Detection of nephritogenic antigen from the Lewis rat renal tubular basement membrane

Immunopathogenicity of trypsin-solubilized or non-solubilized renal tubular basement membrane (TBM) of the Lewis (LEW) rat was investigated. Autoimmune tubulointerstitial nephritis (TIN) was induced in BALB/c mice by immunization with trypsin-solubilized LEW rat TBM, while immunization with non-solubilized TBM did not produce the disease. Based on this preliminary experiment we studied the characterization of immunogenic and nephritogenic TBM antigen of the LEW rat. TIN was characterized by severe mononuclear cell infiltrates with multi-nucleated giant cells in the interstitium, tubular destruction and intensive IgG and C3 deposits along the TBM. Anti-TBM antisera and eluate from the nephritic mouse kidneys reacted with the TBM of normal LEW rat kidney by immunofluorescence. LEW rat TBM was also detected immunofluorescently by using antisera from BALB/c mice immunized with autologous trypsin-solubilized TBM. A competitive inhibition test revealed a higher titer of anti-TBM antibody in the eluate than in the adsorption-treated antisera per microgram IgG. Immunoblotting showed one reactive band with a molecular weight of 45,000 daltons, and the blotting patterns in tryptic TBM of the Brown Norway (BN) and LEW rats appeared similar. Amino acid analysis of nephritogenic LEW rat tryptic TBM showed that it contained no hydroxyproline and hydroxylysine, suggesting that this TBM preparation was not collagenous. These findings suggest that tryptic digestion contributes to the release of nephritogenic antigen from the LEW rat TBM and that this antigen system might participate in the immune system involved in the anti-TBM associated TIN that is well known to be induced by non-digested TBM of TBM antigen positive animals.     

Induction, characterization, and cell transfer of autoimmune tubulointerstitial nephritis

Autoimmune tubulointerstitial nephritis (TIN) was induced in Lewis (LEW) rats by immunization with homologous Brown-Norway (BN) rat renal basement membrane (RBM), complete Freund's adjuvant and Bordetella pertussis vaccine. The BN strain has a tubular basement membrane (TBM) antigen (Ag+) detectable by immunofluorescence which is lacking in unmodified LEW rat TBM. Development of TIN in LEW rats correlated with TBM Ag+ immunogens from homologous and heterologous RBM preparations. By day 14 after immunization TIN developed characterized by elevated serum creatinine levels and by tubular destruction with focal, circumscribed lesions containing epithelioid cells, giant cells and mononuclear cell infiltrates. Approximately 60% of the mononuclear cells bore T cell antigens with most cells expressing Ia markers. Immunofluorescence and elution studies revealed no selective IgG fixation to TBM at day 14 despite high titers of circulating alloantibody reactive with the immunizing TBM. Intravenous transfer of LNC and/or splenic cells (3.5 to 7 X 10(8)) to naive LEW rats resulted in less severe but histologically identical TIN in seven days with T cell subpopulations similar to those seen in the active model. This model strongly suggests an initiating role for cell-mediated immunity in TIN in the rat and may provide a parallel to human TIN.     

Experimental gold nephropathy in guinea pigs: detection of autoantibodies to renal tubular antigens

Renal tubular dysfunction was induced in Hartley guinea pigs by injection of sodium aurothiomalate (gold) as manifested by excretion of tubular basement membrane (TBM) antigen and renal tubular epithelial (RTE) antigen in urine and tubular proteinuria. Following the tubular dysfunction, autoimmune tubulointerstitial nephritis (TIN) and/or immune complex nephropathy (ICN) developed in a large proportion of animals. TIN was associated with anti-TBM antibodies, and the histological features were characterized by tubular lesions with interstitial mononuclear cell infiltration, destruction of tubules, and interstitial fibrosis. In ICN, the glomerular lesions consisted of partial thickening of capillary walls and mesangial cellularity, and granular immune deposits were seen in the mesangial area and on capillary walls. Furthermore, electron-dense deposits were demonstrated in the mesangial area and in the glomerular basement membrane (GBM) by electron microscopy. Anti-RTE antibodies were detected in the sera and eluates from the kidney of animals with ICN. RTE antigens were also detected in the glomerular deposits by indirect immunofluorescence using anti-guinea pig RTE antibody. These results suggest that TBM and RTE antigens released from renal tubules damaged by a direct toxic action of gold may lead to antibody formation against these antigens and induce TIN and/or ICN.     

