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Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disease due primarily to mutations in the type I procollagen genes, COL1A1 and COL1A2, causing bone deformity and numerous lifetime fractures. OI murine (oim) model mice carry a mutation in the col1a2 gene causing aberrant production of homotrimeric type I collagen [α1(I)(3)], leading to bone fragility and glomerular accumulation of type I collagen. Previous studies demonstrated that heterozygous (+/oim) and homozygous (oim/oim) mice have elevated tibiae fluoride concentrations but reduced femoral biomechanics. However, it is unclear whether these 2 variables are causally related, because impaired renal function could reduce urinary fluoride excretion, thus elevating bone fluoride concentrations regardless of disease status. Our goal in this study was to determine whether dietary fluoride restriction would improve femoral biomechanics in oim mice. Wild-type, +/oim, and oim/oim mice were fed a control (5 mg/kg fluoride) or fluoride-restricted diet (0 mg/kg fluoride) for ∼13 wk, at which time plasma and femora were analyzed for fluoride concentrations and bone biomechanical properties. In wild-type, +/oim, and oim/oim mice, dietary fluoride restriction reduced femoral fluoride burden by 54-74%, respectively (P < 0.05), without affecting glomerular collagen deposition. Oim/oim mice fed the fluoride-restricted diet had reduced material tensile strength (P < 0.05) compared with oim/oim mice fed the control diet. However, dietary fluoride restriction did not affect stiffness or whole bone femoral breaking strength, regardless of genotype. These data suggest that oim mice have reduced bone strength due to homotrimeric type I collagen, independent of bone fluoride content.

Aim: We tried to establish the significance of quantifying urinary type IV collagen (IV-C) excretion for the evaluation of renal involvement of type 2-diabetic patients., Methods: Twenty patients (13 males and 7 females; age range 31 to 69 years) with type 2 diabetes mellitus had undergone renal biopsy and relationship between the severity of morphological alteration, IV-C expression and urinary IV-C excretion were examined., Results: Urinary IV-C excretion significantly correlated with mesangial expansion score (p = 0.49, p < 0.05) and tubulointerstitial injury score (p = 0.56, p < 0.05). Furthermore, urinary IV-C excretion significantly correlated with both glomerular (r = 0.56, p < 0.01) and tubulointerstitial IV-C expression areas. Urinary protein excretion also correlated with mesangial expansion score and tubulointerstitial injury score. However, it did not correlate with the expression of IV-C in the kidney., Conclusions: These results suggest that urinary IV-C excretion reflects the pathogenetic process of diabetic nephropathy, which urinary protein excretion alone cannot do sufficiently. It can be concluded that urinary IV-C excretion could be a more useful marker for the evaluation of renal involvement of type 2-diabetic patients.

The ultrafiltration function of the glomerular basement membrane (GBM) of the kidney is impaired in genetic and acquired diseases that affect type IV collagen. The GBM is composed of five (alpha1 to alpha5) of the six chains of type IV collagen, organized into an alpha1.alpha2(IV) and an alpha3.alpha4.alpha5(IV) network. In Alport syndrome, mutations in any of the genes encoding the alpha3(IV), alpha4(IV), and alpha5(IV) chains cause the absence of the alpha3. alpha4.alpha5 network, which leads to progressive renal failure. In the present study, the molecular mechanism underlying the network defect was explored by further characterization of the chain organization and elucidation of the discriminatory interactions that govern network assembly. The existence of the two networks was further established by analysis of the hexameric complex of the noncollagenous (NC1) domains, and the alpha5 chain was shown to be linked to the alpha3 and alpha4 chains by interaction through their respective NC1 domains. The potential recognition function of the NC1 domains in network assembly was investigated by comparing the composition of native NC1 hexamers with hexamers that were dissociated and reconstituted in vitro and with hexamers assembled in vitro from purified alpha1-alpha5(IV) NC1 monomers. The results showed that NC1 monomers associate to form native-like hexamers characterized by two distinct populations, an alpha1.alpha2 and alpha3.alpha4.alpha5 heterohexamer. These findings indicate that the NC1 monomers contain recognition sequences for selection of chains and protomers that are sufficient to encode the assembly of the alpha1.alpha2 and alpha3.alpha4.alpha5 networks of GBM. Moreover, hexamer formation from the alpha3, alpha4, and alpha5 NC1 monomers required co-assembly of all three monomers, suggesting that mutations in the NC1 domain in Alport syndrome may disrupt the assembly of the alpha3.alpha4.alpha5 network by interfering with the assembly of the alpha3.alpha4.alpha5 NC1 hexamer.

