Harutaka Yamada

Quantitative analysis of extracellular-superoxide dismutase in serum and urine by ELISA with monoclonal antibody

The superoxide anion has been implicated in a wide range of diseases. The major protector against superoxide anion in the extracellular space is extracellular-superoxide dismutase (EC-SOD). EC-SOD is the major SOD isozyme in plasma and forms an equilibrium between the plasma phase and heparan sulfate proteoglycan on the surface of the endothelium. An ELISA method for the measurement of human EC-SOD with monoclonal antibody was established. The proposed method had a high sensitivity (assay range, 0.05-50 ng/ml), good recovery (recovery percentage, 96.9 +/- 5.6%) and reproducibility (within-day assay, C.V. = 8.6-10.2%; between-day assay, C.V. = 6.5-11.7%). EC-SOD levels in sera from healthy persons are clearly divided into two groups: a lower group (Group I, below 120 ng/ml, n = 146) and higher group (Group II, above 400 ng/ml, n = 10). The EC-SOD in Group I were almost normally distributed and the mean level was 55.8 +/- 18.8 ng/ml. The serum EC-SOD level assayed by ELISA correlated well with serum SOD activity. The serum EC-SOD in Group I is heterogeneous with regard to affinity for heparin-Sepharose and could be separated into three approximately equal fractions, whereas the EC-SOD in Group II is mainly one fraction with a high affinity for the column. The apparent molecular weight and carbohydrate structure of serum EC-SOD in Group II are identical to those in Group I. The high EC-SOD level in sera from some individuals may reflect the excessive stimulation of EC-SOD synthesis in vivo or the growth of selected cells in vivo, because EC-SOD is known to be expressed by a few cell types in vivo as a high-heparin-affinity subtype.

Protective Role of Extracellular Superoxide Dismutase in Hemodialysis Patients

Background: The superoxide anion and other oxygen radicals have been implicated in the progression of chronic renal failure, and are removed by extracellular superoxide dismutase (EC-SOD) in the extracellular space on the surface of the endothelium. A single-base substitution of the EC-SOD gene which reduces the binding capability to endothelial cells resulting in an increased serum concentration, has been identified in healthy persons and hemodialysis patients. Results: The proportion of patients with this mutation among hemodialysis patients in each 20 months’ duration after the initiation of hemodialysis was retrospectively studied. The percentage of substitution-positive patients declined 80 months after the start of hemodialysis in non-DM patients. In contrast, in DM patients, the rapid decrease was obvious as early as 40 months after the initiation of hemodialysis. By prospective study for 5 years, there were significant differences in the survival rate between patients with and without R213G in DM, but not in non-DM patients. Among those who died, the incidence of ischemic heart disease and cerebrovascular disease in cases with R213G was significantly higher than in cases without R213G. Conclusion: These results suggest that the presence of a substitution in the EC-SOD gene at the heparin-binding domain could be a prognostic marker of dialysis patients.

MOLECULAR ANALYSIS OF EXTRACELLULAR-SUPEROXIDE DISMUTASE GENE ASSOCIATED WITH HIGH LEVEL IN SERUM

SummaryExtracellular-superoxide dismutase (EC-SOD) is one of the SOD isozymes mainly distributed in the extracellular fluid. In the vascular system, it is located on the endothelial cell surface according to studies on the heparin binding capacity. By measurement of serum EC-SOD levels of Japanese in healthy persons (n=103) and hemodialysis patients (n=150), 7 healthy subjects and 24 hemodialysis patients were classified into group II associated with high EC-SOD levels. By molecular analysis of the EC-SOD coding region from the group II individuals in Sweden, a single nucleotide substitution of G to C generating an amino acid change of arginine to glycine has been identified in the region associated with the heparin affinity of the enzyme. The same mutation was detected in the Japanese as a homozygote in both alleles of 2 hemodialysis patients and as a heterozygote in one allele of all the healthy group II individuals and 17 hemodialysis patients. The amino acid substitution may result in the decrease of the heparin affinity which is favorable for the existence of EC-SOD in the serum.

