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The production of nidogen by four renal cell carcinoma (RCC) and three pancreatic adenocarcinoma (PAc) cell lines has been studied in cell culture and in xenografted tumours in nude mice. In RCC cells, immunoreactivity for nidogen was seen only after exposure to monensin to induce cytoplasmic accumulation of secretory proteins. In PAc cells, immunoreaction was also detectable in control cells. Immunoblotting of control and monensin-exposed cells and immunoprecipitation of culture media of radioactively labelled cells demonstrated the production of nidogen polypeptide of Mrca. 150000 by six of the seven cell lines. Basement membranes (BMs) and stroma of the xenografted tumours derived from these six cell lines demonstrated immunoreactivity for both human and mouse nidogen, as revealed with species-specific antibodies. The ability of the cells to produce nidogen in vitro and deposit in vivo was positively correlated with high histological grade of the xenografted tumours, although the small number of cell lines studied calls for further studies to confirm this. The distribution of nidogen in human RCC and PAc specimens was also studied by immunohistochemistry. There was strong immunoreactivity for nidogen in tumour stroma, BM of carcinoma cell nests, and endothelial basal lamina, but no conclusions could be drawn regarding histological grade and immunostaining patterns, because stromal production could not be ruled out. The results show that nidogen is produced by human carcinoma cells both in vitro and in vivo. Copyright © 1999 John Wiley & Sons, Ltd.

Tubulointerstitial nephritis antigen (TIN-ag) is a 58 kDa glycoprotein restricted within the kidney to basement membranes underlying the epithelium of Bowman's capsule and proximal and distal tubules. Autoantibody formation against this component has been described in association with primary immune-mediated tubulointerstital nephritis, membranous nephropathy and anti-glomerular basement membrane nephritis. In the present report, the ontogeny of this protein was studied in human fetal kidney tissue by immunohistochemical analysis of immature and developing nephrons using a panel of monoclonal and polyclonal antibodies. TIN-ag is first detected in basement membranes underlying the epithelium of Bowman's capsule of early capillary loop stage glomeruli and the primitive proximal tubule. No detectable expression is observed in the basement membranes of the branching ureteric bud, nephrogenic vesicle, or comma shape and s-shape stages of nephrogenic development. Increased staining of the proximal tubular basement membrane is associated with outgrowth of the primitive tubule from the urinary pole of the developing glomerulus. In more mature fetal tubules, TIN-ag expression closely resembles that of previously reported observations in mature tissue where it is present in high amounts in the basement membranes of proximal tubules, and to a lesser extent in Bowman's capsule and distal tubules. Our results suggest that TIN-ag expression is developmentally regulated in a precise spatial and temporal pattern throughout nephrogenesis.

Tubulointerstitial nephritis antigen (TIN-ag) is a 58-kDa basement membrane glycoprotein that is recognized by human autoantibodies in certain forms of tubulointerstitial nephritis. To further characterize this macromolecule and isolate cDNAs encoding TIN-ag, amino acid sequences from tryptic peptides were used to design and synthesize primers in order to amplify a probe for screening a rabbit kidney cortex cDNA library. A cDNA encoding TIN-ag was cloned and sequenced. The predicted amino acid sequence deduced from this cDNA includes the chemically determined sequences of peptides derived from TIN-ag, supporting its authenticity. The predicted amino acid sequence also shows that the carboxyl-terminal region of the molecule exhibits a 30% homology with human preprocathepsin B, a member of the cysteine proteinase family of proteins. A domain in the amino-terminal region of TIN-ag contains an epidermal growth factor-like motif that shares homology with laminin A and S chains, alpha 1 chain of type I collagen, von Willebrand's factor, and mucin, suggesting structural and perhaps functional similarities among these molecules. Immunoprecipitation of in vitro generated recombinant protein using a TIN-ag-specific monoclonal antibody (A8), confirms the identity of the isolated TIN-ag cDNA. In this report the cDNA and predicted amino acid sequences of TIN-ag are presented. Knowledge of the primary structure of TIN-ag will facilitate our understanding of the molecular structure of this novel basement membrane component and may provide clues toward understanding its functional role.

