Ultrastructural localization of anionic sites in the rat skin demonstrated with cationic probes.
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Biomedical subjects
T Oite
Publications and source records attributed to T Oite.
Studies on the 'linear pattern' in renal glomeruli demonstrated with immunofluorescence.
We have experimentally induced the 'linear pattern' in immunofluorescence: the linear deposition of endogenous immunoglobulin (Ig) along the glomerular basement membrane (GBM) in rats injected with both protamine and nephrotoxic serum. This Ig was demonstrated to have no specific antibody activity against GBM or rabbit serum. Our findings could be of value in the analysis of the mechanism and pathological meaning of the 'linear pattern' of endogenous Ig in immunofluorescence.
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Long-term observation of passive Heymann nephritis.
Chronic renal lesion with membranous changes and massive proteinuria similar to human membranous nephropathy was induced in rats through repeated intravenous injections of gamma-globulin fraction of rabbit anti-rat F X 1A antisera (GRAFA) 0.37-1.5 mg/100 g body weight. The injections were administered every 2 or 3 days over a 68-day period for a total of 26 times. It is suggested that a sufficient amount of GRAFA was given to the rats to induce renal lesion, judging from the result that no remarkable differences were observed between the severity of the histopathological and clinical changes in each rat given the various doses of GRAFA. Furthermore, this renal lesion is considered to correspond to human membranous nephropathy morphologically and clinically, judging from the fact that the protein excretion was kept at a plateau, and the capillary lumen was kept almost intact with advanced membranous changes.
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Genetic factors influence level of proteinuria in cationic antigen-induced immune complex glomerulonephritis in the rat.
The influence of genetic factors on the susceptibility of the rat to cationic antigen-induced in situ immune complex glomerulonephritis was investigated. The levels of proteinuria developing in 11 inbred strains of rats differing in MHC and in genetic background varied markedly. Susceptibility was not MHC associated but resided in the genetic background.
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A nephropathy induced by immunization of rats with EHS tumour.
Immunization of rats with Engelbreth-Holm-Swarm tumour induced a nephropathy immunohistopathologically similar to Heymann nephritis except that IgG was deposited along the tubular basement membranes. Autoantibodies against both brush border and basement membranes of proximal tubules were found in the sera. Immunoperoxidase staining of the tumour cells and absorption studies suggested that the tumour cells produced the FX1A-related antigens. Immunodiffusion analysis suggested that the antigens which induced this nephropathy differed from gp 330 and gp 600 which have been reported to be responsible for Heymann nephritis.
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Preservation of fixed anionic sites in the GBM in the acute proteinuric phase of cationic antigen mediated in-situ immune complex glomerulonephritis in the rat.
Cationic antigens have been observed to bind with the negatively charged glomerular basement membrane (GBM). Using the cationic reagent polyethyleneimine (PEI), the distribution of glomerular anionic sites was evaluated ultrastructurally in the early stage (2 h-day 7) of cationic antigen mediated in-situ immune complex formation type glomerulonephritis (GN) in the rat. Renal perfusion via the renal artery with 100 micrograms of cationized human IgG(pI greater than 9.5), followed by the i.v. injection of specific antibodies, led to an initial increase in urinary albumin excretion, subsequent massive globulinuria and the formation of numerous subepithelial deposits on day 7. The most striking alteration in glomerular anionic sites was observed on the epithelial cell surface coat; the PEI deposition on the epithelial cell surface was almost identical to that in control glomeruli at 2 and 4 h after the induction of GN; thereafter, on day 7, a broad loss of anionic sites was observed on flattened epithelial foot processes. In contrast, fixed anionic sites of the laminae rarae of the GBM showed no apparent alterations in the distribution and number from 2 h to day 7 and did not disappear even in the lamina rara externa adjacent to subepithelial deposits. These findings not only show that fixed anionic sites of the GBM, in contrast to the rapid decrease in those of the epithelial cell surface, are not completely neutralized or destroyed even in GN, in which cationic antigen participates in the in-situ formation of GBM-deposits.(ABSTRACT TRUNCATED AT 250 WORDS)
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Restoration of antigenicity of tissue antigens, cell-bound immunoglobulins and immune deposits in paraffin-embedded tissue. The influence of fixation and proteolytic enzymatic digestion.
