抗肾小球基底膜抗体的抗原决定簇与患者肾损伤程度相关

目的研究抗肾小球基底膜（GBM）抗体的抗原决定簇及其与临床病理表现的关系。方法用辣根过氧化物酶标记的亲和层析纯化的抗GBM自身抗体（APab-HRP）和抗α1、3、5（IV）NC1单克隆抗体（Mab1、3、5）作为识别GBM上不同抗原决定簇的探针,应用竞争性ELISA法测定抗GBM抗体的抗原决定簇。结果59例患者中,58（98．3％）例可抑制Mab3,20（33．9％）例可抑制Mab1,均不抑制Mab5。同一血清对APab．HRP和对Mab3的抑制率呈平行关系（P〈0．01）。不同患者血清抗体对APab．HRP的抑制率与该患者确诊时的血肌酐浓度呈正相关（P〈0．05）。出现少尿或无尿的患者血清抗体对APab-HRP的抑制率较高（P〈0．01）。单纯抗GBM抗体阳性组较合并ANCA阳性组对APab．HRP的抑制率较高（P〈0．05）。是否出现咯血、肾脏病理新月体所占比例、肾转归以及其他临床表现不同的患者,对各种探针的抑制率均无差异。结论抗GBM抗体的主要抗原决定簇位于α3（IV）NC1,但并不完全一致。抗原决定簇的差异可能与肾损害的程度相关。     

足细胞抗原与肾小球疾病

肾小球足细胞的损伤及其相关蛋白的表达异常是蛋白尿形成的主要原因, 足细胞表面抗原在肾小球疾病发病机制中发挥了重要作用.人类膜性肾病第一个足细胞抗原-中性内肽酶以及第一个裂孔隔膜组成分子Nephrin的发现开辟了足细胞研究的新纪元.随着足细胞抗原在肾小球疾病及蛋白尿产生机制中的深入研究, 将为临床进一步揭示肾小球疾病的发病机制和寻找特异有效的防治蛋白尿的新的分子靶点提供理论依据.     

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。     

中和抗肾小球基底膜抗体的单链抗体的筛选与鉴定

目的 制备人抗肾小球基底膜(GBM)抗体的特异性人源化单链可变区抗体.方法 采用噬菌体表面展示技术,获得一个与人抗GBM抗体结合活性较强的单链可变区抗体片段的阳性克隆,并对该克隆进行DNA序列测定分析.结果 对噬菌体单链可变区抗体库经过3轮筛选后,与第1轮相比富集了137倍.噬菌体抗体与人抗GBM抗体的结合活性其中有35株克隆ELISA的吸光度较高.对这些噬菌体抗体进行交叉反应后,确定其中有10株交叉反应较弱.确定1株(C31)阳性克隆提取质粒,进行DNA序列测定,大小为750 bp,并符合人源化单链可变区抗体的序列结构.结论 应用噬菌体展示技术成功获得人-抗GBM抗体的单链可变区抗体基因,为临床上治疗Goodpasture综合征奠定实验基础.     

IL-18在大鼠抗肾小球基底膜肾炎凋亡中作用的研究

目的:探讨IL-18在大鼠抗肾小球基底膜肾炎细胞凋亡中的作用。方法:复制大鼠抗GBM肾炎模型,分别于实验第2、7、14、21和28天,行以下处理:收集24小时尿、血清样本检测大鼠24小时尿蛋白、血肌酐及血尿素氮含量;取肾组织经光镜、电镜观察肾组织病理变化;采用TUNEL法检测大鼠肾组织细胞凋亡情况;RT-PCR、免疫组织化学和Western blot法分别检测肾组织中IL-18、Fas mRNA和蛋白表达情况。结果:与正常对照组相比,肾炎模型组24小时尿蛋白、血肌酐和血尿素氮含量均明显升高(P0.01);光镜和电镜观察到肾小球内可见新月体形成、GBM不规则增厚、足突融合等病变;肾小球内凋亡细胞于第2天开始轻度增加,随后继续增加,于第28天有所减少,但仍维持较高水平;血清IL-18含量第2天开始升高,此后一直维持较高水平;肾组织中IL-18、Fas mRNA和蛋白表达均显著增加(P0.01);且肾炎模型组血清、肾组织IL-18表达与Fas表达均呈正相关(P0.01)。结论:IL-18在大鼠抗GBM肾炎凋亡中发挥重要的促进作用。     

蛋白尿大鼠肾小球基底膜阴离子位点变化

采用多聚亚胺染色的方法，并经电镜下计数肾小球基底膜上ＰＥＩ染色颗粒，研究了阿霉素肾病大鼠及抗肾毒血清肾炎大鼠ＧＢＭ上阴离子位点的变化，结果：两种具有蛋白尿的大鼠与正常大鼠相比，ＧＢＭ外疏松层阴离位点数量明显减少，大小也有显著改变，在上皮细胞足突融合的部位毛失更加显著，结果表明：肾小球阴离子位点的毛失，电荷屏障功能的损害是这些动物模型蛋白尿发生的重要原因。     