Laminin a non-collagenous glycoprotein is a major component of the renal glomerular basement membrane and mesangium. Thus far eleven distinct chains have been described, permutations of which make up 15 laminin isoforms. Laminin molecules interact with cells and other matrix molecules during organ development and differentiation. We studied the distribution of laminin isoforms in patients with type 1 diabetic nephropathy, membranous nephropathy, membranoproliferative glomerulonephritis and IgA nephropathy/ Henoch-Schonlein purpura. Immunofluorescence microscopic studies with laminin-chain-specific antibodies to the α1, α2, α5, β1, β2 and γ1 chains detected α2, β1 and γ1 chain expression in the normal mesangium and α5, β2 and γ1 in normal glomerular basement membrane. Significantly, constituents of the glomerular basement membrane, α5, β2 and γ1 chains were overexpressed in kidneys with diabetic nephropathy. Initially the constituents of the mesangium increased commensurate with the degree of mesangial expansion and degree of diabetic nephropathy. Reduction in α2 chain intensity was observed with severe mesangial expansion and in the areas of nodular glomerulosclerosis. In addition, with late disease aberrant expression of α2 and β2 chains was observed in the mesangium. Glomerular basement membrane in renal disease overexpressed molecules normally present in that location. In summary, the alterations in basement membrane composition in various renal diseases seem to not only reflect the balance between synthesis and degradation of normal basement membrane constituents, but also their aberrant expression.

Tubular basement membrane changes during induction and regression of drug-induced polycystic kidney disease. Defective cell-extracellular matrix (ECM) biophysiology is considered a factor in the development of polycystic kidney disease (PKD). Altered biosynthesis of various ECM components may result in tubular dysmorphogenesis and uncontrolled tubular cystic expansion. In this study, expression of certain ECM components was investigated in a diphenylthiazole (DPT)-induced rat model of PKD. DPT induces cystic change in all the collecting tubules, most severe in the outer medulla and inner cortex, and following withdrawal of DPT, cystic tubules return to normal with persistence of focal interstitial fibrosis. SDS-PAGE analyses of isolated tubular basement membranes (TBMs) of control and PKD kidneys revealed overall similar electrophoretic migratory bands. However, in PKD, there were relative increases in components with M r ˜ 380,000, 250,000 and 145,000, and a decrease in the component with M r ˜ 55,000. Immunoblot analyses revealed that the major components of TBM (type-IV collagen, laminin β 1 and β 2 chains and entactin) were present in the same relative concentrations in control and PKD. The expression of tubulointerstitial (TIN) antigen was decreased. Also, the relative concentrations of type-I collagen and fibronectin were increased in the PKD group. Following recovery, the expressions of TIN and fibronectin returned to normal, whereas type-I collagen remained elevated. ELISA determinations revealed increased expression of interstitial collagens type-I, -V and -VI in PKD vs control and they remained elevated following recovery, while that of type-III was unchanged. Since cell-matrix integrity is vital in the maintenance of normal biophysiology of the renal tubule, the observed altered expressions in various ECM glycoproteins may be relevant to the pathogenetic mechanisms involved in the development and progression of PKD.

Tubulointerstitial nephritis (TIN) antigen has been recently identified as a novel basement membrane macromolecule. It consists of a single chain of 58 kDa and exhibits a restricted distribution. The interaction between TIN antigen and laminin or type IV collagen has been studied using solid-phase binding assays and found to be for both macromolecules specific, saturable, and with an affinity in the low micromolar range. In similar assays, TIN antigen did not interact with heparin. In turbidimetry assays, it was found that the presence of TIN antigen did not affect the polymerization of type IV collagen but had a concentration-dependent inhibitory effect on laminin polymerization and on preformed laminin polymers. TIN antigen was able to promote adhesion of epithelial cells derived from kidney tubules and of endothelial cells derived from aorta. The data suggest that TIN antigen may be a macromolecule of importance both for basement membrane ultrastructure and cellular adhesion.

Two antigenically and structurally related heparan sulfate proteoglycans (HSPG), with masses of 200 and 350 kDa, have been isolated and characterized from bovine renal tubular basement membranes (BTBM) using DEAE-Sephacel, octyl-Sepharose CL-4B, and Propac PA-1 chromatography. Heparitinase treatment revealed core proteins of 145 and 125 kDa, with corresponding core proteins after trifluoromethanesulfonic acid treatment of 88 and 82 kDa, from the 200- and 350-kDa HSPGs, respectively. The separated HSPGs produced similar tryptic peptide maps, had similar amino acid compositions, and had similarly sized GAG chains. The 200-kDa HSPG had 2.1 mg of protein/mumol of hexuronic acid compared with 1.1 mg/mumol for the 350-kDa HSPG. Anti-BTBM HSPG monoclonal antibody (mAb A12) reacted with core proteins derived from the 200- and 350-kDa HSPGs, whereas anti-perlecan polyclonal and monoclonal antibodies did not bind to the BTBM HSPG core proteins described above but reacted with a 230-kDa core protein, which was nonreactive with mAb A12. Immunohistochemical studies of the kidney demonstrated differences in the distribution of BTBM HSPG and perlecan. Comparison of amino acid sequences from BTBM HSPG tryptic peptides with the sequence of perlecan revealed similarities but not extensive identity. Two tryptic peptides show homology to rat agrin, a basement membrane component of synaptic junctions. These data suggest that the two BTBM HSPGs are immunologically and structurally related and that differences in these molecules may arise from alternative splicing or posttranslational modifications. In addition, the two BTBM HSPGs are immunologically and structurally distinct from perlecan but may share homology with agrin.

