Expression changes of TRPC6 and podocalyxin in human membranous nephropathy

目的 检测膜性肾病(membranous nephropathy,MN)患者肾活检组织中肾小球足细胞瞬时受体电位阳离子通道蛋白6(transient receptor potential cation channel 6,TRPC6)和podocalyxin的表达和分布,探讨足细胞蛋白TRPC6和podocalyxin在MN蛋白尿发生中的作用.方法 采用常规组织病理以及免疫组化检查,并行免疫荧光双套色染色于激光共聚焦显微镜下观察TRPC6、podocalyxin在各组肾组织中表达及分布的改变,以计算机图像分析系统进行半定量分析.结果 对照组肾组织podocalyxin沿肾小球毛细血管壁连续均匀分布,阳性荧光信号表达强,MN组表达减弱,且分布不均或节段性缺失,部分呈点状、短线状不连续分布,肾病综合征组较非肾病综合征组减弱更明显;TRPC6在对照组肾小球有一定的表达,沿肾小球基膜呈均匀连续线型分布,MN组TRPC6荧光强度有不同程度增强,且呈点状、团块状不均匀分布,肾病综合征组较非肾病综合征组更明显.结论 TRPC6和podocalyxin在正常肾组织沿肾小球基膜呈连续线状均匀分布;MN患者肾小球内TRPC6表达增加,podocalyxin表达减少,且分布形式亦发生变化,提示TRPC6和podocalyxin等足细胞相关蛋白表达改变及分布异常可能是MN蛋白尿发生的重要病理机制.     

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。     

层粘连蛋白α2亚链在膜性肾病肾小球基膜的异常分布

目的观察Ⅳ型胶原和层粘连蛋白(laminin)亚链在膜性肾病(membranous nephropathy,MN)肾小球基膜(glomerular basement membrane,GBM)中的异常分布。方法收集52例MN肾组织,按照Ehrenreich和Churg提出的基于电镜形态特征的分期方法进行分期,同时收集10例微小病变肾组织作为正常GBM的对照。采用间接免疫荧光法检测不同分期MN肾组织GBM正常存在的代表性α5(Ⅳ)链及lamininα5、β2链的分布形态,以及可能异常存在的laminin α2、β1链的表达。结果在微小病变肾组织中α5(Ⅳ)链、lamininα5和β2链均沿GBM呈连续线状阳性;laminin α2、β1链在GBM中无表达。在Ⅰ期MN肾组织中α5(Ⅳ)链、lamininα5和β2链均呈连续线状表达。Ⅱ期MN肾组织中α5(Ⅳ)链在连续线状表达基础上形成大量钉突;lamininα5、β2链表达不规则增多,节段在连续线状表达基础上形成钉突。在Ⅲ期MN肾组织中α5(Ⅳ)链表达不规则增多,节段双轨状表达;lamininα5、β2链表达不规则增多,节段双轨、链环状表达。laminin α2链在Ⅰ期MN肾组织GBM未见明显阳性表达;在Ⅱ期和Ⅲ期MN肾组织GBM呈颗粒状阳性。lamininβ1链在各期MN肾组织GBM均未见明显阳性表达。结论MN患者GBM增厚不仅源自GBM固有Ⅳ型胶原亚链和laminin同种型的表达增多,还有正常仅表达于肾小球系膜区的laminin α2链的参与。     

不同类型肾小球疾病中MMP-9的表达及其在鉴别诊断中的意义

目的探讨基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)在多种肾小球疾病中的表达及意义。方法采用免疫组化Polink-2增强法检测MMP-9在增生性肾小球病变、基膜性肾小球病变和肾小球足细胞病变中的表达,分析表达位置及其在各种病变中的意义。结果 MMP-9在增生的系膜细胞、内皮细胞及新月体形成细胞胞质中呈强阳性,而系膜基质增生和纤维细胞性新月体形成时仅见少量弱阳性;MMP-9在基膜增厚病变为主的膜性肾病和Alport综合征肾小球足细胞胞质内呈强阳性;在薄基膜肾病肾小球中呈阴性;MMP-9在足细胞病肾小球中未见明显表达。结论 MMP-9参与炎症增生和肾小球基膜重建,辅助诊断肾小球基膜性病变。     

