Detection of myeloperoxidase in membranous nephropathy-like deposits in patients with anti-neutrophil cytoplasmic antibody-associated glomerulonephritis

Anti-neutrophil cytoplasmic antibody-associated glomerulonephritis is usually classified as a pauci-immune type. However, it sometimes shows immune complex deposition of unknown origin. We examined the glomerular localization of myeloperoxidase by double immunofluorescence and immunoelectron microscopy in cases of anti-neutrophil cytoplasmic antibody-associated glomerulonephritis with membranous nephropathy-like immunoglobulin G deposition to investigate the immune complex antigens in these cases. Six (35%) of the biopsy samples from 17 cases with anti-neutrophil cytoplasmic antibody-associated glomerulonephritis showed granular deposition of immunoglobulin G along the glomerular capillary walls. Light microscopy revealed necrotizing crescentic glomerulonephritis with segmental thickening of the glomerular basement membrane. Electron microscopy showed electron-dense deposits in intramembranous and mesangial areas. However, the size and distribution of the deposits were irregular and segmental in the examined cases, unlike typical global and subepithelial lesions of membranous nephropathy. Double immunofluorescence using Alexa Fluor 594-labeled anti-myeloperoxidase antibody and fluorescein isothiocyanate-labeled anti-immunoglobulin G antibody, as well as immunoelectron microscopy using anti-myeloperoxidase antibody labeled with 25-nm gold particles revealed partial colocalization of myeloperoxidase and immunoglobulin G within the glomerular basement membrane and mesangium. In some cases of anti-neutrophil cytoplasmic antibody-associated glomerulonephritis, myeloperoxidase may form immune complexes and develop membranous nephropathy-like lesions.     

Ultrastructural changes in acute nonstreptococcal glomerulonephritis associated with mixed cryoglobulinemia

A kidney biopsy specimen obtained from a patient with mixed cryoglobulinemia and glomerulonephritis was studied by electron microscopy. The glomeruli showed focal proliferations of endothelial and mesangial cells, a focal increase of the mesangial matrix, and various electron-dense deposits, including discrete subepithelial deposits. To our knowledge, such deposits have not previously been illustrated in this syndrome. The finding of subepithelial deposits indicates that immune complexes are implicated in the pathogenesis of the glomerulonephritis. The cryoglobulins may have constituted the immune complexes. The differential diagnostic significance of subepithelial deposits is discussed.     

Experimental proliferative glomerulonephritis in the cat.

A model of chronic serum sickness was used to induce immune-complex glomerulonephritis in seven experimental cats, by daily intravenous inoculation of an increasing dose (5 to 35 mg) of human serum albumin (HSA). At week four, two of the seven animals developed anterior uveitis. At week 23, two different animals developed the subcutaneous oedema characteristic of the nephrotic syndrome (NS), whilst the other five cats appeared clinically normal. The kidneys were examined at necropsy by light microscopy and by transmission electron microscopy. The glomeruli of four animals (three with both proteinuria and uraemia, and one with proteinuria only) showed morphological changes under light microscopy. The abnormalities suggested that a diffuse mesangial proliferative glomerulonephritis (GN) had been induced in three cats and diffuse membranoproliferative GN induced in another. Ultrastructural studies revealed electron-dense deposits (immune-complexes) in six of the seven cats. Two cats without glomerular abnormalities by light microscopy had mesangial deposits and three cats with mesangial proliferative GN had deposits at mesangial, subendothelial and/or subepithelial sites. The single cat with membranoproliferative GN had deposits at mesangial, subendothelial, subepithelial and intramembranous sites. Immunohistological examination (peroxidase-antiperoxidase technique) showed that HSA and immunoglobulin (IgG and IgM) were deposited in the glomeruli of these cats. Deposits were the most dense in cats with more severe renal lesions. Deposits of IgM were most abundant. An extensive cellular infiltrate, comprising macrophages, neutrophils and plasma cells, was observed only in the four animals which showed abnormalities in glomerular ultrastructure. The disease induced in these cats thus appears to differ from the membranous nephropathy previously described in the cat and bears a close resemblance to immune complex (IC) disease in man. In view of the relatively few specific animal models of IC-mediated proliferative GN, this model has potential for application to the study of human IC disease.     