X-chromosome inactivation patterns were investigated in livers of nine spfash female heterozygous ornithine transcarbamylase (OTC)-deficient mice. Quantitative morphometric analysis of cellular mosaicism was performed on sections of frozen liver reacted with purified anti-OTC antibody and prepared for immunofluorescent microscopy. Analysis of enzymatic OTC activity was performed on sections of these livers using a radiochromatographic technique. Several areas of cellular mosaicism were seen in each of the histological sections that were studied. The distribution of the volume fraction of the liver tissue cells having cells with normal OTC content among the nine mice ranged from 20 to 70% and it correlated (r = 0.8, P = 0.005) with the enzymatic activities of the respective livers. The extreme variegation of mosaic patches in the liver suggests the high probability that a single needle biopsy will be diagnostic in females heterozygous for an OTC mutation. This study also suggests that at the time of X inactivation, the number of primordial liver embryonic cells is small and the observed variegation of liver mosaicism probably results from complex migration patterns of liver cells during fetal development. This study shows that the spfash mouse is a suitable animal model for quantitative studies of X-chromosome inactivation in liver using immunohistochemical staining of OTC protein.

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.

Entactin: A possible auto-antigen in the pathogenesis of non-Goodpasture anti-GBM nephritis. It has recently been demonstrated that many patients with various types of glomerulonephritis have antibodies to the 6M guanidine-HCl extract of glomerular basement membrane (Bygren et al, Nephrol Dial Transplant 4:254–261, 1989). In the present study a 150K protein was isolated from the guanidine extract of bovine glomerular basement membrane utilizing ion exchange and gel filtration chromatographic procedures. Amino acid analysis and size of the isolated protein revealed similarity to that of entactin/nidogen. The identity of this protein as entactin/nidogen was further suggested by its precipitation with two different antibodies in a radioimmunoassay and by its reaction with four different antibodies in a sandwich ELISA. Inhibition of the antibodies to 150K by bovine entactin, which was isolated separately and sequenced for amino acids, confirmed the identity of the 150K protein as entactin/nidogen. Furthermore, it was shown that about one third of those patients who show antibodies to the crude guanidine extract have circulating antibodies directed against entactin. This was further confirmed by the competitive inhibition of antibodies to the crude guanidine extract in one of the positive serum by entactin in an ELISA inhibition and by immunoblotting experiments. These observations propose entactin as a possible non-Goodpasture glomerular basement membrane antigen that could be involved in the pathogenesis of certain forms of autoimmune glomerulonephritis (non-Goodpasture anti-GBM glomerulonephritis) in man. Most of these patients have a granular pattern of the immunoglobulin deposition along the glomerular basement membrane. This suggests the possibility that anti-GBM glomerulonephritis in human beings can have non-linear immunoglobulin deposits along the GBM.

Alport syndrome, an inherited disorder of the kidney, eye and ear, has fascinated nephrologists, pathologists, and geneticists for nearly a century. With the recent application of molecular biochemical and genetic techniques, this mysterious disease has begun to yield some of its secrets. Alport syndrome can now be viewed as a generalized disorder of basement membranes that appears to result from mutations in an X-chromosome-encoded basement membrane collagen chain. This chain, along with two other novel collagen chains, is absent from Alport basement membranes, in contrast to the classical chains of collagen IV. Phenotypic heterogeneity in Alport syndrome probably arises from allelic mutations at a single genetic locus. The phenomenon of post-transplant anti-glomerular basement membrane nephritis may be a manifestation of specific mutations at the Alport locus that prevent synthesis of the gene's protein product and the establishment of immunological tolerance.

