Subepithelial Electron-dense Deposits Research Articles

Active Heymann nephritis in complement component C6 deficient rats

The mechanisms of renal injury that result in proteinuria in active Heymann nephritis (AHN) remain unclear, though data suggest that in analogy of the passive form of the disease the membrane attack complex C5b-9 may be involved. AHN was induced in an inbred strain of PVG/c-rats that are totally deficient in the C6 component of complement and are unable to form the lytic C5b-9 complex, as well as in non-complement deficient PVG/c+ rats that are immunologic identical to the deficient strain. In both groups of animals comparably high titers of anti-Fx1A autoantibodies were found after three weeks and persisted at 40 weeks. Proteinuria was also similar in both groups, and was first evident at six weeks. High levels of urinary protein, ranging from 200 mg/24 hr to 500 mg/24 hr, were found after 10 weeks and persisted up to one year. Renal biopsy findings at various times post-immunization were identical in both groups, including immunofluorescence staining for Ig and C3 deposits, and also EM findings of subepithelial electron-dense deposits were not different. The injection of heterologous rabbit complement, that partially and temporarily restored the CH50 activity in PVG/c- rats did not alter or hasten the disease. Long-term follow-up showed that all rats in both groups continued to have severe proteinuria and that most animals died between 8 to 12 months after disease induction, without renal impairment. EM findings in serial biopsies demonstrated that the growth of the subepithelial deposits as measured by surface area occurred between weeks 4 and 12. A positive correlation (r = 0.94) between the size of the deposits and the level of proteinuria was found. These studies demonstrate that the membrane attack complex of complement does not play a major role in AHN. The relationship of the size of the immune deposits to the level of proteinuria suggests that the growth of the immune deposits on itself initiate secondary mechanisms that damage the permselective characteristics of the glomerular membrane.

Electron microscopic autoradiographic study on immune complex formation in rat glomerulus with anti-heparan sulfate proteoglycan antibody

Using electron microscopic autoradiography, we studied the mechanism of the formation of immune complex in the subepithelial aspect of glomerular basement membrane in rats, as a model for glomerulonephritis in man. Rats were immunized with normal rabbit IgG, and were administered intravenously with radioiodinated rabbit anti-heparan sulfate proteoglycan (HS-PG) antibody. Two weeks later the center of the grains appeared to correspond with the center of the subepithelial electron dense deposits, thus suggesting a topological superimposition of the radioiodinated anti-HS-PG antibody over the immune complex deposits located in the subepithelial space. From our present study it has become clear that the HS-PG site may serve as the corenidus for the formation of immune complex deposit. Electron microscopic autoradiography is very helpful in the demonstrating ofin situ immune complex formation in glomerulonephritis.

Factors contributing to the outcome in 100 adult patients with idiopathic membranous glomerulonephritis.

Follow-up data on 100 patients with idiopathic membranous glomerulonephritis (IMGN), in whom renal biopsy has been performed, are discussed. The period of follow-up ranged from 3 to 24 years (median 7.8 years). Sixteen of the patients had end-stage renal disease. Complete remission of renal disease occurred in 41 patients. The remaining 43 patients exhibited persisting renal functional abnormalities at the end of the follow-up period. Using Cox's proportional hazard model, six parameters were found as predictive factors for prognosis at the time of biopsy: serum cholesterol level > 260 mg/dl, serum creatinine concentration > 1.4 mg/dl, the presence of tubulointerstitial (TI), vascular, mesangial, and Bowman's capsular lesions. No effect was observed in the size of subepithelial electron dense deposits (SED) on prognosis. A weakly positive correlation was found between the thickness of the glomerular basement membrane (GBM) and poor outcome. Treatment with steroid and/or immunosuppressive agents was not found to improve survival. Assessment of these factors thus allows the pathologist to make a relevant statement concerning the probable course and prognosis of the disease at the time of the diagnostic biopsy.

Ultrastructural Localization of the Three Major Basement Membrane Components-Type IV Collagen, Heparan Sulf ate Proteoglycan and Laminin – in Human Membranous Glomerulonephritis

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.