TRPC6 突变致足细胞病变机制的研究进展

瞬时受体电位阳离子通道蛋白6 (transient channel receptor potential cation 6, TRPC6) 是肾脏足细 胞裂孔隔膜 (slit diaphragm, SD) 的重要组成蛋白之一, 与肌动蛋白、 足细胞表面蛋白以及其他裂孔隔膜 蛋白分子相互作用, 共同维持足细胞的正常功能。 TRPC6 过表达或过度活动导致细胞内钙超载而损伤足细 胞。 目前报道 TRPC6 基因致病突变可导致局灶节段性肾小球硬化症。 近来研究发现, TRPC6 与以蛋白尿为 主要表现的肾小球疾病的发生发展密切相关, 但尚无特效靶向药物应用于临床。 文章主要对 TRPC6 基因突 变致足细胞病变的分子机制以及 TRPC6 基因型与临床表型之间的关系进行了综述。     

细胞骨架蛋白巢蛋白在人肾足细胞中的表达及意义

目的 检测细胞骨架蛋白巢蛋白在成人正常及病理状态肾组织中的表达,并观察其表达水平与患者尿蛋白的关系,初步探讨巢蛋白与足细胞损伤的关系.方法 正常对照肾组织(外科手术切除)6例,应用免疫组织化学(SP法)和免疫电镜检测巢蛋白的表达;病理状态肾组织:IgA肾病(无蛋白尿,电镜观察无足突融合,IrA-np)4例;IgA肾病(有蛋白尿,IgA-P)17例;膜性肾病8例;局灶性节段性肾小球硬化3例.应用免疫组织化学及即时RT-PCR的方法检测巢蛋白在肾组织中的表达并进行半定量、定量分析,与患者尿蛋白水平进行比较,分析两者之间相关关系.结果 巢蛋白表达在成人正常肾组织的足细胞初级足突中;免疫组织化学半定量及即时RT-PCR结果显示:IgA-np中巢蛋白的表达水平与正常肾组织差异无统计学意义;IgA-P、局灶性节段性肾小球硬化、膜性肾病中,巢蛋白的表达比正常肾组织显著降低(P<0.05),并与24 h尿蛋白呈负相关(r=-0.43,P<0.05).结论 巢蛋白在足细胞中低表达与肾小球中足细胞的损伤有关.     

TRPC6在TGF-β1诱导的体外培养肾小球足细胞损伤中的表达及其高表达对nephrin、desmin表达的影响

目的体外研究经典瞬时受体电位通道6(TRPC6)在TGF-β1诱导的肾小球足细胞损伤中的表达变化及其高表达对足细胞nephrin、desmin表达的影响。方法以肾小球足细胞株(MPC5)为研究对象,以不同质量浓度(0、4、8、12 ng/ml)TGF-β1刺激足细胞,干预72 h后用免疫荧光、Real-time PCR、Wstern blot检测TRPC6的分布及m RNA和蛋白表达。再将MPC5细胞分组,应用12 ng/ml TGF-β1处理各组细胞,分别于0、24、48、72 h应用免疫荧光、Real-time PCR、Western blot检测TRPC6的分布及m RNA和蛋白表达。用脂质体法将小鼠TRPC6真核表达载体p EX-3-TRPC6及对照质粒空载体p EX-3-NC转染足细胞,48 h后应用Western blot检测转染后TRPC6蛋白水平评估传染效率;同时应用免疫荧光、Real time RT-PCR及Western印迹方法检测转染后nephrin、desmin分布及表达的变化。结果与正常对照组相比,倒置显微镜下TGF-β1干预后足细胞足突融合、回缩,当TGF-β1增加至16 ng/ml时足突甚至消失。TGF-β1作用能使足细胞TRPC6表达及分布发生变化,当TGF-β1质量浓度为4 ng/ml,干预72 h后与对照组相比TRPC6 m RNA和蛋白含量即升高,但差异无统计学意义,当TGF-β1质量浓度提高到为8、12 ng/ml时TRPC6 m RNA和蛋白含量明显升高(P0.05),并呈剂量依赖性。应用12 ng/ml干预24 h,与对照组相比,TRPC6m RNA和蛋白含量即升高,但差异无统计学意义,当干预时间延长至48、72 h时TRPC6 m RNA和蛋白含量明显升高(P0.05),并呈时间依赖性。p EX-3-TRPC6转染48 h后足细胞TRPC6蛋白表达水平明显增高(P0.05),nephein分布未发生变化,但表达量在m RNA及蛋白水平分别下降25%及42%左右(P0.05),desmin分布发生改变,表达量在m RNA及蛋白水平分别升高132%及116%左右(P0.05)。结论 TGF-β1可诱导足细胞TRPC6分布变化且表达上调,TRPC6高表达可影响nephrin、desmin表达及desmin的分布,这可能是TRPC6参与足细胞损伤的机制之一。     

