Differential expression of basement membrane collagen chains in diabetic nephropathy

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

Quantitive changes in the glomerular basement membrane components in human membranous nephropathy

In membranous nephropathy (MN), the glomerular basement membrane (GBM) is thickened due to accumulation of GBM material between and around the subepithelial immune deposits. Alterations in the GBM components in relation to subepithelial deposits and GBM thickening are not clearly defined. The GBM distribution of classical and novel [α4(IV)] chains of type IV collagen, laminin, and fibronectin have been studied in seven patients with MN and in three normal controls by a quantitative immunogold technique. In normal kidneys, the labelling of type IV collagen or fibronectin was distributed predominantly along the endothelial side of the GBM; α4(IV) was found in the lamina densa; and laminin was concentrated in the epithelial zone of the GBM (P<0·01). In MN, there were increased immunogold densities for classical and novel type IV collagen chains, laminin, and fibronectin in the spikes of MN patients compared with controls (P<0·05). Furthermore, gold particle labelling for the α4(IV) collagen chain was increased in the middle zone (P<0·01) and that for fibronectin was increased in the endothelial and middle zones of the GBM (P<0·05) compared with normal controls. These findings suggest that subepithelial immune deposits stimulate glomerular epithelial cells (GEC), resulting in enhanced secretion of classical and novel type IV collagen chains, laminin, and fibronectin, forming spikes in MN; of these newly formed components, only novel type IV collagen appears to migrate towards the middle zone of the GBM, contributing to thickening of this zone. The results also suggest that fibronectin, possibly derived from the circulation, is related to thickening of the endothelial zone of the GBM, which in turn might be related to progressive glomerulosclerosis. © 1997 by John Wiley & Sons, Ltd.     

Chemical estimation of interstitial and basement membrane collagen in obstructive nephropathy

Analyses of total 3-hydroxyproline and 4-hydroxyproline in tissue samples can be used to estimate the quantity of two kinds of collagen: that of basement-membrane and that of the several interstitial Types. Applied to an experimental model for obstructive nephropathy in the rabbit, such data show that the large increase in collagen in the renal cortex (at the 32nd day following unilateral ureteral ligation) is primarily of interstitial type. However there is also an appreciable increase in basement membrane collagen. Both findings are in agreement with the qualitative impression from electronmicrographs. The chemical method offers an approach to quantitation of these two kinds of collagen (basement membrane and interestitial) in normal and diseased tissues.     

Glomerular and serum immunoglobulin G subclasses in membranous nephropathy and anti-glomerular basement membrane nephritis

The distribution of human IgG subclasses among the glomerular deposits of 53 patients with glomerulonephritis was examined by immunofluorescence (IF) with subclass-specific monoclonal antibodies (Mab). A subclass restriction was observed in idiopathic membranous nephropathy (MN) with glomerular deposits predominantly containing IgG4 (81% of the studied biopsies) and IgG1 (75%). In de novo MN, occurring after transplantation, the restriction was markedly different, with a predominance of IgG1 (100%) and IgG2 (69%). In anti-glomerular basement membrane (a-GBM) nephritis the restriction was considerable with deposits containing almost exclusively IgG₁ (91%) and IgG4 (73%). The same restriction was observed for circulating anti-GBM antibodies detected by indirect IF assay. By contrast IgG1, IgG2, and IgG3 deposits were identified in lupus proliferative glomerulonephritis. Serum IgG subclass levels were measured in 29 patients with idiopathic MN and a-GBM nephritis by an indirect competitive immunoenzymatic assay using Mab. Mean percentage of IgG₂ serum level was significantly lower in patients. In spite of high variations from patient to patient, a serum IgG subclass imbalance was clearly present in 10 cases with low IgG₂ and high IgG₁ and IgG₃ levels. The imbalance in these patients was not due to urinary loss since it was observed with a similar frequency in hypo- and normoimmunoglobulinemic patients. In 5 out of these 10 patients IgG₂ levels were very low, analogous to those observed in selective IgG₂ deficiency. Whether the important subclass restriction of glomerular IgG (in which patterns differed according to the type of glomerulonephritis) and the serum subclass imbalances were due to a clonally restricted antibody response to a particular antigen or to a host immune response defect, or both, remains to be elucidated.     

Human tissue distribution of novel basement membrane collagen.

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

Diffuse glomerular basement membrane lamellation in post-transplant IgA nephropathy

Diffuse glomerular basement membrane (GBM) lamellation, reminiscent of Alport's syndrome, has rarely, and exclusively, been reported in renal allografts from pediatric donors to adult recipients. We report on a similar lesion, identified in a 42-year-old male, who received a kidney from an unrelated 21-year-old living male donor. The disease of the recipient was unknown. Renal allograft biopsies were performed 3.5 and 4.8 years after the renal transplantation, due to massive proteinuria and serum creatinine elevation. The histological features of both biopsies were similar, but more advanced in the second biopsy. Glomerular mesangium was widened and had an IgA deposit in the first biopsy. In addition to the presence of mesangial electron dense deposits, the GBM showed diffuse lamellation and splintering on the subepithelial side, but no definite deposits. In the second biopsy, IgA deposits were extended to the peripheral capillary walls, but electron microscopic examination was not available. Two months after the second biopsy, the patient returned for hemodialysis.     

