Nephritogenic antigen determinants in epidermal and renal basement membranes of kindreds with Alport-type familial nephritis.

We probed epidermal basement membranes (EBM) of acid-urea denatured skin from members of kindreds with Alport-type familial nephritis (FN) for the presence of antigens reactive with Goodpasture sera (GPS) and serum (FNS) from an Alport patient who developed anti-glomerular basement membrane (GBM) nephritis in a renal allograft. By immunoblotting, GPS reacted primarily with the 28,000 molecular weight (mol wt) monomer but also the 24,000 mol wt and 26,000 mol wt monomers of the noncollagenous globular domain (NC1) of type IV collagen from normal human GBM, while FNS identified only the 26,000-mol wt monomer. FNS reacted with EBM of 12 controls and nine unaffected male kindred members but not EBM of eight affected males. Five affected females exhibited interrupted reactivity of FNS with EBM. GPS showed variable reactivity with EBM and was not discriminating with respect to Alport-type FN. FNS did not stain renal basement members of five affected males. However, the EBM, tubular basement membrane, and Bowman's capsules of affected males contained antigens reactive with GPS. These immunochemical studies suggest that the FNS antigen is distinct from Goodpasture antigen(s). The expression of FNS antigen located on the NC1 domain of type IV collagen is altered in basement membranes of patients with Alport-type FN, and the distribution of this antigenic anomaly within kindreds suggests X-linked dominant transmission of a defective gene.

Alport familial nephritis. Absence of 28 kilodalton non-collagenous monomers of type IV collagen in glomerular basement membrane.

Alport-type familial nephritis (FN), a genetic disorder, results in progressive renal insufficiency and sensorineural hearing loss. Immunochemical and biochemical analyses of the non-collagenous (NC1) domain of type IV collagen isolated from the glomerular basement membranes (GBM) of three males with this disease demonstrate absence of the normally occurring 28-kilodalton (kD) NC1 monomers, but persistence of the 26- and 24-kD monomeric subunits derived from alpha 1 and 2 (both type IV) collagen chains, respectively.

Comparison of non-collagenous type IV collagen components in the human glomerulus and EHS tumor.

A method for the isolation of the NC1 domain of type IV collagen has been developed using the EHS sarcoma, a basement membrane-producing mouse tumor. This NC1 domain has been compared to the NC1 of human glomerular basement membrane (hGBM) to assess its usefulness in the biochemical characterization of the Goodpasture antigen which is associated with NC1. Both NC1 isolates appeared to migrate by gel filtration as hexamers (Mr 160,000) and in SDS-polyacrylamide gel electrophoresis as dimers and monomers (Mr 54,000 and 26,000), and were shown to share biochemical identity by amino acid analysis. The hGBM NC1 showed greater complexity in the monomer region, and when compared by two-dimensional gel electrophoresis was found to contain more components in both regions than EHS NC1. Anti-GBM autoantibodies from patients with Goodpasture's syndrome reacted with the EHS NC1 by immunoblotting of two-dimensional gels. The EHS NC1 isolated by reverse phase HPLC partially inhibited the reactivity of the anti-GBM autoantibodies against hGBM NC1 by inhibition ELISA assay. Reverse phase HPLC elution of EHS and hGBM NC1 showed differences in subunit composition and interaction; complete dissociation of the EHS monomers and dimers in 0.1% trifluoroacetic acid was observed, whereas hGBM monomers and dimers eluted together. Rotary shadowing of hGBM NC1 domains revealed size heterogeneity of globular domains, compared with greater uniformity of EHS NC1 hexamers. We conclude that EHS NC1 contains an epitope(s) that is reactive with human autoantibodies to hGBM NC1. However, the immune response in Goodpasture's syndrome may involve antibodies directed against epitopes which are present in greater density and on a more complex array of peptides in the hGBM NC1 than in EHS NC1.

Expression of novel basement membrane components in the developing human kidney and eye.

