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We report a case of idiopathic immunotactoid glomerulopathy with IgA2, kappa light chain deposition, ameliorated by steroid therapy. A 28-year-old male patient was admitted to our hospital due to exacerbation of nephrotic syndrome. The onset of his renal disease was at 24 years of age and the renal biopsy revealed membranoproliferative glomerulonephritis with moderate-degree deposition of IgA, IgG, IgM, C3 and C1q. Prednisolone therapy was started at the dose of 50 mg/day and effective for nephrotic syndrome and renal dysfunction. Two years later, the proteinuria and microscopic hematuria gradually exacerbated during reduction of prednisolone. The second renal biopsy showed mesangioproliferative glomerulonephritis with predominant deposition of IgA and C3. The glomerular proliferative changes were successfully suppressed by steroid treatment. On electron microscopy, a microtubular deposit with an average width of 40 nm and double-tracked appearance was observed in the mesangial and subendothelial areas. Immunohistochemical examination revealed that the deposit was predominantly composed of IgA2 subclass and kappa light chain. Selective deposition of IgA2 subclass and kappa light chain indicated that the glomerular lesion should be induced by monoclonal immunoglobulin, although it could not be detected in the serum and urine clinically. Immunoglobulin subclass staining of renal biopsy specimens provides an important clue for understanding the pathogenesis of immunotactoid glomerulopathy or fibrillary glomerulonephritis.

The present study was designed to elucidate whether glomerular epithelial cell (GEC) injury is associated with the mechanism of proteinuria in biopsy-proven IgA nephropathy (IgAN). Twenty-four IgAN patients were divided into 4 groups based on their grade of proteinuria. Light microscopic examination revealed that GEC injury appeared prior to glomerular lesions such as sclerosis, crescent and adhesion. Reduced glomerular expression of C3b receptor (CR1), an indicator of GEC injury, was detected initially at the portion of damaged GEC and around the lesions of sclerosis, crescent and adhesion. CR1 expression eventually disappeared in the group IV patients who had nephrotic range proteinuria. Moreover, the grade of proteinuria in IgAN patients was associated with GEC injury and reduced glomerular expression of CR1. Taken together, GEC injury might be an important pathological finding which appears initially in the glomeruli, suggesting that GEC injury is a predictor of proteinuria in IgAN patients.

There is a strong association between epithelial crescent formation and the outcome of patients with Henoch-Schönlein purpura nephritis (HSPN), but little is known about the mechanisms of crescent formation in this disease. In this study, we examined whether or not glomerular endocapillary proliferation, fibrinoid necrosis and infiltrating inflammatory cells are involved in the process of crescent formation. Renal biopsy specimens were obtained from 19 patients (11 males and 8 females, 7.8 +/- 3.8 years old). The biopsies were performed within the initial 3 months after the onset of urinary abnormalities in all patients. Twelve patients had crescents (% glomeruli with crescents; 27.5 +/- 20.4%), and the other 7 did not. Renal tissues biopsied from 10 patients with asymptomatic hematuria served as controls for quantitative studies of glomerular leukocyte infiltration. Prominent endocapillary proliferation as well as fibrinoid necrosis were encountered in patients with crescents in contrast to patients without crescents. A positive correlation between crescents and endocapillary proliferation or fibrinoid necrosis was also observed. Significant increases in the number of glomerular CD68+ macrophages, CD45RO+ T cells and NP57+ neutrophils were observed in HSPN patients compared with the controls. When patients with or without crescents were compared, patients with crescents had a greater number of inflammatory cells. Although all kinds of inflammatory cells appeared in the endocapillary proliferative lesion, only CD68+ macrophages and NP57+ neutrophils were present in the fibrinoid necrotizing lesion. In conclusion, the present study demonstrated that necrotizing lesions with capillary rupture may be essential in the process of crescent formation seen in HSPN. Although the mechanism by which capillary wall breaks occur is still unknown, this study indicates that infiltrating macrophages as well as neutrophils may be involved in capillary rupture leading to epithelial crescent formation.

