Hiroyoshi Wada

Roles of active oxygen species in glomerular epithelial cell injury in vitro caused by puromycin aminonucleoside

The mechanism of puromycin aminonucleoside (PAN)-induced nephrosis has not yet been well defined. In the present study, we examined the protective effect of active oxygen scavengers on the PAN-induced injury of cultured rat glomerular epithelial cells (GECs) and the generation of active oxygen species in PAN-treated GECs. When exposed to PAN (greater than or equal to 25 micrograms/ml), cellular damage occurred in a time- and dose-dependent manner as evaluated by both the LDH release and MTT colorimetric assays. Concomitant addition of either the hydrogen peroxide (H2O2) scavenger, catalase, or the iron chelating agent, deferoxamine, to the culture medium caused a striking reduction of cellular injury. This suggested a role for H2O2 and for hydroxyl radicals (OH.) generated via the iron-catalyzed breakdown of H2O2 in PAN nephrosis. Using the scopoletin fluorescence assay, the release of H2O2 into the culture medium by GECs exposed to PAN (greater than or equal to 50 micrograms/ml) was shown to increase dose-dependently (greater than or equal to 57 +/- 11 pmol/4.4 x 10(6) cells per h, P less than 0.01) as compared with control cells (14 +/- 2 pmol/4.4 x 10(6) cells per h). These results strongly suggested that active oxygen species, especially H2O2 and OH., might play an important role in PAN-induced GEC injury in vitro as well as in vivo.

Mechanism of Hematuria in Glomerular Disease

From an electron-microscopic study in a case of diffuse membranous glomerulonephritis, we would like to propose a possible mechanism of hematuria in glomerular disease. There are several factors we sh

EXPERIMENTAL ANTI‐GBM GLOMERULONEPHRITIS INDUCED IN RATS BY IMMUNIZATION WITH SYNTHETIC PEPTIDES BASED ON SIX α CHAINS OF HUMAN TYPE IV COLLAGEN

The anti‐glomerular basement membrane (GBM)‐nephritis‐inducing activity of six synthetic peptides having an amino acid sequence consisting of the six α chains of human type IV collagen was examined by injecting the peptides into rats. The peptides consisted of 27 amino acid residues from the non‐collagenous domain (NC1) of the α1 to α6 chains and were non‐consensus sequences sandwiched between two consensus sequences near the carboxyl terminus. Each peptide was coupled to keyhole limpet haemocyanin and injected with adjuvant into the footpads of 20 female WKY–Crj rats. The number of rats with proteinuria (over 10·0 mg of urinary protein/15 h) and haematuria was 2 with the α3 peptide, 8 with the α4 peptide, and 1 with the α5 peptide. Histological changes seen in the glomeruli were characteristic of those in anti‐GBM nephritis. Linear deposition of rat IgG along the GBM was observed in five rats injected with the α4 peptide. A nephritogenic monoclonal antibody against the α4 peptide was established using lymph node cells from a rat injected with the α4 peptide. The results indicate that α4(IV)NC1 is a potent nephritogenic antigen like α3(IV)NC1, which has already been recognized as a primary target antigen in Goodpastures syndrome.

Subglottic neurofibroma in a child.

Reported here for the first time is a case of subglottic neurofibroma in an infant which was removed by laryngofissure. Neurofibromas are ubiquitous in distribution, but very rare in the larynx and also extremely rare in infancy. Only 9 cases had been reported in children under 9 years of age. In the present case, H.T., a boy aged 2 years and 7 months, complaining of inspiratory stridor since the beginning of December 1985, was admitted to our hospital on Jan. 21, 1986. The tumor was completely removed by laryngofissure and pathological diagnosis showed it to be a neurofibroma. Laryngeal neurofriboma cases are reviewed and discussed.

Granulocyte‐macrophage colony‐stimulating factor abrogates transforming growth factor‐β1‐mediated cell cycle arrest by up‐regulating cyclin D2/Cdk6

The role of positive and negative cytokine interactions in G1 cell cycle regulation of haemopoietic cells was analysed by determination of the expression patterns of D‐type cyclins and cyclin‐dependent kinases (cdks) in SKM‐1 myelodysplastic syndrome (MDS) cells incubated with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and/or transforming growth factor‐β1 (TGF‐β1). TGF‐β1 inhibited SKM‐1 cell proliferation due to the cell cycle arrest in G1 phase. GM‐C18SF abrogated the TGF‐β1‐mediated G1 arrest in these cells. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis indicated that TGF‐β1‐mediated G1 arrest correlated with the down‐regulation of cdk4, cdk6 and cyclin D2, and that abrogation of TGF‐β1‐mediated G1 arrest by GM‐CSF correlated with the constitutive over‐expression of cyclin D2 and cdk6 but not cdk4. These results suggest the importance of cyclin D2/cdk6 levels in abrogating G1 arrest in cells exposed to TGF‐β1, and raise the possibility that the GM‐CSF‐mediated up‐regulatory pathway of signal transduction through cyclin D2/cdk6 differs from the TGF‐β1‐cdk4‐mediated pathway in SKM‐1 cells. This signal transduction pathway through cyclin D2/cdk6 might play an important role in haemopoietic regulation by the cytokine network.

Retinoic Acid Enhances the Number of Epidermal Growth Factor Receptors in Rat Glomerular Epithelial Cells in vitro

The renal epithelium appears to be an important target tissue for retinoic acid and epidermal growth factor (EGF). We report here that retinoic acid enhances the proliferative effect of EGF on glomerular epithelial cells (GEC) in vitro and also increases EGF binding to GEC. When GEC were exposed to EGF (> or = 1 ng/ml), cellular DNA synthesis was markedly increased. Moreover, the stimulating effect of EGF was synergistically increased by retinoic acid at 5 micrograms/ml. 125I-EGF binding to cultured GEC was increased approximately 3-fold after addition of retinoic acid to cultures for 48 h. Analysis of 125I-EGF binding revealed 8.1 x 10(4) receptors per control (untreated) cell, while retinoic acid-treated cells demonstrated an increase to 14.3 x 10(4) receptors per cell with no detectable change in receptor affinity. These findings suggest that interactions between retinoic acid and EGF may play an important role in the regulation of GEC growth.

Immunocytochemical Characterization and Identification of SGE1 a Rat Glomerular Epithelial Cell Line

Glomerular epithelial cells (GEC) in culture facilitate the study of glomerular physiology and pathology. However, characterization and identification of GECs in culture have been difficult due to the absence of markers specific to them. We compared the immunocytochemical characteristics of a rat normal GEC line (SGE1) and glomerular cells from rat kidney sections using a lectin and commercially available and newly raised monoclonal and polyclonal antibodies. Antivimentin, anti-dipeptidyl-peptidase-IV (gp 108) and monoclonal antibody 5-1-6 antibodies and Limax flavus lectin bound to visceral GECs, anticytokeratin antibody and antibody against common acute lymphocytic leukemia antigen bound to parietal GECs, and anti-SGE1 cell membrane and monoclonal antibody PHM 5 antibodies bound to both visceral GECs and parietal GECs in normal rat kidney sections, and all of these antibodies and L. flavus lectin consistently bound to SGE1 cells in culture. The pattern of antigenic expression on SGE1 cells indicates that SGE1 cells possess phenotypic characteristics of visceral GECs and parietal GECs, and it further suggests that SGE1 cells may be stem cells or cells undergoing differentiation.