Renal Biopsy Pathology in Wegener''s Granulomatosis

Eighteen renal biopsies from 10 patients with active generalized Wegener's granulomatosis (GWG), with GWG in remission on therapy, and with active localized Wegener's granulomatosis (LWG) have been examined by light and electron microscopy. In all 9 patients with active GWG, light microscopy revealed focal and segmental glomerulonephritis. Electron microscopy revealed subepithelial basement membrane densities resembling immune complex deposits in two biopsies from patients with active GWG. In biopsies from patients on cytotoxic therapy, there was no active inflammatory process, but focal glomercular obsolescence and segmental tuft sclerosis provided evidence of prior focal and segmental glomercular disease. Discrete zones of basement membrane crimping and increased mesangial material along the capillary wall were observed in some patients with apparent LWG, as well as proven GWG, possibly representing foci of previous glomerular injury. The appearance of dense deposits on the epithelial side of the basement membrane suggests that immune complex deposition in the glomeruli may be at least partially responsible for the renal injury in Wegener's granulomatosis.     

Glomerulonephritis induced by high doses of ovalbumin

Summary Experimental glomerulonephritis was produced in 16 rabbits by intravenous injections of ovalbumin in high doses (0.1 g/day during the first week, 0.2 g×6/day during the second). The animals were killed on day 14. At that time all animals had 2–4+ proteinuria and a serum C3 level reduced to about 50% of the control level; 11 animals had a significantly raised blood urea level. In all rabbits the antigen had induced severe proliferative glomerulonephritis. Electron microscopy showed that many of the cells accounting for the hypercellularity were monocytes. Surprisingly, electron dense deposits were few and small, mainly on the subendothelial and subepithelial aspects of the glomerular basement membrane. In all the animals ultrastructural immunoperoxidase technique revealed deposits containing ovalbumin, rabbit IgG and C3. With immunofluorescence sparse deposits were occasionally seen. It is concluded that a severe experimental glomerulonephritis can be produced in a state of antigen excess, with the deposition of immune complexes being minimal. Immuno-electron microscopy is essential, however, in detecting even the smallest amounts of deposited immune reactants.     

Localization of glomerular 'deposits' in Henoch-Schönlein nephritis

Twenty-five renal biopsies from patients with Henoch-Schönlein nephritis were examined using light microscopy, immunofluorescence, I μm plastic-embedded sections and electron microscopy. The I μm plastic-embedded sections and electron microscopy showed deposits in mesangial, subendothelial and subepithelial sites. Some of the latter were very large and similar to those which have been described as 'humps' in acute proliferative glomerulonephritis. Immunofluorescence showed the mesangial deposition of IgG, IgA and C₃ with extension into a peripheral position in some cases. Fibrin was frequently found associated with crescents. The case for Henoch-Schönlein disease being mediated, in part at least, by immune complex deposition, is presented.     

The role of H-2 in apoferritin-induced murine immune complex glomerulonephritis

The role of the major histocompatibility complex in the development of apoferritin induced immune complex glomerulonephritis was studied in H-2 congenic B10 mice. The glomerular lesions varied strikingly among the three different strains studied. The B10 (H-2b) mice had minimal mesangial expansion or no lesions at all. The B10.BR (H-2k) mice had mesangial expansion and proliferative glomerulonephritis without crescents or interstitial mononuclear cell infiltration. In contrast, the B10.D2 (H-2d) mice had necrotizing glomerulonephritis with crescents and an interstitial mononuclear cell infiltrate. Immunofluorescence and electron microscopy demonstrated only minimal mesangial deposits in B10 (H-2b) mice, predominantly mesangial deposition in the B10.BR (H-2k) mice, and mesangial and subepithelial immune complex deposits in B10.D2 (H-2d) mice. These morphologic differences correlated with functional abnormalities. Only the B10.D2 (H-2d) mice developed proteinuria, hematuria, and elevated blood urea nitrogen. They also had the most elevated antiapoferritin IgG levels. These experiments demonstrate that differences in the pathologic lesions and susceptibility to immune complex glomerulonephritis can be seen in animals that differ only at the H-2 locus. This model will lend itself to the study of the mechanisms by which the major histocompatibility complex influences the development of immune complex glomerulonephritis.     

