Membranous glomerulopathy with Bowman's capsular and tubular basement membrane deposits.

Bowman's capsular and tubular basement membrane (TBM) deposits are an extremely unusual finding in non-lupus membranous glomerulopathy (MGN). We report three atypical cases of MGN with abundant Bowman's capsular and TBM deposits. In two cases, MGN was idiopathic; in the third case, MGN occurred in the renal allograft in the setting of HCV seropositivity. In addition to the usual glomerular capillary wall deposits, immunofluorescence and electron microscopy revealed extensive immune deposits within Bowman's capsule and TBMs, predominantly at the base of parietal and tubular epithelial cells. These cases suggest a potential pathomechanism of autoantibody to secreted epithelial antigens shared by visceral, parietal, and tubular epithelial cells. In all three cases, indirect immunofluorescence was unable to detect autoantibody to normal renal epithelial or matrix constituents. Furthermore, ELISA was unable to demonstrate circulating antibody to major extracellular matrix components. The implications of these findings for the pathogenesis of MGN are explored.

Characterization of a tubular basement membrane component reactive with autoantibodies associated with tubulointerstitial nephritis.

A kidney tubular basement membrane (TBM) component that is bound by antibodies from individuals with anti-TBM antibody-associated tubulointerstitial nephritis (TIN) was purified and characterized (TIN antigen). TIN antigen was prepared from rabbit TBM by extraction with guanidine and purified by ion-exchange, gel filtration, and reversed-phase chromatography. Based upon yields of protein and antibody reactivity, TIN antigen accounts for about 9% of the mass of TBM and thus is a major component of this basement membrane. A predominant 58-kDa form comprises about 90% of purified TIN antigen, and a 50-kDa form accounts for the remainder. The two forms share the amino-terminal sequence Ser-Ile-Phe-Gln-Gly-Gln-Tyr-X-Arg-Ser-Phe-Gly- and give similar tryptic peptide maps, indicating that they are structurally related. Their amino acid compositions overall are similar to laminin and entactin/nidogen. The absence of hydroxyproline and hydroxylysine and the low levels of glycine in TIN antigen indicate that it is noncollagenous. No similarities were found between other known proteins and sequences of tryptic peptides and the amino terminus of TIN antigen, suggesting that it is distinct from other characterized basement membrane components. A goat polyclonal antibody toward rabbit TIN antigen showed the same kidney distribution as human antibodies and was completely inhibited in enzyme-linked immunosorbent assay by purified TIN antigen. These data further support the idea that TIN antigen is the primary target for anti-TBM antibodies associated with TIN. This research presents methods to prepare TIN antigen for biochemical studies and investigations of its role in anti-TBM autoimmune TIN.

Circulating immune complexes and kidney lesions following 131I administration in mice with thyroglobulin antibodies.

Immune complexes containing thyroglobulin have been described in kidneys of some patients with thyroid disease. We investigated the circulating immune complexes (with the Raji cell radioassay) and the kidney histopathology (by immunofluorescence and electron microscopy) in mice that received radioiodine to release thyroglobulin in the circulation, 2 or 4 weeks after immunization with mouse thyroglobulin in Freund's complete adjuvant. Circulating immune complexes and thyroglobulin, antibodies were found in all mice. Granular deposition of IgG, IgM, C3, and thyroglobulin, mainly in the mesangium but also in the capillary walls of the glomeruli, were observed in most of the mice. These experiments suggest that circulating immune complexes composed of thyroglobulin are responsible for the glomerular lesions. Hyperthyroid patients should be tested for thyroglobulin antibodies before treatment with radioiodine to avoid formation of thyroglobulin-containing circulating immune complexes.

Studies on cell proliferation and tracer localization in the kidneys of guinea pigs with experimental autoimmune anti-tubular basement membrane nephritis.