The distribution of two novel human, basement-membrane (BM) collagens has been characterized by immunohistochemical analysis of developing and mature tissue using monoclonal antibodies specific for the non-collagenous (NC1) domain of each molecule. A distribution more restricted than that of type IV collagen was observed. In the kidney, the 28K parent molecules appear relatively late, at the early capillary-loop stage of glomerular development, whereas type IV collagen is present in all BM, including those of the ureteric bud, S-form, primitive glomerulus, and vessels. Antibody to the Alport familial nephritis antigen (a 26K peptide), which is missing from epidermal BM and glomerular BM in Alport syndrome, reacted with the ureteral bud BM and all stages of glomerular BM development from the early capillary-loop stage onward, but not with BM of more primitive glomeruli (vesicles and S forms). In the human fetal eye, the collagen molecules from which the 28K NC1 peptides are derived appear later in development than type IV collagen. They are present in trace amounts in Bruch's membrane but are not detected until after birth in the retinal internal limiting membrane and cuticular and non-pigmented epithelial BM of the ciliary process. In contrast, the BM of the lens capsule and Descemet's membrane were reactive with anti-28K antibodies early in development. In all instances, the 28K peptides are detected in BM that also contain the Alport antigen, although the later is present in some BM not containing the 28K peptides. The distribution of Alport antigen and type IV collagen in developing eye is similar to that observed in the mature eye. The 28K parent molecules appear to be expressed in concert with the maturation of the BM, coincident with fusion of glomerular endothelial and epithelial BM, whereas the lens capsule BM and Descemet's membrane contain these restricted components much earlier in gestation.

Antibody specificity of human glomerular basement membrane type IV collagen NC1 subunits. Species variation in subunit composition.

NC1 subunits were purified from gel filtration pools of acid-extracted, collagenase-digested human glomerular basement membranes (hGBM). This methodology, which enriches 28-kDa monomers (M28) in the total digest, allowed purification of these monomers and 24-kDa (M24) and 26-kDa (M26) monomers free from dimers. Reactivity of these subunits with Goodpasture autoantibodies using immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional nonequilibrium pH gradient electrophoresis gels showed strong reactivity with the purified M28 subunits. Inhibition enzyme-linked immunosorbent assay, used to quantitate the reactivity of the purified NC1 subunits, indicated that M28 had a greater than 10-fold increase in ability to inhibit binding to NC1 than NC1 itself. Comparison of hGBM NC1 components were made with those obtained from collagenase digests of salt and acid-extracted bovine and sheep GBM and Englebreth-Holm-Swarm tumor similarly purified by gel filtration and reverse-phase high performance liquid chromatography. Two-dimensional gel analysis of these NC1 isolates revealed absence of the very cationic M28 monomers. Reactivity with antibodies eluted from diseased kidneys of sheep immunized with hGBM (Steblay nephritis) was compared with Goodpasture autoantibody reactivity by immunoblotting two-dimensional gels of hGBM NC1. We conclude that a very cationic M28 monomer (M28 ) found only in hGBM is the probable target in Goodpasture syndrome, that the epitope is present on most NC1 components from extracted and unextracted hGBM, and is exposed by urea denaturation which is enhanced by acid treatment. A weakly cationic M28 monomer (M28+) is present in GBM from other species and is the probable target in Steblay nephritis. Differential recognition of the two M28 components by these antibodies points to different genetic origins or possibly distinct post-translational modifications for these components. This is supported by their presence or absence in different species and tissues, as well as biochemical differences from the M24/26 monomers which presumably are derived from alpha 1(IV) and alpha 2(IV) collagen chains.

Analysis of nephritogenic antigens in human glomerular basement membrane by two-dimensional gel electrophoresis.