Immunochemical studies of the Alport antigen.

The Alport antigen, a component of normal glomerular basement membranes (GBM) which is absent in Alport familial nephritis, is characterized as a 26 kD non-collagenous (NC1) peptide identified by a monoclonal antibody (Mab A7) and an Alport alloantibody. Both antibodies discriminate X-linkage of the Alport defect using indirect immunofluorescence of hemizygous and heterozygous Alport GBM and epidermal basement membrane (EBM). Immunoblotting of SDS-PAGE gels of collagenase-digested Alport renal BM shows absence of monomeric and dimeric components of the Alport antigen, alpha 3(IV) NC1, and alpha 4(IV) NC1. By immunoprecipitation experiments with specific antibodies, the Alport antigen is distinct from the 26 kD NC1 peptide derived from alpha 1(IV). The monoclonal antibody to the Alport antigen and rabbit antiserum to a non-consensus sequence of alpha 5(IV) NC1 react similarly by immunofluorescence with normal kidney and both fail to bind to Alport renal BM. Two dimension Western blots of collagenase-digested BM show that the anti-Alport antigen and the ant-alpha 5(IV) NC1 react similarly with monomeric and dimeric components of BM collagen. These studies are consistent with the likelihood that the Alport antigen and alpha 5(IV) NC1 are the same or are highly homologous molecules. The precise relationship will require characterization of alpha 5(IV) NC1 protein and determination of the nucleotide sequence of the Alport antigen. The associated absence of alpha 3(IV) NC1 and alpha 4(IV) (NC1) from Alport BM is consistent with other observations for a molecular association of these chains in a novel collagen network.     

Distribution of Goodpasture antigens within various human basement membranes

Collagenase-digested basement-membrane preparations from human kidney glomeruli, kidney tubules, lung, choroid plexus, aorta, intestine, and placenta were analysed according to their reactivity to anti-glomerular basement membrane (anti-GBM) antibody-positive Goodpasture sera. Sodium dodecylsulphate polyacryl gel electrophoresis (SDS-PAGE), and immunoblotting were performed after antigen enrichment by passage of the collagenase digests through an anion-exchange column. Reactivity of anti-GBM antibodies with one to three monomers (24, 26 and 28 kD) and two dimers (44 and 50 kD) were demonstrated in basement membrane preparations of kidney glomeruli, kidney tubules, lung, placenta, and aorta. In basement membranes of choroid plexus reactivity with only the 28 kD monomer and the 50 kD dimer were identified. In intestinal basement membrane, reactivity was restricted to the 50 kD dimer. Analysis of the amounts of Goodpasture antigen by inhibition ELISA demonstrated that the highest concentration were in glomerular basement membrane, while the lowest were found in aortic basement membrane. The results indicate that Goodpasture antigens are common to all the basement membranes investigated. The differences in antigen concentration and in reactivity on immunoblotting may indicate different antigen amounts, a heterogeneity of collagen IV within the various basement membranes, or differences in antigen accessibility within the membranes. We conclude that the primary clinical restriction of the anti-GBM disease to lungs and kidneys is not explained by a preservation of the antigen to this basement membrane. Rather, the clinical pattern may be influenced by differences in the molecular composition of the basement membranes as well as by non-immunological mechanisms.     

Renal antigens in mercuric chloride induced, anti-GBM autoantibody glomerular disease.

Two antibody probes were used to characterize the putative renal antigens of HgCl2-induced antiglomerular basement membrane renal disease in Brown Norway (BN) rat. The first probe was the linear immunofluorescence imparting, in vivo bound, nephritogenic antiglomerular-basement-membrane autoantibody (anti-GBM-Ab). The second probe was a rat monoclonal antibody to the B subunit of laminin that was obtained from fusion of spleen cells of HgCl2 injected BN rat. By enzyme-linked immunosorbent assay (ELISA) the anti-GBM-Ab reacted with laminin, type IV collagen, collagenase-resistant noncollagenous portion of glomerular basement membrane (GBM), saline soluble proteins of kidney cortex homogenate and fibronectin. Western blot analysis of laminin indicated that the reactive epitopes detected by both probes were on the B chain subunit but not the A subunit. In nonreduced collagenase-digested GBM the epitopes were present on 27 kD and 42 to 48 kD polypeptides. A similar pattern was seen on collagenase-digested human GBM. On rat and human GBM the patterns obtained with rat autoantibody and autoantibody from a patient with Goodpasture syndrome were similar, suggesting that some of the in vivo bound anti-GBM autoantibodies in HgCl2-induced disease in rat are directed against epitopes which are similar to the Goodpasture antigen of human. Reactive epitopes were also detected on saline soluble proteins of kidney cortex homogenate with the predominant antigen being a 31 kD polypeptide. In the saline soluble proteins the reactive polypeptides including the major 31 kD polypeptide did not originate from laminin, type IV collagen, or the collagenase-resistant noncollagenous part of GBM. The precise structural origin of soluble proteins was not defined.(ABSTRACT TRUNCATED AT 250 WORDS)     