The mitotic activity in kidneys of guinea pigs with experimental autoimmune anti-tubular basement membrane (TBM) nephritis was investigated using autoradiographic techniques to determine the uptake of [3H]thymidine by actively dividing cells. It was observed in these animals that cells of proximal tubules, distal tubules, cortical and medullary interstitium, medullary collecting ducts, and loops of Henle took up significantly greater amounts of [3H]thymidine when compared with normal animals. In addition, the behaviour of horseradish peroxidase (HRP) and goat anti-HRP IgG in extraglomerular sites in the kidneys of these animals was studied. Contrary to what was expected, these tracers appeared to be less concentrated in the tubules and interstitium of animals with anti-TBM disease, with tracer movement restricted in areas of disrupted TBM. The significance of these observations is discussed.

The glomerular distribution of type IV collagen and laminin in human membranous glomerulonephritis.

The glomerular distribution of type IV collagen and laminin, the major collagenous and noncollagenous components of the glomerular basement membrane, was studied by immunofluorescence microscopy in idiopathic and lupus membranous glomerulonephritis. Affinity-purified antibodies against type IV collagen reacted preferentially with the inner aspect and irregularly with the adjacent outer area of the thickened basement membrane. In contrast, laminin was detected along the inner aspect of the glomerular basement membrane, in subepithelial basement membrane protrusions ("spikes"), and in the newly formed basement membrane layer above the immune deposits. We conclude that type IV collagen and laminin do not codistribute in the newly formed matrix. This aberrant antigenic distribution may reflect a loss of coordinate biosynthesis or degradation of these matrix components by visceral epithelial cells.

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.

Identification of a target antigen in human anti-tubular basement membrane nephritis.

Sera from two patients with primary anti-tubular-basement-membrane-mediated tubulointerstitial nephritis, one a renal allograft recipient and the other with spontaneous anti-tubular-basement-membrane disease, were analyzed for the specificity of their autoantibodies. Both sera had circulating antibodies that reacted by ELISA with extracts of tubular basement membrane from several species, but failed to react significantly with extracts of glomerular basement membrane. Reactive antigen was solubilized with 6 M guanidine-HCl, 6 M urea, with reduction and alkylation, and with sodium dodecylsulfate. Digestion of the basement membrane with collagenase released relatively small quantities of antigen from the membrane, and trypsin and pepsin destroyed its antigenicity. The antigenic activity was characterized with respect to its size distribution by gel filtration and by immuno-overlay analysis of protein blots. Collectively, the results indicate that the major reactivity of both sera is directed towards a Mr 58,000 component that is unique to the tubular basement membrane. Minor reactivities toward high molecular weight components common to both glomerular and tubular basement membranes were detected by immuno-overlay analysis. This study identifies an antigen that is involved in human anti-tubular-basement-membrane-mediated tubulointerstitial nephritis, and demonstrates an advantage of the use of denaturing extraction over proteolytic methods to prepare the antigen.

Isolation of the target antigen of human anti-tubular basement membrane antibody-associated interstitial nephritis.

Using a monoclonal anti-tubular basement membrane antibody (alpha TBM-Ab) affinity column, we isolated from collagenase-solubilized human renal tissue (HSRTA) a predominantly 48,000-mol-wt moiety (H3M-1) which is selectively recognized by antisera from two patients with alpha TBM-Ab-associated interstitial nephritis (alpha TBM disease). Whereas both antisera had alpha TBM-Ab titers of 1:64-1:128 by immunofluorescence on tissue sections, their reactivity with H3M-1 in a solid-phase radioimmunoassay was demonstrable at dilutions up to 1:10,000. While these sera displayed some reactivity with pre-column HSRTA, this was markedly less than with H3M-1. HSRTA depleted of H3M-1 by passage over the alpha TBM-Ab affinity column was almost completely depleted of reactivity. Neither pooled normal human sera nor sera from patients with a variety of renal lesions not associated with alpha TBM-Ab (including interstitial nephritis and antiglomerular basement membrane disease) were reactive with H3M-1. Both patient antisera containing alpha TBM-Ab were also highly reactive with R3M-1, the 48,000-mol-wt rabbit glycoprotein antigen of experimental alpha TBM disease. Furthermore, a competitive inhibition radioimmunoassay revealed that alpha TBM-Ab from rodents with experimental alpha TBM disease could inhibit 45-98% of the R3M-1 binding reactivity of patient antisera and 85% of the H3M-1 binding reactivity of patient antisera, thus suggesting paratypic cross-reactivity. We conclude, therefore, that tubular basement membrane target epitopes and their paratypic recognition are highly conserved among mammals.