Cobalt chloride decreases EC-SOD expression through intracellular ROS generation and p38-MAPK pathways in COS7 cells.

It is known that cells suffer a chronic hypoxic condition during the development of proximal tubulointerstitial disease. However, it is accepted that extracellular-superoxide dismutase (EC-SOD) protects the cells from oxidative stress. The purpose of this study was to elucidate the regulation of EC-SOD expression in cells under hypoxia. The results show that the expressions of EC-SOD mRNA and protein in cobalt chloride (CoCl2)-treated COS7 cells decreased in a dose- and time-dependent manner, whereas the expressions of other SOD isoforms (Cu/Zn-SOD and Mn-SOD) were not changed. The down-regulation of EC-SOD mRNA was suppressed by pre-treatment with the antioxidant trolox and the p38 mitogen-activated protein kinase (p38-MAPK) inhibitor SB203580. It is concluded that the expression of EC-SOD is decreased through ROS and p38-MAPK signalling cascades and that the down-regulation of EC-SOD leads to a decrease in the resistance to oxidative stress of COS7 cells under hypoxia induced by CoCl2.

Heparin-stimulated expression of extracellular-superoxide dismutase in human fibroblasts

Extracellular-superoxide dismutase (EC-SOD) is the major SOD isozyme in the arterial wall and may be important for antioxidation capability of the vascular wall and normal vascular function. EC-SOD is expressed in various cell types in the vascular wall such as fibroblasts, smooth muscle cells and macrophages, and the synthesis of EC-SOD by human fibroblasts is known to be highly responsive to various inflammatory cytokines, although there is no response to oxidative stress. Heparin is a highly sulfated glycosaminoglycan with many functions such as antithrombotic, antilipemic and antiatherosclerotic effects. Another less well-known function of heparin is regulation of protein synthesis. In this study, we measured the induction of EC-SOD after treatment with heparin to understand the role of heparin in the antiatherosclerotic response of fibroblasts. Heparin induced EC-SOD expression at both the mRNA and protein levels. Heparin showed the greatest stimulatory effect and heparan sulfate showed moderate effects. The effect of chondroitin sulfate A was not clear. In contrast, desulfated heparin and chondroitin sulfate C did not increase EC-SOD expression. The stimulatory effect seemed to increase roughly with the degree of glycosaminoglycan sulfation. The enhanced expression of EC-SOD by heparin must contribute to the antiatherosclerotic effect of heparin.

Polymorphism of extracellular superoxide dismutase (EC-SOD) gene: relation to the mutation responsible for high EC-SOD level in serum.

SummaryExtracellular superoxide dismutase (EC-SOD) with amino acid substitution R213G generated by the nucleotide substitution 760C→G in the heparin binding domain is responsible for the high EC-SOD level in serum. We identified the two DNA polymorphic sites in the coding region of EC-SOD gene related to the 760C→G and determined the allele frequencies. The polymorphisms were A and G at nucleotide position (nt.) 241 and C and T at nt. 280 near the N-terminal. The haplotype frequencies in Japanese were 241A280C: 0.45, 241G280T: 0.37, and 241G280C: 0.18. The haplotype of 241A280T did not exist. The mutation 760C→G must occur on the allele having the haplotype of 241G280T.

Extracellular superoxide dismutase and glomerular mesangial cells: its production and regulation.

Extracellular superoxide dismutase (EC‐SOD) is synthesized in mesenchymally derived cells and prevents the oxygen radical‐induced injury. We studied whether kidney mesangial cells (MCs) produce EC‐SOD and how its production is associated with chemokine secretion. Under unstimulated condition, MCs produced EC‐SOD, and its production was correlated positively with cyclic adenosine monophosphate (cAMP), but negatively with interleukin (IL)‐6 or IL‐8 production. By prednisolone or phorbol myristate acetate treatment, EC‐SOD levels were correlated negatively with levels of IL‐6 and IL‐8. The presence of adenylate cyclase inhibitor 2′,3′‐dideoxyadenosine lost the prednisolone effect. The stimulation of EC‐SOD production might be one of the important effects of prednisolone via cAMP pathway in MCs.