The glomerular basement membrane defect in Alport-type hereditary nephritis: absence of cationic antigenic components

Alport-type hereditary nephritis is a familial disorder which results in progressive renal insufficiency and sensorineural hearing loss. It is thought to result from a biochemical defect affecting basement membranes. To study this further, non-collagenous components of type IV collagen were prepared from the glomerular basement membrane (GBM) by collagenase digestion from three male patients with hereditary nephritis. The normal Goodpasture antigenicity of the 28 and 26 kD monomers and 54 and 50 kD dimers which may be isolated from the GBM was absent on one-dimensional immunoblots. Two-dimensional electrophoresis and immunoblotting studies showed absence of Goodpasture antigenicity of these molecular weight components as well as all cationic monomeric and dimeric spots. It is concluded that the expression of the Goodpasture antigen is altered in basement membranes of hereditary nephritis patients. The altered antigenicity thus acts as a marker for the underlying abnormality.     

Childhood membranous nephropathy, circulating antibodies to the 58-kD TIN antigen, and anti-tubular basement membrane nephritis: an 11-year follow-up

Childhood membranous nephropathy (MNP) with anti-tubular basement membrane (anti-TBM) nephritis is a rare disorder that may have extrarenal manifestations. This article describes a new case to be added to the 10 previously reported. A renal biopsy specimen from a 1-year-old white boy with nephrotic syndrome, microhematuria, and hypertension showed MNP (granular global IgG, IgA and C3, and segmental IgM and C1q) associated with hypercellularity and granular deposits of IgM and C1q in the mesangium, arteriolar IgA, and linear TBM IgG, IgA, and C3. A biopsy at age 4 years showed MNP (IgG and C3) and linear IgG and C3 along the TBM. Six months later, temporary glucosuria suggested a mild tubular dysfunction. Biopsy at age 8 years showed sclerosing MNP (IgG and C3), linear TBM IgG and C3, and chronic active tubulointerstitial nephritis (TIN). Indirect immunofluorescence showed circulating anti-TBM antibodies, and the enzyme-linked immunosorbent assay (ELISA) approach verified strong reactivity with the 58-kd TIN antigen. Despite trials with steroids, chlorambucil, azathioprine, and cyclosporine, end-stage renal disease developed by the age of 9 years. At age 10 years, the patient received a cadaveric kidney transplant. With the patient now aged 12 years, the graft is still functioning well, without any clinical evidence of disease recurrence. Neurological, ocular, and abdominal symptoms, including nonbacterial diarrhea, were observed during the follow-up period. The pathophysiology of these extrarenal symptoms remains unclear. Serotyping and genotyping of HLA antigens (A2, A10, B12, B41, DR5 [1101, 1103-4, 1106 or 1108-1113], DR6 [1303, 1312, or 1413], DRB3 [*0101 and 0201-2 or 0301], DQA1 [*0501 homozygous], and DQB1 [*0301 homozygous]) did not indicate any HLA association similar to those described previously in childhood MNP with anti-TBM nephritis (HLA-B7 in four patients, HLA-DR8 in two patients). The presented case is the fifth in the literature that displays reactivity with the 58-kd TIN antigen, and for which data on HLA antigens are reported.     

