川芎嗪对大鼠被动型Heymann肾炎病变的影响

建立ＳＤ大鼠典型的被动型Ｈｅｙｍａｎｎ肾炎（ＰＨＮ）模型,观察川芎嗪对肾内血栓素Ａ２（ＴｘＡ２）．前列环素Ｉ２（ＰＧＩ２）及ＴＸＡ２－ＰＧＩ２平衡的影响。结果表明：川穹嗪能够降低大鼠尿中的ＴＸＡ２而提高ＰＧＩ２的含量。此外,对于减少尿蛋白的分泌及肾组织学的损伤也有一定的作用。提示川芎嗪可用于ＰＨＮ病变的防治。     

雷帕霉素减缓大鼠被动Heymann肾炎的进展

 目的：观察雷帕霉素对大鼠被动Heymann肾炎（PHN）的影响,并探讨自噬在其中的作用。方法：雄性SD大鼠随机分为3组,即对照组、PHN模型组和雷帕霉素治疗组。以造模后第21天为观察结点,采用全自动生化分析仪测定24 h尿蛋白总量、血尿素氮和血清肌酐,过碘酸-六次甲基四胺银染色观察肾脏病变,Weibel-Gomez点计数方法计数足细胞数量,免疫荧光染色检测肾小球内C5b-9的沉积,免疫组化染色观察caspase-3的表达,Western blotting检测肾小球LC3的表达。结果：雷帕霉素明显减轻PHN模型大鼠的蛋白尿排出（P<0.05）,同时各组大鼠的肾功能均正常,其间无显著差异;雷帕霉素使PHN大鼠肾小球基底膜增厚的程度和范围有所减轻;雷帕霉素明显改善PHN大鼠足细胞缺失情况,减少足细胞凋亡;雷帕霉素可增强肾小球内固有细胞的自噬水平。结论：在PHN的病变过程中,适度增强自噬减少足细胞凋亡,减轻肾脏病变和缓解蛋白尿,可能是雷帕霉素减缓大鼠PHN进展的重要机制之一。     

Progressive passive Heymann nephritis: induction of autologous antibodies to rat brush border by multiple injections of heterologous antiserum.

Multiple i.v. injection of a heterologous anti-rat kidney fraction 3 antiserum and guinea pig anti-rabbit F3 into rats produced a progressive immune complex glomerulonephritis. The kidney disease was characterized by diffuse beaded deposition of rat gammaglobulin along the glomerular capillaries and proteinuria. Gammaglobulin eluted from the kidneys reacted with the brush border region of normal rat kidney frozen sections. Control animals did not develop the progressive disease. It appears that multiple i.v. injections of the heterologous antisera are capable of inducing an autoimmune kidney disease which is similar to Heymann nephritis.     

Stimulation of circulating autoantibody levels in the rat with established progressive passive Heymann nephritis.

Rats with established progressive passive Heymann nephritis (PPHN) were stimulated with tubular nephritogenic antigen derived from rat kidney fraction 3 (rKF3) or heterologous antibody to the eKF3 antigen. Rats stimulated with antigen had elevated levels of circulating autoantibody and increased amounts of rat IgG in a beaded pattern around the glomerular capillaries. The brush border (BB) region of the proximal convoluted tubules also stained for rat IgG. Rats stimulated with antibody had similar changes, but in addition the injected antibody was demonstrated in the glomerular deposits and in the BB region of the proximal convoluted tubules. Proteinuria was markedly increased in the antibody injected rats. This study indicates that the cells of the 'primed' immune system of rats with PPHN can be stimulated by 'additional' rKF3 antigen or antibody to it, to produce increased levels of circulating autoantibody. It is suggested that the progression of PPHN is dependent on the availability and access of the nephritogenic autoantigen to the immune system and that autoantigen may be released by autoantibody.     