Collagenase digests of GBM were partially purified by column chromatography and analyzed by 2-D gel electrophoresis. Silver staining of 2-D gels showed charge- and size-related heterogeneity of proteins in the 45 to 50 kDa and 25 to 27 kDa regions. These components were transferred to nitrocellulose sheets and reacted with 10 human anti-GBM autoantibodies. Detection of bound anti-GBM autoantibodies to blotted proteins was carried out with peroxidase-labeled goat anti-human IgG and revealed binding predominantly to the cationic (pI 8 to 9.0) 45 to 50 kDa and 25 to 27 kDa components. Positive-staining patterns of blotted proteins were similar with all anti-GBM autoantibodies except that three sera additionally identified neutral (pI 5.5 to 6.5) protein components. One anti-GBM autoantibody, which developed following renal transplantation, lacked reactivity with the most cationic components in the 25 to 27 kDa region. These findings suggest heterogeneity of nephritogenic GBM antigens. The cationic 45 to 50 kDa components were sensitive to reduction, while one neutral 45 to 50 kDa component was resistant; a complex array of 25 to 30 kDa proteins (pI 5.5 to 7.5) were observed by silver staining postreduction. None of the reduced protein components reacted with anti-GBM antibodies, suggesting that epitopes on nephritogenic GBM antigens may be related to disulfide-bonded regions. Although there is variable immunohistochemical reactivity of anti-GBM autoantibodies with the GBM of infant kidneys, 2-D gels of collagenase-digested human infant GBM blotted and reacted with anti-GBM autoantibodies and showed staining patterns similar to that of adult GBM. These studies demonstrate the presence of nephritogenic antigens in the GBM of immature human kidney which are not detectable by immunohistochemical analysis.

Immunohistologic studies of type IV collagen in anterior lens capsules of patients with Alport syndrome.

BACKGROUND: Alport syndrome is an inherited disorder affecting the kidney, eye and ear arising from mutations in the gene COL4A5, which encodes the alpha 5 chain of type IV collagen. Structural defects of glomerular basement membranes in Alport syndrome are associated in most instances with failure to detect the alpha 3, alpha 4, and alpha 5 chains of type IV collagen as well as the Alport antigen that is identified in normal tissues by a genetically discriminating alloantibody and monoclonal antibody. Anterior lenticonus is an ocular abnormality pathognomic of Alport syndrome that is associated with marked thinning of the anterior lens capsule (ALC). The reactivity of Alport ALC with type IV collagen antibodies has not previously been reported. EXPERIMENTAL DESIGN: ALCs were obtained at the time of cataract extraction from two unrelated males with Alport syndrome and anterior lenticonus, and stained with antibodies against the alpha 1, alpha 2, alpha 3 and alpha 4 chains of type IV collagen, as well as an antibody against the alpha 5 (IV) chain. Controls consisted of ALCs from a normal individual and from a patient with diabetes mellitus. RESULTS: Normal and diabetic ALCs reacted with antibodies against the alpha 1, alpha 2, alpha 3, and alpha 4 chains of type IV collagen and the alpha 5 (IV) chain. In one of the Alport patients, ALC showed no reactivity with antibodies against the alpha 5 (IV) chain and the alpha 3 and alpha 4 chains of type IV collagen. In the second patient, ALC reactivity with these antibodies was preserved. Epidermal basement membranes from this second patient also showed reactivity with antibody against the alpha 5 (IV) chain, unlike most males with Alport syndrome. In both Alport patients, ALCs reacted with antibodies against the alpha 1 (IV) and alpha 2 (IV) chains. CONCLUSIONS: These findings suggest that anterior lenticonus in patients with Alport syndrome may be associated with absence of the alpha 3 and alpha 4 chains of type IV collagen, as well as the alpha 5 (IV) chain, from anterior lens capsule. On the other hand, these chains may be present in Alport patients with anterior lenticonus. The precise structural basis for mechanical weakness of the anterior lens capsule in patients with Alport syndrome remains to be determined.

Collagen distribution in human membranous glomerulonephritis.

In membranous glomerulonephritis (MGN), thickening of the glomerular basement membrane (GBM) is partly due to the accumulation of basement membrane material between and around immune deposits located on the epithelial aspect of the GBM. We investigated the distribution of type IV collagen chains (alpha 1/alpha 2, alpha 3, alpha 4, alpha 5, alpha 6) and of types I, III, V, and VI collagen in the glomeruli from 16 patients, by indirect immunofluorescence in 13 and the high-resolution immunogold technique in 6. No changes were detected in stage I MGN. The spiky projections of the GBM in stage II MGN and the basement membrane layers encircling immune deposits in stage III contained the alpha 3, alpha 4, and alpha 5 chains of type IV collagen. In contrast, the alpha 1/alpha 2 chains of type IV, as well as type VI collagen accumulated in the subendothelial aspect of the GBM. No significant staining for types I, III, and V collagens or for the alpha 6 chain of type IV collagen was detected. The results show that, as in the normal glomeruli, the different chains of type IV collagen are not co-distributed in the glomerular extracellular matrix in MGN. They also indicate that type IV collagen chains and type IV collagen play an important role in the thickening of the GBM in human MGN.