Immunopathology of glomerulonephritis associated with chronic woodchuck hepatitis virus infection in woodchucks (Marmota monax).

Retrospective analysis of necropsy findings of 705 woodchucks was performed to determine the prevalence and morphology of immune-mediated glomerulonephritis, its relationship to woodchuck hepatitis virus (WHV) infection, and the presence of major WHV antigens. Twenty-six woodchucks had glomerular lesions. Renal tissue of the 26 animals was evaluated histologically and immunohistochemically for immune-mediated glomerulonephritis. Of these 26 animals, immune-mediated glomerulonephritis was diagnosed in six, all of which were chronic WHV carriers. Membranous glomerulonephritis was identified in three animals, two of which also had mesangial proliferation. Host immunoglobulin was present within the mesangium and along capillary loops in all three. Woodchuck hepatitis virus core antigen (WHcAg) was present along capillary loops of two of these animals, one membranous and one mixed, and in the mesangium of all three. Woodchuck hepatitis virus surface antigen (WHsAg) deposition was similar to WHcAg deposition but was only present along capillaries in those animals with mixed nephritis. The remaining three animals had mesangial proliferation. WHsAg and host immunoglobulin deposition were predominately mesangial; WHcAg was not detected. Transmission electron microscopy showed thickening of the capillary loop basement membranes and subepithelial electron-dense deposits in animal one, and deposits in the mesangium in animal six.     

Effect of cyclosporin on immune complex deposition in murine glomerulonephritis.

Chronic glomerulonephritis (GN) was induced in N/M mice by daily injections of human serum albumin (HSA). The glomerular lesion was similar to that observed in human membranous GN and was characterized by intense mesangial and capillary loop immunofluorescent staining for HSA, IgG and C3. Electron microscopic examination revealed numerous electron-dense deposits in the mesangium and along the subepithelial side of the glomerular basement membrane, the latter deposits being associated with membranous spikes. Chronically injected mice that had been treated with cyclosporin (CsA) from Day 1 had different patterns of immune complex deposition. Mesangial deposition was apparently unaltered but no subepithelial deposits or spikes were evident. In addition, only two out of 21 HSA-injected mice which began CsA treatment on Day 21 had subepithelial deposits. There was no significant difference in serum levels of HSA-specific IgG between the three groups of mice. CsA treatment would therefore appear to ameliorate the immunopathology of antigen-induced glomerulonephritis in this model without affecting serum antibody levels, and may be of therapeutic value in the treatment of human membranous GN.     

Kidney involvement in Behçet's syndrome

Summary The finding of a focal and segmental glomerulonephritis (GN) in a patient with a Behçet's syndrome led us to perform systematic renal biopsies in ten other patients with the disease. Renal biopsy specimens of 11 patients with Behçet's syndrome (followed for 6 months to 15 years) have been studied by light, electron and immunofluorescence microscopy. In all cases, blood pressure and renal function were normal. Proteinuria was present in five patients. By light and electron microscopy, amyloidosis could not be demonstrated in any case. In one patient, the focal and segmental GN was associated with fibrinoid, electron dense, mesangial and irregular subepithelial deposits. These deposits were also detected in seven other patients but to a lesser degree. Arteriolosclerosis was present in all cases. By immuno fluorescence, small scattered granules of C3 were observed in 10 patients in the mesangium and along the capillary basement membrane. They were diffuse in six cases and focal in four. Small focal deposits containing IgA and/or IgG, Clq were also observed in four cases. Circulating immune complexes found in six out of seven patients in whom they were sought. Rare cases of focal and segmental GN and amyloidosis have been reported in Behçet's syndrome. To our knowledge, glomerular C3 deposits have not been yet reported. These findings with the presence of circulating immune complexes suggest that renal symptoms occasionnally observed in Behçet's syndrome could be related to immune complex deposition.     