A monoclonal antibody to brush border and passive Heymann nephritis

An IgG2a monoclonal antibody (MoAb) reacting with the brush border of the renal proximal tubule and glomerular capillary wall was produced by fusion of NS1 myeloma cells with spleen cells from BALB/c mice immunized with renal brush border preparations from rat kidney cortex. This antibody reacts with a 90,000 mol. wt protein which can be isolated by immunoprecipitation of radiolabelled brush border or glomerular preparations and localized on these structures by immunoperoxidase electron microscopy, thus demonstrating the presence of common antigenic determinants. Survey of various organs showed that the MoAb reacted with the brush border of the gut, but also with antigens associated with the distal vascular system. In the liver antigenic determinants were located along the sinusoid walls but mainly on bile canaliculi. Specific glomerular binding could be demonstrated in vivo by immunofluorescence after an intravenous injection of 2 mg of antibody or by paired label methodology using tracer amounts. Kinetics however were dramatically different from those observed in classical passive Heymann nephritis since glomerular binding was transient during the first hours after injection. Binding was also found in tubular structures, as well as in lung, liver, spleen and heart. These results identify a well defined antigen-antibody system responsible for the formation of transient extramembranous glomerular deposits and may be relevant to some human cases of glomerulonephritis. They may also provide new models to study glomerular and tubular transfer of membrane bound antibodies.     

The effect of pre-injection of rat kidney F3 antigen on the development of passive Heymann nephritis

The effect of repeated i.p. injections of a small dose of rat kidney F3 antigen, in an aqueous solution, on the development of passive Heymann nephritis in the rat has been investigated. The test animals receiving the antigen before the i.v. injection of the heterologous antibody developed more numerous and larger immune complexes on the epithelial side of the glomerular basement membrane than the control rats. It is suggested that the injected antigen is trapped in the glomerular basement membrane at sites where the pre-existing nephritogenic antigen is present making it possible for the more numerous and larger deposits to form.     

The effect of pre-injection of rat kidney F3 antigen on the development of passive Heymann nephritis.

The effect of repeated i.p. injections of a small dose of rat kidney F3 antigen, in an aqueous solution, on the development of passive Heymann nephritis in the rat has been investigated. The test animals receiving the antigen before the i.v. injection of the heterologous antibody developed more numerous and larger immune complexes on the epithelial side of the glomerular basement membrane than the control rats. It is suggested that the injected antigen is trapped in the glomerular basement membrane at sites where the pre-existing nephritogenic antigen is present making it possible for the more numerous and larger deposits to form.     

Preparative polyacrylamide gel electrophoresis in the isolation of the nephritogenic proteins of passive Heymann nephritis

To study kidney antigens involved in the formation of glomerular subepithelial immune deposits in passive Heymann nephritis polypeptides of 500, 130 and 105 kDa were isolated from rat kidney brush border (BB) membrane fraction using preparative polyacrylamide gel electrophoresis. Polyclonal antibodies raised against these proteins were specific for their respective antigens in immunoblotting. All three antisera bound to proximal tubular BB of kidney and to apical surfaces of several other epithelia as shown by indirect immunofluorescence on frozen sections of normal rat tissues. The anti-500 kDa and anti-105 kDa, but not the anti-130 kDa, antibodies also stained glomeruli and the anti-105 kDa antibodies also endothelial cells. After injection into rats the anti-500 kDa IgG bound to kidney glomeruli forming diffuse, granular deposits of rabbit IgG along the glomerular capillary walls, as shown by direct immunofluorescence. In electron microscopy the immune deposits were subepithelial and electron dense. The deposits remained in glomeruli for at least 60 days and increased with time. Deposits of C3 were not detected and proteinuria did not develop. The anti-130 kDa and the anti-105 kDa IgGs did not form glomerular deposits after in vivo injections. The results suggest that the 500 kDa and the 105 kDa proteins or related antigens are present in glomeruli and the 500 kDa protein is located on the epithelial side of the glomerular basement membrane. Circulating antibodies can bind to the 500 kDa protein forming immune complexes which rearrange and form electron dense deposits. The results further demonstrate that preparative gel electrophoresis is a useful technique for the isolation of kidney proteins of immunopathologic interest.     

Antibodies to glycolipids activate complement and promote proteinuria in passive Heymann nephritis.