小儿肾小球薄基膜病的临床病理研究

目的 :探讨小儿肾小球薄基膜病 (TBMN)的临床病理特征。方法 :对 11例TBMN患儿进行了临床、病理及超微结构的系统观察 ,测量了肾小球基膜 (GBM)及致密层的厚度 ,对小儿薄基膜病的临床病理特征进行了分析。结果 :11例小儿TBMN临床主要表现为单纯血尿 ,无其它明显的阳性体征 ,光镜下肾小球无明显改变或轻微异常 ,未见蛋白管型 ,包曼囊内及肾小管腔内可见渗出的红细胞 ,电镜下GBM广泛变薄 ,平均厚度 <2 0 0nm ,致密层厚度 <10 0nm。结论 :小儿肾小球薄基膜病的诊断主要依靠电镜 ,同时必须强调与临床病史、生化检查及病理组织学、免疫组化紧密结合方可确诊。     

Approach to the diagnosis of thin basement membrane nephropathy in females with the use of antibodies to type IV collagen

CONTEXT: Thin basement membrane nephropathy is recognized by a diffusely thin glomerular basement membrane (GBM) ultrastructurally. In contrast to Alport syndrome (AS), there is no GBM thickening, lamellation, or granular inclusions. Morphologically, there is overlap between thin basement membrane nephropathy and AS in female patients in whom there might be only thin GBM and no pathognomonic findings of AS. OBJECTIVE: To determine if the use of antibodies to collagen IV is helpful in making the distinction between thin basement membrane nephropathy and AS in female patients with primarily thin GBMs. DESIGN: We examined renal biopsies from 9 adult female patients with thin GBMs for the presence of alpha1, alpha3, alpha4, and alpha5 chains of type IV collagen by immunofluorescence. RESULTS: In 2 patients with segmental GBM staining, no suggestion for AS was found on physical examination or in their family history. In the remaining 7 patients with normal GBM staining, 4 had family members with end-stage renal disease of unknown etiology, raising the suspicion of X-linked or autosomal-recessive AS. Three patients were presumed to have thin basement membrane nephropathy. CONCLUSION: Segmental GBM staining for alpha3, alpha4, and alpha5 chains of type IV collagen raises the suspicion of AS in the presence of adequate controls and other supporting evidence. Normal GBM staining for alpha3, alpha4, and alpha5 chains of type IV collagen, however, does not exclude AS.     

A quantitative study of glomerular basement membrane changes in IgA nephropathy

A quantitative study of glomerular basement membrane (GBM) changes in 16 cases of IgA nephropathy was carried out on two whole glomeruli per case using a wide-field electron microscope. We found that GBM changes are observed more frequently than previously reported, although some changes are small and uneven in distribution. Changes include (a) attenuation, (b) lytic attenuation, (c) garland-shaped widening of the GBM, (d) dome-shaped widening of the GBM, and (e) disruption of the GBM. There was some correlation between the severity of light microscopic findings and the percentage of GBM affected by complicated changes ((b)-(e)). The percentage of complicated GBM changes correlated with the amount of proteinuria at biopsy. The frequent occurrence of GBM changes in IgA nephropathy suggests that they might be playing a role in the pathogenesis of IgA nephropathy.     

Comparison of non-collagenous type IV collagen subunits in human glomerular basement membrane, alveolar basement membrane, and placenta

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.     

Monoclonal antibody to human basement membrane collagen type IV

We have developed a hybridoma cell line which secretes monoclonal antibody to human basement membrane type IV collagen. The monoclonal antibody secreted by this hybridoma has been obtained in large amounts by either concentrating it from culture supernatants or from the ascites fluid of mice bearing the hybridoma tumour. This monoclonal antibody to type IV collagen does not cross-react with other types of collagens, including types I, III and V, as determined by an enzyme-linked immunosorbent assay (ELISA) and by immunohistochemical staining of corneal and lens sections. Descemet's membrane of mouse, rabbit and human corneal endothelium and lens capsule, both rich in type IV collagen, bind the antibody when stained immunohistochemically. By the indirect precipitation technique, the antibody is found to bind more than three peptides in the basement membrane collagen-rich fraction of human placenta. Based on the observations of other investigators, these peptides are probably derived by proteolysis of the larger polypeptides, since the purification steps in involve extensive pepsin digestion.     