Expression of CD2AP on podocytes with different pathological types of nephrotic syndrome

目的 观察不同病理类型的原发性肾病综合征(nephrotic syndrome,NS)患者肾小球足细胞中CD2相关蛋白(CD2AP)的表达,探讨其与足细胞损伤的关系.方法 选取原发性NS患者54例,10例同期肾肿瘤切除患者正常肾组织作为对照.肾活检后常规染色观察肾脏组织病理改变,肾组织行免疫荧光法CD2AP和肾小球上皮细胞蛋白-1(GLEPP1)双重标记,对肾小球CD2AP的表达进行定位;分别用real time PCR和免疫组化SP法检测组织中CD2AP的表达,采用real time PCR检测nephrin的表达,透射电镜观察足细胞的结构变化,并定量测量足突密度.结果 (1)NS患者肾小球中CD2AP的表达及nephrin的表达下调,足细胞足突不同程度融合,足突密度降低.(2)病理表现为微小病变性肾病(minimal change disease,MCD)、局灶性节段性肾小球硬化(focal segmental glomerulosclerosis,FSGS)和膜性肾病(membranous nephropathy,MN)的NS患者CD2AP表达及nephrin表达较对照组明显降低,且CD2AP与nephrin表达呈正相关,病理表现为MCD和FSGS的NS患者CD2AP表达与足突密度呈正相关.结论 本研究首次发现原发性NS患者肾小球足细胞中CD2AP的表达降低,且在MCD和FSGS中与足细胞病变程度相关,提示CD2AP低表达在足细胞病变为主的肾小球疾病中发挥重要作用.CD2AP有利于诊断足细胞病变的早期检测,对CD2AP表达减低进行早期干预可能有助于延缓疾病进展.     

Podocalyxin与糖尿病肾病的关系

足细胞标记蛋白(podocalyxin)作为足突顶端质膜的主要构成部分,是足突顶膜区主要的带负电荷的唾液酸蛋白,参与维持足细胞的正常结构和滤过屏障。糖尿病肾病早期以肾脏肥大和肾小球高滤过为特征。在肾小球足细胞的检测中,podocalyxin作为最常用的标记蛋白之一,对监测肾小球疾病的发生和发展起到极其重要的作用。深入研究podocalyxin对糖尿病肾小球病变的早期诊断及治疗有着重要的指导意义。     