<bold>Pathogenesis of trypanosomiasis-induced glomerulonephritis in mice</bold>

We investigated the pathogenesis of glomerulonephritis in mice that had been infected with Trypanosoma brucei. Polyclonal B cell activation was evaluated, with special attention to the presence of autoantibodies, which are known to be involved in other models for glomerulonephritis. The BALB/c mice studied developed severe albuminuria. Light-microscopy of the kidneys showed increase of mesangial matrix and thickening of the glomerular capillary walls. Immunofluorescence studies showed immunoglobulins in a mixed linear-granular pattern along the glomerular capillary wall and in the mesangium. Trypanosomal antigens were not found in these aggregates. Electron-microscopy revealed mesangial, subendothelial, and subepithelial electron-dense deposits. During the course of this infection, significant levels of antibodies directed against known nephritogenic renal autoantigens, i.e. GBM, laminin, RTE, and gp330, were measured in serum and glomerular eluates. These findings led us to conclude that mice infected with T. brucei and treated with diminazene aceturate develop an autoimmune-mediated glomerulonephritis, characterized by mesangial, subendothelial, and subepithelial immune aggregates. This murine model seems to offer a useful tool for the study of immunological aspects of polyclonal B cell activation in infection-related glomerular disease.

Prognostic Indicators in Childhood IgA Nephropathy

A number of clinical, laboratory and pathologic parameters were assessed for their prognostic significance in 200 children aged less than 15 years with IgA nephropathy, who had shown normal renal function at the time of initial biopsy and were followed for more than 2 years thereafter. After a mean follow-up period of 5.0 years from the initial biopsy, 93 patients had no demonstrable abnormality, 76 had minor urinary abnormalities, 21 had persistent heavy proteinuria and 10 had developed chronic renal impairment. A poor outcome was found to be correlated with heavy proteinuria at biopsy, diffuse mesangial proliferation, a high proportion of glomeruli showing sclerosis, crescents or capsular adhesions, the presence of moderate or severe tubulointerstitial changes, and the presence of subepithelial electron-dense deposits and lysis of the glomerular basement membrane by electron microscopy. The percentage of glomeruli displaying crescents, sclerosis and adhesions appeared to be the most reliable prognostic indicator. Nine of the 27 patients (33%) in whom greater than or equal to 30% of glomeruli showed crescents, sclerosis and adhesions developed chronic renal impairment, and only 14% of these patients had normal urine at follow-up. In contrast, only 1 of the 173 patients in whom less than 30% of glomeruli showed such lesions developed chronic renal impairment (p less than 0.001) and 51% of these patients showed complete remission at follow-up (p less than 0.001). These results demonstrate that an accurate prediction of the outcome based on the initial renal biopsy findings is possible early in the course of children with IgA nephropathy.

Predictors of One Year Outcome in Lupus Nephritis: The Importance of Renal Biopsy

The short-term prognosis of lupus nephritis was evaluated by assessing serum creatinine 12 months after renal biopsy in 87 patients with lupus nephritis. On univariate analysis, significant clinical and laboratory predictors of this outcome included clinical signs of renal injury (serum creatinine, 24-hour urinary protein, prolonged renal disease, nephrotic syndrome, serum albumin), as well as thrombocytopenia, older age, and coexisting illness or hypertension at the time of biopsy. On renal biopsy, diffuse proliferative nephritis, higher activity, chronicity, or tubulointerstitial scores, or subendothelial or subepithelial electron dense deposits predicted a higher serum creatinine 12 months after biopsy. A clinical predictive model was developed which included as independent predictors serum creatinine, age, platelet count and 24-hour urinary protein. Any one of three biopsy variables added information to the clinical prediction model: a marked quantity of subendothelial deposits (p = 0.02), a higher activity index score (p = 0.02), or the presence of diffuse proliferative lupus nephritis (p = 0.05). However, the relative predictive accuracy of the clinical model did not improve with the addition of any of the biopsy variables. The value of renal biopsy in lupus nephritis is discussed based on the ability of biopsy information to confirm the prognosis, to add new predictive information for a group of subjects, and to improve predictive accuracy for individual patients.