Renal tubular dysgenesis and tubulointerstitial nephritis antigen in juvenile nephronophthisis

Aim: The relationship between abnormalities of tubular architecture and tubulointerstitial nephritis antigen (TIN‐ag) in juvenile nephronophthisis (J‐NPH) was evaluated. Methods: Sixteen J‐NPH patients were examined. Nephrocystin‐1, TIN‐ag, type IV collagen, Fas antigen and the C5b‐9 complement complex were stained by immunohistochemical methods. Results: Renal tubules of patients with J‐NPH showed morphological abnormalities of tubular basement membranes (TBM) and frequent apoptosis of tubular epithelial cells. Additionally, the C5b‐9 complement complex was deposited within the TBM in the absence of immunoglobulin deposition, suggesting complement‐dependent TBM injury. Localization of TIN‐ag in the TBM of J‐NPH patients disclosed a partial defect or discontinuity in 14 of the 16 patients, while type IV collagen immunoreactivity was relatively preserved. These findings suggest that tubulogenesis is disturbed during nephronogenesis in J‐NPH patients because of a defect in nephrocystin, an NPHP gene product. TBM defects induce further morphological abnormalities such as cystic dilation of tubules; as tubular function impairment advances, the incomplete tubules may be injured by C5b‐9 complement complexes, followed by apoptotic cell death. Conclusion: TIN‐ag, which is important in early nephrogenesis, lacks normal activity, and vulnerable and incomplete tubules with deficient TIN‐ag expression are formed. Removal of these defective tubules by apoptosis combined with the C5b‐9 complement complex could be the primary reason for progression to end‐stage renal disease in J‐NPH patients.     

Linear glomerular IgG fixation in renal allografts: incidence and significance in Alport's syndrome

Twelve of 767 renal allograft recipients developed linear fixation of IgG along the glomerular basement membrane (GBM) by direct immunofluorescence technique. This was associated with linear fixation along the tubular basement membrane in 7 of them. Circulating anti-GBM antibodies were not detected by indirect immunofluorescence or radioimmunoassay in any patient whereas anti-TBM antibodies were found in 2 of 4 with linear TBM fixation. Among the 12 patients with linear GBM fixation, 5 had Alport's syndrome; the 7 others had various renal diseases, excluding anti-GBM nephritis. Among the 767 patients, 34 had Alport's syndrome or variants (i.e., 4.5%). The incidence of linear GBM fixation is much higher in Alport's syndrome than in other renal diseases. Linear GBM fixation was not clearly related to anti-GBM antibodies and was not accompanied by significant deterioration of graft function. These findings may be relevant, however, to the missing GBM antigen in Alport's syndrome.     

Characterization of the NC1 domain of collagen type IV in glomerular basement membranes (GBM) and of antibodies to GBM in a patient with anti-GBM nephritis

Anti-glomerular basement membrane (GBM) nephritis is associated with production of antibodies to the Goodpasture antigen (GPA) component of the NC1 domain of collagen type IV. We studied a patient with anti-GBM nephritis with regard to 1) reactivity of the anti-GBM antibodies in his serum, plasmapheresis fluid (PPF), and an eluate prepared from GBM of his nephrectomy specimen, and 2) electrophoretic and immunologic properties of the NC1 domain extracted by collagenase digestion from GBM of his nephrectomy specimen. Antibodies to different NC1 determinants in serum, PPF and eluate were detected by immunofluorescence of glomerular capillaries of normal kidney. In addition, the antibody in PPF, but not in the eluate, reacted strongly in a plate-binding radioimmunoassay with NC1 domain extracted from normal human GBM, and bound by Western blotting to both dimer (46 kD and 49 kD) and monomer (24 kD, 26 kD and 28 kD) components of the NC1 domain. Analysis of the NC1 domain in the patient's GBM by SDS-PAGE showed a number of abnormalities, including an absence of monomer bands. Moreover, there was diminished reactivity of the patient's NC1 domain by the radioimmunoassay and Western blotting, using his PPF and a rabbit anti-NC1 antiserum. These findings indicated that there were different types of antibodies to NC1 domain in PPF and eluate, associated with an abnormal NC1 domain in GBM. These results have allowed us to speculate on the pathogenesis of anti-GBM nephritis in this patient.     

Immunohistochemical study of a tubular basement membrane antigen in normal human urinary sediment by a monoclonal antibody

This preliminary study concerns murine monoclonal antibodies (Mabs) against tubular basement membranes (TBM) of human kidney which were produced by the classical hybridization technique. One of these Mabs (PP8-1) specifically recognized an antigen present in the tubular basement membranes of various portions of tubule and in the Bowman's capsule. By immunocytochemical techniques, this Mab was used to study the presence and the distribution of the related antigen in urinary sediment from 12 normal healthy subjects. This TBM and Bowman's capsule antigen was demonstrated along hyaline urinary cast surface and, in some cases, in free extracellular fibrillar structures morphologically unrelated to the casts. This suggests that, in some normal conditions, catabolism of TBM and/or Bowman's capsule could release material in urine, as it has been described for glomerular basement membrane (GBM) constituents. Such an immunocytochemical approach could be useful to detect components from tubular renal basement membranes in urinary sediment during pathological conditions.     