Passive Heymann nephritis is an experimental rat model of human membranous nephropathy induced by injection of antisera against crude renal cortical fractions such as Fx1A or rat tubular microvilli. This results in the formation of subepithelial immune deposits, the activation of the C5b-9 membrane attack complex of complement, and severe proteinuria. While the formation of immune deposits is attributed to in situ immune complex formation with antibodies specific for the gp330-Heymann nephritis antigenic complex (HNAC), activation of complement and proteinuria appear to be caused by at least one additional antibody species present in anti-Fx1A sera. We have separated by affinity absorption polyspecific antisera against Fx1A and rat microvilli into one IgG fraction directed specifically against microvillar proteins (anti-Fx1A-prot) and another IgG fraction specific for glycolipids (ant-Fx1A-lip) of tubular microvilli. When injected into rats, the anti-Fx1A-prot fraction induced immune deposits but failed to activate complement or produce proteinuria, similar to results obtained with affinity-purified anti-gp330 IgG. When the antibodies of the anti-Fx1A-lip fraction were injected alone they did not bind to glomeruli. By contrast, when the IgGs specific for the Fx1A-prot fraction (or for gp330-HNAC) were combined with those directed against the Fx1A-lip glycolipid preparation, immune deposits were formed, in situ complement activation was observed, and also proteinuria was induced. It is concluded that within anti-Fx1A and anti-microvillar sera there are at least two IgG fractions of relevance for the development of PHN: one directed against the gp330-HNAC complex which is responsible for the development of immune deposits, and a second specific for glycolipid antigen(s) which activate(s) the complement cascade.     

Lymphocyte subsets in Heymann nephritis

Altered ratios of T lymphocyte subsets have recently been reported in some forms of glomerulonephritis, including membranous glomerulonephritis. Heymann nephritis is a model of membranous glomerulonephritis that can be induced in susceptible strains of rat by a single subcutaneous injection of renal tubular antigen in Freund's complete adjuvant. Monoclonal antibodies were used to identify cytotoxic/suppressor and helper/inducer T cells in the blood, spleen, peripheral lymph nodes, and, where relevant, the lymph node draining the antigen injection site in susceptible and nonsusceptible rat strains before and after immunization with renal tubular antigen. A marked interstrain variation in the proportions of T lymphocyte subsets was found, but this did not segregate strains that are susceptible to Heymann nephritis induction from those that are resistant. Neither the development of Heymann nephritis in susceptible strains or immunization of resistant strains with renal tubular antigen was associated with any specific alteration in the T lymphocyte subpopulations.     

异搏停和山莨菪碱对大鼠被动型Heymann肾炎病变的影响

本实验应用抗大鼠肾小管抗原的抗血清制做大鼠被动型Ｈｅｙｍａｎｎ肾炎（ＰＨＮ）动物模型，并选用异搏停和山莨菪碱进行处理，观察两药对其病变的影响，结果表明：异搏停及山莨菪碱处理的大鼠尿蛋白均明显低于ＰＨＮ组，病理组织损伤亦有一定程度的改善。提示：上述两药均对大鼠ＰＨＮ病变有一定的治疗作用。     

Sequential localization of antibody to multiple regions of the glomerular capillary wall in passive Heymann nephritis

Passive Heymann nephritis was produced in rats by injection of the multispecific anti-Fx1A antibody. At time points 1 hour, 1 day, 2 days, 3 days, and 4 days groups of rats were sacrificed and their kidneys fixed by retrograde perfusion with paraformaldehyde lysine periodate. The antibody was visualized by direct immunofluorescence and by 125I-Protein-A electron microscopic autoradiography. Localization of the antibody in the lamina rara interna, lamina densa, lamina rara externa and the glomerular epithelial cell was determined by electron microscopic autoradiography according to the method of Saltpeter, Fertuck, and Saltpeter (Saltpeter MM, Fertuck MC, Saltpeter EE: J Cell Biol 72:161, 1977). At least 100 grains/kidney were analyzed. At one hour the antibody was localized in a linear, discontinuous pattern by immunofluorescent microscopy. Ultrastructurally, the antibody was present in all regions of the capillary wall although predominantly in the lamina rara interna. At later time points the immunofluorescence staining changed to the typical granular pattern with majority of the grains localizing to the lamina rara externa and the cell body of the glomerular epithelial cell. The importance of these observations is several-fold. (a) It suggests the involvement of multiple antigens in the pathogenesis of Heymann nephritis. (b) The initial reaction to the lamina rara interna may be potentiating the eventual formation of deposits in the lamina rara externa by locally permeabilizing the capillary wall and allowing passage to other antibodies. (c) The immune complexes formed at the various sites in the capillary may be getting shed and trapped in the lamina rara externa resulting in coalescence and genesis of the nephritogenic electron-dense deposits.     