Contraction of the pre- and postglomerular arteries play an important role in the regulation of glomerular blood flow. Mesangial cells may also be involved in the mechanism of this regulation, but it has not been clarified yet whether or not mesangial cells and vascular smooth muscles show an identical phenotype, especially in terms of their contractile proteins. In this study, in order to elucidate any difference in the cellular phenotypes between mesangial cells and renal vascular smooth muscles, we investigated the localization of myosin heavy chain isoforms using a monoclonal antibody against SM1 and SM2. Both SM1 and SM2 are specific to smooth muscles. SM1 is specifically expressed in smooth muscles from early development and SM2 appears after birth. In normal renal tissues, SM1 and SM2 were expressed only in the smooth muscle cells of the arterioles and small arteries. However, glomerular cells, including mesangial cells, were not stained with either anti-SM1 antibody or anti-SM2 antibody. Localization of SM1 and SM-2 was similar to that of alpha-smooth muscle actin (SM alpha-actin). Staining for SM-1 was not observed in the mesangial areas of renal tissues with glomerular disease. These results clearly indicate that mesangial cells have a different phenotype from that of vascular smooth muscle cells in terms of their contractile proteins.

To investigate the mechanism of glomerular endothelial response to high glucose, an in vitro model of the glomerular endothelial barrier was established in which transfer of fluorescein-labeled albumin across confluent monolayers of immortalized bovine glomerular endothelial cells (GEN) grown on polycarbonate membranes was measured. We first examined the effects of increased concentrations of D-glucose on albumin permeability across GEN monolayers and further investigated the role of protein kinase C (PKC) in the regulation of glucose-induced changes in endothelial barrier function. Incubation with 30 mM D-glucose increased albumin permeability more than those with 10 mM D-glucose. Albumin permeability incubated with 10 mM D-glucose plus 20 mM mannitol was not significantly different from those with 10 mM D-glucose, indicating that the increase in albumin permeability induced by 30 mM D-glucose was not due to high osmolar stimuli. A protein kinase C (PKC) inhibitor, H-7 (25 microM) significantly reduced the permeability-increasing effects of D-glucose. Lactate dehydrogenase release from endothelial cells was not significantly increased above baseline after incubation with 10 mM or 30 mM D-glucose. These results suggest that elevated concentrations of glucose activates PKC, resulting in an increase in albumin permeability across GEN.

Infiltration of leukocytes and mononuclear cells into the glomeruli is an early pathological finding in human and experimental glomerulonephritis. However, the cellular and molecular basis for cell infiltration into the glomeruli is not yet completely understood. In addition, there is little information on the expression of intercellular adhesion molecule-1 (ICAM-1) on glomerular cells. In the present study, we investigated the expression of ICAM-1 on cultured bovine glomerular endothelial cells (GEN) and its regulation by the pro-inflammatory cytokines, interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and lipopolysaccharide (LPS). Immunocytochemical staining showed that ICAM-1 molecules were constitutively expressed on the surface of GEN. In flow cytometric and ELISA analyses, ICAM-1 molecule expression on GEN was significantly upregulated by IL-1 beta, MCP-1 and LPS in a dose-dependent manner, but not by IL-6. LPS was the most potent inducer of ICAM-1 molecule expression on GEN. The effects of IL-1 beta, MCP-1 and LPS were observed as early as 4 h and reached a maximal level by 18 h. These results suggest that ICAM-1 on GEN can participate in the infiltration of mononuclear cells into glomeruli in human and experimental glomerulonephritis.

A 49-year-old woman suffering from nephrotic syndrome (NS) was admitted for renal biopsy and treatment of NS. Light microscopy demonstrated that the glomerular capillary wall was markedly thickened with diffuse accumulation of periodic acid Schiff- and periodic acid methenamine-positive materials, leading to lobular accentuation of glomerular tufts. By electron microscopy, numerous collagenous fibers were observed in the mesangium and subenodothelial area. The fibers were peculiarly curved and frayed, as reported in nail-patella syndrome. These materials were thought to be type I and type III collagen as a result of immunohistochemical studies. No laboratory data or pathological findings were found to be compatible with previously described glomerulonephritis. The primary glomerular fibrosis in the present patient seemed to be a case of collagenofibrotic glomerulonephropathy.