Effect of leukocyte stimulation on rabbit immune complex glomerulonephritis

Phytohemagglutinin (PHA), a leukocyte mitogen, induces a lymphocyte and blast cell glomerulonephritis in rat renal allografts (Cell Immunol 13:146, 1974). The aim of this study was to assess whether PHA similarly enhances rabbit monocyte-dependent experimental, acute immune complex glomerulonephritis, and whether this effect is associated with local release of interleukin-1 (IL-1) and tumor necrosis factor (TNF). Rabbits with experimental acute serum sickness (AcSS: Group I) had focal proliferative and exudative glomerulonephritis with immune deposits, scattered subepithelial electron-dense deposits (humps), mild and transient proteinuria, normal creatinine clearance and slightly increased production of IL-1 and TNF from isolated glomeruli. Rabbits with AcSS and injected with PHA (Group II) developed severe lymphocyte and blast cell glomerulonephritis with diffuse endothelial damage; immune deposits were significantly reduced, focal subepithelial electron-dense deposits were absent, proteinuria was increased, creatinine clearance was decreased and production of IL-1 and TNF was markedly augmented as compared to rabbits in Group I. Rabbits with AcSS and injected with IL-1 beta and TNF alpha (Group V) had lesions comparable to those seen in Group II. These results show that PHA, IL-1 and TNF enhance the severity of acute immune complex glomerulonephritis, presumably by activating glomerular endothelial and mesangial cells and resident or infiltrated leukocytes.

Experimental immune complex glomerulonephritis and the nephrotic syndrome in cats immunised with cationised bovine serum albumin

Membranous nephropathy was induced in four cats by repeated intravenous injections of 120 mg cationic bovine serum albumin ( bsa, pI 9·5). All four cats developed diffuse granular deposits of IgG and C3 along the glomerular capillary walls as early as five weeks which persisted until the end of the experiment at 17 weeks. Ultrastructural studies revealed many subepithelial electron dense deposits. Two cats developed severe proteinuria and the nephrotic syndrome characterised by hypoalbuminaemia and oedema. An additional four cats received repeated injections of unmodified native BSA (pI 4·5) and remained basically normal. This is the first report of membranous nephropathy and the nephrotic syndrome in an experimental animal model which, unlike other animal models, is subject to the spontaneously occurring disease.

Heymann nephritis: Mechanisms of renal injury

The Heymann nephritis (HN) models of rat membranous nephropathy are extraordinarily valuable tools to investigate the immunopathology of glomerular subepithelial immune deposit formation and the mechanism by which such deposits injure glomeruli. The immunopathology of HN is reviewed by Brentjens and Andres in this volume [1]. In this review we will largely confine ourselves to a discussion of the mediators, mechanisms and pathophysiological consequences of renal injury in rat membranous nephropathy. The primary manifestation of glomerular injury in HN is proteinuria; however there are also subtle, but not trivial, changes in glomerular hemodynamics and tubular function, and alterations in renal hormone production. The alterations in glomerular permselectivity that lead to proteinuria closely resemble the defect in human membranous nephropathy, and the rat model has been used to study mechanisms of renal sodium retention, edema formation and metabolic abnormalities in nephrotic syndrome. Investigators have also used the HN models to study the impact of systemic hypertension on pre-existing glomerular disease, in terms of both altered glomerular function and progressive renal injury. Mostly, the mechanisms and effects of glomerular injury in rat membranous nephropathy have been investigated in the standard active and passive (PHN) models of HN; however, both models have also been extensively modified to elucidate specific mechanisms or mediators. Such modifications, which include the use of the isolated perfused kidney, renal transplantation, partial renal ablation and, more recently, cell and tissue culture, will be discussed in context later in this review. Active HN is induced by immunizing susceptible strains of rats (such as, Lewis, Fisher) with certain fractions of homologous or proximal tubular brush border and is characterized by granular glomerular capillary wall deposits of rat IgG and subepithelial electron-dense deposits after three to four weeks. Proteinuria develops in 30 to 80% of rats within eight to ten weeks of immunization. PHN is induced by a single intravenous injection of anti-brush border antiserum (anti-Fx1A) that produces IgG deposits which accumulate in glomeruli over hours and days. When an appropriate complement-fixing antiserum is used, proteinuria occurs in almost all animals within five days [2, 3]. This heterologous is then followed by an autologous phase during which rat IgG antibodies with specificity for the glomerular-bound IgG are deposited and induce a further increase in proteinuria. HN more closely resembles human membranous nephropathy in its slow evolution than does PHN and it is truly an autoimmune disease. In those HN rats that become proteinuric, urine protein excretion and glomerular dysfunction tend to be more severe than in PHN; however, the variability between animals and the duration of onset make it a less suitable model than PHN for studying the mediators of injury.