This study examines the similarities and differences in the noncollagenous domain (NC1) of type IV collagen from human glomerular basement membrane (hGBM), alveolar basement membrane (hABM), and placenta (hPBM). Following collagenase digestion, NC1 domain was isolated on Bio-Gel A-0.5m or by cation exchange chromatography on S-Sepharose. NC1 from each source was characterized by SDS PAGE, and two dimension NEPHGE/SDS PAGE. Immunoblotting and ELISA inhibition was performed using antibody probes specific for M28 , M28+, M26 and M24 monomer subunits of human NC1. It was observed that all NC1 subunits were present in hGBM and hABM derived material, however M28 and M28+ monomers were absent in hPBM NC1. These findings indicate that while alpha 1(IV) and alpha 2(IV) collagen chains are present in hGBM, hABM and hPBM, alpha 3(IV) and alpha 4(IV) collagen chains are only found in hGBM and hABM but are absent in hPBM. It can now be appreciated that heterogeneity of alpha (IV) chain composition exists in basement membranes from various organs.

A method is presented for the preparation of tubular basement membrane from renal cortex. The procedure utilizes a Polytron tissue disrupter, differential sieving, and for some species, sucrose density centrifugation. The procedure is especially useful for obtaining highly purified rabbit and bovine tubular basement membranes. Human tubular and glomerular basement membranes can be prepared with some cross contamination. The method offers the capability of rapidly generating large quantities of the basement membranes that retain the Goodpasture antigen and a TBM antigen associated with anti-TBM nephritis.

Tubulointerstitial nephritis antigen (TIN-ag) is a recently described basement membrane component reactive with autoantibodies in some forms of autoimmune mediated tubulointerstitial nephritis. Immunofluorescence studies using polyclonal and monoclonal antibodies have indicated a restrictive tissue distribution for TIN-ag, with the site of most prominent expression the kidney tubular basement membrane. However, Lewis rat does not demonstrate any immunoreactivity for TIN-ag and does not develop tubuloinsterstitial nephritis after injection of tubular basement membrane material. As TIN-ag would appear to be a molecule of biological significance, experiments were designed to explore the presence or absence of this macromolecule in the Lewis rat model. Southern blotting of Lewis rat genomic DNA revealed the presence of gene sequences corresponding to TIN-ag. RTPCR analysis of Lewis rat kidney cortex total RNA illustrated the expression of a TIN-ag gene product. Western blotting demonstrated the presence of TIN-ag protein forms in kidney cortical homogenates of Lewis rat. The data suggest either extensive epitope masking or expression polymorphism of TIN-ag in the Lewis rat.

Publisher Summary This chapter focuses on tubular basement membrane autoantibodies. Antibodies to kidney tubular basement membrane (TBM) are present in tubulointerstitial nephritis (TIN), a common disease leading to renal insufficiency. The antibodies are usually detected in the clinical setting by direct immunofluorescence during evaluation of renal biopsy specimens. A sensitive radioimmunoassay was used in studies of TBM antibodies in patients with anti-GBM nephritis. The common renal disease associations with anti-TBM disease include membranous glomerulonephritis and antiglomerular basement membrane disease. Anti-TBM occurs in membranous glomerulonephritis or in drug-induced interstitial nephritis in male patients.

Monoclonal antibodies were used to study the expression of three recently characterized basement membrane components and two carbohydrate antigens in 11 renal-cell carcinomas, using immunohistological and biochemical techniques. The expression of several site-specific kidney antigens in renal-cell carcinoma were studied to determine the origin of the carcinoma and if it is possible further classify this type of carcinoma. Tubulointerstitial nephritis antigen (TIN) and two alpha-chains of type IV collagen, alpha 1 (IV) and alpha 3 (IV) were studied. In addition the expression of carbohydrate antigens Lex and SLex, which also exhibit site-specific distribution were characterized. Lex and SLex antibodies stained the majority of the tumours. TIN was expressed in 9 of 11 tumours, the alpha 1 (IV) chain was present in all 11, and the alpha 3 (IV) chain in two of the 11 tumours. Interestingly, the two alpha 3 (IV)-positive tumours were the same two that were negative for TIN. In normal tissue alpha 3 (IV) is found in distal tubules while TIN is found in proximal tubules. Our results are consistent with earlier observations that the proximal tubule is the origin of most renal-cell carcinomas, but the results also indicate that renal-cell carcinoma may originate from the distal tubule.

