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We aimed to develop an image analysis software that enabled measurement of glomerular basement membrane (GBM) thickness.With this software, we evaluated the range of GBM widths found in a cohort of Asian patients diagnosed with a spectrum of renal diseases including minimal change/IgM nephropathy, focal and segmental glomerulosclerosis, IgA nephropathy, systemic lupus erythematosus nephritis, diabetic nephropathy, pauci-immune crescentic glomerulonephritis, thin basement membrane disease, and tubulointerstitial nephritis. Measurements were taken from a minimum of five glomerular capillary loops of each glomerulus. For each loop, at least 10 different points of the GBM were measured.The average GBM width measured for minimal change disease was 347.4 +/- 9.0 nm, with the highest value being 403.9 nm and lowest being 214.7 nm. No association was found between GBM width and gender. We found a significant increase in GBM width in pathological states like lupus nephropathy (p0.0001), diabetic nephritis (p0.001) and tubulointerstitial nephritis (p0.01) as compared with minimal change disease. Only one case of thin membrane nephropathy (198.7 nm) was available for analysis and we found a significant thinning of the GBM.These observations provide insights into the range of GBM thickness in several disease states and support the use of this novel software in the daily diagnostic laboratory setting.

The basement membranes of the glomerulus, thyroid and adrenal all contain the Goodpasture antigen, the target of autoantibodies in antiglomerular basement membrane (GBM) disease. Antithyroid antibodies can be associated with antiGBM disease, and there have been occasional reports of antithyroid antibodies in Alport syndrome, an inherited kidney disease where the GBM lacks the Goodpasture antigen. The aim of this study was to determine how often antithyroid and antiadrenal autoantibodies occurred in antiGBM disease, Alport syndrome and a related condition, thin basement membrane disease (TBMD). Sera from patients with antiGBM disease (n = 19), Alport syndrome (n = 5) or TBMD (n = 13) were tested for antithyroglobulin, antithyroid microsomal and antiadrenal antibodies. Five of the patients with antiGBM disease (5/19, 26%, P NS) had antimicrosomal, and one had antithyroglobulin, antibodies (1/19, 5%, P NS). No patient with Alport syndrome had antithyroid antibodies. One with TBMD (1/13, 8%, P NS) had antithyroglobulin and antimicrosomal antibodies at titres of 1/400 and 1/25,600, respectively. Both patients with antithyroglobulin antibodies had previously been diagnosed with hypothyroidism. No one with antiGBM disease, Alport syndrome or TBMD had antiadrenal antibodies. Antithyroid microsomal antibodies do not occur significantly more often in patients with antiGBM disease than in normals, and antithyroid and antiadrenal antibodies are not associated with Alport syndrome or TBMD.

The glomerular and tubular basement membranes are the principal barriers to filtration and re-absorption of water and molecules in the nephron. They are composed primarily of type IV collagen, laminin, fibronectin, sulphated proteoglycans and collagen type I. Three common inherited diseases are associated with abnormalities of basement membrane proteins: Alport's syndrome, thin basement membrane disease (TBMD) and adult polycystic kidney disease. In this review we describe the application of molecular biological techniques to the study of these conditions. Classic Alport's syndrome is an X-linked disorder with a lamellated glomerular basement membrane (GBM) which typically results in renal failure in males. Studies with sera from patients with Goodpasture's syndrome, or monoclonal antibodies specific for the Goodpasture antigen, show that the Goodpasture antigen is absent or masked in the kidneys of individuals with Alport's syndrome. There is some evidence to suggest that the Goodpasture antigen is best represented by the non-collagenous domain of the alpha 3 chain of type IV collagen, but that other non-collagenous regions may also contribute to the antigen. It is through these non-collagenous regions that the type IV collagen chains form the typical network, and the abnormality in Alport's syndrome interferes with this network formation. However, we have recently demonstrated that the gene for the non-collagenous domain of the alpha 3 collagen chain is present in individuals with Alport's syndrome. Furthermore, other groups have shown a defect in a novel type IV collagen chain, the alpha 5 chain, in 3 unrelated cases of Alport's syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

