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ABSTRACT: Thin basement membrane disease (TBMD) is characterized histologically by diffuse thinning of glomerular basement membrane (GBM). Although recent genetic analysis has shown that TBMD might be included within type IV collagen disorders, conventional immunohistochemical studies demonstrated normal labeling of type IV collagen alpha chains in the GBM. We have, however, successfully used confocal laser scanning microscopy to demonstrate a significantly reduced signal of type IV collagen alpha5 chain (alpha5(IV)) along capillary walls in TBMD. In order to further understand the association of type IV collagen with TBMD, we used immunoelectron microscopy to examine renal biopsies from 6 children with TBMD and six control children with minimal change nephrotic syndrome.
Ultrathin sections of LR gold resin were incubated with a rat monoclonal antibody against human alpha1(IV), alpha2(IV), alpha3(IV), alpha4(IV) alpha5(IV) or alpha6(IV) followed by colloidal gold conjugated goat anti-rat IgG. After taking electron micrographs, the labeling was quantitatively evaluated in the area occupied by the segments of basement membrane. The basement membrane was divided into three equal segments viz. subepithelial side, central portion and subendothelial side.
In control subjects, the number of gold particles for alpha1(IV) or alpha2(IV) was significantly greater in the subendothelial side and central portion than in the subepithelial side of the GBM, whilst alpha3(IV), alpha4(IV) or alpha5(IV) labeling was significantly more prominent in the central portion compared to the subepithelial and subendothelial side of the GBM. TBMD samples showed a similar distribution pattern except that the subepithelial side and central portion of the GBM had a significantly reduced amount of alpha5(IV) antigen compared to control subjects.
This is the first report demonstrating a diminished labeling intensity of alpha5(IV) in the central portion and subepithelial side of the GBM in renal biopsy specimens from patients with TBMD. These findings suggest that an abnormality of alpha5(IV) might possibly be associated with the pathogenesis of TBMD.
Clinical nephrology 12/2005; 64(5):329-36. · 1.17 Impact Factor
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ABSTRACT: Although infiltrating macrophages found in renal biopsy specimens have been accepted as a useful marker for evaluating the activity of IgA nephropathy (IgAN), it is difficult to perform renal biopsies repeatedly, especially in children. To establish a more convenient and noninvasive method for estimating the degree of macrophage infiltration we examined the number of macrophages in urinary sediments.
Ten ml of morning urine were collected from 30 children with IgAN, 10 with thin basement membrane disease (TBMD), 8 with idiopathic renal hemorrhage (IRH) which was defined as nonglomerular hematuria due to nutcracker phenomenon revealed on ultrasonography, and 10 healthy children as controls. Ten of the 30 children with IgAN were treated with combination therapy comprising prednisolone, warfarin and dipyridamole and urine samples were collected weekly during the period of treatment. Two microl of the urine sediment were smeared on glass slides, dried and stained with a monoclonal antibody to human macrophages (anti-CD68, PG-M1) followed by a FITC-conjugated secondary antibody. After staining with propidium iodide (PI), the cells were examined by fluorescence microscopy with cells stained with both FITC and PI being counted as macrophages. In addition, anti-CD68 staining was used to quantify macrophage infiltration in renal biopsies from the same group of IgAN patients.
The number of urine macrophages in children with IgAN was significantly higher than in children with TBMD and IRH as well as the control group (p < 0.01), whereas that was similar among TBMD, IRH and healthy children. In IgAN, there was a significant correlation between urine macrophage number and the activity index (p < 0.01), proteinuria (p < 0.01) and urine WBC count (p < 0.01). In addition, there was also a significant correlation between urine macrophage number and glomerular (p < 0.05) as well as interstitial macrophage infiltration (p < 0.01). In children with IgAN who received combination therapy, urine macrophage number decreased significantly (p < 0.01) in the 1st week of treatment whilst the degree of proteinuria decreased significantly (p < 0.01) in the 4th week.
Urinary macrophage number may represent a noninvasive and straightforward estimate of the pathological activity evident in renal biopsy specimens, and may also be a more sensitive indicator than proteinuria of the therapeutic effect of interventional treatments in childhood IgAN.