Glomerular basement membrane (GBM) plays a crucial function in the ultrafiltration of blood plasma by the kidney. This function is impaired in Alport syndrome, a hereditary disorder that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the present study, the supramolecular organization of type IV collagen of GBM was investigated. This was accomplished by using pseudolysin (EC 3.4.24.26) digestion to excise truncated triple-helical protomers for structural studies. Two distinct sets of truncated protomers were solubilized, one at 4 degrees C and the other at 25 degrees C, and their chain composition was determined by use of monoclonal antibodies. The 4 degrees C protomers comprise the alpha1(IV) and alpha2(IV) chains, whereas the 25 degrees C protomers comprised mainly alpha3(IV), alpha4(IV), and alpha5(IV) chains along with some alpha1(IV) and alpha2(IV) chains. The structure of the 25 degrees C protomers was examined by electron microscopy and was found to be characterized by a network containing loops and supercoiled triple helices, which are stabilized by disulfide cross-links between alpha3(IV), alpha4(IV), and alpha5(IV) chains. These results establish a conceptual framework to explain several features of the GBM abnormalities of Alport syndrome. In particular, the alpha3(IV). alpha4(IV).alpha5(IV) network, involving a covalent linkage between these chains, suggests a molecular basis for the conundrum in which mutations in the gene encoding the alpha5(IV) chain cause defective assembly of not only alpha5(IV) chain but also the alpha3(IV) and alpha4(IV) chains in the GBM of patients with Alport syndrome.

A 66-year-old Japanese male, who suffered from persistent proteinuria and leg edema, underwent renal biopsy. Light microscopy revealed marked narrowing of the glomerular capillary lumen with a diffuse accumulation of weakly PAS-positive material. By electron microscopy, abundant abnormal collagen fibers were observed predominantly in the subendothelial space and occasionally in the mesangial matrix. The fibers had a periodicity of about 60 nm and were immunoreactive for anti-type III collagen. Subendothelial electron-dense deposits were also found in some of the capillary walls. The serum level of procollagen III peptides was elevated and changed in parallel with the amount of proteinuria during the patient's clinical course. On the basis of these findings, a diagnosis of the collagenofibrotic glomerulonephropathy was made. A review of the literature, including 29 similar or identical cases, failed to reveal the etiology and pathogenesis of this disease. We suggest that this disease may be divided into two different clinical subtypes, an adult-onset type and a pediatric type.

Type VI collagen was revealed by high-resolution immunocytochemistry in renal glomeruli from short- and long-term streptozotocin-injected hyperglycaemic rats and from their age-matched normoglycaemic controls. The labellings obtained over the glomerular basement membrane and the mesangial matrix were assessed by quantitative evaluations. The labellings over the glomerular basement membrane were low and sparse in the young normoglycaemic animals but became consistent and increased in intensity with age and in both the short- and long-term diabetic animals. For the mesangial matrix, this was labelled more systematically, and its intensity increased with age and in the short-term hyperglycaemic animals. For the long-term hyperglycaemic animals, the intensities of labelling resembled those of their age-matched controls. These results indicate that type VI collagen appears to be a minor constituent of the extracellular matrix of the rat glomeruli, rather concentrated in the mesangial area in the young control animal. Concomitant with the general modifications of the extracellular matrix occurring with age and diabetes, this component increases, but apparently not with the length of the hyperglycaemic state.

Collagen IV is a major structural component of all basal laminae (BLs). Six collagen IV alpha chains are present in mammals; alpha 1 and alpha 2(IV) are broadly expressed in embryos and adults, whereas alpha 3-6(IV) are restricted to a defined subset of BLs. In the glomerular BL of the kidney, the alpha 1 and alpha 2(IV) chains are replaced by the alpha 3-5(IV) chains as development proceeds. In humans, mutation of the collagen alpha 3, alpha 4, or alpha 5(IV) chain genes results in a delayed onset renal disease called Alport syndrome. We show here that mice lacking collagen alpha 3(IV) display a renal phenotype strikingly similar to Alport syndrome: decreased glomerular filtration (leading to uremia), compromised glomerular integrity (leading to proteinuria), structural changes in glomerular BL, and glomerulonephritis. Interestingly, numerous changes in the molecular composition of glomerular BL precede the onset of renal dysfunction; these include loss of collagens alpha 4 and alpha 5(IV), retention of collagen alpha 1/2(IV), appearance of fibronectin and collagen VI, and increased levels of perlecan. We suggest that these alterations contribute, along with loss of collagen IV isoforms per se, to renal pathology.

Nephritogenicity (anti-GBM-nephritis-inducing activity) and alpha-chain composition of globular-do-main (NCI) fractions of type IV collagen from bovine renal, pulmonary, and placental basement membranes (BMs) was examined by injecting these fractions with adjuvant into WKY/NCrj rats and by Western blotting using epitope-defined monoclonal antibodies to the six different alpha chains of type IV collagen. A purified nephritogenic fraction from renal BM contained alpha 1-alpha 6(IV)NCI, whereas a non-nephritogenic fraction contained only alpha 1-alpha 2(IV)NCI. Renal and pulmonary NCI had strong nephritogenic activity: placental NCI had weak activity. The renal and pulmonary fractions contained alpha 1-alpha 6(IV)NCI, and the placental fraction had a large amount of alpha 1-alpha 2(IV)NCI and a very small amount of alpha 3-alpha 6(IV)NCI. Immunohistochemical study of bovine renal BM with the monoclonal antibodies revealed that bovine glomerular BM contained alpha 1-alpha 5(IV) chains, but not the alpha 6(IV) chain. The absence of alpha 6(IV) chain in glomerular BM in bovine and in humans indicates that alpha 6(IV) chain is not a target antigen of anti-GBM nephritis. Nephritogenicity is apparently a property of alpha 3-alpha 5(IV)NCI.

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.