基于siRNA技术的TRPC6基因沉默对血管紧张素Ⅱ诱导的足细胞自噬和凋亡的影响

目的采用小分子干扰RNA(siRNA)干扰技术沉默足细胞相关分子瞬时受体电位阳离子通道蛋白6(TRPC6)对血管紧张素Ⅱ(AngⅡ)诱导的自噬和凋亡的影响。方法设计、构建siRNA,转染足细胞,使TRPC6基因沉默,采用流式细胞术、激光共聚焦、透射电镜及Western Blot技术检测不同浓度siRNA转染后TRPC6基因表达,优化获得TRPC6基因沉默最佳效果的条件。体外培养小鼠肾小球足细胞,设立对照组、AngⅡ组、空载体组、沉默TRPC6基因组和AngⅡ+沉默TRPC6基因组。对照组用含0.02%DMSO的RPMI 1640培养液培养;AngⅡ组加入AngⅡ(10~(-8)M)刺激足细胞;空载体组加入空载体至足细胞;沉默TRPC6基因组运用siRNA干扰技术沉默TRPC6基因;AngⅡ+沉默TRPC6基因组同时加入AngⅡ(10~(-8)M)及沉默TRPC6基因。处理12、24 h和48 h后分别收集细胞,采用流式细胞仪检测足细胞凋亡率,Western Blot检测TRPC6及自噬相关蛋白的表达,透射电镜及激光共聚焦电子显微镜观察自噬相关蛋白的分布。结果自噬在正常足细胞的表达量很微弱,AngⅡ组足细胞凋亡率明显升高,沉默TRPC6使足细胞凋亡率明显降低,与对照组比较差异有统计学意义(P0.05)。透射电镜下AngⅡ组示足细胞超微结构发生改变,自噬表达增加,胞质中出现独立双层膜结构,胞质成分及溶酶体等细胞器形成双层及多层膜结构的自噬体。沉默TRPC6使自噬表达下降,与AngⅡ组比较差异有统计学意义(P0.05)。激光共聚焦检测AngⅡ组LC3-Ⅱ的表达增加,沉默TRPC6使LC3-Ⅱ表达趋于稳定,与对照组比较差异有统计学意义(P0.05)。结论AngⅡ促进足细胞的凋亡,沉默TRPC6基因能有效保护AngⅡ诱导的肾小球足细胞损伤,发挥保护足细胞的作用。     

Angiotensin II contributes to podocyte injury by increasing TRPC6 expression via an NFAT-mediated positive feedback signaling pathway

The transient receptor potential channel C6 (TRPC6) is a slit diaphragm-associated protein in podocytes involved in regulating glomerular filter function. Gain-of-function mutations in TRPC6 cause hereditary focal segmental glomerulosclerosis (FSGS), and several human acquired proteinuric diseases show increased glomerular TRPC6 expression. Angiotensin II (AngII) is a key contributor to glomerular disease and may regulate TRPC6 expression in nonrenal cells. We demonstrate that AngII regulates TRPC6 mRNA and protein levels in cultured podocytes and that AngII infusion enhances glomerular TRPC6 expression in vivo. In animal models for human FSGS (doxorubicin nephropathy) and increased renin-angiotensin system activity (Ren2 transgenic rats), glomerular TRPC6 expression was increased in an AngII-dependent manner. TRPC6 expression correlated with glomerular damage markers and glomerulosclerosis. We show that the regulation of TRPC6 expression by AngII and doxorubicin requires TRPC6-mediated Ca(2+) influx and the activation of the Ca(2+)-dependent protein phosphatase calcineurin and its substrate nuclear factor of activated T cells (NFAT). Accordingly, calcineurin inhibition by cyclosporine decreased TRPC6 expression and reduced proteinuria in doxorubicin nephropathy, whereas podocyte-specific inducible expression of a constitutively active NFAT mutant increased TRPC6 expression and induced severe proteinuria. Our findings demonstrate that the deleterious effects of AngII on podocytes and its pathogenic role in glomerular disease involve enhanced TRPC6 expression via a calcineurin/NFAT positive feedback signaling pathway.     

Characterization and in vivo transfer of nephritogenic autoantibodies directed against dipeptidyl peptidase IV and laminin in experimental lupus nephritis

The present studies dealt with the pathogenesis of renal involvement in murine chronic graft-versus-host disease, which is a model for human systemic lupus erythematosus. The disease was induced in (C57BL10xDBA/2)F1 hybrids by injection of DBA/2 lymphocytes. The animals developed systemic disease accompanied by deposition of autoantibodies in the glomeruli and a lupus type of nephritis. Antibodies were eluted from glomeruli isolated during various stages of the disease by magnetic extraction from iron-perfused kidneys. For assessment of the specificity of the antibodies, we used indirect immunofluorescence, an enzyme-linked immunosorbent assay, and immunoblotting. In glomeruli from week 4, autoantibodies were found to be directed against several antigens, among which were the glomerular basement membrane component laminin and the glomerular enzyme dipeptidyl peptidase IV, whereas week 8 glomeruli also showed antibodies directed against nuclear antigens. Both laminin and dipeptidyl peptidase IV are known nephritogenic antigens occurring in renal tubular epithelial brush border preparations. Antibodies eluted from isolated glomeruli of diseased animals bound in a granular pattern along the glomerular capillary wall after in vivo transfer. Anti-renal tubular epithelial antibodies in the sera of diseased animals were affinity purified and injected into naive mice, which induced immune complex glomerulonephritis and proteinuria, thus confirming the nephritogenic role of these autoantibodies in this model.     

