stract). Proceedings of the Fifth International Workshop on Polycystic Kidney Disease, June 19th-20th, 1992, Kansas City, Mo. Polycystic Kidney Disease Research Foundation, Kansas City, p 178 62. The European Polycystic Disease Consortium (1994) The polycystic kidney disease 1 gene encodes 14 kb transcript and lies within a duplicated region on chromosome 16. Cell 77:881-894 63. Kimberling WJ, Kumar S, Gabow PA, Kenyon JB, Connolly CJ, Somlo S (1993) Autosomal dominant polycystic kidney disease: 767

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.

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 (α1/α2, α3, α4, α5, α6) 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 α3, α4, and α5 chains of type IV collagen. In contrast, the α1/α2 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 α6 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 VI collagen play an important role in the thickening of the GBM in human MGN.

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.

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.

The nephropathy of Alport syndrome is associated with unique abnormalities of glomerular basement membranes and is caused in many families by mutations in the X-chromosomal gene COL4A5, which encodes the alpha 5 chain of type IV collagen. We have previously reported that Alport epidermal and glomerular basement membranes fail to bind a monoclonal antibody, Mab A7, that reacts with normal epidermal and glomerular basement membranes, and that this abnormality is unique to Alport syndrome. The molecule in normal tissues that reacts with Mab A7 was termed the "Alport antigen". In the present study we used recombinant carboxyterminal noncollagenous (NC1) domains of the alpha 1, alpha 2, alpha 3, alpha 4 and alpha 5 chains of type IV collagen to determine the molecular identity of the Alport antigen. Mab A7 was found to bind specifically to the NC1 domain of the alpha 5 chain of type IV collagen, by ELISA and immunoblotting studies. This finding provides a molecular explanation for the utility of Mab A7 as a marker for the Alport basement membrane defect. Mab A7 can identify the Alport basement membrane defect in those patients in whom COL4A5 mutations prevent incorporation of alpha 5(IV) into basement membranes.

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.

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.

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.

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.

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.

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.

This report describes the development of anti-glomerular basement membrane (GBM) glomerulonephritis after kidney transplantation in related males with Alport syndrome. Antibodies in sera from one of these patients stained normal GBM, Bowman's capsule, tubular basement membranes, and epidermal basement membranes but did not stain tissues from an unrelated Alport male. The target antigen was found to be a 26 kd peptide of the noncollagenous domain of basement membrane collagen. This study provides further evidence of the importance of abnormalities of basement membrane collagen in the pathogenesis of the Alport nephropathy. We speculate that certain mutations at the Alport locus, such as large intragenic deletions or frame-shift mutations, may be associated with failure to develop immune tolerance to epitopes on this 26kd peptide. In the setting of permissive immune response and regulation, transplantation of a normal kidney may result in the generation of anti-GBM antibodies.

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.