Glomerular expression of extracellular matrices at the protein and mRNA levels was examined in rats with mesangial proliferative glomerulonephritis induced by the intravenous administration of a monoclonal anti-rat Thy-1 antibody. In close association with the mesangial proliferative lesion, type I collagen was immunostained at day 8 but not demonstrated at day 28 in the glomeruli of the kidneys. Type I collagen mRNA expression prominently increased in the nephritic glomeruli at day 4, prior to the appearance of type I collagen protein. In addition, fibronectin expression was also elevated in the diseased glomeruli at both the protein and mRNA levels. These results indicated that glomerular, probably mesangial cells, change their phenotypes during this disease, to synthesize abnormal extracellular matrices that lead to the progression of glomerular sclerosis.

Passive Heymann nephritis (PHN) is a model of human membranous nephropathy that is characterized by formation of granular subepithelial immune deposits in the glomerular capillary wall which results in complement activation. This is causally related to damage of the filtration barrier and subsequent proteinuria. The local accumulation of injurious reactive oxygen species (ROS) is a major effector mechanism in PHN. ROS may induce tissue damage by initiating lipid peroxidation (LPO). In turn, this leads to adduct formation between breakdown products of LPO with structural proteins, such as formation of malondialdehyde (MDA) or 4-hydroxynonenal-lysine adducts. To examine the role of LPO in the development of proteinuria we have localized MDA and 4-hydroxynonenal-lysine adducts in glomeruli of PHN rats by immunofluorescence microscopy, using specific monoclonal antibodies. By immunogold electron microscopy, MDA adducts were localized to cytoplasmic vesicles and cell membranes of glomerular epithelial cells, to the glomerular basement membrane (GBM), and also to immune deposits. Type IV collagen was specifically identified as being modified by MDA adducts, using a variety of techniques. Collagenase pretreatment of GBM extracts indicated that the NC-1 domain of type IV collagen was a site of adduct formation. When LPO was inhibited by pretreatment of PHN rats with the antioxidant probucol, proteinuria was reduced by approximately 85%, and glomerular immunostaining for dialdehyde adducts was markedly reduced, even though the formation of immune deposits was not affected. By contrast, lowering of the serum cholesterol levels had no influence on the development of proteinuria. These findings are consistent with the premise that ROS-induced glomerular injury in PHN involves LPO and that this results not only in damage of cell membranes but in modification of type IV collagen in the GBM as well. The close temporal correlation of the occurrence of LPO with proteinuria and the ability of probucol to inhibit proteinuria support a causal role for LPO in the the alteration of the glomerular permselectivity which results in proteinuria.

Two-dimensional (2-D) electrophoresis of collagenase-digested human glomerular basement membrane (GBM), containing the Goodpasture antigen, revealed a range of monomeric (24-30 kD) and dimeric (43-56 kD) subunits present across a pI range of 3-10. Five distinct alpha(IV)-chains were identified by amino-terminal sequence analysis of 18 of these components transferred to polyvinylidene difluoride membrane. The positions of the well-characterised 26-kD alpha 1(IV)-chain and 24-kD alpha 2(IV)-chain were confirmed. A highly cationic 28-kD monomer was identified as the alpha 3(IV)-chain, while more neutral 28-kD monomers were found to contain the alpha 4(IV)-chain. Sequences from neutral 26-kD monomers corresponded to the known cDNA sequence of the alpha 5(IV)-chain. The presence of charge isoforms of the alpha 1(IV)- and alpha 4(IV)-chains was confirmed by identification of several monomers with different pI but the same sequence. Sequence analysis of dimeric components demonstrated homodimers of alpha 1(IV), alpha 2(IV) and alpha 4(IV), and suggested the presence of heterodimers of alpha 3/alpha 5 and alpha 1/alpha 5. 2-D Western blots of human GBM, with anti-GBM autoantibodies, a monoclonal antibody (P1) to the Goodpasture antigen and a monoclonal antibody to the alpha 3(IV)-chain, demonstrated that the major autoantigenic epitope was localised to the alpha 3(IV)-chain, but that there was also reactivity with the alpha 4(IV)-chain.