Rat bovine serum albumin (BSA) nephritis. VI. The influence of chemically altered antigen.

Bovine serum albumin (BSA) used to incite chronic serum sickness glomerulonephritis in rats was chemically modified to study the effect of antigenic alteration. The BSA was used in its native form (n-BSA) as well as anionic (a-BSA), cationic (c-BSA) or glycosylated (g-BSA) forms. Spontaneously hypertensive rats (SHR) preimmunized 8 weeks earlier received daily intravenous injections of the respective BSA preparations for the ensuing 3 weeks. Histological examination of their kidneys revealed that c-BSA given for 2 weeks induced a severe diffuse proliferative glomerulonephritis and profound proteinuria. Electron-dense deposits localized preferentially in the subepithelial spaces of renal glomeruli from these rats, but a few in the mesangia. Quite differently, rats receiving n-BSA or g-BSA developed a less severe form of glomerulonephritis even after 3 weeks of injections. Besides the massive mesangial deposits, the subepithelial deposits were conspicuous in the glomeruli from rats given g-BSA for 2 weeks, but deposition in glomerular capillaries was rare in rats given n-BSA for the same duration. In contrast, the administration of a-BSA resulted in minimal abnormalities visible by light microscopy and a few immune deposits in the mesangia even at the third week. The antibody response in rats given c-BSA or a-BSA was apparently different from n-BSA treated rats. The present study shows the important role of the antigen's electric charge in the pathogenesis of proliferative glomerulonephritis. The foregoing results also foster our proposal that the carbohydrate content of the antigen influences the development of this renal disease.     

Lupus nephritis--light, electron and immunofluorescent microscopy (a clinicopathological study)

Kidney biopsy specimens from nine patients of systemic lupus erythematosus were studied for the purpose of correlating the findings of light and electron microscopy and immunofluorescent studies with the kidney functions and the presence of proteinuria. Morphologically, three were mesangiocapillary glomerulonephritis, three mesangioproliferative glomerulonephritis and one membranous nephropathy. Subendothelial deposits were observed in eight cases, subepithelial deposits in seven cases and mesangial deposits in eight cases. Four cases with massive subendothelial deposits had massive proteinuria and decrease in renal functions. Most of the patients with mild subendothelial deposits were considered to be useful in assessing the prognosis and severity of lupus nephropathy, in addition to the morphologic types of lesions, as mesangial pattern had shown insignificant proteinuria. Study of semithin sections by light microscopy could demonstrate the deposits which were observed on electron microscopy and immuno-fluorescent microscopy.     

Stimulation of mesangial phagocytes does not influence the removal of established glomerular immune complex deposits

To test the hypothesis that mesangial phagocytes are involved in the removal of established glomerular immune complex deposits, either puromycin aminonucleoside (PAN) or polyvinyl alcohol (PVA) was given to rats as they recovered from chronic serum sickness glomerulonephritis. Both these substances increase the number and activity of mesangial macrophages. The nephritis was induced with radiolabelled cationized bovine serum albumin (BSA). After 10 days of such treatment, the animals were killed and the amount of isotope in whole renal cortex and in isolated glomeruli was measured. The volume fraction of subepithelial and mesangial electron-dense deposits was assessed by morphometric methods. Neither PAN nor PVA caused any significant alteration in any of these parameters. These results suggest that the methods by which well established glomerular immune complex deposits are removed do not involve mesangial macrophages to any major extent, and therefore differ from the systems which handle particulate matter and immune complexes as they arrive at the glomerular filter. The morphologic appearances of the deposits at the end of the experiment suggest extracellular dissolution in situ.     