A 41-year-old woman was discovered to have enteric infection of Aeromonas caviae and was treated with antibiotics. Subsequently she developed nephrotic syndrome. Renal biopsy showed a non-argentaffine hole in the glomerular basement membrane (GBM) in PAM stain with IgG and C3 deposition. This finding was similar to membranous glomerulonephritis. However, electron microscopy showed no electron dense deposits but microspheres or microtubular structures associated with podocytic infolding into the GBM. Corticosteroid treatment resulted in rapid remission. 1 This case corresponds to a new disease entity of podocytic infolding glomerulopathy, which was proposed by a working group of The Japanese Society of Nephrology on the basis of 25 cases corrected from 17 institutions all over Japan. 2 The question arises whether these morphological changes are specific for a new disease entity or they could reflect a nonspecific cellular response of the podocyte to an injury.

Introduction The clinical manifestations of HIV Infection sometimes resemble those of autoimmune diseases (AD). Immune dysregulatfon with perturbed B cell function and hypergammaglobullnaemla is an established consequence of HIV Infection. Polyclonal B cell activation is thought to contribute to the development of self-reactive antibodies In AD such as systemic lupus erythematosus. Previous studies have demonstrated the high prevalence of cardlolipln antibodies (aCLa) in HIV Infection but have yielded conflicting results regarding antlnuclear antibodies (ANA). However, the spectrum of autoantibody (AAb) production in HIV Infection is uncertain. This study sought to estimate the point prevalence of organ specific and nonorgan specific AAbs, and to determine the relationship of AAb production and the polyclonal gammopattiy of HIV Infection. Method All patients attending the HIV clinics at RPAH were invited to participate in the study. Informed consent was obtained. Patients responded to a questionnaire regarding symptoms; blood was drawn for measurement of Immunoglobulin (lg) levels, protein electrophoretogram, and AAbs. Results 90 patients (88 males) were enrolled. The mean CD4 T cell count was 179 × 10 6 /L. There was significant elevation of the Ig levels compared with the healthy population (mean IgG 17.0 g/L; pc.00/ t-tesf). 9 patients had three AAbs, 27 had 2 and 32 had 1, while 24 patients had no AAbs. aCLa (52/90) and smooth muscle antibodies (SMA, 45/90).accounted for the majority of the positives. Only 8/90 were ANA positive. C-ANCA occurred in 4 patients and glomerular basement membrane antibodies (GBMa) In 3. Antibodies to SS-A, SS-B, dsDNA, RNP, Sm, Jo-1 parietal cells and mitochondria were not detected. The relationship between the number of AAbs and Ig levels was not statistically significant (one-way ANOVA). Conclusion Although the overall prevalence of AAbs is high in HIV Infected individuals, the spectrum of AAb production is narrow. aCLA and SMA are common, C-ANCA. GBMa and ANA are uncommon. 8 of the AAbs sought did not occur at all. The Infrequency of most AAbs and the lack of relationship with Ig levels suggests that polyclonal B cell activation selectively generates aCLas and SMA rather than a random spectrum of AAbs.