Distribution of tubulointerstitial nephritis antigen and evidence for multiple forms.

A monoclonal antibody (A8) to a basement membrane component (TIN antigen), which is associated with autoimmune tubulointerstitial nephritis, was developed and utilized to characterize tissue distribution and properties of TIN antigen by immunofluorescence microscopy and immunoblotting. Results were confirmed with polyclonal goat anti-rabbit and human autoantibodies. TIN antigen was found in basement membranes of kidney cortex, small intestines, skin, and cornea, but was not detected in the renal medulla. Within the kidney cortex proximal tubular basement membrane (TBM) showed the strongest staining. TIN antigen was also detected in Bowman's capsule, distal TBM, peritubular capillaries, and focally in the interstitium, but not in glomerular basement membrane or mesangial matrix. Immunoblotting of SDS-extracted human, rabbit, mouse, and Brown Norway rat TBM with A8 revealed predominantly a 58 kD TIN antigen; however, other reactive components were detected in minor quantities. Bovine TBM contained components of 52 kD, 45 kD and 35 kD in varying concentrations. Immunoblotting of isolated rabbit TIN antigen revealed the major 58 kD component that was characterized previously, and minor components of 300 kD, 175 kD, 160 kD and 50 kD. TIN antigen was not detected in Lewis rat TBM by immunofluorescence or immunoblotting. These studies suggest the following: 1) TIN antigen may be synthesized as a high molecular weight glycoprotein that is processed to smaller forms; 2) it may be covalently associated with other basement membrane components; 3) the antibody reactive epitope may be present on multiple TBM components; and 4) high molecular weight forms may represent aggregates of TIN antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Collagen distribution in focal and segmental glomerulosclerosis: an immunofluorescence and ultrastructural immunogold study.

Focal and segmental glomerulosclerosis (FSGS) is a non-specific scarring process of the glomerulus, initially described in idiopathic nephrotic syndrome. The distribution of types I, III, IV, V, and VI collagen and of the alpha 1, alpha 3, alpha 4, alpha 5, and alpha 6 chains of type IV collagen was studied by immunohistochemistry in sclerotic lesions of nine nephrotic children. Dual immunofluorescence and high-resolution immunogold labelling were used to determine the precise distribution of the antigens. No changes were detected in normal glomeruli of patients compared with controls. In FSGS, type IV collagen [alpha 1(IV)2 alpha 2(IV)], and to a lesser degree type VI, accumulates in the two components of the lesion: the enlarged mesangial matrix and the material deposited between the pushed-out podocytes and the alpha 3-alpha 5(IV)-positive glomerular basement membrane. Staining for alpha 6(IV) and types I, III, and V collagen was practically negative. These results suggest that the matrix components of the sclerotic lesion are produced solely by glomerular cells. Changes in the relative distribution of type IV collagen chains, characterized by the presence of collagen [alpha 1(IV)2 alpha 2(IV)] in close contact with the podocytes, strongly suggest a switch in the podocyte programme of collagen synthesis.

Enzyme immunoassay of anti-glomerular basement membrane antibodies.

An immunoassay has been developed to detect anti-glomerular basement membrane (GBM) antibodies in human sera. Various plating conditions, types of microtiter plates, and the use of biotinylated or peroxidase-labeled secondary antibodies were examined. The described assay is reliable, fast, and convenient. Sera with positive reactivity in anti-GBM nephritis and Goodpasture's syndrome are readily discriminated from sera obtained from normal individuals or patients with a variety of other diseases.

Basement membrane collagen in the kidney: regional localization of novel chains related to collagen IV.