Demonstration of Antigenic Sites in Glomeruli of Patients with Acute Poststreptococcal Glomerulonephritis by Immunofluorescein and Immunoferritin Technics

The presence and localization of antigenic sites in glomeruli of 14 patients with acute poststreptococcal glomerulonephritis (AGN) were studied by immunofluorescein and immunoferritin technics. Labeled IgG fractions from the same patients were used for the identification of antigenic sites. The staining capacity of these IgG fractions depended on the time when sera were obtained. Staining was minimal during the first week, and increased up to the fourth or fifth week. Glomeruli, however, stained only when renal tissue was obtained during the early phase of the disease. Precise localization of antigenic sites was determined with ferritin-conjugated patients' IgG. Segmental deposition of ferritin was observed in the mesangial matrix and on the endothelial side of the glomerular basement membrane. Subepithelial electron-dense deposits contained no or very few ferritin particles. In contrast, ferritin-conjugated antihuman IgG was distributed diffusely in the mesangial matrix, on the endothelial side of the basement membrane and in subepithelial deposits. These findings suggest that, during the early stage of acute poststreptococcal glomerulonephritis, free antigen is present in the glomeruli of patients with this disease.     

Morphological changes in the kidneys during nonspecific ulcerative colitis]

Light microscopy, immunohistochemical examinations and electron microscopy of kidney biopsies showed the different localization of immune complexes in the glomeruli (subepithelial, intramembraneous, subendothelial, and mesangial immune complexes) and various forms of materialization of their harmful effect (induction of immune inflammation or immunosuppression) to be determined by the involvement in phagocytosis and reparation reactions of either podocytes or mesangial and endothelial cells and also hematogenous elements. Therefore, podocyte, mesangioendothelial and podocyte-mesangioendothelial ways of morphogenesis of immunocoplex glomerulonephritis may be distinguished. Each of them is associated with the development of certain clinico-morphological forms of immunocomplex glomerulonephritis which should be considered as distinct entities.     

Glomerular and vascular IgG deposits in HgCl2 nephritis: role of circulating antibodies and of immune complexes

The respective roles of circulating anti-glomerular basement membrane antibodies and of circulating immune complexes in the appearance of glomerular linear and granular IgG deposition during HgCl2-induced glomerulonephritis in the Brown-Norway rat has been studied. Syngeneic kidney transplantations have been performed at various phases of the disease. Results show that circulating antibodies are responsible for linear IgG deposition which did not change to granular deposits during the course of the disease. Electron-dense subepithelial deposits occurred only when circulating immune complexes were detected. These experiments strongly suggest that, in the mercury model, circulating immune complexes are responsible for granular IgG deposits observed in arteries and in the subepithelial space of glomeruli.     

Simultaneous anti-glomerular basement membrane and membranous glomerulonephritis: case report and literature review

A 20-year-old male experienced a sore throat, fever, and lumbar pain. Examination revealed haematuria, proteinuria, and transiently impaired renal function. Renal biopsy revealed minor mesangial widening and small cellular crescents in 20% of the glomeruli under the light microscope, whereas immunofluorescence showed bright, linear staining of IgG along the glomerular basement membrane (GBM). Ultrastructural analysis showed minute subepithelial deposits analogous to early membranous glomerulonephritis (MGN). Anti-GBM antibodies were detected in the patient's serum. These findings were suggestive of simultaneous anti-GBM and immune complex glomerulonephritis in a patient with a mild, reversible renal illness.     

Glomerulonephritis with organized deposits: a mesangiopathic, not immune complex-mediated disease? A pathologic study of two cases in the same family.