Late stage diabetic nephropathy is histologically characterized by either diffuse or nodular expansion of the glomerular matrix. This is presumed to represent the morphological correlate for the functional impairment of the kidney. The exact matrix composition of the nodular glomerulosclerosis lesion of end-stage diabetic nephropathy is not known. Biochemical studies have provided evidence that the microfibrillar collagen type VI is increased in diabetic nephropathy. Consequently, this immunohistochemical study was designed to evaluate the extent and exact morphologic location of increased collagen VI deposition at various stages of diabetic glomerulosclerosis (GS). An irregular, sometimes spot-like staining of collagen VI was observed in diffuse GS in the mesangial portion. The uninterrupted staining which was evident along the glomerular basement membrane in normal glomeruli was discontinuous in diffusely sclerotic glomeruli. In nodular GS, the markedly increased deposition of collagen VI appeared to be evenly distributed throughout the entire nodular lesion. At the same time, mesangial staining for collagen IV was reduced in nodular GS, suggesting that in the expanded mesangial matrix collagen IV is progressively substituted by collagen VI during the transition from diffuse to nodular GS. The colocalization of PAS staining with collagen VI deposition in nodular GS suggests that the typical Kimmelstiel-Wilson lesions at least in part consist of collagen VI. Biochemical analysis confirmed the increased collagen VI deposition in glomeruli extracted from diabetic patients with nodular GS. Application of two antisera, recognizing primarily the alpha 1(VI)- and alpha 2(VI)-chains and the N-terminal part of alpha 3(VI)-chain, respectively, revealed no difference in staining pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of glomerular sclerosis in benign nephrosclerosis (BNS) was studied. We investigated the intraglomerular expression of type III and IV collagens and their mRNAs by immunohistochemistry and by the in situ hybridization method. Formalin-fixed paraffin sections from 28 patients with BNS and 10 control cases were stained by the avidin-biotin complex (ABC) method using monoclonal antibodies for human type III and IV collagens. In the course of the sclerotic process of the glomerulus in BNS, the glomerular staining intensity of type IV collagen increased. The strongest staining was observed in the glomerulus at the early sclerotic stage, and intensity decreased slightly at the later stages. Although type III collagen was absent in normal and nonsclerotic glomeruli, peripheral regions of the sclerotic glomeruli were positive at the early sclerotic stage. Later, type III collagen was diffusely observed in the completely hyalinized glomeruli. The expression of type III and type IV collagen mRNAs was detected in the glomeruli of BNS by the non-radioactive in situ hybridization method using thymine-thymine (T-T) dimerized synthetic oligonucleotides. The number of mRNA positive cells for type III and type IV collagens increased at the presclerotic and early sclerotic stages. But these cells gradually decreased in number as glomerular sclerosis developed. We concluded that type III collagen was presumably synthesized by the intraglomerular cells and may contribute to the development of glomerular sclerosis in BNS along with type IV collagen.

Membranous glomerulonephritis (MN) is characterized by the presence of subepithelial immune complexes and thickening of the glomerular basement membrane (GBM). Immune complexes are recognized as subepithelial electron-dense deposits (EDDs) by electron microscopy. We used immunogold electron microscopy to detect the GBM components--type IV collagen, heparan sulfate proteoglycan (HS-PG) and laminin--in thickened GBM, and studied the relationship between immune complexes and these GBM components. We demonstrate that the three major basement membrane components are distributed throughout the newly synthesized GBM. These findings suggest that type IV collagen, HS-PG, and laminin together comprise the spike-like structures and the newly synthesized GBM-like matrix in the thickened GBM of idiopathic MN and membranous lupus nephritis. The newly constructed matrix in the GBM appears to be composed of nearly normal GBM. In type IV collagen, the alpha 1-chain was rarely present on the newly synthesized basement membrane in the lamina rara externa, while alpha 3-chain was present on the subepithelial newly synthesized basement membrane. HS-PG was found within EDDs in membranous lupus nephritis. This suggests that anti-DNA antibody may cross-react with the HS-PG component of the GBM and thus form a subepithelial immune complex.

The target of autoantibodies in Goodpasture's disease, the Goodpasture antigen has recently been characterized as the NC1 domain of the alpha 3 chain of type IV collagen. In order to study the Goodpasture antigen in different organs, NC1 domains were isolated from basement membranes (BM) of human glomeruli (GBM), tubules (TBM), alveoli (ABM), placenta (PBM) and aorta (VBM). NC1 preparations were separated by 2-D electrophoresis, and silver stained or immunoblotted to determine the subunit structure and antigenicity of different basement membranes. All basement membranes contained monomeric components of MW 26 kDa and 24 kDa, and associated dimers, corresponding to the 2-D location of alpha 1(IV) and alpha 2(IV) chains respectively. However, GBM, ABM, and to a lesser extent TBM possessed an extra set of monomeric components of MW 28 kDa and associated dimers corresponding to the proposed location of alpha 3 (IV) and alpha 4 (IV) chains. 2-D-separated polypeptides were Western blotted with autoantibodies from patients with Goodpasture's disease, a monoclonal antibody to the Goodpasture antigen (P1) and a monoclonal antibody to the bovine alpha 3 (IV) chain. The predominant binding of all these reagents was to cationic 28 kDa monomers of GBM, ABM and TBM, corresponding to the alpha 3 (IV) chain, although autoantibodies and Pl also bound to neutral 28 kDa monomers, corresponding to the alpha 4 (IV) chain. Autoantibodies bound weakly to more neutral components of PBM and VBM, but neither monoclonal antibody bound to these basement membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro glomerular collagen synthesis and its response to various concentrations of transforming growth factor-beta 1 were studied in normal and diabetic rats. TGF-beta 1 increased collagen synthesis in normal glomeruli in a dose-dependent manner up to 5 ng/ml. Concentrations > 5 ng/ml showed a gradual decline in collagen synthesis. Basal collagen synthesis was increased in diabetic glomeruli, but addition of TGF-beta 1 had no effect. Antibody to TGF-beta 1 prevented this increase in collagen synthesis. Both circulating TGF-beta 1 concentration and its glomerular expression of mRNA were higher in diabetic than in normal rats. Although addition of TGF-beta 1 did not increase synthesis in vitro, the absence of an effect is consistent with downregulation of its receptors attributable to the high circulating levels. This study clearly indicates a regulatory role of TGF-beta 1 in renal glomerular collagen synthesis in the normal rat, and suggests a possible causal role for enhanced collagen synthesis in the diabetic rat.