We used immunogold electron microscopic (IEM) techniques with periodate-lysine-paraformaldehyde-fixed and Lowicryl-embedded or cryopreserved tissues to study the distribution of alpha 1(IV) and alpha 3(IV) chains of Types IV and VI collagen in glomerular basement membrane (GBM) and mesangial matrix of glomeruli in normal human kidneys. Monoclonal antibodies to alpha 1(IV) and alpha 3(IV) collagen chains and Type VI collagen could be detected only with cryoultramicrotomy, whereas polyclonal anti-Type IV collagen antibody was detectable in Lowicryl-embedded tissue. Ultrastructural detail was better preserved in the Lowicryl-embedded tissue. IEM labeling provided more detailed information as to the site-specific array of these extracellular matrix molecules in glomeruli than did immunofluorescent microscopy. The labeling of alpha 1(IV) collagen chain was distributed mainly along the endothelial side of glomerular basement membrane and the mesangial matrix. Mesangial GBM was relatively poorly labeled compared with that of mesangial matrix. In contrast, the alpha 3(IV) chain was detected throughout the thickness of the GBM, but there was no labeling of mesangial matrix. Type VI collagen distribution was identical to that of the alpha 1(IV) chain within the glomerulus but was also associated with interstitial collagen fibrils. This study documents and details the heterogeneous distribution of Type IV and VI collagen chains within the normal human glomerulus and provides the framework for the study of these matrix components in human glomerular diseases.

Glomerular distribution of type IV collagen in diabetes by high resolution quantitative immunochemistry. We examined type IV collagen distribution and density in human diabetic kidneys by quantitative immunogold electron microscopy. We studied normal kidney transplant donors and “slow-track” and “fast-track” insulin dependent diabetic (IDDM) patients. The “slow-track” patients had IDDM for ≥ 20 years and mesangial volume fraction (VvMes/glom) of ≤ 0.32. The “fast-track” patients had IDDM for ≤ 20 years and VvMes/glom ≥ 0.37. Renal biopsies were embedded in Lowicryl, reacted with polyclonal anti-type IV collagen (in the distribution of the classical α1(IV) and α2(IV) collagen chains) and monoclonal anti-α4(IV) collagen chain antibody followed by gold conjugated secondary antibody. We found, by morphometric techniques, a decrease in the immunogold densities of anti-type IV collagen in the subendothelial zone of the GBM in the “fast-track” IDDM patients. There was a trend towards a decrease in mesangial matrix (MM) particle density in the “fast-track” (P = 0.07) but not in the “slow-track” patients. However, because of the marked increase in MM in the “fast-track” patients, the per glomerulus estimated quantity of these antigens in MM was increased. In contrast, the density of α4(IV) collagen chain was increased in the epithelial zone of the GBM in the “fast-track” IDDM patients. It is not known whether these changes in glomerular type IV collagen represent markers of advanced diabetic lesions or whether these changes might be detected earlier in diabetic patients destined for the later development of serious lesions.

Goodpasture's syndrome is a very severe and aggressive autoimmune kidney disease. The patients' autoantibodies, which are pathogenic, are restricted to the C-terminal region of the alpha 3-chain of type IV collagen. In this paper we characterize an anti-type IV collagen antibody from a patient with a non-progressive form of glomerulonephritis. ELISA and immunoblotting were used to study the specificity of this patient's antibodies. The patient had high titres of antibodies restricted to the C-terminal region of the alpha 1-chain of type IV collagen. The antibody recognized an epitope hidden in the NC1 molecule which was fully exposed after denaturation or reduction. It was an IgG3 antibody composed of only lambda light chains, indicating that it has a potential to induce inflammatory damage and that it is probably monoclonal. This patient also had MPO-ANCA which were of IgG1 subclass. Our patient had no disease progression during the 5 years of treatment. Even though the anti-alpha 1 (IV) antibodies react with the same domain, but of a different chain of type IV collagen compared to the Goodpasture's antibodies, they do not induce any severe damage. It is thus uncertain if the anti-alpha 1 (IV) antibodies have any pathogenic role; the kidney damage might have been caused by the MPO-ANCA. The findings support the theory that the anti-alpha 3 (IV) antibody causes disease in Goodpasture's syndrome and that antibodies restricted to other subunits of the C-terminal region of type IV collagen are less harmful.