A knowledge of the components comprising the glomerular basement membrane (GBM), their distribution, and dynamics, is essential to a proper understanding of renal pathology. Furthermore, the traditional concept of GBM composition has been overturned recently by the finding that, in addition to the known collagen IV subtypes α 1 and α 2 , there are further subtypes in the glomerulus designated α 3 (Goodpasture’s epitope), α 4 , and α 5 . To investigate the distribution of laminin and the collagen IV subtypes α 1 , α 2 , and α 3 , normal human tissue from nephrectomies was processed, unfixed, directly into low-acid glycol methacrylate by ethanediol dehydration, a procedure which allows maximum antigenic retention. Subsequently, the epitopes were localised by colloidal gold electron immunocytochemistry using monoclonal antibodies to collagen IV subtypes α 1 and α 3 , polyclonal antibodies to subtypes α 1 and α 2 combined, and polyclonal antibodies to laminin. With polyclonal antibodies, collagen IV α 1 /α 2 was found as a narrow subendothelial band within capillary loops and in the central area of mesangia. With monoclonal antibodies α 1 could be demonstrated in the centre of mesangia only. In glomeruli undergoing sclerosis, material accumulating in the subendotheliai zone, or causing occlusion, was also positive for α 1 /α 2 . In contrast, collagen IV α 3 and laminin were demonstrated across the main thickness of loops and around the periphery of mesangia. No α 3 or laminin were found in association with α 1 /α 2 in sclerotic areas. These results indicate that GBM components have a heterogeneous distribution within loops and appear to be organized in discrete concentric zones with collagen IV α 3 a major constituent. Thus, thinning events are likely to involve a reduction in α 3 , rather than α 1 /α 2 while thickening may involve any subset.

Summary Human glomerular basement membrane (GBM) emulsified in Freund's complete adjuvant was injected into 16 Holtzman rats. Eleven (69%) of them subsequently developed glomerulonephritis. This disease was characterized by glomerular changes which included focal lobular hypercellularity due to localized hyperplasia of intracapillary (mesangial or endothelial) cells which often obstructed glomerular capillaries. Later, affected lobules showed necrosis of intracapillary cells, and capillary lumina were filled with homogeneous eosinophilic material. An immunoperoxidase technique revealed autologous anti-GBM antibody in all 3 layers of the GBM and in some regions it was present in greater amount in the lamina rara interna and externa than in the lamina densa. Experimental autoimmune glomerulonephritis in the rat is thus characterized morphologically by a focal proliferative glomerulonephritis which proceeds to focal glumerulosclerosis.

Summary A series of monoclonal antibodies to human glomerular antigens was prepared by immunisation of a mouse with isolated whole glomeruli, followed by boosting with particulate glomerular basement membrane and fusion of murine spleen cells with the NSI-myeloma line. Hybridoma supernatants were screened jointly by a radioimmunoassay involving binding to isolated glomeruli, and by a 4-layer immunoperoxidase technique applied to polyester wax-embedded sections. Seven monoclonal antibodies with different specificities (PHM7-PHM13) were established and repeatedly cloned. Each antibody displayed a distinctive distribution within the glomerulus, including different patterns of staining of mesangial cells, mesangial matrix and glomerular basement membrane, in addition to extra-glomerular basement membranes and extracellular matrix. All antibodies also stained cellular outgrowths of isolated glomeruli cultured in vitro , and showed additive binding to cultured cells by radioimmunoassay. Physical characterization using absorptions with purified substrates, plus specific chemical and enzymatic digestions, indicated that PHM12 is directed against type IV collagen. PHM13 is directed against fibronectin as shown by absorption with purified fibronectin and immunoprecipitation of a 220 000 MW glycoprotein. The remaining 5 monoclonal antibodies, which react with carbohydrate (PHM7) or protein (PHM8-PHM11) determinants, were shown to be nonreactive with type IV collagen, fibronectin or other known glomerular components including sialic acid, laminin, amyloid P-component or various glycosaminoglycans. These monoclonal antibodies therefore appear to define a new series of human glomerular antigens, or possibly closely related antigenic determinants, which are synthesized by glomerular cells and incorporated into the mesangium and glomerular basement membrane. These antibodies, by providing markers for at least 2 antigens known to be important in glomerular cell-matrix interactions, should prove useful in research into the mechanisms involved in renal pathology.

