Norihisa Akano

Cellular localization of inflammatory cytokines in human glomerulonephritis.

We evaluated the expression of inflammatory cytokines in renal tissues obtained from 45 patients with several types of glomerulonephritis. Immunofluorescence studies with specific antibodies to interleukin (IL)-1α, IL-1β, IL-6, tumour necrosis factor (TNF)-α, and TNF-β showed intense cytoplasmic staining in the glomeruli and interstitium. Cells positive for these cytokines were found frequently in tissue from patients with lupus nephritis (WHO Class IV) and membranoproliferative glomerulonephritis, and, to a lesser extent, in tissue from patients with mesangial proliferative glomerulonephritis, Henoch-Schönlein purpura nephritis, and minimal change nephrotic syndrome. Most of these cells were dual-stained with a monoclonal antibody to monocytes-macrophages. In situ hybridization for cytokine mRNA, combined with immunoperoxidase staining for monocytes-macrophages, detected IL-1α, IL-6, and TNF-α mRNA in monocytes-macrophages infiltrating the glomeruli and interstitium. Occasionally, there was weak or moderate immunostaining for IL-1α, IL-6, and TNF-α in the glomerular mesangial and epithelial cells, but in situ hybridization signals were rarely found in these loci. These findings suggest that infiltrating monocytes-macrophages, rather than resident glomerular cells, are the major source of inflammatory cytokines in human glomerulonephritis.

Acute interstitial nephritis and uveitis syndrome: activated immune cell infiltration in the kidney

Infiltrating cells were analysed in renal biopsy tissue obtained from a 15-year-old girl with acute interstitial nephritis and uveitis using monoclonal antibodies specific for mononuclear cell surface markers. The interstitial infiltrates consisted mainly of T cells and monocytes/macrophages. A considerable proportion of the infiltrating cells were identified by a monoclonal antibody against the interleukin-2 receptor, indicating that a majority of those immune cells are activated. This observation documents the participation of cell-mediated immune injury in interstitial nephritis associated with uveitis.

Immunohistochemical localization of C3d fragment of complement and S-protein (vitronectin) in normal and diseased human kidneys: association with the C5b-9 complex and vitronectin receptor

The localization of C3d, a fragment produced by C3 activation and S-protein (vitronectin), a regulatory factor of C5b-9, was studied immunohistochemically in normal human kidney and renal biopsies from patients with several types of glomerulonephritis. Immunofluorescent staining of the normal kidneys showed that C3d was present along the glomerular basement membrane (GBM), tubular basement membrane (TBM) and arterioles, and that S-protein was present in the GBM, mesangium, TBM, and arterioles. Immunoelectron microscopy of isolated basement membranes showed that C3d was localized exclusively on the epithelial side of the GBM, and that S-protein was present along both the epithelial and endothelial sides. In nephritic tissues, glomerular staining of C3d, C5b-9, and S-protein was increased when compared with that in normal tissues. S-protein, frequently co-localized with C3d and C5b-9 neoantigen, was intensely positive in the immune deposits of glomerular capillaries and the mesangial area, overlapping the background staining of GBM and mesangial matrix. S-protein and its receptor were occasionally co-localized in the glomeruli. These findings indicate that C3d and S-protein are normally present in the glomeruli. Co-staining of C3d, C5b-9 neoantigen, and S-protein within the immune deposits of nephritic kidneys suggests in situ binding of S-protein to locallyformed C5b-9 complex, or merely co-distribution of S-protein with the complex, rather than trapping of large molecular SC5b-9 complex from the circulation.

Immunohistochemical study of the membrane attack complex of complement in IgA nephropathy

The localization of the membrane attack complex of complement (MAC) was examined in the normal human kidneys and in biopsy specimens from patients with primary IgA nephropathy by immunofluorescent and immunoelectron microscopies. Immunofluorescent staining for MAC was significantly more intense than in the normal kidneys, and was observed in the mesangium and occasionally along the glomerular capillary walls of 22 of 30 patients with IgA nephropathy. By dualstaining, the MAC deposits were generally concordant with the deposits of IgA, C3, C5 and C9, or of IgG, when present. C1q or C4 was infrequently observed in the glomeruli. Immunoelectron microscopy revealed various staining patterns of glomerular MAC deposition; homogeneous fine-granular staining beneath the glomerular basement membrane (GBM) in the paramesangial zone, patchy staining within the mesangial electron dense deposits (EDD), and ring-shaped or ribbon-like staining, associated with the striated membrane structures (SMS), in the matrix of the mesangium, GBM and tubular basement membrane (TBM). This study suggests that the terminal complement system is activated, mainly by an alternative complement pathway mechanism, in the mesangium of IgA nephropathy, and is associated with the paramesangial lesion and EDD. MAC deposition in glomerular SMS may also result from in situ activation rather than trapping from the circulation. There was little correlation between glomerular MAC deposition and proteinuria or renal histology of patients with IgA nephropathy.