Immunoelectron microscopic localization of membrane attack complex and hepatitis B e antigen in membranous nephropathy

Immunoelectron microscopy was used to localize membrane attack complex (MAC) and hepatitis B e (HBe) antigen in renal tissue specimens from a total of 9 patients with membranous nephropathy (MN); 6 with MN associated with a hepatitis B virus (HBV) infection, 2 with idiopathic MN, and 1 with lupus nephritis. All the patients were proteinuric, and 2 patients were classified as stage I-II, 6 as stage II, and 1 as stage IV. MAC, along with IgG and C3, was distributed within the subepithelial electron dense deposits in all the stages. MAC was also stained in the striated membranous structures within the glomerular basement membrane and mesangial matrix of some patients. In HBV-associated MN, HBe antigen was localized in the subepithelial electron dense deposits of 5 patients, while it was absent from the subepithelial deposits in a patient that was sero-positive for hepatitis B s antigen but negative for HBe antigen. This patient also lacked MAC deposition in these loci. These results suggest that MAC is associated with the formation of subepithelial deposits and proteinuria in MN. In HBV-associated MN, HBe antigen-antibody immune complex makes up the subepithelial deposits and is likely to activate the terminal components of complement in situ.

Immunoelectron microscopic localization of fibrin-related antigen in human glomerular diseases.

The distribution of fibrin-related antigen (FRA) in glomeruli was examined by immunoelectron microscopy in 9 patients with idiopathic membranous nephropathy (MN), 8 patients with minimal-change nephrotic syndrome, and 10 patients with IgA nephropathy (IgA-N), using antisera against human gamma--chain, alpha-chain, mu-chain, and fibrinogen. Electron-dense reaction products of FRA were observed in the endothelium, subendothelium, and/or in electron-dense deposits (EDD). Among the three glomerular diseases, the amount of electron-dense reaction products of FRA in the endothelium was highest in MN. This suggests that coagulation occurs on the endothelium in MN. Although the mesangial EDD of IgA-N were intensely stained with reaction products of FRA, the staining was weak in the subepithelial EDD of MN. This suggests that FRA hardly penetrates into the subepithelial EDD in MN.

Immune complex-induced defects in glomerular basement membrane charge can be "repaired" despite continuing glomerular disease.

There have been conflicting reports over whether or not glomerular immune complex deposits cause proteinuria by neutralizing the glomerular basement membrane anionic charge. We investigated whether the reported discrepancies could arise as a result of repair of the charge layer, in the face of continuing antigen deposition, and how rapidly this repair could be effected. We used a chronic serum sickness model of glomerulonephritis which consistently produces a nephrotic syndrome stable over 8 weeks. Charge distribution was demonstrated by the polyethyleneimine (PEI) method. Subepithelial electron dense deposits were associated with defects in the charge layer of the lamina rara externa at 2 and 4 weeks, but at 8 weeks many of the deposits had become surrounded by a new charge layer. This finding is of relevance to the interpretation of similar studies on human material.

Altered glomerular localization of heparan sulfate proteoglycan in experimental nephritides.