We conducted a study to investigate whether expression of the contractile proteins in cultured rat mesangial cells (MC) was associated with the cell cycle and protein kinase C (PKC). When growth-arrested MC were stimulated with 100 nM phorbol myristate acetate (PMA) for 24 hours, an increased expression of smooth muscle alpha-actin and vimentin was detected by immunocytochemistry. A proportion of the S- and G2/M-phase in MC was increased in accordance with the enhanced expression of contractile proteins on flow cytometry. Immunoblot analysis revealed that 100 nM PMA stimulated expression of alpha-actin and vimentin as a single band. These results indicate that expression of contractile proteins, such as alpha-actin and vimentin, is dependent on the cell cycle and PKC, suggesting a phenotypic change in which MC assume smooth muscle cell characteristics.

Bovine subcultures (second passage) of glomerular endothelial cells (GEN) isolated from one-year-old kidney were successfully transfected by recombinant plasmids containing the simian virus (SV)-40 T antigen (Tag) using a lipofectin-mediated procedure. One cell clone was selected, propagated and characterized. This clone can be grown in RPMI 1640 medium supplemented with 10% fetal calf serum. The advantage of this cell line is the cultivation of bovine GEN without the addition of fibroblast growth factor or a coating of fibronectin or gelatin on the culture plate. More than 80 passages were achieved and the doubling time was 32 h. The Tag was easily identified in transfected-GEN by indirect immunofluorescence. These cells weakly expressed factor VIII-related antigen, slightly took up acetylated-low density lipoprotein and secreted a detectable amount of angiotensin-converting enzyme. Immunocytochemical staining for UAE-1 was also positive. Moreover, oncoproteins, such as Ki-67 and p53, were expressed in these cells. Cell cycle analysis by flow cytometry revealed that the percentages of G1, S, and G2/M stages in cycling transfected-GEN culture in RPMI 1640 medium supplemented with 10% fetal calf serum were 34%, 52.9%, and 13.1%, respectively. The conditioned medium from confluent transfected-GEN stimulated [3H]thymidine incorporation into glomerular mesangial cells. This cell line may provide a useful tool for examining modulators of mesangial cell growth. Thus this cell line is the first immortalized bovine GEN that retain the morphologic, phenotypic, and functional characteristics of bovine GEN.

A new monoclonal antibody (MAb) was raised against human glomerular components to investigate the possible existence of glomerular antigens reflecting cellular adaptation in glomerulonephritis. The MAb recognized a podocyte antigen as well as those expressed on renal arterial endothelial cells and smooth muscle cells by indirect immunofluorescence. An additional, but weaker immunoreaction was found in epithelial cells of the Bowman's capsule. This MAb recognized a 30-kD protein on western blotting of glomerular extracts under non-reducing and reducing conditions. Immunoperoxidase electron microscopy revealed that this antigen is present within the cytoplasm, but not on the cell membrane of the podocytes. Moreover, the antigen was found to be reduced in the glomeruli of patients with minimal change glomerulonephritis. These results suggest that the 30-kD protein is a novel protein, which we hypothesize is involved in maintenance of the structural and functional integrity of the podocytes. In addition, reduced expression of the 30-kD protein in the podocytes may be related to the increasing proteinuria in minimal change glomerulonephritis.

Because of difficulties of propagating and cultivating glomerular endothelial cells (GEN), the role of glomerular mesangial cells (GMC) in the regulation of GEN growth is still unknown. We cloned individually GEN and GMC from the same adult bovine kidneys, and a direct coculture system was established to examine the interactions between GEN and GMC. When cocultured with GMC which was growth arrested by mitomycin C, the GEN growth was inhibited by 63% after 8 days of the beginning of coculture. No inhibitory activity was detected when media conditioned by individual culture of GMC. In contrast, media conditioned by GEN cocultured with GMC inhibited bromodeoxyuridine (BrdU) uptake of GEN detected by flow cytometry. Addition of anti-transforming growth factor (TGF)-beta antibody to media conditioned by coculture blocked the inhibitory actions of conditioned media on GEN growth. Western blotting analysis of conditioned media by coculture revealed an existence of TGF-beta in the media. These results indicate that latent TGF-beta is produced by these cells and activated in coculture of GEN and GMC.