Albino Holtzman, albino Wistar and hooded HS rats were injected fortnightly for 14 weeks with human glomerular basement membrane (GBM) emulsified in Freund's complete adjuvant. Half of the rats were pretreated with Freund's complete adjuvant and some were unilaterally nephrectomized. Anti-GBM antibody glomerulonephritis, characterized by proteinuria (greater than 100 mg/16 h) and a diffuse linear deposition of host immunoglobulin along the glomerular basement membrane, was first detected in Holtzman rats 4 weeks after treatment with GBM had begun, and had developed in 69% of these rats by 15 weeks. In contrast, none of the similarly treated Wistar or HS rats became proteinuric at any time, although a few showed weak glomerular fluorescence at the end of the experiment. Thus Holtzman rats are susceptible, and HS and Wistar rats are resistant to experimental anti-GBM antibody glomerulonephritis. Pretreatment with Freund's complete adjuvant apparently shortened the induction period of the experimental disease in the Holtzman rats whereas unilateral nephrectomy appeared to decrease their susceptibility to it.

Summary Structural models of the glomerular basement membrane (GBM) have been based solely on the localization of antigens in animal kidneys. These models depict a type IV collagen lattice as the structural skeleton along the central portion of the membrane, with the glycoprotein laminin attached predominantly in the laminae rarae where it is thought to be involved with endothelial and visceral epithelial cell attachment. The human GBM is also known to contain type IV collagen and laminin. The present study localizes both of these structural antigens in normal human GBM using the ultrastructural immune-gold technique. Type IV collagen is situated in the sub-endothelial third of the GBM and is continuous with the type IV collagen within the mesangial matrix. Laminin is localized throughout the entire thickness of the GBM and mesangial matrix. These results indicate that the structure of human GBM is significantly different from that proposed in animal models. A structure for the normal human GBM is discussed, depicting a thin type IV collagen lattice which is asymmetrically placed in the sub-endothelial third of the membrane, with laminin distributed as an integral component of the membrane in addition to its role in cell attachment.

Injection of rats with large doses of bovine serum albumin causes proteinuria which may persist long after the period of overload has ended. In order to assess in this model of proteinuria the relative importance of podocytic epithelial changes versus alterations in anionic groups in the glomerular capillary wall a morphological study has been made of animals in which the kidneys were fixed by vascular perfusion or by in situ drip fixation. By transmission electron microscopy, podocytes showed protein droplets, cytoplasmic vacuoles, spreading of epithelial cytoplasm with loss of foot processes, and focal separation of epithelium from the glomerular basement membrane, occasionally with cytoplasmic disruption. Staining with colloidal iron showed no reduction in the density of anionic groups per unit area on epithelial cell surfaces or elsewhere in glomeruli. However, the reduced surface area of epithelial cells caused by the changes to their structure accounts adequately for the less intense glomerular colloidal iron staining evident by light microscopy. Changes in podocyte structure, particularly those leading to focal cytoplasmic defects on the outer surface of the glomerular basement membrane, appear to be more important than loss of glomerular anionic groups for the development of proteinuria in protein overload nephropathy.

Using an ultrastructural immunoperoxidase technique, the distribution of endogenous albumin and immunoglobulin G was examined in superficial glomeruli of Munich-Wistar rats with aminonucleoside nephrosis. In glomerular capillaries in which the external surface of the glomerular basement membrane (GBM) was completely covered with spread expanses of epithelial cytoplasm, albumin and IgG were distributed normally, with no detectable penetration beyond the endothelial fenestrae. However, at sites of focal loss of the epithelial covering of the GBM, both albumin and IgG were found to penetrate the GBM. These results suggest that, in this experimental model, plasma proteins leak into the urine at sites of glomerular epithelial denudation.

Administration of 6 mM silver nitrate in the drinking water of mice resulted in deposition within the glomerular basement membrane of silver granules which were detected on electron microscopy after 12 days. Larger aggregates were detected in the basement membrane and mesangium when silver ingestion was extended to 14 weeks. The silver deposits did not alter significantly over a period of 21 weeks after silver nitrate ingestion was stopped.