Human glomerular basement membrane (GBM) is known to contain type IV collagen, heparan sulfate proteoglycan (HS-PG) and laminin in the extracellular matrix. In this study, ultrastructural distribution of these antigens was investigated in normal human glomeruli using the immunogold technique. Type IV collagen was present throughout the GBM, mainly on the lamina densa, and all over the mesangial region. HS-PG was present on the laminae rarae, predominantly on the lamina rara externa, and periphery of the mesangial region. Laminin was present throughout the GBM and peripheral region of the mesangial matrix. Our study demonstrated the preferential localization of each component in the GBM.

Background: Scarring in the interstitial compartment of the renal cortex heralds a poor prognosis in many forms of renal injury, however, the mechanism through which glomerular inflammation leads to interstitial scarring is not understood. In a model of anti-GBM disease in the rabbit, development of crescentic glomerulonephritis is associated with marked interstitial fibrosis and decreased renal function. We previously demonstrated that collagen accumulation in the model was preceded by increases in collagen I and IV mRNA and that these changes were primarily extraglomerular at early time points when inflammation was predominantly intraglomerular. In order to identify the cellular origins of extraglomerular collagen synthesis in this model, in situ hybridization using an alpha 2(I) procollagen probe was performed., Experimental Design: A 602 bp rabbit alpha 2(I) procollagen cDNA was cloned using a PCR strategy and sequenced. The nucleotide sequence of the coding region was 94% identical with the human alpha 2(I) procollagen sequence. Northern blots were performed to define conditions of specific hybridization of the anti-sense riboprobe. Tissue sections from normal rabbit kidneys and from kidneys 4, 5, 7, 10 and 14 days after injection of anti-GBM antibody were hybridized with 35S-labeled sense and anti-sense riboprobes. Cells containing alpha 2(I) mRNA were identified by autoradiography and mRNA abundance was quantitated by grain density., Results: No specific hybridization was detected with the sense probe at any time. alpha 2(I) mRNA was undetectable with the anti-sense probe in normal kidney sections. In contrast, the anti-sense probe hybridized specifically at all time points after induction of anti-GBM disease. In agreement with previous filter hybridization studies, on day 4, when inflammation was predominantly intraglomerular, cells in the periarterial adventitial compartment of renal cortex hybridized strongly. At later time points, labeling was also present in the interstitial spaces, the periglomerular region, in Bowman's space and in the glomerular tuft itself., Conclusions: We conclude that perivascular adventitial cells are among the first to respond to glomerular inflammation and represent a pool of cells that subsequently contribute to interstitial and glomerular scarring.

The subunits of the collagen IV hexameric, noncollagenous NC1 domain obtained from bovine aorta, glomerular basement membrane, alveolar basement membrane and placental basement membrane are predominantly dimers. A large fraction of the dimers had been thought to be linked by nondisulfide bonds because they were resistant to cleavage by 5% (v/v) 2-mercaptoethanol, 2% (w/v) SDS, at 100 degrees C. However, if an unusually high concentration of 2-mercaptoethanol, e.g., 40% (v/v), is used, complete conversion of dimers into monomers is achieved, indicating the lack of intersubunit nondisulfide cross-links. Electrophoresis patterns indicate that some of the intermolecular disulfide bonds of the dimers are more resistant to reduction in aqueous SDS than are some of the intramolecular disulfide bonds.

A patient with collagenofibrotic glomerulopathy associated with hepatic persinusoidal fibrosis is described. Renal biopsy revealed that the glomerular tufts contained homogeneous material that was proved by electron microscopy to be collagen fibers. The material was reactive to anti-type III collagen monoclonal antibody. Liver biopsy also showed an increase of type III collagen fibers in the perisinusoidal area. Since the serum procollagen III peptide level was elevated in this patient, fibrosis may have been simultaneously activated in kidney and liver by some unknown condition.

Type IV collagen, which has long been assumed to contain two alpha 1(IV) and one alpha 2(IV) chains, also contains alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains. Stoichiometry of collagenous alpha(IV) chains differs among tissues, suggesting the existence of subclasses of type IV collagen, each with a unique chain composition. This study seeks to define, by characterization of subunit compositions of NC1 domain populations, the structural organization of type IV collagen from bovine glomerular basement membrane. NC1 hexamers from type IV collagen were separated on two affinity chromatography columns, one containing monoclonal antibodies to the alpha 3 chain, and another, to the alpha 1 chain. SDS-polyacrylamide gel electrophoresis, immunoblotting, reversed phase high-performance liquid chromatography, and enzyme-linked immunosorbent assay identified three NC1 hexamer populations: 1) a hexamer composed of (alpha 1)2 and (alpha 2)2 homodimers; 2) a hexamer composed of (alpha 3)2 and (alpha 4)2 homodimers; 3) a hexamer containing all four alpha chains connected in heterodimers, alpha 1-alpha 3 and alpha 2-alpha 4. Results suggest that there are two distinct type IV collagen molecules, one composed of alpha 1(IV) and alpha 2(IV) chains and another composed of alpha 3(IV) and alpha 4(IV) chains. Furthermore, polymerization occurs between molecules with the same chain composition and between molecules with different chain composition. Moreover, crosslinking between different alpha chains is restricted, thus limiting the number of possible macromolecular structures.