Clinical nephrology 12/2004; 62(5):336-43. · 1.17 Impact Factor
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ABSTRACT: Analysis of glomerular anionic charge in human renal biopsy specimens has been restricted previously to staining of sites at the electron microscopic level, which is a product that needs skills and precludes a wide observable area. The introduction of a new tool, confocal laser scanning microscopy together with FITC conjugated poly-L-lysine as a cationic tracer, which demonstrates fixed anionic sites in thin sections from routinely formalin-fixed and paraffin-embedded renal biopsy tissue, has now enabled glomerular charge at light microscopic level. In this method, the patterns of staining in tissue showing minimal change nephrotic syndrome (MCNS) indicate that the intensity of anionic charge in 4 children with heavy proteinuria was significantly less than that in 7 children without proteinuria at remission, supporting previous observations using electron microscopy. Furthermore, staining the serial sections after methylation or saponification revealed that carboxyl components such as sialic acid may be responsible for proteinuria. We anticipate that this method may facilitate the investigation of the participation of charged components in the pathogenesis of MCNS and their role in relation to glomerular proteinuria.
Nippon Jinzo Gakkai shi 02/2000; 42(1):16-23.
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ABSTRACT: Precise localization of cytokines such as transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 and IL-6 was observed in glomeruli using immunogold electron microscopy in 21 children with various types of renal diseases. The distribution pattern of these cytokines, as well as immunoglobulins, C3c and fibrinogen (Fg), was essentially confined to the electron-dense deposits (EDDs) regardless of their location. Frequency of positive labelling of each cytokine was different among various types of renal disorder, that is, TGF-beta was found mainly in lupus nephritis (LN), membranous nephropathy and IgA nephropathy, TNF-alpha in LN, and IL-1 in Henoch-Schönlein purpura nephritis. IL-6 was detected only in 1 case of LN. TNF-alpha was also found in the cytoplasm of glomerular epithelial cells. Furthermore, in order to evaluate the relation of cytokines to mesangial expansion, extracellular matrix components such as type IV collagen, laminin and fibronectin were stained. The result was that there was no significant correlation between the signal intensity or distribution pattern of cytokines and that of extracellular matrix components. These findings indicate that these cytokines could be associated with the formation of EDDs together with immunoglobulins, C3c and Fg. The involvement of each cytokine in renal pathophysiology might also depend upon the type of renal disease. They also raise the possibility that the glomerular epithelial cells might produce or absorb TNF-alpha. However, these results did not show significant correlation between cytokine involvement and mesangial expansion.
Nephron 02/1999; 83(2):132-8. · 13.26 Impact Factor
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ABSTRACT: The relationship between the activation of platelets (PLT) in urine and renal histological findings in children with IgA nephropathy (IgAN), thin basement membrane disease (TBMD), and minimal-change nephrotic syndrome (MCNS) was examined. The ratio of activated PLT to total PLT (activated and nonactivated PLT) was examined by double immunofluorescence using rhodamine-conjugated P selectin antibody (activated PLT) and fluorescein isothiocyanate conjugated PLT membrane glycoprotein antibody (GPIIb/IIIa, total PLT); the effect of urine on activation on PLT was also investigated. The number of activated PLT and the ratio of activated PLT to total PLT in urinary sediments were significantly higher in children with IgAN with diffuse mesangial proliferation than in those with TBMD or MCNS. PLT were activated by addition of urine in 13 out of 27 children with IgAN, and the activity was higher in the urine of those with active glomerular or interstitial lesions, while the urine of children with TBMD or MCNS had no effect. The presence of activated PLT and the effect of urine on PLT activation may be associated with the active glomerular or interstitial lesions in IgAN.
Nephron 02/1998; 78(2):162-7. · 13.26 Impact Factor
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ABSTRACT: In 22 children with steroid responsive nephrotic syndrome (SRNS), we examined platelets aggregability to ristocetin, and the data obtained wre compared with negative charge on platelet membrane based on the binding of cationic dye alcian blue 8GX (AB) or plasma levels of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor (Rcof) activity. At the initial or relapse stage of SRNS, the enhanced platelet aggregation to ristocetin was observed, and correlated with the decreased alcian blue binding to platelets. Ristocetin-induced vWF:Ag binding to platelets by using 125I-vWF was significantly increased. In addition, ristocetin-induced platelets aggregation (RIPA) using washed nephrotic platelets still enhanced as found in the patient's platelets rich plasma (PRP), even when it was resuspended into normal plasma. These results suggest that the decrease of platelet surface negative charge play an important role of heighten RIPA found in children with SRNS.
Nippon Jinzo Gakkai shi 07/1990; 32(6):659-66.