小儿肾小球薄基膜病超微病理的研究

一、材料与方法1.材料 :收集我院 1989～ 1999年住院并经肾活检、临床表现主要或单纯表现为血尿的患儿 83例 (不包括紫癜性肾炎 ) ,9例肾小球薄基膜病 (ｔｈｉｎｂａｓｅｍｅｎｔｍｅｍｂｒａｎｅｎｅｐｈｒｏｐａ ｔｈｙ ,ＴＢＭＮ)患儿均具有完整的临床及病理资料 ,其中男 5例 ,女 4例 ,年龄 4～ 12岁 ,中位年龄为 8.9岁。常规进行尿常规、尿蛋白定量、尿沉渣及肾功能等检查 ,入院后均行肾穿刺活检。 9例ＴＢＭＮ患儿 ,表现为单纯血尿者 8例 ,其中 6例为持续性镜下血尿 ,2例为肉眼血尿 ,病史半年至 3年半不等 ,另 1例镜下血尿患儿偶有轻度…     

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。     

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。     

Molecular characterization of the target antigens of anti-glomerular basement membrane antibody disease

The abnormal immune response to renal antigens is a significant cause of progressive glomerulonephritis and end-stage renal disease, leading to the need for dialysis or kidney transplantation. Type IV collagen of the glomerular basement membrane (GBM), an important component of the blood filtration barrier, is the target of pathogenic antibodies in two forms of anti-GBM antibody nephritis. Type IV collagen is a family of six chains that assemble into three networks with distinct composition and tissue-specific distribution. The GBM contains an α3·α4·α5(IV) network essential for the maintenance of kidney ultrafiltration function: the absence of this network in patients with Alport’s syndrome leads to progressive glomerulonephritis. In some Alport patients that receive a kidney transplant, anti-GBM alloantibodies develop against the non-collagenous (NC1) domains of the α3·α4·α5(IV) collagen network, which is present in the renal allograft but absent in the Alport kidneys, causing Alport post-transplant nephritis. In Goodpasture’s (GP) syndrome, anti-GBM autoantibodies target the NC1 domain of the α3 (IV) chain in the GBM, causing rapidly progressing glomerulonephritis. The GP epitopes have been localized to two homologous regions of the α3 NC1 domain, E_A and E_B, and several populations of autoantibodies with distinct epitope specificity were purified and characterized. The epitopes of GP autoantibodies are sequestered in the NC1 hexamer that connects two adjoining triple-helical molecules. Hydrophobic amino acids have been identified in the epitope of the immunodominant GP_A autoantibodies, suggesting that the cryptic nature of the GP epitopes is due to interactions among NC1 domains in the NC1 hexamer. Experimental anti-GBM nephritis can be induced in animal models by passive transfer of anti-GBM antibodies or by active immunization with NC1 domains of the α3·α4·α5(IV) network. RID="*" ID="*" Present address: Vanderbilt University Medical Center, Department of Medicine, Nashville, TN 37232, USA RID="*" Correspondence to: D.-B. Borza     

Antimitochondrial autoantibodies in anti-glomerular basement membrane disease.

Anti-glomerular basement membrane (GBM) disease is characterized by the production of an autoantibody with very restricted specificity, with no evidence of polyclonal B cell activation. It was therefore surprising to find that in a solid-phase ELISA a proportion of anti-GBM sera showed significant binding to pyruvate dehydrogenase (PDH), a reactivity usually associated with the antimitochondrial autoantibodies (AMA) found in primary biliary cirrhosis (PBC). The specificity of this reactivity was confirmed by inhibition and competition experiments. The AMA found in anti-GBM sera were of much lower affinity than those found in PBC sera, and recognized a more restricted set of species (mainly the 55-kD and occasionally the 74-kD component of PDH). However, it was possible to block the binding in a Western blot of an anti-GBM serum to both the 55-kD and 74-kD species with F(ab')2 fragments prepared from a PBC serum. Although AMA have been found in diseases other than PBC, such diseases have usually been characterized by polyclonal B cell activation. The stimulus to the production of AMA in anti-GBM disease, and their significance in pathogenesis (if any), are unknown.     

Coexistent Wegener's granulomatosis and anti-glomerular basement membrane disease

Wegener's granulomatosis and Goodpasture's syndrome represent two major causes of a pulmonary-renal syndrome. We describe the clinical course and morphologic features of a patient in whom pulmonary manifestations of Wegener's granulomatosis developed and were followed six months later by anti-glomerular basement membrane disease. Although we regard this as a unique and probably fortuitous association, a genetic predisposition or a secondary form of anti-GBM disease cannot be excluded.     