Ultrastructural localization of collagen type IV and laminin expression in the epithelial basement membrane of oral carcinomas

Epithelial basement membrane in oral carcinomas was examined by immunoelectron microscopy using anticollagen type IV and antilaminin antibodies. Intense and irregularly increased immunostaining was observed at the boundary between carcinoma cells and stromal tissue. Heterotopic immunostaining was found in the enlarged intercellular spaces of peripheral carcinoma cells. In addition, rough endoplasmic reticulum (rER) and vesicles in these cells were frequently stained. These findings suggest an increased synthesis and secretion of basement membrane components at the invading edge of the tumor, and also suggest that some oral carcinomas may spread into the surrounding tissue without apparent loss of the basement membrane.This study was presented in part at the 26th annual meeting of the Clinical Electron Microscopy Society of Japan, Kochi, October 6, 1994     

Ultrastructure of a model basement membrane lacking type IV collagen

Basement membranes (BMs) are specialized extracellular matrices which have important roles in cell attachment, migration, growth, and differentiation. The major components of these matrices include type IV collagen, laminin, entactin, and heparan sulfate proteoglycan. The framework or scaffold of BMs has been proposed to be type IV collagen (Yurchenco et al., 1986, J. Histochem. Cytochem., 34:93-102). However, a murine teratocarcinoma cell-line, M1536-B3, has been described which produces an extracellular matrix (ECM) composed of some of the known components of BM, e.g., laminin, entactin, and sulfated proteoglycan, but lacking type IV collagen (Chung et al., 1979, Cell, 16:277-287). With the use of morphological techniques, we have found that the ECM assembled by these cells is composed of multiple layers of electron-dense cords arranged in an interweaving meshwork with short 2-4 nm-diameter cylindrical rods embedded throughout. This organization closely resembles that reported for naturally occurring BMs, e.g., Reichert's membrane (Inoué et al., 1983, J. Cell Biol., 97: 1524-1537). The previous identification of known in vivo BM components in M1536-B3 ECM and the correspondence in morphological appearance of M1536-B3 ECM with that present in naturally occurring BMs suggests that a BM-type of ECM can be assembled without a type IV collagen framework, thus indicating that other components of BMs have a critical role in BM organization and assembly.     

The use of the radioimmunoassay in the characterization of antibodies to basement membrane collagen.

This study describes the use of the radioimmunoassay for the characterization of antibodies to basement membrane (type IV) collagen from bovine anterior lens capsule. The immunogen was extracted from calf anterior lens capsules by limited pepsin digestion and injected into rabbits. The antisera were characterized using gel diffusion, haemagglutination and the radioimmunoassay in which 125I-labelled types I, II, III, and IV bovine collagen were employed. In the direct radioimmunoassay there was no reaction with either native or denatured types I, II or III bovine collagen, whereas there were high titres towards both native and denatured type IV bovine collagen. Radioimmune inhibition studies using unlabelled types I, II, III and IV bovine collagen, collagenase digested and repepsinized type IV collagen showed that there was marked inhibition by either native, denatured or repepsinized type IV collagen, and slight inhibition by native type I collagen; native type II and type III, denatured types I, II and III, and collagenase digested type IV collagen had no inhibitory effects.     

Hormonal requirements for basement membrane collagen deposition by cultured rat mammary epithelium.

Alveoli and ducts isolated from virgin rat mammary glands synthesize basement membrane collagen (typeIV) in primary culture. Using purified antibodies to type IV collagen, prominent intracellular and extracellular fluorescence is observed in the epithelium. No fluorescence is observed with antibodies to collagen type I and III. From quantitation of the incorporation of [14c]proline-labeled proteins, 1.5 to 2.5 per cent of the newly synthesized proteins are collagen. Type IV collagen from these cultures was biochemically identified on the basis of (1) the high ratio of labeled 3-hydroxyproline to 4-hydroxyproline (1:10), (2) the gel electrophoretic pattern of the collagenase-sensitive proteins precipitated with 1.7 M NaCl, (3)the failure of the collagen to bind to diethylaminoethyl-cellulose, and(4)the immunologic cross-reactivity with mouse tumor type IV is identical with that of type IV collagen from other sources. When the supportive hormones, insulin, prolactin, hydrocortisone, progesterone, and estradiol are removed from the cultures, there is a 90 per cent reduction in the amount of [3H]proline recovered in collagen synthesis coincides with only a 30 percentdrop in the growht rate and a 20 per cent drop in total protein synthesis of the sells over the 24-hour period without hormones. Pulse-chase experimout hormones. Pulse-chase experiments revealed an enhanced turnover of collagen following hormone withdrawal.This system may be an in vitro model of collagen turnover in mammary gland in involution.     

Coexistence of IgA nephropathy and membranous nephropathy

Renal biopsy findings of a 27-year-old female with asymptomatic proteinuria and hematuria revealed two distinct glomerular alterations compatible with both IgA nephropathy and membranous nephropathy. The concomitant presence of two different primary glomerular diseases is very rare.