例肾小球轻微病变患者。收集患者临床资料,免疫组化方法检测CaN A亚基α亚单位（CnAα）在肾小球的表达,免疫荧光染色观察足细胞骨架蛋白synaptopodin表达,分析CnAα与临床病理表现及预后的关系。结果 29例抗GBM病患者肾小球内均有不同程度的CnAα表达阳性,阳性区域占肾小球面积百分比显著高于轻微病变者（21.63%±14.27% vs 2.21%±1.41%,p<0.01）,同时伴有synaptopodin缺失。CnAα的表达量与抗GBM抗体峰值、血肌酐水平、血红蛋白水平、新月体比例及细胞/细胞纤维新月体比例呈现显著相关性,并与预后相关。结论 首次观察到抗GBM病患者肾小球内CnAα表达增强,表达量与疾病活动、严重程度及预后相关,提示足细胞可能参与抗GBM病新月体形成,其作用机制仍有待进一步研究。     

普乐可复防治大鼠抗肾小球基底膜肾炎的实验研究

目的:观察普乐可复(FK506)防治大鼠抗肾小球基底膜(GBM)肾炎的疗效。方法:复制大鼠抗GBM肾炎模型。实验分3组:肾炎+FK506组、肾炎对照组及正常对照组。大鼠一次性尾静脉注射抗GBM抗血清后6 h内皮下注射FK506注射液(0.5 mg·kg^-1·d^-1), 至第21 d。对照组则给予等量的生理盐水。定期于第4 d、第14 d和第21 d, 检测大鼠尿蛋白、血清肌酐和尿素氮水平, 观察肾组织病理学改变, 以及检测T淋巴细胞转化功能。结果:肾炎对照组大鼠注射抗血清后于第4d即出现异常蛋白尿, 血清肌酐和尿素氮亦持续上升;肾小球内可见细胞数增加和新月体形成, 肾小管内大量蛋白管型, GBM呈不规则增厚, 足突大片融合;T淋巴细胞转化功能异常。而肾炎+FK506组大鼠上述病变均明显较轻。结论:FK506能够明显改善大鼠抗GBM肾炎的肾功能。     

膜性肾病合并IgA肾病的临床病理特点

目的探讨膜性肾病合并IgA肾病的临床病理特点。方法回顾性研究北京大学第一医院肾内科和北京大学肾脏病研究所1998年1月—2006年4月问的肾活检病例9572例,对11例膜性肾病合并IgA肾病的临床病理特点进行分析,结合免疫电镜标记方法,对其病理诊断及发病机制进行探讨。结果11例患者以中年为发病高峰,平均年龄39．9岁,女性多于男性（男：女为1：2．9）,临床表现为蛋白尿,其中7例（63．6％）出现肾病综合征水平的蛋白尿,7例（63．6％）合并镜下血尿,肾功能均正常,除外了肝炎病毒感染、系统性红斑狼疮等继发性疾病。光镜下可见肾小球基底膜空泡变性和增厚,系膜细胞和基质轻度增生,2例可见少数肾小球伴有新月体形成。免疫荧光检查见IgG和c3颗粒样沿肾小球毛细血管壁沉积;IgA团块状在肾小球系膜区沉积。电镜检查可见肾小球上皮细胞下多数块状电子致密物沉积,系膜区可见团块状电子致密物沉积。免疫电镜标记结果显示,IgG定位于肾小球上皮细胞下的电子致密物,IgA定位于肾小球系膜区的电子致密物。结论膜性肾病合并IgA肾病兼具有膜性肾病和IgA肾病的临床病理特点,其发生过程可能为各自独立发生的两种疾病的叠加所致。     

小儿肾小球薄基膜病的临床病理研究

目的 :探讨小儿肾小球薄基膜病 (TBMN)的临床病理特征。方法 :对 11例TBMN患儿进行了临床、病理及超微结构的系统观察 ,测量了肾小球基膜 (GBM)及致密层的厚度 ,对小儿薄基膜病的临床病理特征进行了分析。结果 :11例小儿TBMN临床主要表现为单纯血尿 ,无其它明显的阳性体征 ,光镜下肾小球无明显改变或轻微异常 ,未见蛋白管型 ,包曼囊内及肾小管腔内可见渗出的红细胞 ,电镜下GBM广泛变薄 ,平均厚度 <2 0 0nm ,致密层厚度 <10 0nm。结论 :小儿肾小球薄基膜病的诊断主要依靠电镜 ,同时必须强调与临床病史、生化检查及病理组织学、免疫组化紧密结合方可确诊。     