Role of a basement membrane glycoprotein in anti-tubular basement membrane. Tubulointerstitial nephritis antigen (TIN antigen) is a basement membrane component which is recognized by human autoantibodies in TIN and has been shown to induce TIN in Brown Norway (BN) rats. Detectable by immunofluorescent microscopy, TIN antigen reacts with monoclonal, polyclonal, and human autoantibodies in basement membranes of kidney cortex, small intestine, skin and cornea. Specific sites of TIN antigen within kidney cortex include basement membranes of proximal tubules, distal tubules, Bowman's capsule and peritubular capillaries, with highest concentration in proximal tubular basement membrane (TBM). TIN antigen is also present in interstitium between tubules and in the periarterial sheath, but not in glomerular basement membrane or mesangial matrix. Immunoblotting of TIN antigen isolated from rabbit TBM reveals a major 58 kDa component with minor components of 300 kDa, 175 kDa, 160 kDa, 100 kDa and 50 kDa. Partial protein sequence analysis indicates that 58 kDa TIN antigen represents a newly defined glycoprotein. The structural relationships between various molecular weight forms are currently being investigated. High molecular weight (HMW) forms of TIN antigen, consisting of a mixture of 300 kDa, 175 kDa and 160 kDa forms, are more efficient than low molecular weight (LMW) forms (58 kDa and 50 kDa forms) in inducing TIN in BN rats. The resultant antibody specificity of rats injected with either HMW TIN antigen or LMW TIN antigen is identical as determined by immunofluorescent microscopy and Western analysis. Higher antibody titers and greater amounts of kidney-bound IgG are found in the HMW TIN antigen-immunized animals. TIN antigen is the primary target of anti-TBM antibodies in human and experimental immunologically-mediated anti-TBM nephritis.

We report three patients, from two unrelated families, with anti-tubular basement membrane (TBM) antibody nephritis associated with membranous nephropathy. This rare disorder is characterized by nephrotic syndrome, tubular dysfunction, and progression to renal failure. Direct immunofluorescent studies in these patients revealed linear IgG deposition along the proximal TBM, while circulating antibodies reacting with proximal TBM but not with glomerular basement membrane were identified by indirect immunofluorescence. Sera from all three patients reacted by enzyme-linked immunosorbent assay and Western immunoblotting with purified 58-kd tubulointerstitial nephritis (TIN) antigen isolated from TBM. Additional reactivity with a 175-kd component, which may be a higher-molecular-weight form of TIN antigen, was observed by immunoblotting. Since recurrent Fanconi syndrome was seen after transplantation in one patient, anti-TBM antibodies were removed by plasmapheresis prior to kidney transplantation in the other two patients. Neither patient has clinical evidence of recurrent anti-TBM nephritis in the allograft despite the post-transplantation reappearance of anti-TBM antibodies in the serum of one patient. Serologic and molecular HLA class I and class II polymorphism analysis has identified the presence of both HLA-B7 and -DRw8 antigens in two unrelated affected individuals (0.3% expected frequency in the white population). We conclude that sera from patients with anti-TBM nephritis associated with membranous nephropathy react with 58-kd TIN antigen previously implicated in the pathogenesis of primary anti-TBM nephritis. This rare autoimmune disorder may be HLA associated with B7 and/or DRw8, providing susceptibility to the disease. Further investigation is needed to understand the pathogenesis of recurrent anti-TBM nephritis in the renal allograft.

The globular domain (NC) of alpha 4(IV) collagen chain was partially sequenced and compared with the NC domain of other collagen IV chains. The alpha 4(IV) NC domain was found to be most closely related to alpha 2(IV) NC domain but distinct from the NC domain of alpha 1(IV), alpha 2(IV), alpha 3(IV) and alpha 5(IV) collagen chains. Partial sequence, representing nearly one half of alpha 4(IV) NC domain, shows 56%, 69%, 51% and 54% identity with the corresponding NC domains of alpha 1(IV), alpha 2(IV), alpha 3(IV) and alpha 5(IV) collagen chains, respectively. A short, highly polar, region of variable sequence is found near the carboxy terminus of alpha 4(IV) NC domain. This sequence corresponds to a non-conserved region among NC domains, suggesting functional specialization at this site. It exhibits high surface probability with predicted structural differences among NC domains. These results confirm uniqueness of alpha 4(IV) NC domain and indicate its structural relatedness to other NC domains of collagen IV.

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. These studies further characterize TIN antigen distribution in kidney, demonstrate its presence in extrarenal tissues, and reveal various molecular weight forms in isolated TBM preparations.