Biology of glomerular visceral epithelial cells ("podocytes") and their role in inflammatory process remain obscure, partly because of the lack of well-differentiated podocyte cultures. We have established a human cell line by transfecting with a replication-defective SV40 plasmid (pSVHB1), a primary culture of podocytes derived from an enriched preparation of unencapsulated glomeruli free of tubule and Bowman's capsule contaminants. Podocyte specificity of the primary culture was assessed by a dual immunomorphological and functional approach. The resulting cell line (HGVEC.SV1) was cloned and the clonal cells were adapted to hormonally defined medium supplemented with only 2% newborn bovine serum. Clone A4 has been exhibiting over 35 passages, a combination of markers unique to podocytes, including expression of vimentin, podocalyxin, ectoenzymes (CALLA antigen and mRNA), heparan-sulfate proteoglycans (molecular mass of core protein = 75 kDa), and production of type IV collagen (alpha 1 and alpha 5 chains) established by immunoprecipitation and Northern blot analysis. Cytokeratin was detected in rare cellular foci and the search of Von Willebrand factor was negative. This clonal cell line has been used to demonstrate: (1) that human podocytes are highly sensitive to atrial natriuretic peptide (ANP) which induced a dose-dependent increase in cGMP production (x20 at 0.5 microM ANP), and (2) that secretion of ANP-stimulated cGMP is dramatically polarized as 93% of extracellular cGMP were released in the apical medium when filter-grown HGVEC. SV1A4 cells were stimulated at their basal pole.

We have undertaken an ultrastructural immunogold investigation of the distribution of type IV collagen and heparan sulphate proteoglycan (HSPG) in glomeruli from the kidneys of one normal control and three patients with diabetes mellitus and proteinuria. The sample included both diffuse and nodular diabetic glomerulosclerosis. In the control and diabetic kidneys, the type IV collagen was present predominantly on the endothelial aspect of the glomerular basement membrane (GBM), and by contrast the HSPG was found mainly on the epithelial side. In the mesangium in both control and diabetic glomeruli, type IV collagen was found predominantly in the central regions, while HSPG was mostly restricted to the region beneath the epithelial cells. Consequently, where there is a marked increase in mesangial matrix with nodule formation in diabetics there is a corresponding increase in the amount of type IV collagen but not of HSPG. Although the three diabetic patients were proteinuric, the HSPG was not decreased in the thickened GBMs.

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 C-terminal globular domain NC1 of collagen IV, which carries the epitopes recognized by anti-GBM antibodies in Goodpasture's syndrome, was isolated from human basement membranes (BM) of glomeruli (GBM-NC1), tubules (TBM-NC1), lung (ABM-NC1), placenta (PBM-NC1), and small intestine (IBM-NC1). All NC1 hexamers were of globular size on electron microscopy. On SDS-PAGE, the hexamers dissociated into monomeric and dimer-sized subunits of similar molecular weights. The following monomer:dimer relationships were identified: GBM-NC1, and PBM-NC1 = 1:3; ABM-NC1 = 1:4; and IBM-NC1 = 1:32. On immunoblot, all dimers of the various NC1 globules showed binding of anti-GBM antibodies. However, monomers stained differently, with three monomers demonstrable in GBM-NC1 and no monomer staining in PBM-NC1. In addition, studies with monoclonal antibodies showed that the C-terminus of the alpha 1(IV) collagen chain was demonstrable in all different NC1 hexamers. In contrast, the alpha 3(IV) chain, to which Goodpasture sera preferentially bind, showed a restricted distribution. One monomer and dimers were demonstrable in GBM-NC1 and ABM-NC1, only a weak dimer staining was seen in TBM-NC1, while no evidence for alpha 3(IV) was found in IBM-NC1 and PBM-NC1. Dissociation by 6 M guanidine-HC1 or treatment by acid increases the apparent number of accessible epitopes for anti-GBM antibodies. In addition, dose-response curves, which were obtained by incubation of increasing concentrations of NC1 with anti-GBM antibody positive sera, indicated that for GBM-NC1 and ABM-NC1 the lowest NC1 protein concentrations were necessary to bind 50% of the antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

A new computerized polarization microscopic method was developed for objective, quick quantitative analysis of anisotropic structures. The paper presents the technology on collagen fibers of kidney and liver preparations. According to the observations the birefringence of one fiber can be characterized not by a single number as it is accepted generally but by a series of data. It suggests that the process of compensation is gradual. One of the explanations of this fact might be that the collagen fibers are inhomogeneous at submicroscopic level. With regard to this assumption, first several birefringent capillary basement membranes of a whole glomerulum were measured simultaneously. In this case, the individual capillaries showed nearly the same retardation. Second, portal zones of normal and cirrhotic livers as functional units were analyzed simultaneously. In the latter case the individual fibers show different birefringence. Comparing the results obtained by their technique with the data of the classic measuring method authors found similarity that proved its validity. Data suggest that the new method seems to be useful in experimental and diagnostic pathology.