糖尿病肾病患者同时测定血、尿Ⅳ型胶原的意义

糖尿病肾病的病理特征是肾小球系膜扩张 ,基底膜增厚 ,导致肾小球硬化 ,出现蛋白尿 ,肾功能衰竭等。Ⅳ型胶原（Ⅳ．ｃｏｌ）是肾基底膜及肾小球膜基质的主要成份。我们同时测定了５９例糖尿病肾病患者的血清、２４小时尿中Ⅳ．ｃｏｌ的含量 ,以探讨其对糖尿病肾病的测定意义。对象和方法一、对象 :（一）正常对照组２５例（男１３ ,女１２） ,年龄５３．２±１７．５岁 ,为我院体检正常人 ,其空腹血糖、血压正常 ,无糖尿病、高血压、肝病及肾脏疾病。（二）糖尿病组 :根据１９８５年ＷＨＯ糖尿病诊断标准选定的Ⅱ型糖尿病病人５９例（男３３…     

真武汤对阿霉素所致大鼠肾损伤的治疗作用

 目的：研究真武汤对阿霉素肾病模型（adriamycin nephropathy,AN）大鼠足细胞裂孔隔膜蛋白分子（podocin和nephrin）表达的影响,探讨其防治阿霉素肾病大鼠蛋白尿的机制。方法：采用常规生化、病理方法（包括HE染色、Masson染色及电镜）观察真武汤对AN模型所致纤维化大鼠肾功能、肾组织形态学变化及羟脯氨酸（Hyp）含量的改善作用;采用蛋白免疫印迹等方法探索真武汤对足细胞标志蛋白podocin和nephrin信号分子表达的影响。结果：模型组大鼠尿蛋白（TP）、血尿素氮(BUN)和血清肌酐（SCr）显著增加,肌酐清除率（CCr）显著下降（P<0.05）;Hyp显著增加（P<0.05）;肾组织podocin和nephrin蛋白表达水平显著降低（P<0.05）;肾小管萎缩,基底膜增厚;足突扁平、融合、消失。肾小球集中现象明显;部分肾小管扩张,肾小管上皮细胞变性,蛋白管型明显;肾间质纤维组织增生和较多炎症细胞浸润。与模型组相比,各治疗组TP、BUN和SCr均有一定程度的下降,CCr显著提高;Hyp明显下降（P<0.05）;真武汤组肾组织podocin和nephrin蛋白表达水平显著提高（P<0.05）;肾组织病变程度轻于模型组。结论：真武汤能减少阿霉素肾病大鼠肾组织羟脯氨酸含量,改善肾功能及减轻病理损伤。 真武汤降低模型大鼠蛋白尿的作用可能与其维持足细胞podocin和nephrin的表达有关。     

Deletion of CD151 results in a strain-dependent glomerular disease due to severe alterations of the glomerular basement membrane

Alterations in CD151 have been associated with primary glomerular disease in both humans and mice, implicating CD151 as a key component of the glomerular filtration barrier. CD151 belongs to the tetraspanin family and associates with cell-matrix adhesion complexes such as α3β1-integrin. Here we show that Cd151-deficient mice develop severe kidney disease on an FVB background but are healthy on a B6 background, providing a new and unique tool for the identification of genes that modulate the onset of proteinuria. To better understand the function of CD151 in the kidney, we studied its expression pattern and characterized early ultrastructural defects in Cd151-null kidneys. CD151 is expressed in podocytes of the mouse kidney and co-localizes with α3-integrin at the base of podocyte foot processes, at the site of anchorage to the glomerular basement membrane (GBM). Interestingly, the first ultrastructural lesions seen at the onset of proteinuria in Cd151-null kidneys were severe alterations of the GBM, reminiscent of Alport syndrome and consisting of massive thickening and splitting of the GBM. These lesions are associated with increased expression of GBM components. Podocyte abnormalities, effacement of foot processes, and podocyte loss appear to occur consequently to the GBM damage. In conclusion, CD151 appears to be involved in the establishment, maturation, and/or maintenance of the GBM structure in addition to its role in integrin-mediated adhesion strengthening.     