Variability in the collagen chain composition of renal basement membranes was demonstrated by immunofluorescent microscopy using polyclonal and monoclonal antibodies and correlating with imaging of the glomerular basement membrane by phase microscopy. Antibodies toward the globular domains of alpha 1(IV) and alpha 2(IV) collagen chains, triple helical and 7S domains of collagen IV bind within the glomerulus to mesangial matrix, along the subendothelial region of the glomerular capillary wall, and to all tubular and vascular basement membranes. The portion of glomerular basement membrane corresponding to the phase dense image is not reactive with these antibodies. A different binding pattern is seen with antibodies against two novel globular regions of basement membrane collagen chains which bind to the phase dense aspect of glomerular basement membrane and to Bowman's capsule. Human tubular basement membrane is not reactive, except along portions of the distal tubule, whereas bovine tubular basement membrane is diffusely reactive; mesangial matrix and extraglomerular vascular basement membranes are not reactive. Although a possible explanation for the regional distribution of basement membrane collagen antigens in the glomerulus may relate to antigen exposure, a more likely reason is that collagen chains are regionally expressed. The staining patterns suggest that the novel collagen chains have a selective tissue distribution compared with alpha 1(IV) and alpha 2(IV) chains and that the glomerular cells of origin of these collagen IV chains may differ.

Distribution of familial nephritis antigen in normal tissue and renal basement membranes of patients with homozygous and heterozygous Alport familial nephritis. Relationship of familial nephritis and Goodpasture antigens to novel collagen chains and type IV collagen.

The glomerular basement membranes (GBM) of Alport familial nephritis (FN) are laminated and split and fail to bind Goodpasture autoantibodies by indirect immunofluorescence. The Goodpasture antigen has been localized to multiple peptides of the noncollagenous C terminal (NC1) domain of type IV collagen. The principal target antigen is a 28-kDa peptide (M28) that coisolates with type IV collagen NC1 and which is derived from a larger collagenous molecule. We have shown that two novel 28-kDa peptides found in normal GBM (M28M28+) are absent from collagenase digests of X-linked dominant Alport FN GBM and that monoclonal antibodies specific for these collagen chains fail to bind to Alport GBM. In normal tissue these chains have a distribution restricted to specific basement membranes of kidney, eye, inner ear, lung, and brain, the former three of which are affected in Alport FN. Epitopes on a 26-kDa NC1 peptide identified by an antibody from a transplanted Alport patient (FN antibody) colocalized with the 28-kDa components in these tissues. The FN antibody did not bind to the GBM of homozygous Alport males. Antibodies to the 28-kDa peptides and the FN antibody colocalized in a segmental pattern in heterozygous Alport GBM by indirect immunofluorescence and were unrelated to the normal distribution of type IV collagen. Three of eight homozygous Alport FN tissues showed the presence of the 28-kDa components in Bowman's capsule in a focal distribution, and in four of eight tissues reactive antigen was present in the cytoplasm of some parietal and visceral epithelial cells. These observations support the hypothesis that the genetic abnormality in Alport FN is a defective parent chain of the 26-kDa peptide, which results in failure of normal 28-kDa collagen chain integration.

Human tissue distribution of novel basement membrane collagen.

The authors have defined the specificity of monoclonal antibodies to collagen fragments of basement membrane (BM) and have used these highly specific antibodies to study the human tissue distribution of two novel 28 kd noncollagenous (NC1) peptides (M28 , M28+) compared with those derived from type IV collagen (alpha 1[26 kd] and alpha 2[24 kd] NC1). A limited distribution of the 28 kd peptides was observed in specialized BM of the kidney, eye, cochlea, lung, and brain, whereas type IV collagen is found in all human BM. These novel peptides, which colocalize with each other, are found in BM that also contain type IV collagen but do not, in all cases, colocalize with type IV collagen. The presence of the 28 kd peptides in the BM of the kidney, cochlea, and eye is in keeping with abnormalities involving these components in BM of patients with Alport familial nephritis (FN), who frequently have hearing loss, anterior lenticonus and retinal flecks in addition to renal disease. These 28 kd peptides are distinct, biochemically and immunochemically, from the alpha 1 and alpha 2 chain NC1 peptides of type IV collagen, and represent either peptide fragments of genetically distinct BM collagen molecules or additional molecules originating from the same gene family as type IV collagen.

Evidence for separate networks of classical and novel basement membrane collagen. Characterization of alpha 3(IV)-alport antigen heterodimer.