The possibility of preservation and restoration of antigenicity of some antigens in paraffin-embedded tissue was evaluated by direct immunofluorescent technique on deparaffinized sections. Fixation with 96% ethanol-1% acetic acid, 10% neutral buffered formalin and p-formaldehyde was useful for the preservation of tissue antigens and immune deposits, whose antigenicity could be easily restored by trypsin digestion. Neutral buffered formalin was also a satisfactory fixative in immunofluorescent staining on lymphocyte/plasma cell-bound immunoglobulins. Fixation with alcohol-Bouin's fluid showed contrast results; feasible for staining of cell-bound immunoglobulins, but poor for that of glomerular immune deposits. After papain digestion, BSA and lysozyme, antigens of immune complexes, were easily detected in experimental chronic serum sickness glomerulonephritis. Pepsin was more efficient than trypsin in restoring the antigenicity of renal tissue antigens such as fibronectin and polyantigenic basement membrane, but the brush border antigen of the proximal renal tubules was frail to the pepsin digestion. In general, the enzymatic digestion time necessary for the restoration of antigenicity was in parallel with fixation time. Results obtained have shown that deparaffinized sections could be used as satisfactory substrate for immunohistochemistry when proper fixation and efficient proteolytic enzymatic pretreatments were performed.
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Experimental glomerulonephritis induced by human IgG in rats.
Experimental immune complex glomerulonephritis was induced with human IgG (HIgG) in two strains of rats, Wistar-King-Aptekman (WKA) and spontaneously hypertensive rats (SHR). The rats were pre-immunized s.c. with 1 mg of HIgG in Freund's complete adjuvant, and 8 weeks later were given a daily i.v. 2 mg of HIgG injections for 4 weeks. Renal tissue, obtained at weekly intervals after the beginning of i.v. injections, was examined by light, immunofluorescence and electron microscopic tests. SHR developed an endocapillary proliferative glomerulonephritis with heavy proteinuria after the administration of HIgG for 4 weeks. They had massive depositions of HIgG, rat IgG, and rat C3 both in the mesangial area and along the capillary wall. On the other hand, in WKA rats, the proliferative lesions were scarcely seen and the immune deposits were observed almost exclusively in the mesangium. Moreover, urinary protein excretion of these rats was within normal range. In comparison with bovine serum albumin (BSA) nephritis in SHR, HIgG-induced glomerular lesion was relatively mild. This difference seemed attributable to the nature of the HIgG, such as its molecular weight and immunogenicity.
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Monoclonal antibodies to rat renal antigens.
We have developed hybridoma cell lines which secrete monoclonal antibodies to some rat renal antigens, namely the brush border of proximal tubular epithelium and the cytoplasm of tubular cells. The immunoglobulin class of the hybridoma was found to be IgG1. Specific antibody activity against either glomerular basement membrane (GBM) and tubular basement membrane (TBM) or Bowman's capsule and a part of TBM was observed, although these hybridoma cell lines have not yet been successfully established. In particular, the hybridoma secreting antibodies to TBM did not remain stable during antibody production, and was lost during the culture and cloning procedures. These monoclonal antibodies should be of value in research on the pathogenesis of human glomerulonephritis.
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Localization of glomerular fibronectin in human and experimental glomerulonephritis.
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Solubilization of immune complex deposits by native 7S IgG molecules in lupus glomerulonephritis--a possible antigen excess effect on rheumatoid factor-IgG complexes.