Ultrastructural changes of kidney in Schistosoma mansoni-infected mice

Schistosomiasis is the second threatening parasitic disease after malaria and among Schistosoma spp., Schistosoma mansoni (S. mansoni) affects about 100 million people in tropic regions in Africa and South America. The current study was carried out to investigate ultrastructural changes of the kidney in mice infected with cercariae of S. mansoni, in which 20 Swiss albino mice of 60-day-old were assigned into two groups (10 each). Control group received 1 ml normal saline by intraperitoneal route. Model group were intraperitoneally infected with 1 ml normal saline containing 40 cercariae of S. mansoni/mouse. After 60 days of infection, specimens from the kidneys of both control and infected mice were obtained and processed for transmission electron microscopy (TEM) examination. The main ultrastructural changes were observed in both glomeruli and tubules. Glomerular findings included irregular thickening and splitting of the glomerular basement membrane (GBM), flattening and effacement of the foot processes of podocytes, and proliferation of mesangial cells. Tubular changes were in the form of swelling, atrophy and vacuolation of tubular epithelial cells, and presence of autophagic vacuoles. In conclusion, adopting TEM shows a number of ultrastructural changes in the kidneys of mice infected with cercariae of S. mansoni, most notably thickening and splitting of GBM and flattening and effacement of foot processes of podocytes and tubular autophagic vacuoles. These changes are still unraveled well in the literature.     

Clinical, light, and electron microscopy findings in idiopathic haematuria

The electron microscopic findings are reported in detail in 20 patients submitted to renal biopsy with the major complaint or clinical finding of gross or microscopic haematuria. The lesions were classified histologically into four groups: group 1, minor glomerular alterations; group 2, focal mesangial thickening and/or cellular proliferation; group 3, diffuse mesangial proliferation; and group 4, other lesions. The major ultrastructural alterations included irregularity in thickness and density of the capillary basement membrane, with apparent discontinuity and bi- or multilaminar splitting of the lamina densa. There were varying degrees of foot process fusion, visceral epithelial polykaryocytosis, and granular deposits related to the capillary basement membrane. Densities were found in the mesangial basement membrane-like material, which was often markedly increased in quantity. A few microtubular aggregates were observed in endothelial cell cytoplasm. Changes consistent with acute diffuse proliferative and membrano-proliferative glomerulonephritis were also seen. The significant clinical findings, histological groups, and ultrastructural changes are correlated.     

Peripheral glomerular capillary wall lesions in IgA nephropathy and their implications

Peripheral glomerular capillary walls were studied in 26 cases of IgA nephropathy by means of the transmission electron microscope. Ultrastructural abnormalities were identified in 11 cases (42%). Abnormalities of the glomerular basement membrane (GBM) were the most frequent change which consisted of localized thinning, lamination, irregular thickening, disruption, membranolysis and aneurysmal dilatation of the GBM. Subendothelial electron dense deposits were seen. Necrosis and detachment of the podocytes from the GBM were also encountered. The changes were correlated with the clinical findings at the time of diagnosis which showed a significant correlation of these peripheral glomerular capillary wall lesions with proteinuria. With light microscopy, crescents were significantly more frequently seen in the cases showing the ultrastructural capillary wall abnormalities than those without. This observation suggested that local peripheral glomerular capillary wall damage was an important factor in the pathogenesis of the extracapillary lesions in IgA nephropathy.     

Changes in thickness and anionic sites of the glomerular basement membrane after subtotal nephrectomy in the rat.

Rats progressively develop proteinuria and glomerular sclerosis with age. These physiologic and histologic changes are accelerated by subtotal renal mass ablation. Alterations in the glomerular basement membrane (GBM), such as thickening and decreased anionic site density, occur during senescence. This study examines the ultrastructural alterations of the GBM affecting remnant glomeruli. Six week-old male Wistar rats underwent a subtotal nephrectomy (70%) and were compared with sham operated rats. Rats were fed a standard diet, and physiologic measurements were performed every 3 weeks. Rats were killed 6 and 12 weeks after surgery. Anionic sites were labeled by polyethyleneimine perfusion before killing. Kidneys were processed for light and electron microscopy. Nephrectomized rats had higher protein-uria than the controls and developed glomerular sclerosis. The GBM of nephrectomized rats were significantly thinner and anionic sites were less numerous 12 weeks after nephrectomy, especially in the lamina densa (LD) of the GBM. The number and distribution of anionic sites were similar to those observed in sham operated rats killed 6 weeks after surgery. These results indicate that glomerular sclerosis and GBM thickening are unrelated phenomena. They also suggest that the number and density of anionic sites in LD are not a prominent factor for increasing proteinuria after subtotal nephrectomy.     

Glomerular epithelial abnormalities associated with the onset of proteinuria in aminonucleoside nephrosis.