Sequence of events in the glomerular capillary wall at the onset of proteinuria in passive Heymann nephritis

Proteinuria in passive Heymann nephritis (PHN) results from complement-mediated glomerular injury, since complement depletion with cobra venom factor (CVF) prevents proteinuria. However, there are no comprehensive morphological studies identifying the sites of injury leading to onset of proteinuria. To address this issue, we attempted to locate sites of injury involved in the onset of proteinuria in PHN. PHN was induced in intact Munich-Wistar rats (PHN-rats, examined at days 3, 5, and 7) and in complement-depleted rats (CVF treated, PHN-CVF-rats, examined at days 3 and 5). The distribution of endogenous albumin in the glomerular basement membrane (GBM) was studied in in situ drip-fixed glomeruli using immunogold immunocytochemistry, and glomerular anionic sites were visualized by polyethyleneimine staining. In addition, the ultrastructural localization of an epitope recognized by a proteinuria-inducing monoclonal antibody (called 5-1-6) directed against the slit diaphragm was examined. Significant proteinuria was seen in intact PHN-rats, starting at day 5. The intensity of gold labeling for endogenous albumin was significantly increased at the outermost site of the GBM (GBM interfacing foot process and the filtration slit, designated area O) at day 3 in both PHN-rats and PHN-CVF-rats in comparison to untreated controls. At day 5, labeling for albumin in area O was decreased in PHN-rats, but not in PHN-CVF-rats, where it was then higher; in PHN-rats, some areas between epithelial cells and subepithelial deposits were almost free of albumin labeling at day 7. There was no evidence of epithelial cell detachment in any group at day 5, but on day 7 limited focal detachment was seen exclusively in PHN-rats. In proteinuric rats, amorphous material that stained for albumin could be seen in the urinary space, without any exocytosis of labeling by glomerular epithelial cells. A significant reduction of intensity of staining for anionic sites was seen in parallel in both groups, but only in the regions of the lamina rara externa adjacent to subepithelial deposits. This local loss of charge might contribute to enhanced permeability to albumin in both PHN- and PHN-CVF-rats. Changes in the appearance of the filtration slits and in the density and distribution of antigen recognised by monoclonal antibody 5-1-6 were similar in PHN- and PHN-CVF-rats at day 5. Complement depletion prevented neither the reduction in anionic sites of the GBM nor the changes in the slit diaphragm observed. These data suggest that albumin leakage between the epithelial cell and the GBM (area O) could occur in PHN-rats, perhaps as a result of epithelial foot-process changes. This may be the final link in the chain of events responsible for the onset of proteinuria in PHN.     

Monoclonal autoantibodies in Heymann nephritis.

We have developed hybridoma cell lines, each of which secretes a monoclonal antibody (MoAb) to rat renal and hepatic tissue antigens, from Lewis rat with Heymann's nephritis. Three antibodies bind to the brush border of proximal tubular epithelium (BB), one in a fine net-like pattern (no. 3-11), another one in a coarse granular pattern (no. 1-8) and the third one in a typical pattern (no. 3-9). Three antibodies bind to glomerulus in characteristic patterns but not to BB. After repeated intravenous injections of MoAb (no. 3-11), granular mesangial deposits of rat IgG were observed and of MoAb (no. 1-8), fine granular deposition along capillary walls. These monoclonal autoantibodies should be of value in research on the mechanism of autoimmune membranous nephropathy.     

被动Heymann肾炎大鼠尿蛋白与氧化,抗氧化的关系

为了解被动Heymann肾炎大鼠的氧化、抗氧化状态在蛋白尿形成中作用,分别测定了大鼠血清和尿中维生素E、丙二醛、黄嘌呤氧化酶和24小时尿蛋白。结果显示被动Heymann肾炎大鼠异体相(7天)和自体相(14天)的24小时尿蛋白、血清和尿中MDA、尿VE均明显升高,血VE显著降低,血清和尿XOD始终无明显变化。此外,尿蛋白与血清、尿中MDA、尿中VE呈正相关,与血清VE呈负相关。提示被动Heymann肾炎大鼠存在脂质过氧化反应亢进和抗氧化能力降低。     

Lipoproteins accumulate in immune deposits and are modified by lipid peroxidation in passive Heymann nephritis.

Proteinuria in passive Heymann nephritis is primarily caused by reactive oxygen species that are produced by glomerular cells. Reactive oxygen species apparently exert their damaging effects on the glomerular filter by lipid peroxidation and subsequent adduct formation on matrix proteins of glomerular basement membranes. This raised the question as to the source of polyunsaturated fatty acids required as substrates for lipid peroxidation. Here we have localized by immunocytochemistry rat apolipoprotein E and apolipoprotein B within subepithelial immune deposits. Moreover, apolipoprotein B extracted from isolated glomeruli of proteinuric passive Heymann nephritis rats shows degradation and lipid peroxidation adduct formation, similar to apoproteins of oxidized lipoproteins in atherosclerotic lesions. These data provide evidence that lipoproteins accumulate within immune deposits and suggest that their lipids generate lipid-peroxidation-derived reactive compounds.     