IgM and IgG class rheumatoid factor (RF) containing glomerular immune complex (IC) deposits were detected in renal biopsy material from 10 out of 123 patients with various forms of glomerulonephritis (GN) by an immunofluorescent assay using FITC conjugated aggregated human IgG(FAHIgG). In lupus GN, six out of 28 cases (23.5%), showed such activity. Solubilization of glomerular IC deposits in frozen sections of lupus GN was observed after a 5 h incubation with native 7S IgG. The binding of FAHIgG to glomerular IC deposits and solubilization of deposits by excess HIgG were also confirmed in sections of two types of rat experimental GN in which HIgG acted as exogenous antigen; in situ IC formation type GN in cationized HIgG planted kidneys and chronic serum sickness type GN. These results suggest a possible antigen excess mechanism contributing to the solubilization of glomerular IC deposits via RF and IgG interactions in lupus GN.
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Alteration of glomerular anionic sites by the development of subepithelial deposits in experimental glomerulonephritis in the rat.
Using highly cationic polyethleneimine, alteration of glomerular anionic sites were evaluated ultrastructurally in two types of rat glomerulonephritis (GN); chronic serum sickness GN and heterologous (passive) or autologous (active) Heymann's GN. Daily i.v. injections of egg white lysozyme in physiologic saline into presensitized rats led to the formation of numerous mesangial and subepithelial deposits. In the non-proteinuric period in which immune deposits were localized predominantly in the mesangium, anionic sites of the laminae rarae and the epithelial cell coat were clearly observed. In the subsequent proteinuric period in which numerous subepithelial deposits were superimposed, a broad loss of anionic sites in the epithelial cell coat was seen. Splitting and focal loss of anionic sites on the lamina rara externa adjacent to the subepithelial deposits were commonly observed both in passive and active Heymann's GN and in lysozyme GN. These findings indicate that the subepithelial deposits are closely involved in the development of proteinuria by injuring the anionic sites, especially those on lamina rare externa of the glomerular basement membrane.
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An active model of immune complex glomerulonephritis in the rat employing cationized antigen.
An active model of in situ immune complex glomerulonephritis involving a cationic antigen was established. Three weeks after immunization with human IgG, the left kidneys of Wistar rats were perfused with 100 micrograms of cationized human IgG (pI greater than 9.5) via the left renal artery. At this time the animals exhibited a mean serum concentration of 0.58 +/- 0.33 mg anti-human IgG antibody per milliliter. Renal tissue was examined at regular intervals by immunofluorescence, light, and electron microscopy thereafter. Cationized human IgG and rat IgG were distributed along the glomerular capillary wall in a pattern that became increasingly granular with time; rat C3 was also present. Histologically, a severe proliferative lesion was seen with crescent formation in 10-20% of the glomeruli; adhesions of glomerular tufts to Bowman's capsule were common and with time spike formation in the glomerular basement membrane became very prominent. Extensive subepithelial dense deposits were seen by electron microscopy. Proteinuria was present in 19 of 26 animals within 24 hours, and in 30 of 31 by Day 7. Protein excretion fell from Day 14 onward, but some animals exhibited chronic proteinuria. The model described here represents another situation in which cationic antigens can induce subepithelial immune complexes, namely, the rapid release of small quantities of antigen into a previously sensitized host. This sequence could well mirror the events occurring in nature more closely than the previously described passive in situ model.
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Nephropathy induced in rats by repeated intravenous injections of rabbit anti-FX1A antibody.
Repeated intravenous injections of small amounts of rabbit anti-rat FXIA antibodies (RAFA) induced a renal damage with the morphological features of epimembranous glomerulonephritis with significant and persistent proteinuria. These findings indicated that repeatedly injected RAFA accumulated in glomeruli and that there seems to be a threshold in quantity of RAFA localized in glomeruli to induce renal damage with proteinuria.
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Quantitative studies of in situ immune complex glomerulonephritis in the rat induced by planted, cationized antigen.