An ultrastructural study of the mechanisms of proteinuria in rat nephrotoxic nephritis.

Nephrotoxic nephritis was induced in Sprague-Dawley and Munich-Wistar rats by the injection of rabbit antirat kidney serum. A biphasic pattern of proteinuria was induced: the heterologous phase with a peak of proteinuria occurring at 10 to 16 hours, and the autologous phase with a peak at 10 to 15 days. For morphologic studies, glomeruli were fixed by perfusion, or by drip-fixation during good blood flow. In the heterologous phase, glomerular endothelial detachment or loss and leukocytic infiltration were prominent. In the autologous phase, focal detachment of glomerular endothelium and epithelium was commonly found. At sites of endothelial loss, in both phases, endogenous albumin (demonstrated by an ultrastructural immunoperoxidase technique), but not intravenously injected ferritin, showed abnormally deep penetration into the glomerular basement membrane. At sites of epithelial loss, found in the autologous phase, both albumin and ferritin were detected throughout the glomerular basement membrane. It is proposed that, in glomerular disease, leakage of plasma proteins may occur across the glomerular basement membrane at sites of endothelial or epithelial detachment.     

Ultrastructural study of nephritis induced in Brown Norway rats by mercuric chloride.

Chronic intoxication by mercuric chloride induces a two-phased nephropathy in Brown Norway rats characterized at first by a linear fixation of antiglomerular basement membrane antibody, then by a granular arteriolar and glomerular fixation of IgG superimposed on the linear one. On the 8th and 9th days, concomitant with the glomerular fixation of antiglomerular basement membrane antibodies, electron microscopy studies demonstrated an influx of monocytes into the glomerular and interstitial capillaries and a focal detachment of the glomerular endothelial cells. On the 14th and 15th days, subendothelial heterogeneous material and scattered subepithelial deposits appeared in glomeruli. By the 60th day, no cellular infiltration nor glomerular endothelial alterations persisted. Subepithelial glomerular deposits were seen in half of the animals, and abnormal deposits situated between muscular cells of the arteriolar walls were seen in all of them. These observations suggest that the glomerular fixation of antiglomerular basement membrane antibodies induces transient alterations of the inner aspect of the glomerular capillary wall. Arteriolar and subepithelial glomerular deposits confirm the idea that an immune-complex disease replaces the antiglomerular basement membrane antibody-mediated process. The morphologic expression of the transition between these two mechanisms is evident around the 15th day.     

Anti-GBM nephritis in the mouse: severe proteinuria in the heterologous phase

Summary Highly reproducible anti glomerular basement membrane (GBM) nephritis has been induced in the mouse after a single injection of rabbit or goat antibody against purified homologous GBM. The severity of albuminuria was closely related to the amount of antibody given. With doses of 4 mg or more, low serum albumin concentrations, sometimes accompanied by ascites and oedema, were observed after 1 week. Glomerular injury was characterized by an initial accumulation of polymorphonuclear granulocytes followed by thrombosis and necrosis, the extent of which defined the outcome of the glomerulonephritis. With high doses of antibody the exudative lesions entered a chronic phase, while at doses lower than 2 mg remission of the lesions occurred. Immunofluorescence studies showed prompt linear fixation of the injected anti-bodies to the glomerular capillary wall, accompanied by immediate binding of C3 in a fine granular pattern. Fibrin deposits appeared at 2 h in some glomeruli, increased thereafter, and were present after one day in more than 90% of the glomeruli in mice that had received 4 mg of antibody. This new reproducible model in the mouse is suited for the study of the relationship between activation of mediator systems, histological lesions, and proteinuria.     

Regional heterogeneity of glycoconjugate distribution in the glomerulus revealed by lectin-gold cytochemistry and SDS-PAGE.