Serial functional affinity of autoantibodies in anti-glomerular basement membrane disease.

Anti-glomerular basement membrane (GBM) disease is caused by an autoantibody directed against an epitope on the alpha 3 chain of type IV collagen. Animal models demonstrate that the higher the affinity of such antibodies, the greater the degree of glomerular injury. Affinity maturation (the process whereby somatic mutation followed by antigen selection leads to an increase in affinity of antibody) might therefore be of pathogenic significance if it occurs in human anti-GBM disease. We have examined serial samples from nine patients with anti-GBM disease and looked for evidence of changing functional affinity by measuring the inhibition of binding produced by the mild chaotrope diethylamine (DEA) in an anti-GBM antibody ELISA. Seven patients showed no change in the inhibition produced by DEA with time. Two patients showed an apparent decrease with time in the inhibition produced by DEA; this apparent increase in functional affinity proved, on further investigation, to represent simply the loss of anti-GBM antibodies. These results may imply that affinity maturation has been completed by the time that patients present with anti-GBM disease. If there had been evidence for a further increase in functional affinity after this point then this might have added extra urgency to the need for removal of these autoantibodies as part of treatment.     

Thrombotic microangiopathy in anti-glomerular basement membrane glomerulonephritis

A review was made of the clinical histories, treatment, and renal histologic features of 12 patients with anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM disease). Two patients had milder histopathologic changes in their kidney biopsy specimens and achieved clinical remission. The other ten patients with severe lesions in their biopsy specimens progressed to renal failure. In addition, vascular lesions of thrombotic microangiopathy were present in six of 12 patients. Six patients had clinical laboratory evidence of microangiopathic hemolytic anemia. There are two important points to be noted from this study. First, the kidney biopsy specimen may be a useful indicator of disease severity and prognosis. Second, clinical laboratory or histologic evidence of thrombotic microangiopathy may occur in 75% of patients with anti-GBM disease.     

Transforming growth factor-beta production in anti-glomerular basement membrane disease in the rabbit.

The purpose of this study was to assay for the presence of collagen synthesis stimulatory activity in the kidney during immune-induced renal injury that results in severe fibrosis in both glomerular and interstitial compartments. A model of antiglomerular basement (anti-GBM) disease in the rabbit was induced on day 0 by the injection of anti-GBM antibody and renal cortex tissues were then sampled at various time points. Only conditioned media prepared from diseased renal cortical samples showed collagen synthesis stimulatory activity when tested on rabbit mesangial cells. The activity had an estimated molecular weight range of 16 to 25 kd and was neutralized by antibody to transforming growth factor-beta (TGF-beta). A standard assay for TGF-beta using a mink lung epithelial cell line confirmed the increase in TGF-beta activity in conditioned media of diseased cortex from day 7 and day 14 animals, which was not significantly activated by previous acidification. This suggests that most of the TGF-beta present in renal conditioned media was in the active form. The increase in renal cortical secretion of active TGF-beta was accompanied by increases in renal cortical TGF-beta mRNA content on days 4 and 7 after induction, with subsequent return to control levels. A similar increase in TGF-beta activity was present in nonacidified conditioned media of purified glomeruli from diseased days 7 and 14 animals, which was also accompanied by significant increases in TGF-beta mRNA. However with acidification no significant differences were noted between control and diseased samples, suggesting the presence of substantial latent TGF-beta activity in control glomerular conditioned media. These same control-conditioned media contained inhibitor activity for added exogenous TGF-beta. These results support the conclusion that the association between increased TGF-beta secretion and increased renal cortical collagen synthesis in this model is consistent with a role for this cytokine in directing fibrogenesis in the kidney.     

Fibronectin is the major fibroblast chemoattractant in rabbit anti-glomerular basement membrane disease.