Cationized human IgG can bind to the rat glomerular basement membrane (GBM), act as planted antigen, and induce in situ immune complex formation accompanied by severe glomerulonephritis. Perfusion of highly cationized human IgG (isoelectric point {more than} 9.5) via the left renal artery resulted in preferential localization within the perfused kidney (up to 56 percent of dose injected); after intravenous administration, only 4 percent was bound to the kidneys. The planted antigen was localized along the glomerular capillary walls and was accessible for antibody administered intravenously 1 h after perfusion, when virtually no antigen remained in the circulation. Persistence of cationized human IgG in the perfused kidney was markedly prolonged when complexed with antibody; one-half the cationized human IgG was still present after 12 d. There was a difference in the disappearance rates of antigen and antibody, as cationized human IgG was removed faster from the kidney than the antibody, the binding of which remained almost unchanged during the first week. Renal perfusion of a minimum of 20 mug of cationized human IgG, followed by intravenous injection of antibody, regularly induced severe glomerulonephritis with a proteinuria of at least 100 mg/24 h. The degree and the persistence of proteinuria induced depended on the dose of cationized human IgG perfused. Experiments using radiolabeled antigen and antibody showed that after renal perfusion of 20 mug cationized human IgG, 11.1 mug was kidney bound at the time of antibody injection. At the onset of proteinuria, 4.0 mug of antigen and 31.9 mug of anti-human IgG antibody were present in the perfused kidney. Immunofluorescence revealed immune deposits consisting of cationized human IgG and rabbit IgG (anti-human IgG) along the GBM. The staining pattern was linear (confluent) during the first 2 d and became granular during the course of the disease. Electronmicroscopically, a prominent finding was the accumulation of dense deposits, mainly in the subepithelial space and beneath the slit pores.
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Masugi nephritis in the nude mice and their normal littermates.
Masugi nephritis was studied in the male nude and their normal littermate BALB/c mice. In the second phase, severe histological changes consisting of numerous thrombi, thickening of the capillary walls, segmental capillary necrosis, tubular atrophy and numerous casts in the tubular lumen were found in the nephritic nude mice. By electron microscopy, dense deposits were found in the subendothelial space, together with massive fibrin deposition in the capillary lumina. Degeneration and disappearance of endothelial cells could also be seen. On the other hand, the nephritic BALB/c mice showed definite hypercellularity of the glomeruli but less exudative changes. In the second phase, granular deposition of mouse IgG was seen in the mesangium of the nephritic nude mice. In contrast, in the nephritic BALB/c mice, mouse IgG was localized in a linear fashion along the capillary walls. In the plaque forming assay, no indirect plaques against rabbit gammaglobulin were developed in the nephritic nude mice.
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The fibrinolytic activity of isolated rat glomeruli by fibrin slide technique. Its application to rat Masugi nephritis.
A method for measurement of rat glomerular fibrinolysis and the results of its application to rat Masugi nephritis are described. The glomerular fibrinolytic activity was measured on isolated glomeruli using a fibrin slide technique. The procedure of this method is simple and the results obtained have a good reproducibility. By applying this method to rat Masugi nephritis, sequential changes in glomerular fibrinolytic activity could be assessed. A decrease in glomerular fibrinolytic activity that was related to the extent of fibrin deposits in the glomeruli was demonstrated in the autologous phase.
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Autoimmune anti-glomerular basement membrane glomerulonephritis induced with isologous renal antigen in rats.
In an attempt to reproduce "glycoprotein nephritis", nephritogenic glycoprotein or glycopeptides were prepared from isologous renal cortices by the method of Shibata et al. Rats of Wistar Imamichi that were given a single injection with glycoprotein or glycopeptides in incomplete Freund's adjuvant (IFA) to the hind footpads developed glomerulonephritis. Light microscopic findings were essentially similar to those in their reports. However, in spite of similar materials and methods as they used, immunofluorescent findings were entirely different; localization of rat IgG and complement showed a "linear pattern" in contrast to their "mesangial pattern." Linear pattern in immunofluorescent study supports a notion that we succeeded in induction of autoimmune anti-glomerular basement membrane glomerulonephritis (Steblay's type nephritis) by immunization with isologous renal antigen.
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