Pseudotumors due to IgG4 immune-complex tubulointerstitial nephritis associated with autoimmune pancreatocentric disease

Autoimmune pancreatitis (AIP) is a mass-forming chronic fibroinflammatory condition centered on the pancreatobiliary system and characterized by predominant immunoglobulin G4 (IgG4)-positive plasma cells. Recent reports have brought to light the multiorgan involvement of this disease. We describe a series of 5 cases of tubulointerstitial nephritis (TIN) associated with AIP and characterize the clinical, pathologic, ultrastructural, and immunopathologic features of TIN. The specimens consisted of 4 biopsies and 1 nephrectomy. The average patient age was 64 years (range 45 to 78) and the male to female ratio was 4:1. All had histologic and/or clinical and radiographic evidence of AIP, mass-forming sclerosing cholangitis, or both. The clinical impression in 4 patients was a renal mass or vasculitis. Two patients had renal insufficiency. Histologic preparations revealed a dense tubulointerstitial lymphoplasmacytic infiltrate. Eosinophils were often numerous. Tubulitis and tubular injury were present, along with tubular atrophy with focally thickened tubular basement membranes (TBMs). The histologic appearance ranged from a cellular, inflammatory pattern without tubular atrophy to a striking expansive interstitial fibrosis with tubular destruction. The nephrectomy specimen demonstrated a masslike nodular pattern of inflammation with normal renal tissue elsewhere. Glomeruli were uninvolved. By immunohistochemistry or immunofluorescence, numerous plasma cells in the infiltrate were positive for IgG4. TBM granular IgG deposits, predominantly of the IgG4 subclass, were detected in 4 of 5 cases by either immunofluorescence or immunohistochemistry. By electron microscopy, corresponding amorphous electron-dense deposits were present in the TBM in these cases. This type of TIN, typically characterized by a masslike lesion consisting of a lymphoplasmacytic infiltrate with eosinophils and prominent IgG4-positive plasma cells and immune-complex deposits in the TBM, may be part of a systemic IgG4-related disease, which we term "IgG4-associated immune complex Multiorgan Autoimmune Disease" (IMAD).     

Autosomal recessive polycystic kidney in rats.

We evaluated the characteristics of renal lesions in rat autosomal recessive polycystic kidney (ARPK). In rat ARPK, small cysts appeared primarily in the medulla 2 months after birth and gradually extended to the cortex, forming large cysts involving the entire layer after 8 months. By immunofluorescence microscopy, type IV collagen was more strongly stained in the epithelial basement membrane of the rat ARPK than in the normal rat tubular basement membrane (TBM). Electron microscopy demonstrated a marked thickening, slight splitting and lamination of the TBM in the ARPK. As peroxidase-labeled lectins, dolichos biflorus very strongly stained the cyst epithelium whereas lens culinaris did not. These findings indicate that cysts in rat ARPK originate in the collecting duct.     

Allograft rejection and glomerular basement membrane antibodies in Alport's syndrome

BACKGROUND: Anti-glomerular basement membrane (GBM) antibodies occasionally occur in Alport patients after renal allograft transplantation. METHODS: We report a patient with Alport's syndrome who lost four transplants each within the first year post transplantation. We searched for the presence of anti-GBM antibodies using recombinant NC1 domains of type IV collagen. Immunoblotting, enzyme linked immunosorbent assay (ELISA), and immunofluorescence were used to detect the presence of antibodies against the glomerular basement membrane. RESULTS: High antibody titers to the alpha3 chain (the Goodpasture antigen) and alpha5 chain of type IV collagen were detected. Review of pathologic specimens showed features of vascular rejection in all specimens. CONCLUSION: The association of high titer anti-GBM antibodies and vascular rejection may be important. When vascular rejection occurs in Alport patients, the presence of anti-GBM antibodies should be sought. Recombinant anti-GBM assays should be used if standard anti-GBM testing is equivocal.     