The distribution of heparan sulfate proteoglycan (HS-PG) was examined electron microscopically by the high iron diamine (HID) method in puromycin aminonucleoside (PAN) nephrosis, accelerated Masugi nephritis (NTN), and serum sickness nephritis induced by bovine serum albumin (BSA nephritis) in the rat. In PAN nephrosis rats, no change was observed in the distribution of HS-PG in the lamina rara externa (LRE) of the glomerular basement membrane (GBM) throughout the experiment. In NTN rats, however, the loss of HS-PG was observed, and it was associated with subepithelial electron dense deposits formed possibly by serum sickness mechanism, but not with inflammatory cell infiltration. In BAS nephritis, immune deposits were seen in mesangial, subendothelial, intramembranous and subepithelial areas. The deposits in the former three areas seemed to have little reciprocity with the loss of HS-PG and proteinuria. Urinary protein increased in accordance with the development of subepithelial deposits and the loss of HS-PG in the area of the deposits in the LRE. These results indicate that HS-PG could be preserved even in marked proteinuric states in morphologically intact basement membrane, but altered and lost distribution of HS-PG associated with subepithelial immune deposits could in turn result in the development of proteinuria.

Autologous Immune Complex Nephritis in Streptozotocin-Induced Diabetic Rats

In order to investigate the influence of diabetes mellitus on immune complex-mediated nephritis , we produced Heymann nephritis in streptozotocin-induced diabetic rats (DM-HN group) in which the clinical course for 24 weeks and histological changes were examined. Nondiabetic rats with Heymann nephritis (HN group) and diabetic rats (DM group) were also examined as controls. The degree of proteinuria, hypoproteinemia, hyperlipidemia and anemia were more pronounced and the mortality rate was higher in the DM-HN group than in the HN group or in the DM group. Histologically, larger and more subepithelial or intramembranous electron-dense deposits as well as a more markedly thickened glomerular basement membrane (GBM) were observed in the DM-HN group than in the HN group. In conclusion, the nephrotic manifestations and histological changes in the GBM in Heymann nephritis were augmented by the association with diabetes mellitus.

Membranous and vascular choroidopathy: Two patterns of immune deposits in systemic lupus erythematosus

Two patterns of immune aggregate localization were demonstrated by immunofluorescence and electron microscopy in the choroid plexus of four young women with fatal systemic lupus erythematosus. The two patients with granular immune aggregates localized to the basement membrane of the choroid epithelium (membranous choroidopathy) had subepithelial and intramembranous electron-dense deposits and membranous glomerulopathy in their kidneys. The two patients with immune aggregates in the walls of choroidal blood vessels (vascular choroidopathy) had subendothelial electron-dense deposits and proliferative glomerulonephritis. Vascular deposits in the choroid plexus were associated with capillary thrombi and extravasation of fibrinoid material, while isolated membranous choroidopathy had no histopathologic evidence of inflammation. The clinical presentation and serological studies of blood and cerebrospinal fluid were compared in an effort to discriminate between patients with membranous and vascular choroidopathy. All patients had variable neuropsychiatric symptoms and major motor seizures. While those with vascular choroidopathy had more evidence of disease activity in their sera, both groups demonstrated elevated titers of immune-complexed antinuclear antibodies in cerebrospinal fluid. Although both patterns of choroidal localization of immune aggregates were associated with neuropsychiatric dysfunction, we were unable to identify discrete clinical-symptom complexes which differentiated patients with membranous and vascular choroidopathy. These contrasting patterns of choroid plexus immunopathology suggest that factors responsible for differential localization of immune aggregates are not restricted to the renal glomerulus.

Role of the terminal complement pathway in experimental membranous nephropathy in the rabbit.