Immunotactoid glomerulopathy in a child with Down syndrome

A 9-year-old girl with Down (21-trisomy) syndrome was found to have proteinuria and microscopic haematuria at age 6 years. Proteinuria gradually increased during the next 3 years, although blood pressure and renal function remained normal. The patient exhibited no underlying systemic diseases, monoclonal gammopathy, cryoglobulinaemia or histological evidence of plasmacytoma. A percutaneous renal biopsy revealed immunotactoid glomerulopathy (fibrillary glomerulonephritis) characterized by thickening of the glomerular basement membrane, diffuse mesangial expansion and various-sized acid-Schiff-positive nodules that were intensely positive for IgG, light chains (κ and λ) and complement components (C3, C4, C1q) along the glomerular capillaries in the mesangium. Congo red dye and amyloid thioflavine T staining were negative. Fibrils (15–17 nm in diameter — larger than amyloid fibrils) were present in the mesangial area and within the glomerular basement membrane. We are not aware of a previous report of immunotactoid glomerulopathy and a patient with chromosomal abnormalities.

Proto-oncogene expression in human glomerular diseases

The expression of the protein products and mRNA of c‐fos, c‐myc, p53, and c‐raf was examined in normal renal tissues and biopsy specimens from 73 patients with various glomerular diseases. Immunofluorescent staining showed that there were cell nuclei stained for c‐Fos, c‐Myc, and p53, and cytoplasm positive for c‐Raf, in the glomeruli of patients with proliferative types of glomerulonephritis, including IgA nephritis and lupus nephritis, and in patients with focal glomerular sclerosis. Glomerular expression of c‐fos and c‐myc mRNA was detected by in situ hybridization. The number of proto‐oncogene‐positive glomerular cells was significantly higher in lupus nephritis, IgA nephritis, and focal segmental sclerosis, as compared with minimal change nephrotic syndrome and normal specimens. In IgA nephritis, the population of glomerular cells positive for c‐Fos and c‐Myc and the grade of c‐Raf immunoreactivity were significantly correlated with the proportion of proliferating cell nuclear antigen (PCNA)‐positive glomerular cells, with histological grading of mesangial hypercellularity and matrix increase, and with the magnitude of proteinuria. These data indicate that proto‐oncogene expression is associated with mesangial proliferation and matrix expansion in proliferative types of glomerulonephritis and in focal glomerular sclerosis.

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 complementin situ.

Urinary Fibrinogen and Fibrin Fragments in Children with Renal Disease

Fibrinogen fragments (X, Y, D and E) and fibrin fragments (D-dimer and E) were examined in the urine of 52 patients with various types of renal diseases. One or more of the fibrinogen fragments were detectable in all the urine specimens. D-Dimer together with fibrinogen fragments was found in 38 of the 52 patients. The clearance ratio of D-dimer to IgG, which indicates D-dimer generated in the kidney, was lower than 1 in all the patients with the minimal changes nephrotic syndrome, and was greater than 1 in the majority of patients with acute glomerulonephritis, rapidly progressive glomerulonephritis, mesangial proliferative glomerulonephritis and membranoproliferative glomerulonephritis. Our results suggest that urinary fibrin/fibrinogen degradation products (FDP) in renal diseases are derived primarily from increased filtration of FDP from the plasma through a damaged glomerular basement membrane, and that the mechanism of lysis of cross-linked fibrin deposited in the glomeruli occurs simultaneously in some types of glomerulonephritis. It seems that the determination of the clearance ratio of D-dimer to IgG may be useful in assessing the activation of the coagulation and fibrinolysis systems in the kidney in patients with renal diseases.

Transient Massive Proteinuria Associated With Mycoplasma pneumoniae Infection

A 5-year-old girl presented with Mycoplasma pneumoniae infection, which was associated with eyelid edema and massive proteinuria. Her clinical manifestations were similar to those of nephrotic syndrome, except for the absence of hypoproteinemia. Light microscopy of renal biopsy tissue showed minor glomerular abnormalities, with no tubulointerstitial changes. Electron microscopy showed sparse fusion of the foot processes, regular nonthickened glomerular basement membrane, and no electron-dense deposits. Immunofluorescent staining of the glomeruli for immunoglobulins (IgG, IgA, IgM) and complement (C3, C4) was negative. Mycoplasma antigen was not detected by indirect immunofluorescence. These findings suggest a causal relationship between M pneumoniae infection and transient renal injury.