Peripheral glomerular capillary walls were studied in 26 cases of IgA nephropathy by means of the transmission electron microscope. Ultrastructural abnormalities were identified in 11 cases (42%). Abnormalities of the glomerular basement membrane (GBM) were the most frequent change which consisted of localized thinning, lamination, irregular thickening, disruption, membranolysis and aneurysmal dilatation of the GBM. Subendothelial electron dense deposits were seen. Necrosis and detachment of the podocytes from the GBM were also encountered. The changes were correlated with the clinical findings at the time of diagnosis which showed a significant correlation of these peripheral glomerular capillary wall lesions with proteinuria. With light microscopy, crescents were significantly more frequently seen in the cases showing the ultrastructural capillary wall abnormalities than those without. This observation suggested that local peripheral glomerular capillary wall damage was an important factor in the pathogenesis of the extracapillary lesions in IgA nephropathy.

Glomeruli from 48 cases of glomerulonephritis (Gn) were isolated and studied by means of the scanning electron microscope (SEM). Overall glomerular loss due to processing was 2.4%. SEM of these isolated glomeruli showed good preservation of glomerular structure. Changes of the podocyte cell body and its foot processes were noted and correlated with light microscopic morphology. Individual podocyte necrosis, variable foot process fusion and cell body swelling were seen in IgA nephropathy. Podocyte degeneration with separation from the glomerular basement membrane was evident in idiopathic minimal change Gn while idiopathic membranous Gn was characterized by globular swelling of the podocyte cell bodies. In focal sclerosing Gn, large spherical bodies arising from swollen podocyte cell bodies were frequently encountered. Innumerable microvilli and small spherical bodies characterized membranoproliferative Gn. Variable podocyte cell body swelling, microvilli and small spherical bodies were seen in cases of lupus nephritis. Taken alone, none of the SEM changes were diagnostic. However, when the type of the change, the severity and the combination of the changes were considered as a whole, they were indicative of what light microscopy might reveal and helpful in differentiating between idiopathic minimal change Gn and focal sclerosing Gn.

Summary Monoclonal antibodies have been raised against components of glomerular immune deposits in experimental glomerulonephritis and idiopathic human glomerulonephritis. An accelerated model of chronic serum sickness in the rat using cationized human serum albumin was employed to obtain renal tissue with capillary loop and mesangial immune deposits. Mice were immunized with isolated rat glomeruli or a preparation of glomerular basement membrane and mouse spleen cells fused with myeloma cells. Anti-human serum albumin monoclonal antibodies were produced from all technically successful fusions irrespective of the size of the deposits in the immunizng tissue or whether whole glomeruli or glomerular basement membrane were used for immunization. Monoclonal antibodies were then produced following immunization with tissue from postmortem human kidneys with idiopathic membranous and mesangiocapillary glomerulonephritis. Sixteen monoclonal antibodies, apparently reactive with glomerular immune deposits, were cloned; most of these were reactive with components of the complement system including a previously undescribed complement-related protein. These studies demonstrate that monoclonal antibody technology may be useful in determining the identity of antigen and non-antigen components of glomerular immune deposits.

Renal autografts and allografts were placed in the necks of nephrectomized sheep. Some of the sheep which received allografts had been previously injected with human glomerular basement membrane to induce an autoimmune antiglomerular basement membrane glomerulonephritis. All previously diseased and normal recipient animals were treated wtih azathioprine and prednisone from the day before transplantation. The autografts and immunosuppressed allografts in normal recipients functioned similarly for the first 6 d, but subsequently the allografts deteriorated. Allografts in untreated recipients functioned for less than 3 d. Most (90%) of the allografts in previously diseased sheep developed heavy proteinuria and haematuria immediately after transplantation and their renal function remained low. These grafts showed severe glomerular lesions by the third day, and by 8 d more than 50% of the glomeruli contained crescents. At corresponding intervals the glomeruli of autografts and allografts in normal recipients showed only minor changes. These results demonstrate that antiglomerular basement antibodies circulating in the host at the time of transplantation are a significant threat to the survival of renal allografts.