Monoclonal antibodies were produced against NC1, the globular noncollagenous domain of collagen IV, isolated from bovine glomerular basement membrane. Cells from eight positive wells were cloned and the resulting monoclonal antibodies were studied in detail by immunofluorescence on human kidney sections, by Western blot and by ELISA against denatured subunits from NC1 hexamers and against native NC1 hexamers from different tissues. The monoclonal antibodies could be divided into two groups. Firstly, those monoclonal antibodies that, in ELISA and Western blot, reacted with peptides related to the alpha 1 chain of collagen IV and stained all basement membranes in the kidney. Secondly, a monoclonal antibody that, in ELISA and Western blot, reacted with peptides related to the Goodpasture antigen, the alpha 3 chain of collagen IV. When this antibody was applied to human kidney sections it stained the glomerular basement membrane very intensively. Bowman's capsule and some tubular basement membrane were also stained, although to a lesser extent. This staining pattern is the same as that observed with sera from patients with Goodpasture's syndrome. An attempt was made to separate different subtypes of the NC1 hexamer. A monoclonal antibody from the first group was used to make an affinity chromatography column. Glomerular basement membrane digested with collagenase was separated on this column and the collected fractions were analyzed by ELISA and SDS-PAGE. The result from this study support the idea that glomerular basement membrane is composed of at least two different subtypes of type IV collagen.

Renal tubular and glomerular basement membranes were isolated from rabbit, rat and mouse kidneys. Methods were developed to obtain the basement membrane from limited numbers of animals. Rabbit and mouse tubular basement membrane as well as rabbit and rat glomerular basement membrane were obtained from single animals in quantities sufficient for electrophoresis and chemical analysis. The chemical composition of all the basement membranes were compared and sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed on rabbit tubular and glomerular basement membranes in order to compare their polypeptide composition. The chemical composition of the tubular basement membranes were similar, each species having nearly equal concentrations of glycine, hydroxyproline and hydroxylysine, respectively. Glomerular basement membranes were also similar to each other and close to tubular basement membranes in chemical composition. Within each species, the glycine, hydroxyproline and hydroxylysine values for tubular basement membrane were about 10% higher than for glomerular basement membrane. The polypeptide composition of a reduced, sodium dodecylsulfate-soluble fraction of the rabbit basement membranes appeared to be alike with two exceptions. A prominent band of Mr = 160,000 seen in gels of tubular basement membrane was present as a lightly staining band in glomerular basement membrane samples and a prominent band of Mr = 140,000 in gels of glomerular basement membrane was seen as a light band in samples of tubular basement membrane.

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 (pattern 1). 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 (pattern 2). 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.

Renal tubules from rabbit kidneys were isolated from thin shavings of the kidney surface. Basement membrane was then prepared following sonication of the isolated tubules. To insure preservation of the integrity of the basement membrane polypeptides, the protease inhibitors, diisopropyl fluorophosphate, ethylenediaminetetraacetic acid, N-ethylmaleimide, and epsilon-amino-caproic acid were used at all stages of the preparations. The optimal conditions of sonication and centrifugation were established and the chemical composition of basement membrane prepared under these conditions was examined in detail. Glycine, hydroxyproline, and hydroxylysine were found in concentrations of 206, 65, and 18 residues per thousand, respectively, in basement membrane from young kidneys. About 38% of the basement membrane was found to be soluble in sodium dodecyl sulfate upon incubation at 90 degrees C, and to possess relatively low amounts of the amino acids characteristic of collagen. Electrophoretic analysis of this fraction revealed that the major subunits ranged in approximate molecular weight from 18,500 to greater than 10(6). When analyzed with disulfide bonds reduced, a molecular weight range from 31,000 to 275,000 was observed for this fraction. The sodium dodecyl sulfate-insoluble fraction could be dissolved upon reduction and alkylation and its composition was enriched in the amino acids characteristic of collagen. Polypeptides from this fraction were analyzed by electrophoresis in agarose and in agarose-acrylamide gels. The approximate molecular weight of the smallest component was 164,000. Additional polypeptides were observed whose molecular weights occurred in multimers of this component, up to 1.1 x 10(6), possibly indicating covalent cross-linked multimers of a basic collagen-like polypeptide(s).