The present study was carried out to examine the effect of L-arginine on advanced stage nonenzymatic glycosylation end products in glomerular basement membrane (GBM) as represented by carboxymethyllysine (CML). Twelve db/db mice were given a solution containing a daily dosage of L-arginine of 50 mg/kg body weight orally. Twelve db/db mice served as controls. At the end of the 4-months study period treated animals had significantly lower concentrations of CML (0.084 +/- 0.008, 0.071-0.098 nmol/mumol OH-proline; mean +/- SD, range, p less than 0.01) compared to untreated controls (0.11 +/- 0.018, 0.095-0.152 nmol/mumol OH-proline). In addition, there was a significant positive correlation between GBM thickness and concentrations of CML (r = 0.86, p less than 0.001). We conclude that reduction of CML concentrations in treated db/db mice possibly reflects a beneficial effect of L-arginine on advanced stage nonenzymatic glycosylation end-products in GBM. In addition, measuring CML concentrations might have future clinical implications as a noninvasive parameter for basement membrane thickening.

Analysis of the Sephacryl S-200 fractionated type IV collagen domains from bovine and human glomerular basement membranes (GBM) and calf anterior lens capsule (ALC) indicated that Asn-linked oligosaccharides are primarily or exclusively localized in the 7 S region, whereas the hydroxylysine-linked Glc alpha 1----2Gal disaccharides (Glc-Gal-Hyl) are present in all the major segments of the molecule (7 S, NC1, and helical domain); no Ser/Thr-linked saccharide were detected. The Asn-linked carbohydrate units observed in the 7 S domain (Mr approximately 300,000) occurred in a number equal to the 12 polypeptide chains constituting this cross-linked region, and this was consistent with lectin blots of the reduced electrophoretically resolved 7 S components. Fractionation of the N-glycanase and endo-beta-N-acetylglucosaminidase-released oligosaccharides by concanavalin A affinity and high performance liquid chromatography indicated that the Asn-linked carbohydrate occurred predominantly in the form of complex tri- and biantennary units, although submolar amounts of polymannose variants (Man5-7GlcNAc2) were also present in calf ALC and bovine GBM. Structural studies of the complex N-linked oligosaccharides employing hydrazine/nitrous acid fragmentation and glycosidase digestions indicated a pattern in which there was complete fucosylation of the innermost GlcNAc residue of the Man3GlcNAc2 core but only sparse substitution with capping groups of the nonrepeating N-acetyllactosamine branches. Whether tri- or biantennary, the oligosaccharides from bovine GBM contained only one capping residue, in the form of either NeuAc or alpha-D-Gal, whereas those from ALC had only a single alpha-D-Gal and no NeuAc; human GBM oligosaccharides were devoid of both NeuAc and alpha-D-Gal. The absence of terminal alpha-D-Gal in the human 7 S domain was reflected in its lack of reactivity with Bandeiraea simplicifolia I and from its failure to yield Gal alpha 1----3Gal beta 1----4 [3H]anhydromannitol after hydrazine/nitrous acid/NaB3H4 treatment. Application of the latter procedure to the collagen domains yielded, in addition to fragments from the N-linked oligosaccharides, a disaccharide (Glc alpha 1----2[3H]galactitol) derived from the Glc-Gal-Hyl units. The localization of Asn-linked carbohydrate units in the evolutionarily conserved 7S domain of type IV collagens suggests that these oligosaccharides may play a role in the assembly of the collagen network of basement membranes.

Protein A-gold immunocytochemistry was applied in combination with morphometrical approaches to reveal the alpha 1(IV), alpha 2(IV), and alpha 3(IV) chains of type IV collagen as well as entactin on renal basement membranes, particularly on the glomerular one, during maturation. The results have indicated that a heterogeneity between renal basement membranes appears during the maturation process. In the glomerulus at the capillary loop stage, both the epithelial and endothelial cell basement membranes were labeled for the alpha 1(IV) and alpha 2(IV) chains of type IV collagen and entactin. After fusion, both proteins were present on the entire thickness of the typical glomerular basement membrane. At later stages, the labeling for alpha 1(IV) and alpha 2(IV) chains of type IV collagen decreased and drifted towards the endothelial side, whereas the labeling for the alpha 3(IV) chain increased and remained centrally located. Entactin remained on the entire thickness of the basement membrane during maturation and in adult stage. The distribution of endogenous serum albumin in the glomerular wall was studied during maturation, as a reference for the functional properties of the glomerular basement membrane. This distribution, dispersed through the entire thickness of the basement membrane at early stages, shifted towards the endothelial side of the lamina densa with maturation, demonstrating a progressive acquisition of the permselectivity. These results demonstrate that modifications in the content and organization of the different constituents of basement membranes occur with maturation and are required for the establishment of the filtration properties of the glomerular basement membrane.

Collagen IV contains an amino-terminal tetramerization domain (7 S) that is involved in aggregation and cross-linking as part of the process of self-assembly of the collagen IV matrix of basement membranes. We determined the structure and location of the Asn-linked oligosaccharides of the 7 S tetramer. Two glycopeptides, GP-1 and GP-2, were isolated from tryptic digests of the 7 S tetramer and were characterized. GP-1 and GP-2 are derived from the alpha 1(IV) chain and the alpha 2(IV) chain, respectively. Each glycopeptide contained one sequence, -Asn-Xaa-Thr-, which was shown to be N-glycosylated at Asn, corresponding to position 126 of the alpha 1 chains and 138 of the alpha 2 chain. 1H NMR spectroscopic analysis of the oligosaccharide is a biantennary N-acetyllactosamine type of N-linked oligosaccharide with a broad heterogeneity in the presence of the sugar residues at their nonreducing termini as indicated. [formula: see text] The location of the Asn-linked oligosaccharide units and Hyl-linked disaccharide units and their orientation with respect to the surface of the triple helix were calculated using two models. We conclude that both units are important determinants in the assembly of the 7 S tetramer.