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ABSTRACT: The ultrastructural localization of immunoglobulins (IgG, IgA, IgM), complement component (C3c), or fibrinogen-related antigen (FRA) was investigated on 5 biopsy samples from 5 children with idiopathic MN or lupus nephritis using protein A-gold (PAG) complex method. The immunoreactivity of IgG was essentially confined to the mesangial and subepithelial electron-dense deposit (EDD) in all of 5 children, and more intense staining of IgG was observed in 3 children with stage II of MN than those with stage I and stage III of MN. Double immunocytochemical staining showed the same distribution of both IgG and IgM or both IgG and FRA in subepithelial EDD in 2 of 5 children. These findings suggest that IgG deposits are associated with the formation of subepithelial EDD in MN, and raise the possibility that IgM and FRA deposits may result from entrapment and/or immunological reaction.
Nippon Jinzo Gakkai shi 12/1989; 31(11):1143-9.
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Acta paediatrica Japonica; Overseas edition 01/1989; 30(6):662-70.
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ABSTRACT: The localization of intraglomerular deposits of fibrin (Fb)/fibrinogen (Fg)-related antigen (FRA) in children with various glomerular diseases was determined by an immunohistopathologic method using an anti-Fg antibody capable of detecting FRA, an anti-D-dimer antibody capable of detecting crosslinked Fb (XLFb) and its derivatives (XLFbDP), and by a method using the effect of monochloroacetic acid (MCA) treatment on kidney sections. In proliferative glomerulonephritis (PGN), XLFbs were detected within the capillaries and extension beyond the mesangium was seen in severe PGN. The FRA within the mesangium of minimal or mild PGN was composed of the non-XLFb substance. The FRA within Bowman's space of most PGN had disappeared after MCA treatment, suggesting a non-XLFb substance. The presence of FRA within electron-dense deposits (EDD) suggested that FRA deposits are associated with immune-complex deposits in the glomeruli.
American Journal Of Pathology 11/1988; 133(1):61-72. · 4.89 Impact Factor
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ABSTRACT: The concentration of cross-linked fibrin degradation products (XLFDP) in urine were determined by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) in patients with several types of glomerulonephritis with crescents (CrGN). In patients with "active" cellular crescents, increased levels of XLFDP correlated with the percentage of glomeruli containing crescents. Dysmorphic erythrocytes, suggestive of glomerular bleeding, were observed in all of the patients with CrGN, and the urinary red cell counts (URCC) also correlated with percentage of glomeruli containing crescents. The absence of correlation between urinary XLFDP and URCC or urinary protein suggested that lysis of fibrin within crescents may contribute to the urinary excretion of XLFDP in CrGN. The measurement of urinary XLFDP in CrGN is likely to be of value in assessing the activity of glomerular lesions but not renal function.
Clinical nephrology 04/1988; 29(3):124-8. · 1.17 Impact Factor
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ABSTRACT: Renal biopsy tissue from 33 children with various glomerular diseases has been investigated by electron microscopy using a postembedding immunostaining technique with a protein A-gold complex in order to establish more precise correlations between immunopathologic and morphologic findings in glomeruli. This technique could detect immunoglobulins (IgG, IgA, and IgM), complement factor (C3c), and fibrinogen-related antigen. The immunoreactivity of these antigens was essentially confined to the mesangial, paramesangial, subendothelial, and subepithelial 'electron-dense deposits' in the glomeruli. Except for IgM and C3c in the case of glomerular sclerosis, the distribution of the mentioned factors was even in the electron-dense deposits, as could be shown by 'double-immunolabeling'. From the above-mentioned findings one can conclude that several of the localized factors are associated with the formation of electron-dense deposits, the ultrastructural hallmarks of glomerular disease.
Nephron 02/1987; 46(2):182-7. · 13.26 Impact Factor
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ABSTRACT: The localization of intrarenal cross-linked fibrin was examined by the effect of monochloroacetic acid treatment on the kidney sections. In acute glomerulonephritis or in mild diffuse or focal proliferative type of nephritis, cross-linked fibrin was observed mainly within glomerular capillary walls. Extension of cross-linked fibrin deposit over the mesangium or sclerotic area was seen in moderate to severe proliferative type of nephritis or in membranoproliferative glomerulonephritis. In hemolytic uremic syndrome or disseminated intravascular coagulation syndrome, cross-linked fibrin was detected within glomeruli and vessels.
Nephron 02/1983; 35(2):94-9. · 13.26 Impact Factor