The authors have used SDS-PAGE and lectin overlay analysis in parallel with lectin-gold cytochemistry to identify Helix pomatia lectin (HPL) binding glycoconjugates in rat kidney glomeruli. Previous work revealed HPL binding sites only beneath podocyte foot process bases, where they contact the glomerular basement membrane. It is shown here that after neuraminidase digestion of thin sections of glomeruli before incubation with HPL-gold complexes, the number of HPL binding sites is markedly increased. These new sites are mainly associated with the podocyte free surface (adjacent to the urinary space) and with capillary endothelial cells. By lectin overlays, this neuraminidase-dependent HPL binding was shown to be due to reaction of the lectin with desialylated podocalyxin. In contrast, HPL binding sites detected prior to neuraminidase digestion are associated with a novel glycoconjugate having a lower electrophoretic mobility than podocalyxin. Although any role for this glycoconjugate is at present speculative, it is strategically positioned at the site of interaction between foot process bases and the glomerular basement membrane. Its presence correlates with normal podocyte architecture, as shown by our previous studies on developmental and aminonucleoside nephrosis-associated changes in HPL binding to podocytes.     

Immune complex glomerulonephritis mediated by thyroid antigens.

Hypothyroidism, microscopic hematuria, and proteinuria developed in an 11-year-old girl. A renal biopsy specimen showed increased mesangial cells and matrix with focal glomerular basement membrane thickening. Three years later, a pronounced increase in proteinuria was detected. Elevated levels of antibody to thyroid microsomal antigen and thyroglobulin were found in the serum. A renal biopsy specimen showed a pronounced increase in mesangial cells and matrix with generalized glomerular basement membrane thickening. Electron microscopic studies demonstrated granular deposits in the capillary walls and mesangium. Immunofluorescent studies revealed granular deposits of IgG, IgM, and C3, primarily on the glomerular basement membrane. By indirect immunofluorescence, granular glomerular basement membrane and mesangial staining were detected with antibody specific for thyroglobulin and thyroid microsomal antigen. These observations suggest development of immune complex glomerulonephritis mediated by thyroid antigens.     

Expression of agrin, dystroglycan, and utrophin in normal renal tissue and in experimental glomerulopathies

The dystrophin-glycoprotein complex, which comprises alpha- and beta-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against alpha- and beta-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane.     

Isologous monoclonal antibodies can induce anti-GBM glomerulonephritis in rats.

Injection of isologous monoclonal antibodies (SR2, SR3) caused anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in WKY/NCrj rats. The antibodies were obtained from hybridoma cells derived from fusion of the spleen of a nephritic WKY/NCrj rat injected with rat solubilized renal basement membranes with adjuvant, and mouse SP2-myeloma cells. They belonged to the rat IgG2a subclass and bound to rat kidney in a linear pattern along the glomerular and tubular basement membranes. Histological changes in glomeruli were detected at day 1 after the injection; proteinuria with haematuria appeared on day 2; and proteinuria became severe and reached a plateau by day 5. These results demonstrate that anti-GBM nephritis can even be induced by an isologous monoclonal antibody and that the rat IgG2a subclass is at least nephritogenic. The experimental model of anti-GBM nephritis with isologous monoclonal antibodies makes it possible and easier to analyse further the mechanism of anti-GBM nephritis.     

Studies on the formation of glomerular immune deposits in brown Norway rats injected with mercuric chloride

Brown Norway rats injected with mercuric chloride (HgCl2) develop autoantibodies which immunolocalize along the glomerular basement membrane at first in a linear pattern and then in a granular pattern. The aim of this study was to characterize the specificity of these antibodies and to investigate the mechanisms responsible for the formation of granular immune deposits in the subepithelial zone of the glomerular basement membrane. The rats were found to develop circulating anti-laminin, anti-type IV collagen, anti-heparan sulfate proteoglycan, and anti-entactin antibodies. Antibodies against laminin and type IV collagen were found in relatively high titers in the sera and were specifically concentrated in the nephritic kidneys. Antibodies eluted from the nephritic kidneys with either linear or granular deposits reacted with basement membrane antigens synthesized and secreted by cultured rat glomerular visceral epithelial cells. Thus, in this model, the interaction of anti-laminin and type IV collagen antibodies with antigens secreted by glomerular visceral epithelial cells might, together with other mechanisms, contribute to the formation of granular immune deposits in the subepithelial part of the glomerular basement membrane.