The COOH-terminal non-collagenous domains (NC1) of type IV collagen from glomerular basement membranes (GBM), lens capsule basement membranes, and Descemet's membrane varied in the distribution of their NC1 subunits. All of these basement membranes (BMs) contained both classical (alpha 1(IV) and alpha 2(IV)) and novel collagen chains (alpha 3(IV), alpha 4(IV) and the Alport antigen). Whereas GBM had a predominance of disulfide-bonded subunits, the lens capsule and Descemet's membrane were primarily monomeric, differences that are likely related to the functional and structural diversity of collagen in various tissues. A heterodimer formed from monomeric subunits of alpha 3(IV) and the Alport antigen exists in human and bovine GBM. This dimer represents an important cross-link of the NC1 domain of novel collagen. Additionally, immunoaffinity methodology showed that the novel BM collagen hexamers segregate into populations containing only novel BM subunits without the participation of the classical subunits (alpha 1(IV) and alpha 2(IV)). These data provided evidence for the presence of two separate networks of BM collagen: one containing alpha 1(IV) and alpha 2(IV), and the other consisting of the novel collagen chains.

Differential expression of basement membrane collagen chains in diabetic nephropathy.

Diabetic nephropathy is characterized by progressive expansion of mesangial matrix and thickening of the glomerular basement membrane (GBM). Kidney tissues from 13 patients with insulin-dependent diabetes mellitus were studied by immunohistochemical techniques for the distribution of three recently described collagen peptides (M28+, M28 [Good-pasture antigen], and Alport antigen) and various components of classical type IV collagen [alpha 1(IV) noncollagenous (NC) globular domain, alpha 2(IV) NC, 7S, triple helix]. Recently M28 and M28+ were designated as NC monomers of alpha 3(IV) and alpha 4(IV) based on limited amino acid sequencing. During the course of the disease, the distribution of the M28 chains and the Alport peptide segregated completely from that of classical type IV collagen. In diabetic kidneys, antibodies to the M28 and Alport peptides reacted intensely with the thickened GBM but not with the mesangium. In contrast, the reactivity of antibodies to various components of classical type IV collagen was prominent within the expanded mesangial matrix with significant decrease in reactivity in the peripheral capillary wall. In hyalinized glomeruli, components of classical type IV collagen virtually disappeared, whereas the M28 and Alport peptides persisted in the collapsed GBM. These studies support the view that expansion of the mesangial matrix and thickening of the GBM involve separate and distinct collagen components. The differential expression of the M28 and Alport peptides compared with that of classical type IV collagen may be a consequence of differing sites of synthesis (classical type IV collagen from endothelial/mesangial cells and M28 and Alport chains from visceral epithelial cells), independent control mechanisms, and/or differences in degradation.

Bull terrier hereditary nephritis: a model for autosomal dominant Alport syndrome.

Bull terrier hereditary nephritis is inherited as an autosomal dominant disease and causes renal failure at variable ages in affected dogs. The aims of this study were to compare the clinical, ultrastructural and immunohistochemical features of bull terrier hereditary nephritis with the characteristics of the human forms of Alport syndrome. Many animals with bull terrier hereditary nephritis have hematuria, and some have anterior lenticonus. However, deafness is not associated with the renal disease, and affected dogs do not have the large platelets that are occasionally seen in patients with autosomal Alport syndrome. The glomerular capillary basement membrane (GCBM) in affected bull terriers has an identical ultrastructural appearance to that seen in X-linked Alport syndrome, with lamellations and intramembranous electron-dense deposits. However, both the Goodpasture and the Alport antigens, which represent parts of the alpha 3(IV) and alpha 5(IV) collagen chains, respectively, are present in the GCBM of affected dogs. Bull terrier hereditary nephritis represents an animal model for autosomal dominant Alport syndrome, and can be used to further examine how genetic mutations affect a basement membrane protein and the corresponding membrane structure.

Autosomal recessive Alport syndrome: immunohistochemical study of type IV collagen chain distribution.