Evidence for developmental changes of type IV collagen in glomerular basement membrane

Digestion of adult glomerular basement membrane (GBM) with collagenase releases a number of peptides of which the noncollagenous region of type IV collagen is detected by antibodies from patients with anti-GBM nephritis. In our study, 17 of 19 sera reacting with GBM, in indirect immunofluorescence, with cryostat sections of adult kidneys were negative with cryostat sections of fetal kidneys. However, after digestion with collagenase, a similar pattern of peptides was released from both fetal and adult GBM. SDS-PAGE immunoblotting revealed comparable antibody binding of all 19 sera with the peptides from either fetal or adult GBM. These findings suggest conformational differences between type IV collagen in fetal and adult GBM or masking of the antigen by other GBM constituents which may shield the antigen.     

Antitubular basement membrane antibodies in renal allograft rejection.

In a patient with an episode of acute renal allograft rejection, antibodies to tubular basement membranes (TBM) were noted by direct immunofluorescence in a renal biopsy and by indirect immunofluorescence in the serum. The serum antibodies decreased gradually and became undetectable 3 months after the rejected kidney was removed. Anti-RBM antibodies eluted from the rejected kidney were capable of binding in vitro to TBM but not to glomerular basement membranes (GBM) in 39 of 40 human kidneys and various animal kidneys. The specificity was confirmed by blocking studies showing inhibition with ultrasonicated human TBM but not with GBM preparations. Passive transfer experiments showed that anti-TBM antibodies were able to bind in vivo to normal rabbit kidneys, although they could not elicit an inflammatory response. The possible mechanisms of production of anti-TBM antibodies and their potential significance in graft loss are discussed.     

Induction of tubular basement membrane (TBM) antigen specific suppressor T cells in BALB/c mice]

Transfer of tubular basement membrane (TBM)-primed thymocytes from BALB/c mice that had been immunized with allogeneic TBM antigen without adjuvant prevented the development of interstitial nephritis (IN) in recipient BALB/c mice that had been immunized with TBM antigen with complete Freund's adjuvant (CFA) to produce IN. TBM antigen was prepared from TBM of normal ddY mice (ddY TBM antigen). BALB/c mice were highly susceptible to IN and showed a high immune response to TBM antigen when they were immunized with TBM antigen in CFA. Development of IN in high responder BALB/c mice was clearly suppressed by transfer with ddY TBM-thymocytes. The anti-TBM antibody response and the proliferative response of splenic T cells to TBM antigen were also depressed by the transfer. The cell extract of ddY TBM-thymocytes had also suppressive activity on the development of IN and on the immune response to TBM antigen. This simple system without any adjuvant for inducing the thymocytes, which has strong suppressive activity on the development of IN and on the immune response to TBM antigen, may allow us to analyse the role of suppressor T cells in negative regulation of IN.     

Abnormalities in the NC1 domain of collagen type IV in GBM in canine hereditary nephritis

Samoyed hereditary glomerulopathy (SHG) in dogs serves as a model for human X-linked hereditary nephritis (HN). We previously showed that glomerular capillaries of affected males did not stain by immunofluorescence (IF) using serum from a patient with Goodpasture's syndrome. Our goal in the present study was to determine whether the NC1 domain of the collagen type IV molecule, which contains Goodpasture antigen (GPA), could be demonstrated in these dogs, and to assess its immunological reactivity. By SDS-PAGE, NC1 in collagenase digests of glomerular basement membranes (GBM) of unaffected and carrier female dogs in the family with SHG showed 24 kilodalton (kD), 26 kD and 28 kD monomer, and 46 kD and 47 kD dimer components, but the 24 kD monomer was diminished in the affected males. By IF, a rabbit antibody to NCl stained glomerular capillaries of unaffected, affected male, and carrier female dogs. In contrast, a human anti-GBM plasmapheresis fluid (PPF) stained glomerular capillaries of only the unaffected and carrier female dogs. By RIA, both antibodies reacted strongly with NCl in collagenase digests of GBM of the unaffected and carrier female dogs, but showed reduced reactivity with NCl of affected males. By Western blotting, both antibodies bound to dimers and 24 kD and 26 kD monomers of the NCl domain in collagenase digests of GBM of unaffected and carrier female dogs. However, in affected males, the rabbit anti-NCl antibody did not bind to the 24 kD monomer, while the human anti-GBM PPF showed weak binding to the 24 kD and 26 kD monomers.(ABSTRACT TRUNCATED AT 250 WORDS)