The incorporation of intravenously injected dextran molecules (molecular weight 500,000 & 2,000,000) by the glomeruli of laboratory rats was studied by electron microscopy. In normal rats some dextran molecules were observed 12 minutes after injection in vesicles and invaginations of both endothelial and mesangial cells. Rats with nephrotoxic serum nephritis showed extensive separation of endothelial cells from the glomerular basement membrane. These subendothelial spaces contained irregular granular material, cytoplasmic extensions of mesangial cells and, in animals injected with dextran, dense accumulations of dextran molecules. There was no apparent difference in distribution of the two test samples of dextran. These findings demonstrated the involvement of both endothelial and mesangial cells in the glomerular localization of circulating material and indicated that immunologically inactive macromolecules can be incorporated into subendothelial deposits following immunological injury to the glomerulus.

Renal allografts were transplanted into 10 sheep with pre-existing experimental autoimmune glomerulonephritis. Serial biopsy samples were examined by light, electron and immunofluorescence microscopy. Sections were stained with naphthyl acetate to demonstrate non-specific esterase activity. Host antiglomerular basement membrane antibody was demonstrated in graft glomeruli only 30 min after transplantation, at which time up to 40% of glomeruli contained many intracapillary neutrophils. Three d after transplantation gaps in the glomerular basement membrane were evident and the urinary spaces contained polymorphonuclear leucocytes, red blood cells, large amounts of fibrin and occasional mononuclear leucocytes. These mononuclear cells had increased greatly in number by the 6th d and most contained phagocytic vacuoles. From the 7th to the 10th d the incidence of glomerular crescents increased to involve 80% of glomeruli, and the majority of cells within crescents showed phagocytic vacuoles. The number of cells exhibiting non-specific esterase activity increased markedly during the post-operative period and most were located within the urinary spaces. These results suggest that the majority of cells in the glomerular crescents were macrophages which were probably derived from blood monocytes.

Electron microscopic studies of the kidneys of NZB x NZW F1 (B/W) mice show the presence of electron-opaque deposits on the endothelial side of the glomerular basement membrane early in the spontaneous renal disease to which these mice are subject. This change is comparable to that seen in the heterologous phase of murine nephrotoxic nephritis. Widespread formation of subepithelial humps is seen in B/W nephritis in the advanced lesions only.Ultrastructural studies of kidneys of young ‘normal’ mice reveal isolated humps of similar electron-opaque material on the epithelial side of the glomerular basement membrane. It would appear, then, that the spontaneously occurring kidney disease in B/W mice is of a more complicated nature than is suggested by simple laboratory models.

Rats of Sprague-Dawley and two Wistar strains were injected fortnightly for 100 days with isolated human glomerular basement membrane emulsified in Freund's adjuvant and then slaughtered. Renal function was monitored by weekly urine protein estimations, and kidneys taken by unilateral nephrectomy and at slaughter were examined by light and electron microscopy and by immunofluorescence. Proteinuria began after 4 wk in Sprague-Dawley rats and continued until slaughter. Glomerular lesions, ranging from focal cellular proliferation and hypertrophy to widespread adhesions and sclerosis, were seen in these rats. By contrast, rats of both Wistar strains failed to develop proteinuria and no glomerular lesions were found. Sections stained with fluorescein-tagged anti-rat IgG antibody revealed intense and consistent linear fluorescence characteristic of anti-glomerular basement membrane antibody along the glomerular capillary walls of Sprague-Dawley rats. Such staining was either weak or absent in the other rats. This observed strain difference in susceptibility to experimental autologous antiglomerular basement membrane antibody glomerulonephritis is thought to be due to genetic variation in the capacity to produce antibody against the introduced foreign glomerular basement membrane.