Alport syndrome (AS) is an hereditary disease of basement membrane collagen. It is mainly transmitted as a dominant X-linked trait and caused by mutations in the COL4A5 gene encoding the alpha 5 chain of type IV collagen. However, autosomal recessive AS due to mutations in the COL4A3 or COL4A4 genes could represent up to 15% of AS. Using the immunofluorescence technique, we analyzed the distribution of the different chains of type IV collagen in renal (12 specimens) and skin (4 specimens) basement membranes of 12 AS patients belonging to 11 unrelated kindreds in which autosomal recessive inheritance had been demonstrated (3 kindreds) or was suggested by clinical and genealogic data (8 kindreds). The renal and skin distribution was normal in one patient with COL4A4 mutations. A peculiar pattern of distribution of the alpha 3-alpha 5(IV) chains was observed in the other patients. It was characterized the co-absence of the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains in the glomerular basement membrane, and the presence of the alpha 5(IV) chain in a series of extraglomerular basement membranes including capsular, collecting ducts and epidermal basement membranes, a combination never observed in X-linked AS. This immunohistochemical pattern is correlated with the specific distribution of the alpha 3-alpha 5 chains of type IV collagen chains within extraglomerular basement membranes. It could be a useful marker for the identification of autosomal recessive AS.

Renal entactin (nidogen): isolation, characterization and tissue distribution.

Entactin/nidogen (E/N) was isolated from bovine renal tubular basement membrane. Apparent molecular weight, amino acid composition, and molecular configuration by electron microscopy rotary shadowing were similar to that of nidogen from EHS mouse tumor. The identity of bovine E/N was confirmed using a thrombin derived peptide, the sequence of which corresponded to a region within mouse and human E/N. Monoclonal and polyclonal anti-E/N antibodies were used to determine the distribution of E/N in human kidney by immunofluorescent and immunoelectron microscopy. E/N was present in all renal basement membranes and was distributed through the full width of the glomerular basement membrane (GBM) with accentuation along its epithelial aspects. E/N distribution was similar to that of novel collagen chain alpha 3(IV) NC domain in the GBM. In the mesangium, E/N was distributed mainly in the peripheral mesangial region that is bounded by the GBM, while classical collagen chain alpha 1(IV) NC as present diffusely throughout the mesangium. In the developing nephron, E/N was present in basement membranes of the ureteric bud, primitive vesicle and S-form. In all instances, E/N co-localized with laminin B2 chain. Prominent E/N detection within the mesangium was observed in diseases where mesangial expansion was present. This process was also seen in early diabetic nephropathy, but disappeared with disease progression. However, all thickened diabetic renal basement membranes showed an increase in E/N which was also present in Kimmelstiel-Wilson lesions. E/N was observed in the GBM "spikes" of membranous glomerulonephritis and in epithelial crescents associated with various disorders. The association between E/N, laminin and type IV collagen chains observed in the normal kidney were maintained in disorders with altered E/N distribution. We could not detect any changes in the distribution of E/N in other acquired and hereditary kidney diseases. These observations reflect the involvement of E/N in the structure and disease alteration of renal basement membranes and mesangial matrix.

Differential expression of basement membrane collagen in membranous nephropathy.

Membranous nephropathy (MN) is characterized by subepithelial immune complex formation and progressive thickening of the glomerular basement membrane (GBM). Kidney tissues from 21 patients stratified according to morphology (stage I: 5 patients; stage II: 5 patients, stage III: 11 patients) were studied by immunohistochemical techniques using antibody probes to matrix components of recently described (novel) chains of type IV collagen [alpha 3(IV)NC, alpha 4(IV)NC, Alport antigen] and of traditional type IV collagen [alpha 1(IV)NC, alpha 2(IV)NC, 7S(IV), triple helix]; as well as laminin B2, nidogen and fibronectin. In Stage I, there were no detectable changes when compared with normal tissue. In Stage II and early Stage III, the subepithelial projections of GBM (spikes) and the thickened GBM consisted predominantly of the novel type IV collagen chains as well as laminin B2 and nidogen, with no detectable changes in traditional type IV collagen. In late Stage III, an increase in the latter was observed in the subendothelial region of the thickened GBM with narrowing of the capillary lumen. At this stage, there was close apposition of novel and traditional type IV collagen molecules. The expression of these two groups of molecules is spatially and temporally distinct during the evolution of MN. It is hypothesized that immune complex formation in the subepithelial region of the GBM leads to increased formation of the novel type IV collagen network by visceral epithelial cells resulting in the formation of spikes and thickening of GBM between and surrounding immune deposits. These changes precede and are distinct from detectable alterations in traditional type IV collagen. With progression and time, the deposits become embedded in the novel collagen network and increased subendothelial formation of traditional type IV collagen molecules occurs with narrowing of the capillary lumen.