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.

A new type of idiopathic glomerular disease is reported in a 49-year-old Italian woman who presented with uncharacteristic renal symptoms, i.e., hypertension and slight proteinuria. Clinical investigation excluded a familial renal disease and more specifically nail-patella syndrome. Diagnostic renal biopsy by light microscopy showed a picture similar to membranoproliferative glomerulonephritis. The enlarged glomeruli were lobulated, the peripheral basement membranes were thickened by the deposition of light-microscopically undefined material, cell proliferation was lacking. By electron microscopy, the material was nonhomogenous, partly granular partly fibrillar, containing typical collagen fibers. The latter were identified as collagen type III, to a lesser extent collagen type I. Review of the literature resulted in 12 similar or identical cases reported from Japan and one additional case reported in a white American female. Evidence of systemic disease is lacking. Etiology and pathogenesis are elusive. A progressive deterioration of renal function must be expected. Collagen type III glomerulopathy is suggested as term of this new type of idiopathic glomerular disease.

Using an indirect immunoperoxidase technique, we tested frozen specimens from one Wilms' tumour composed of numerous glomeruloid bodies devoid of blood vessels, with monoclonal antibodies directed against vimentin, cytokeratin, CALLA/CD10, CD24, CR1/CD35, endothelium factor VIII, class I and II MHC molecules, laminin, fibronectin, and non-collagenic domain NC1 of type IV collagen. Two reagents against Goodpasture determinants were used: P1 monoclonal antibody and serum IgG (GP antibodies) from a biopsy-proven Goodpasture patient. Glomeruloid bodies comprised two cell types: a peripheral layer of parietal epithelial cells (cytokeratin and CD24-positive) and central cell clumps of podocytes (vimentin and CALLA-positive). The basal lamina surrounding the glomeruloid bodies contained laminin and NC1 domain of type IV collagen, while that present between the podocytes reacted strongly with laminin, and P1 and GP antibodies. Endothelium factor VIII was not detected within the glomeruloid bodies and CR1 molecules bound to the basement membrane material within them. These data favour the hypothesis that podocytes produce the basement membrane material which bears Goodpasture determinants recently identified as a novel chain, named the alpha 3 chain, of type IV collagen.

The protein A-gold immunocytochemical technique was applied to reveal the monomeric elements M1, M2* and M3 from the non-collagenous globular domain (NC1) of type IV collagen over various renal basement membranes from control and long-term streptozotocin-induced diabetic rats. This study includes the basement membranes of the proximal tubule, the Bowman's capsule and the glomerulus as well as the extracellular matrix of the mesangium. The labellings obtained were confined to basement membrane material. The quantitative analysis demonstrated changes in labelling intensities and distribution between tissues from normal and diabetic animals. Increased labelling intensities were observed for M1 and M2* monomers in all the basement membranes studied except for the mesangial matrix which remained unchanged. In addition, the labelling for M1 monomers, present on the endothelial side of the glomerular basement membrane of control animals, was found to be distributed throughout the entire thickness of the basement membrane of diabetic animals. In contrast, neither the intensity of the labelling, nor the distribution of M3 monomers were altered in diabetic animals. Since M1 monomers are markers of the alpha 1(IV) and alpha 2(IV) chains of type IV collagen while M2* and M3 mark alpha 3(IV) and alpha 4(IV) chains respectively, the present results demonstrate changes in the nature of the collagenous elements of basement membranes during diabetes. Furthermore, the results indicate that the alpha 3(IV) and the alpha 4(IV) chains are not necessarily present in the same molecule. The modifications of the collagenous elements of the basement membranes during diabetes must alter the structural characteristics of these matrices which in turn might influence their functional properties.

Human and bovine glomerular basement membrane (GBM) preparations, representing the extracellular matrix of the renal filtration units, were found to contain type VI collagen. This protein was solubilized by guanidine and guanidine-dithiothreitol extractions and characterized after polyacrylamide gel electrophoretic resolution by immunoblotting with an antiserum directed against the alpha 1(VI) and alpha 2(VI) polypeptide chains and by its insensitivity to collagenase digestion in the nonreduced state. In contrast to GBM, which is the product of three distinct cells, type VI collagen could not be detected in extracts from calf lens capsule, an epithelial cell-derived basement membrane. Quantitation by radioimmunoassay of the type VI collagen content of GBM from 17 diabetic and 15 nondiabetic human subjects indicated a 2.8-fold higher level (P less than 0.001) in the diabetic preparations. Because in the glomerulus type VI collagen is considered on the basis of immunohistochemistry to be localized to the mesangium, we believe that measurement of this protein in GBM preparations can provide a valuable index of mesangial expansion in diabetic and other glomerulopathies.