The increase of antiglomerular basement membrane antibody following pauci-immune-type crescentic glomerulonephritis.

A 50-year-old woman was admitted because of high fever and fatigue. Proteinuria, hematuria, and elevated BUN (47.8 mg/dl) and creatinine (3.4 mg/dl) suggested rapidly progressive glomerulonephritis. The serological study revealed all negative results for rheumatoid factor, antinuclear antibody, serum cryoglobulins, MPO-ANCA, PR3-ANCA, and anti-streptolysin O. Antiglomerular basement membrane (GBM) antibody, as assessed by ELISA, was 11 EU (normal, <10). Kidney biopsy on the eighth hospital day demonstrated pauci-immune-type crescentic glomerulonephritis without ANCA. Methylprednisolone pulse therapy (500 mg/day, 3 days) and 45 mg/day prednisolone orally were started. At 3 weeks after kidney biopsy, the anti-GBM antibody value increased from 11 EU/ml to 116 EU/ml, and MPO and PR3-ANCA were still negative. HLA type was DR8 and DR 15(2), with a genotype of HLA-DRB1*08021 and HLA-DRB1*15011. The present case suggests that HLA-DR15 plays an important role on antibody production against alpha 3(IV) NC1 autoantigen after severe nephritis or tissue damage.

Increase of Urinary Extracellular-Superoxide Dismutase Level Correlated with Cyclic Adenosine Monophosphate.

Extracellular superoxide dismutase (EC‐SOD) is a secretory protein that is the major SOD isozyme in extracellular fluids. Plasma EC‐SOD levels are distributed in two discrete groups with the rare group having an enzyme with glycine instead of arginine‐213, which causes a 10‐fold higher serum level. Within the common phenotype group, the urinary EC‐SOD level was significantly correlated with the urinary excretion of N‐acetyl‐β‐D‐glucosaminidase (NAG), but not with serum EC‐SOD. EC‐SOD appears not to be leaked from the plasma by glomerular filtration, but rather to be secreted from the renal tubule or its surrounding tissues. The urinary EC‐SOD level was also significantly correlated with the urinary cyclic adenosine monophosphate (cAMP) level. cAMP analogues and adenylate cyclase modulators significantly stimulated the expression of EC‐SOD but not other SOD isozymes in cultured fibroblast cell lines. Moreover, injection of parathyroid hormone, in Ellsworth‐Howard tests, increased urinary EC‐SOD accompanied with the elevations of urinary cAMP and NAG. Together these observations suggest that factor(s) that stimulate the adenylate cyclase‐cAMP system regulate the urinary EC‐SOD level.

Membranous nephropathy (bubbling appearance and spike formation) without immunoglobulin deposition in a patient with systemic lupus erythematosus.

A 53-year-old Japanese man with systemic lupus erythematosus developed proteinuria and hematuria after a urinary stone episode. A light microscopic study of a kidney biopsy specimen demonstrated a bubbling appearance and spike formation of the basement membrane. Immunofluorescent studies revealed that there were no significant depositions of immunoglobulins, such as IgG (−), IgA (−), IgM (±), κ light chain (±), λ light chain (±), or C3 (−) in the glomerular capillary wall, though C1q was present as one-plus positive staining in mesangial areas. Electron microscopic studies showed that the thickness of the basement membrane varied from thin to thick without electron dense deposits, and that the cellular components of the podocyte were irregularly present in the basement membrane. Urinary protein decreased after the usage of prednisolone and mizoribine; however, proteinuria aggravated after an episode of urinary stone during the same treatment.