A sequential ultrastructural study has been made of glomerular podocytic epithelium before and after the onset of proteinuria induced by daily subcutaneous injections of low doses of puromycin aminonucleoside (PAN). At 4 days, before the onset of proteinuria, the principal change was extensive replacement of podocytic foot processes by broad expanses of epithelial cytoplasm. At 5 days, when proteinuria had developed, the epithelial cells showed in addition multiple cytoplasmic droplets, many large balloon like vacuoles, some of which were ruptured, and many foci of epithelial detachment from the glomerular basement membrane (GBM). Of particular significance, the onset of proteinuria coincided precisely with the development of areas of epithelial detachment that led to direct continuity between externally denuded GBM and the urinary space. It seems likely that these areas are the primary sites of protein leakage across the GBM in this experimental model.     

Renal allografts with glomerulonephritic change and proteinuria

Eight cases of renal allografts with glomerulonephritic change and proteinuria were classified into three groups according to the morphological features of the glomerular lesions. Group I (3 cases): By light microscope, remarkable reduplication of glomerular basement membrane (GBM), widening of mesangial region, and slight increase in mesangial cells, were observed. Electron microscopy revealed thickening of subendothelial space by deposition of electron-lucent material, mesangial interposition, and dense deposits in various regions (mainly in the subendothelial space). Group II (3 cases): By light microscope, crescent formation and reduplication of GBM were observed, while by electron microscope, changes of GBM similar to group I, but less remarkable, were seen. Group III (2 cases): Light microscope revealed spike formation in one case, but not in the other. With an electron microscope, subepithelial dense deposits were observed in both cases. Thickening of subendothelial space by deposition of electron-lucent material was noted in one case, while thickening of lamina densa was observed in the other case. Morphological change caused by rejection was observed in all eight cases, with six cases showing massive proteinuria and the other two showing slight proteinuria.     

Ultrastructural changes of glomerular basement membrane in IgA nephritis: relationship to hematuria

Ultrastructural changes in the glomerular basement membrane (GBM) of 415 samples from 344 patients with IgA nephritis, were examined for potential relationship to hematuria. The GBM showed various alteration: splitting, thinning, membranolysis with swelling of the lamina rara externa and interna, forming of small projections, and rupture. These lesions were present in 48% of IgA nephritis and in 16% of the controls. In the IgA nephritis group, the patients with hemispherical mesangial dense deposits had the highest rate (60%) of capillary wall abnormalities. Such lesions were more frequent in patients biopsied during severe hematuria (80%) than in those biopsied without hematuria (33%), (p less than 0.01). It is assumed that the ultrastructural abnormalities of GBM may contribute to the clinical evidence of severe hematuria.     

DAUNOMYCIN-INDUCED NEPHROPATHY IN RATS

A single intravenous injection of daunomycin into rats Induced severe glomerular injury with massive proteinuria. Mesangial thickening due to an increase in the matrix appeared as early as 5 weeks after injection. Focal and segmental glomerular tuft distortion developed by 10 weeks associated with a progressive mesangial change, which was accompanied by detachments of endothelial cells and podocytes from the glomerular basement membrane (GBM) resulting in obliteration of the affected tufts. After 20 weeks, the lesion ultimately progressed to cause diffuse and global glomerular obliteration. Scattered glomeruli also showed frank shrinkage with a mild obliterative change. By observing a number of isolated glomeruli in scanning electron microscopy, it was revealed that podocyte alterations were variable from case to case and foot processes remained discrete in some cases until 10 weeks, despite the presence of marked proteinuria. Anionic sites distributed throughout the GBM and on the surface of podocytes were usually preserved in proteinuric rats as far as evaluated by ruthenium red and colloidal iron stainings. Our results indicate that loss of foot processes and of glomerular anionic sites are not causative factors but consequences of proteinuria.     

Daunomycin-induced nephropathy in rats

A single intravenous injection of daunomycin into rats induced severe glomerular injury with massive proteinuria. Mesangial thickening due to an increase in the matrix appeared as early as 5 weeks after injection. Focal and segmental glomerular tuft distortion developed by 10 weeks associated with a progressive mesangial change, which was accompanied by detachments of endothelial cells and podocytes from the glomerular basement membrane (GBM) resulting in obliteration of the affected tufts. After 20 weeks, the lesion ultimately progressed to cause diffuse and global glomerular obliteration. Scattered glomeruli also showed frank shrinkage with a mild obliterative change. By observing a number of isolated glomeruli in scanning electron microscopy, it was revealed that podocyte alterations were variable from case to case and foot processes remained discrete in some cases until 10 weeks, despite the presence of marked proteinuria. Anionic sites distributed throughout the GBM and on the surface of podocytes were usually preserved in proteinuric rats as far as evaluated by ruthenium red and colloidal iron stainings. Our results indicate that loss of foot processes and of glomerular anionic sites are not causative factors but consequences of proteinuria.