Gametes contain angiotensin converting enzyme (kininase II).

The localization of angiotensin converting enzyme (ACE) in the gonads of the normal rabbit was studied by immunofluorescence and immunoelectron microscopy. The enzyme is present in the cytoplasm of testicular spermatids and of epididymal and ejaculated spermatozoa, and on the surface of follicular and tubal oocytes. These findings support the hypothesis that ACE has a role in gamete maturation and in fertilization.

Immunopathogenesis of Heymann's nephritis.

Renal localization of the C5b-9 membrane attack complex (MAC) in relation to other complement components and immunoglobulins was studied at different stages of Heymann's nephritis and following reimmunization of Lew rats with stage IV disease. Trace amounts of the MAC were found during stages I and IV of disease, whereas during stages II to III, a period of active glomerular and tubular injury, moderate deposits of MAC were observed in glomeruli, periluminal cytoplasm of proximal tubular epithelial cells, and desquamated intraluminal brush border material. Following reimmunization of stage IV animals, a striking increase in the amount of MAC was observed. Moderate to marked deposition of IgG and C3 was also found during stages II and III at sites containing the MAC. Although a slight decrease in detectable IgG was noted during stage IV, a bright, confluent ribbon-like staining pattern was present in reimmunized animals. These data suggest that the MAC is a mediator for acute in situ immune-complex-induced glomerular and tubular cytoplasmic injury, although subsequent proteinuria may persist without continued complement pathway activation and assembly of the MAC.

Abdominal pain associated with IgA nephropathy. Possible mechanism.

A 36-year-old man presented with IgA nephropathy (Berger's disease) and acute abdominal pain. Surgical biopsy of the ileum revealed deposits of IgA, C3, and fibrin in segments of the wall of submucosal arteries. The immune deposits appeared associated with areas of fibrinoid necrosis. These findings support the hypothesis that Berger's disease is a systemic disease, and provide a possible explanation for the abdominal pain associated with IgA nephropathy.

The macrophage-phagocyte system in rabbits with experimentally induced serum sickness: functional studies.

The objective of this study was to measure the phagocytic functions of the spleen and the liver and, if possible, to correlate them with the morphology and the immunohistology of these organs in rabbits with serum sickness. Serum sickness was induced by repeated intravenous injections of bovine serum albumin (BSA). Rabbits with acute serum sickness (stage A of serum sickness), with chronic serum sickness (stage C), rabbits in a stage of immunization between stage A and stage C (stage B), and rabbits that did not respond to the antigenic challenge [nonresponders (NR)], as well as nonimmunized (NI) rabbits were used. The function of the spleen and of the liver was measured by the clearances of 51Cr-labeled heat damaged (HE), periodate-treated (PE), or immunoglobulin G (IgG)-coated (IgGE) autologous erythrocytes. In NR rabbits the functions of the spleen and liver were normal. In rabbits in stage A of serum sickness the clearance of HE, the only one measured in this stage, was decreased. In rabbits in stage B the clearances of HE and IgGE were determined; both clearances were decreased. In rabbits in stage C, the clearances of HE and IgGE were decreased, while the time of clearance of 50% (T 1/2) of PE was not significantly prolonged. In addition, in rabbits in stages A, B, and C the ratio of whole spleen vs liver radioactivity was increased. In rabbits in stage B the clearance of HE returned to normal 3 days after discontinuation of BSA injections. While all animals with detectable morphologic and immunohistologic changes of the spleen and the liver had prolonged clearances of treated erythrocytes, some animals with similarly prolonged clearances lacked evidence of pathology of these organs. The results obtained indicate that: (1) In NI and in NR rabbits, treated erythrocytes are rapidly removed by the macrophage-phagocyte system (MPS) and preferentially by the liver. (2) In rabbits in stages A, B, and C of serum sickness the clearances of treated erythrocytes are significantly prolonged. Moreover, treated erythrocytes appear to be preferentially removed by the spleen, indicating that the ability of the spleen to clear treated erythrocytes is less impaired than that of the liver. (3) In stages A, B, and C of serum sickness the nonimmunologic clearance of autologous erythrocytes is as altered as the clearance that occurs via Fc receptors. (4) In rabbits in stage B of serum sickness recovery of MPS function occurs after the interruption of BSA injections, suggesting reversible damage.(ABSTRACT TRUNCATED AT 400 WORDS)

Passively transferred anti-brush border antibodies induce injury of proximal tubules in the absence of complement.