Glomerular T cells in Heymann nephritis.

Active Heymann nephritis (HN) is a rat model of human membranous nephropathy. The appearance of T cells within the glomeruli of HN rats suggests a role for these cells in the pathogenesis of the disease. The aims of this study were to investigate T cells infiltrating the glomerulus in HN in Lewis rats by polymerase chain reaction (PCR) of their Vbeta chains, CDR3 spectratyping and sequencing. HN was induced in Lewis rats by immunization with renal tubular antigen (Fx1A) in CFA. Kidneys were collected between 4 and 10 weeks. The glomeruli were separated, homogenized and RNA extracted. RT-PCR, CDR3 spectratyping and sequencing were used to further characterize the infiltrating T cells. Multiple Vbeta families showed restriction of their CDR3 spectratypes in each animal. Several TCR Vbeta families had identical-sized restricted spectratypes across several different animals. Four Vbeta families were sequenced. In three of those four families, the dominant clones showed identical sized CDR3 regions and a striking over-expression of Jbeta2.6. Further analysis of the CDR3 regions of the Jbeta2.6 clones showed a significant restriction of the amino acids at four of the six CDR3 positions. Glomerular T cells bearing similar CDR3 sequences, using Jbeta2.6 and expressing at least two, and possibly more, Vbeta genes are involved in the pathogenesis of HN.     

Circulatory antigen of Heymann nephritis. II. Isolation of a 70,000 MW antigen from normal rat serum which cross-reacts with Heymann nephritis antigen.

Antigens cross-reactive with crude Heymann antigen, FXIA and purified antigen, gp600, were isolated from rat serum by immunoaffinity chromatography using polyclonal anti-gp600 antibodies. The eluted antigens predominantly contained a 70,000 molecular weight (MW) polypeptide. An antiserum was raised against this preparation which, when tested against rat serum by Protein A immunoblotting, showed that the antibodies were directed to 70,000 MW antigens. By immunodiffusion in gel, the antiserum produced three precipitin lines against the antigen preparation, three lines against FXIA and a single line against gp600. All three lines of the serum antigen were continuous with the three lines of FXIA showing complete identity of the isolated serum antigens with FXIA. One of the three common lines of the serum antigen and FXIA was in continuity with the line of gp600 showing cross-reaction of gp600 with the isolated 70,000 MW antigen. Testing of Western blots of gp600 and FXIA against this antiserum by protein A immunoblotting showed reaction with the 70,000 MW subunit of gp600 and FXIA indicating cross-reaction of the serum antigen with 70,000 MW antigen of FXIA and gp600. The antiserum reacted with structures in the glomerulus by indirect immunofluorescence. Patchy brush border staining was visible. When injected into an isolated perfused kidney, these antibodies localized in fine granular pattern along the glomerular capillary wall by direct immunofluorescence. Ultrastructural immunogold technique revealed that the 70,000 MW antigen was located in the brush border and in the GBM-endothelial interface. These results demonstrate that the 70,000 MW antigen present in rat serum is also present in the glomerular capillary wall.     

Passive Heymann nephritis in pre- and post-natal rats

Passive Heymann nephritis (PHN) was induced in pre- and post-natal rats by a single intra-peritoneal injection of 0.2 ml of a rabbit anti-rat kidney fraction 3 (rKF3) antibody. Immune complex formation occurred only in those glomeruli or parts of glomeruli which were open to the circulation. Double staining of kidney sections for the glomerular nephritogenic antigen and rabbit IgG, 2 days after the injection of the antibody showed an identical distribution of both components in the glomeruli. In rats killed more than 4 days after the injection of the anti-rKF3 antibody, the nephritogenic antigen could be demonstrated in the subcapsular glomeruli, in the absence of rabbit IgG; and the same applied when the kidneys had reached maturity. When the injected antibody was expected to be present in the circulation, no nephritogenic antigen was demonstrated in the glomeruli in the absence of the heterologous IgG. These observations indicate that the nephritogenic antigen appears in the glomerulus at the same time as the glomerular capillary loops open to the circulation. Unlike PHN in the adult rat, the immune complexes in the glomeruli of neonatal rats do not persist longer than 84 days.