Our recent observations of a complement-mediated, cell-independent mechanism of altered glomerular permeability in rat membranous nephropathy suggested a possible role for the terminal complement pathway in the mediation of proteinuria in certain forms of glomerular disease. To directly determine whether the membranolytic terminal complement components (C5b-C9) are involved in glomerular injury, we studied the development of proteinuria in normal and C6-deficient (C6D) rabbits, in both of which a membranous nephropathy-like lesion develops early in the course of immunization with cationized bovine serum albumin (cBSA) (pI 8.9-9.2). C6 hemolytic activity of C6D was 0.01% that of control rabbits. After 1 wk of daily intravenous injections of cBSA, proteinuria developed in 71% of controls (median 154, range 1-3,010 mg/24 h, n = 24), whereas none of C6D were proteinuric (median 6, range 2-12 mg/24 h, n = 12, P less than 0.01). After 1 wk of cBSA, both groups had qualitatively identical glomerular deposits of BSA, rabbit IgG, and C3 on immunofluorescence microscopy, predominantly subepithelial electron-dense deposits on electron microscopy, and minimal glomerular inflammatory cell infiltration of glomeruli. Glomeruli were isolated from individual animals after 1 wk of cBSA and deposits of rabbit IgG antibody were quantitated by a standardized in vitro assay using anti-rabbit IgG-125I. Rabbit IgG deposits were found to be similar in control (29.8 +/- 13.2, range 12.7-48.6 micrograms anti-IgG/2,000 glomeruli, n = 6) and C6D rabbits (32.6 +/- 13.8, range 16.8-48.8 micrograms anti-IgG/2,000 glomeruli, n = 5, P greater than 0.05). After 2 wk, coincident with a prominent influx of mononuclear cells and neutrophils, proteinuria developed in C6D rabbits. These results document, for the first time, a requirement for a terminal complement component in the development of immunologic glomerular injury. Since the only known action of C6 is in the assembly of the membrane attack complex, these observations suggest that the membranolytic properties of complement may contribute to glomerular damage.

Precipitating antigen-antibody systems are required for the formation of subepithelial electron-dense immune deposits in rat glomeruli.

This study was conducted to determine whether multivalent, precipitating antigens are required for formation of subepithelial electron-dense immune deposits in glomeruli. 2-nitro-4-azidophenyl (NAP) was conjugated with variable density to human serum albumin (HSA) to yield nonprecipitating (NAP3.1 X HSA and NAP11.4 X HSA) and precipitating (NAP19.7 X HSA) antigens with antibodies to the hapten. These antigen preparations were cationized with ethylene diamine to enhance deposition in renal glomeruli due to interaction with the fixed negative charges in the glomerular capillary wall. Following injection into the left renal artery of rats these antigens alone persisted in the glomeruli for a relatively short time by immunofluorescence microscopy. When antibodies to NAP were injected intravenously after the antigen injection, the nonprecipitating antigens and antibodies were detectable in the glomeruli by immunofluorescence microscopy up to 8 h, comparable to antigen alone. Electron-dense deposits were not formed in these glomeruli. In contrast, when the precipitating antigen was injected and followed by antibodies to the hapten, antigen and antibody were detected by immunofluorescence microscopy through 96 h. In these specimens electron-dense deposits were present from 40 min through 96 h and after 24 h the deposits were present only in the subepithelial area. The same results were obtained when the nonprecipitating hapten-carrier conjugates were followed with antibodies to the carrier molecule. These data indicate that the persistence of immune deposits by immunofluorescence microscopy and the formation of electron-dense deposits in the subepithelial area require a precipitating antigen-antibody system.

Membranous Glomerulonephritis Associated with the Guillain-Barré Syndrome

A 63-year-old white man had simultaneous onset of the Guillain-Barre syndrome and the nephrotic syndrome, three weeks after having an upper respiratory tract infection. Many granular casts were noticed in the urine sediment. Renal biopsy specimens showed early membranous glomerulonephritis and acute interstitial nephritis by light microscopy, with fine granular deposition of IgG, IgM, and C3 along the basement membrane by immunofluorescence. Electron microscopy revealed subepithelial electron-dense deposits (Stage 1 membranous glomerulonephritis). The association of these two syndromes is rare, although evidence of immunologic dysfunction has been described in both. The recent literature is reviewed and possible pathogenetic mechanisms are discussed.

Concurrent Antiglomerular Basement Membrane Antibody and Immune Complex Mediated Glomerulonephritis

A teenage male, with Goodpasture's syndrome and serum antiglomerular basement membrane (anti-GBM) antibodies, had a focal proliferative glomerulonephritis with crescents. Immunofluorescence microscopy of his glomeruli using anti-IgG antibodies demonstrated both intense linear GBM staining, and granular subepithelial staining. Electron microscopy revealed numerous subepithelial electron-dense deposits. Identical IgG subclass restriction (dominance of IgG1 and IgG4) of both types of glomerular deposits in this patient supports, but does not prove, a postulate that the linear staining was due to anti-GBM antibodies bound to intact GBM, and that the granular staining was due to anti-GBM antibodies complexed with freed GBM antigens.