In active Heymann nephritis, antibodies directed against the brush border membrane of proximal tubules are able, when deposited in vivo, to cause substantial damage to the tubule epithelium. Prominent features of the lesion include fragmentation and loss of microvilli and proliferation of epithelial cells. Passive transfer of anti-brush border serum to appropriate proteinuric recipients also leads to proximal tubule pathology. In experiments reported here, full expression of the damage was observed in complement deficient recipients of passively transferred anti-brush border serum. A complement-independent process initiated by cross-linking of membrane determinants, which is analogous to the stimulation of B cell proliferation following cross-linking of Ig receptors by appropriate ligands, could account for the pathogenicity of anti-brush border serum.

Transplacental transmission of antibodies to tubular basement membrane in guinea-pigs with autoimmune tubulointerstitial nephritis.

The offspring of female guinea-pigs with tubulo-interstitial nephritis were studied for possible passive transfer of disease. Whereas no immune deposits were seen on or before day 30 of gestation, IgG was detected in the tubular basement membrane (TBM) of fetuses at and after day 44. Serum of offspring contained antibodies to TBM, albeit in much lower titres than found in circulation of the mother guinea-pigs. No histopathological changes were seen in fetal kidneys. Thus, autoantibodies induced by heteroimmunization of pregnant guinea-pigs may be transmitted to offspring in the last third of the gestation period and can bind to fetal TBM. However, this transfer of antibodies does not cause disease.

Factors influencing susceptibility of LEW rats to Heymann nephritis.

Although most LEW rats develop the proteinuria of Heymann nephritis (HN) within 2 months after immunization with Fx1A, protein excretion of some animals remains normal. We have compared nonproteinuric rats with those that developed HN in order to identify factors that influence susceptibility to immunologically medicated kidney disease. In the primary response to Fx1A, immunofluorescence tests showed that antibrush border titers in serum and immunoglobulin deposition in vivo were similar in all rats. However, complement was detected only in rats with proteinuria. Reimmunization with Fx1A at 30 weeks stimulated anamnestic antibody responses in all rats. Following reimmunization, 60% of nonproteinuric rats developed severe HN with an unusually rapid (1 week) onset. Once again, complement was present only in glomeruli of rats with proteinuria. It appears that titers of antibodies to brush border, measured by immunofluorescence tests, are not an index of the pathogenicity of the immune response to Fx1A. Immunological memory, leading to rapid expression of autoimmune disease upon reexposure to antigen, can be established by a primary immunization that does not produce clinical symptoms. Abnormal urine protein composition may provide a clue to subclinical immunopathology of the kidney.

Effects of prolonged administration of D-penicillamine or captopril in various strains of rats. Brown Norway rats treated with D-penicillamine develop autoantibodies, circulating immune complexes, and disseminated intravascular coagulation.

D-Penicillamine and captopril, two drugs that induce autoimmune manifestations in man, were administered orally for 5 to 10 months to various strains of rats. Three to eight weeks after D-penicillamine administration in a dosage of 20 to 50 mg per day, 73% of Brown Norway (BN) rats became ill. The disease was characterized by weight loss, dermatitis, and a high mortality presumably caused by disseminated intravascular coagulation. Microscopy revealed widespread granulomatous and necrotic lesions. The plasma of these animals contained antinuclear antibodies and immune complexes. In the kidney deposits of IgG were found in a linear pattern along the glomerular basement membrane. IgG eluted from diseased kidneys bound both "in vitro" and "in vivo" to kidney basement membranes. BN rats initially receiving 5 mg of D-penicillamine per day and subsequently 20 and 50 mg per day did not develop disease. No adverse effects were noted in Lewis (LEW) and Sprague-Dawley (SD) rats treated with 20 or 50 mg of D-penicillamine per day, nor in BN and LEW rats treated with 20 mg of captopril per day.