Similar glomerular changes (marked widening of the mesangial stalk, irregular basement membrane thickening, and presence of mesangial and subendothelial deposits) were observed by light microscopy in renal biopsy specimens from two patients (mother and daughter) affected by nephrotic syndrome. Electron microscopy disclosed huge glomerular electron-dense deposits containing 12-nm fibrils in both patients. Immunohistochemical investigations performed with antisera anti-immunoglobulin (Ig) and anti-complement fractions, anti-laminin, anti-collagen IV, and anti-fibronectin (FN) showed scant and focal Ig and complement deposits and strong deposits of FN in the mesangium and along glomerular basement membranes. Most glomerular FN was plasma-derived, as shown by immunohistochemical tests with monoclonal antibodies specific for both plasma and cell-derived FN (IST-4) and for cell-derived FN (IST-9). Electron-dense deposits with fibrillar component could hardly correspond to the Ig and complement deposits, whereas they could be related to FN deposits. Since it is known that in glomeruli FN binds to Ig and immune complexes, and the latter seem to be too scant to justify light and electron microscopic lesions and clinical findings, the hypothesis of a primary mesangiopathic glomerulonephritis in some way connected with abnormal plasma FN deposition within the glomeruli and subsequent non-specific immune reactant entrapment could be considered. We could be dealing with a peculiar form of fibrillary glomerulonephritis with rather indolent evolution, as shown by a slow decrease of glomerular function and the scarcely modified glomerular changes found in the second biopsy performed in the mother 8 years after the first investigation.     

Chronic antigen-antibody-complex glomerulonephritis in mice

Daily injection of human serum albumin into mice genetically selected to produce low-affinity antibody to protein antigens resulted in a more severe antigen-antibody-complex-induced glomerulonephritis than in mice producing high-affinity antibody. The low-affinity mice had higher levels of circulating antigen-antibody complexes, greater impairment of renal function and reduced reticuloendothelial clearance of 125I-PVP compared to high-affinity mice. Electron microscopy of glomeruli revealed the presence of subepithelial electron-dense deposits in low-affinity mice and predominantly subendothelial-mesangial deposits in high-affinity mice which corresponded to the predominantly capillary staining in low-affinity mice and mesangial staining in high-affinity mice by immunofluorescence. Auto-radiography of electron-microscopy sections demonstrated the presence of antigen in the electron-dense deposits, indicating that these deposits indeed contained antigen-antigen complexes.     

Interstrain variations in nephritogenicity of heterologous protein in mice

Swiss albino, BALB/c, and eight substrains of C3H mice were given daily intraperitoneal injections of horse apoferritin (HAF) for up to 56 days. At varying intervals, renal tissue was examined by light, immunofluorescence, and electron microscopy. Swiss mice developed proliferative glomerulonephritis after 7 to 14 days of HAF, and 45 per cent progressed to severe crescentic glomerulonephritis after from 21 to 56 days of HAF. In Swiss mice, glomerular immune deposits evolved from predominantly IgM mesangial deposits at 7 days to mesangial IgG at 14 days to capillary wall IgG after 21 or more days of HAF injections. BALB/c mice given identical HAF doses never developed severe crescentic glomerulonephritis but rather an extensive global necrotizing glomerulonephritis most prevalent after from 9 to 18 days of HAF. The distinct evolution of glomerular immune deposits observed in Swiss mice was less clear-cut in BALB/c mice, with greater persistence of mesangial deposits and IgM over time. Only 11 per cent of C3H mice (confined to two substrains) developed glomerular lesions by light microscopy after 2 to 3 weeks of HAF administration. No C3H/HeN mice developed glomerulonephritis even after up to 47 days of HAF injection. From 7 days on, 45 per cent of HAF-injected C3H mice had low level IgM mesangial immune deposits but did not manifest the evolution from mesangial to capillary deposition observed in BALB/c and Swiss albino mice. F1 hybrid and congenic mice carrying BALB/c H-2 genetic information developed glomerular lesions similar to those produced in BALB/c mice. These data (1) indicate an interrelated morphologic and immunohistologic evolution of heterologous protein induced glomerular lesions in mice, (2) demonstrate morphologic and immunohistologic differences in glomerular lesions development between genetically disparate mouse strains given identical antigen exposures, and (3) support the genetic control of heterologous protein-induced glomerulonephritis and suggest a role for the major histocompatibility region in this genetic regulation.