Tubular basement membrane changes in 2-amino-4,5-diphenylthiazole-induced polycystic disease. Polycystic renal disease was induced in rats by feeding 2-amino-4,5-diphenylthiazole. Tubular (TBM) and glomerular basement membranes (GBM) were purified and analyzed for possible structural changes that may be a factor in the development of the tubular dilations and cysts. Changes in the relative quantities of TBM polypeptides were detected by sodium dodecylsulfate polyacrylamide gel electrophoresis. An overall increase in the concentration of high molecular weight components and a decrease in concentration of those of low molecular weight components were observed. Changes which were particularly notable included a twofold increase in a component of M r = 380,000 and a decrease in one of M r = 55,000 as analyzed without reduction of disulfide bonds. With reduction of disulfide bonds, the M r = 380,000 component dissociates, whereas the M r = 55,000 polypeptide does not, and polypeptides of M r = 245,000 and 145,000 are observed to increase about twofold in concentration (approximate molecular weights were determined using globular protein standards). These changes take place most rapidly from 4 to 8 weeks of drug administration and remain relatively constant between 8 and 16 weeks. If feeding of the drug is discontinued, the distribution of TBM polypeptides returns to normal. These results indicate that tubular basement membrane from animals with 2-amino-4,5-diphenylthiazole-induced polycystic renal disease is abnormal, and this should be considered as a possible contributing factor in the formation of cysts.

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 immunofluo-rescent 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 α3(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 α1(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 Kimmel-stiel-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.

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.

Methods were developed to obtain tubules and glomeruli and their respective basement membranes from mouse kidneys. While the procedures are especially useful for preparing tubules, one method can be used to simultaneously prepare both tubules and glomeruli. Tubules can be obtained from single animals, while a minimum of five animals are required in order to prepare glomeruli. Either minced whole kidneys or dissected cortex tissue is dispersed using a polytron and the desired fractions are obtained by sucrose density gradient centrifugation. discontinuous gradients consisting of 49.5, 53, 57, and 60% sucrose are used in the first method. Following centrifugation, tubules are collected on the 49.5% sucrose layer and glomeruli are pelleted on the bottom of the tube. In the second method, disrupted tissue is mixed with 57% sucrose and after centrifugation a layer of pure tubules is obtained from the top of the sucrose solution. Basement membranes are then obtained by a sonication method and the average yields per kidney are 0.1 mg and 0.02 mg for tubular and glomerular basement membranes respectively. Their chemical compositions are similar to the respective basement membranes from other species.

SDS gel electrophoretic analysis of the products of human prothrombin (IIH) activation reveal a similar product distribution to that observed for bovine prothrombin (IIB). However, some subtle differences are noted when the amino-terminal sequences of the activation intermediates are determined. The amino-terminal sequences of IIH and HH-intermediate 3 are identical (ALA, ASN, PRO, PHE, LEU, GLU, GLU, VAL, ARG, LYS) and homologous to the sequence for IIB. Similarly the amino-terminal sequences of IIH-mtermediate 1 and IIH-intermediate 4 (SER, GLU, GLY, SER, SER, VAL, ASN, LEU, SER, PRO) are entirely homologous with their bovine counterparts. On the other hand IIH-intermediate 2 and α-thrombin A chain have the sequence THR, PHE, GLY, SER, GLY, GLU, ALA, ASN and this sequence is homologous to the IIB intermediate 2, α IIa A chain beginning with residue 14. Studies of the cleavage of IIH-intermediate 1 with factor Xa in the presence of DFP and Hirrudin and with thrombin in the presence of soybean trypsin inhibitor indicate that the IIH intermediate 2 originally produced by factor Xa is secondarily cleaved by thrombin. Thrombin cleavage of the factor X- produced IIH-intermediate 2 removes the amino-terminal 13 residues of this fragment. The amino-terminal sequence of the factor Xa produced intermediate 2 is THR, SER, THR, -, GLU. Supported by NIH grant HL 16150 and the American Heart Association.

Collagenous polypeptides have apparent low electrophoretic mobilities compared to those of typical globular proteins when their molecular weights are examined as a function of mobility. If, however, the number of amino acid residues epr polypeptide chain is examined as a function of electrophoretic mobility, both collagenous polypeptides and globular proteins obey the same empirical linear relationship. The relationship suggests that the number of residues in collagenous polypeptides from sources such as basement membranes, which may contain both collagenous and noncollagenous regions, may be estimated accurately from SDS-gel electrophoresis. Calf skin collagen and its naturally occurring cross-linked multimers, as well as polypeptides derived from it, provide useful molecular standards in a range from 150 residues to 11,500 residues per polypeptide chain (corresponding to a molecular weight range from 13,500 to about 1,000,000 for collagen). The number of residues can in turn be translated into molecular weight values with a knowledge of the mean residue weight of the polypeptide of interest. In contrast, the molecular weight of collagenous chains can be directly estimated by gel chromatography in Sepharose CL-4B, since collagen chains give the correct molecular weight when compared to globular protein standards.

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 M r 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.