The mechanism for fibroblast recruitment in renal fibrosis due to anti-glomerular basement membrane (anti-GBM) disease is unknown. Since fibroblast recruitment can occur via chemotaxis, assessment of the possible production of fibroblast chemotactic activity by affected renal tissue and its identification could provide important clues. Anti-GBM disease was induced by injection of guinea pig anti-rabbit GBM immunoglobulin G into rabbits previously sensitized to guinea pig immunoglobulin G. On days 4, 7, and 14 after induction, renal tissue was harvested and glomeruli isolated. Overnight serum-free conditioned media from whole cortex and glomeruli were prepared and assayed for fibroblast chemotactic activity. The results show low level activity in both conditioned media from control animals. In contrast, conditioned media from anti-GBM-treated animals at all time points showed significantly elevated fibroblast chemotactic activity peaking on day 4 with subsequent reduction thereafter. The magnitude of increase in cortical conditioned media was significantly higher than that for glomerular conditioned media, suggesting that most of the activity was derived from extraglomerular sources. Gel filtration analysis revealed the activity to be heterogeneous, consisting of at least four major species with estimated molecular weights ranging from 10 to > 100 kd. Acidification of conditioned media failed to increase chemotactic activity significantly, whereas protease digestion abolished it. Treatment of conditioned media with antifibronectin inhibited > 85% of the chemotactic activity, whereas antibodies to platelet-derived growth factor and transforming growth factor-beta did not have a significant effect. These findings taken together suggest that fibronectin-derived peptides represent the predominant fibroblast chemoattractant produced by renal cortex in anti-GBM disease.     

Avidity of anti-glomerular basement membrane autoantibodies was associated with disease severity

Anti-glomerular basement membrane (GBM) antibody-mediated diseases are characterized by the binding of autoantibodies to GBM, leading to rapidly progressive glomerulonephritis that often results in irreversible loss of renal function. The nephrotoxic potential of anti-GBM antibodies has been demonstrated in animal experiments. We questioned whether high avidity leads to persistent deposition of anti-GBM antibodies, thereby perpetuating inflammation and renal damage. To address the hypothesis, sera from 32 patients and serial samples from 11 patients with anti-GBM disease were collected. Purified bovine alpha chain non-collagen 1 domains of type IV collagen [alpha(IV)NC1] were employed to exam avidity of anti-GBM antibodies using antigen-inhibition enzyme-linked immunosorbent assay. The amount of alpha(IV)NC1 needed for 50% inhibition of antibody binding was compared among patients with different clinical and pathological parameters. After the sera were diluted to give equivalent concentration of anti-GBM antibodies, the amount of alpha(IV)NC1 used for 50% inhibition was prominently different among patients, from 0.02 microg to 20 microg, with an average at 0.666 microg. A significant correlation was observed between the amount of alpha(IV)NC1 used and the percentage of glomeruli which had crescents (P=0.001). Higher avidity of anti-GBM antibodies predicted higher percentage of glomerular crescents (R2=0.58, P<0.001). No obvious change of avidity was observed in the serial samples. The results suggested that affinity maturation might have been completed by the time that patients presented with anti-GBM disease. The avidity of anti-GBM antibodies was associated with the degree of renal damage and might play a key role in the pathogenesis of anti-GBM disease.     

Endogenous glucocorticoids modulate experimental anti-glomerular basement membrane glomerulonephritis

The influence of endogenous glucocorticoids (GC) on glomerular injury was studied in a rat model of heterologous anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Sprague-Dawley rats underwent adrenalectomy (ADX) or sham-operation 3 days prior to i.v. administration of both nephritogenic (100 microgram/g) and subnephritogenic (50 microgram/g) doses of sheep anti-rat GBM globulin. Administration of a subnephritogenic dose of anti-GBM globulin resulted in GN in adrenalectomized animals only. Similarly, ADX performed prior to administration of anti-GBM in the nephritogenic dose range resulted in exacerbation of GN compared with sham-operated animals (24 h protein excretion: 190.8 +/- 32.8 versus 42.5 +/- 2.6 mg/24 h; P < 0.005). In ADX animals receiving subnephritogenic doses of anti-GBM injury was manifested by abnormal proteinuria (62.7 +/- 5.8 mg/24 h), accumulation of neutrophils which peaked at 6 h (7.2 +/- 1.37 neutrophils per glomerular cross-section (neut/gcs)) and macrophage accumulation in glomeruli at 24 h (6.8 +/- 1.2 macrophages/gcs). Sham-adrenalectomized animals given the same dose of anti-GBM globulin developed minimal or no glomerular injury: urinary protein excretion (8.7 +/- 1.5 mg/24 h, P < 0.001); neutrophils (0.2 +/- 0.04 neutrophils/gcs, P < 0.001); macrophages (1.2 +/- 0.5 macrophages/gcs, P < 0.001). The increased cellular recruitment to glomeruli in adrenalectomized animals was associated with glomerular endothelial P-selectin expression. P-selectin expression was not detected in sham-operated rats after anti-GBM injection. Complement deposition in glomeruli was minimal in both groups. Physiologic GC replacement of ADX rats receiving subnephritogenic-dose anti-GBM reversed the observed susceptibility to GN development, with urinary protein excretion (7.8 +/- 1.12, P < 0.005) and no detectable P-selectin expression or leucocyte accumulation in glomeruli. These results suggest that endogenous GC modulate heterologous anti-GBM nephritis in rats and that this may be attributable, in part, to regulation of P-selectin expression.