Hydrogen peroxide increases extracellular matrix mRNA through TGF-beta in human mesangial cells.

BACKGROUND: Reactive oxygen species (ROS) are excessively produced in pathologic states, including many renal diseases. Transforming growth factor-beta (TGF-beta) may mediate renal fibrotic injury, and ROS may act through the TGF-beta pathway to exert a profibrotic effect. METHODS: The expression of TGF-beta1 and extracellular matrix (ECM) components were assessed in cultured human mesangial cells (HMCs) incubated with glucose oxidase (GO), an enzyme that continuously generates hydrogen peroxide from glucose. A neutralizing anti-TGF-beta antibody was added to test the hypothesis that hydrogen peroxide acts through activation of the TGF-beta pathway to stimulate ECM expression. RESULTS: Northern blot analysis revealed significantly increased steady-state levels of TGF-beta1 and ECM proteins (collagen types I, III, and IV, and fibronectin) by approximately twofold. While no significant effect on mRNA stability after treatment with GO was observed, other studies employing promoter-reporter assays, competitive-quantitative reverse transcription-polymerase chain reaction, mink lung epithelial cell proliferation assay, and TGF-beta1 enzyme-linked immunosorbent assay all demonstrated significant stimulation by GO (>1.5-fold) of TGF-beta1 promoter activity, mRNA level, bioactivity, and protein production, respectively. Catalase pretreatment prevented the GO-induced stimulation of TGF-beta1 mRNA. When incubations were performed with a panselective neutralizing anti-TGF-beta antibody, the GO-stimulated expression of ECM molecules was prevented. CONCLUSIONS: GO-induced hydrogen peroxide production induces TGF-beta1 synthesis and thereby increases ECM gene expression in cultured HMCs. These cellular responses may underlie the development and progression of renal diseases characterized by oxidative stress.

Age-related progressive renal fibrosis in rats and its prevention with ACE inhibitors and taurine.

Our previous studies demonstrated an increased reactive oxygen species (ROS) production, as well as transforming growth factor-beta1 (TGF-beta1) expression in the rat kidney with aging. In the present study, we examined the effect of aging on extracellular matrix (ECM) accumulation and the effects of treatment with angiotensin-converting enzyme inhibitors (captopril and lisinopril) and taurine, an antioxidant amino acid. Age-related increases in types I and IV collagen and fibronectin mRNA expression were found at 24 and 30 mo of age. In contrast, type III collagen only increased in 30-mo-old rats. Captopril-, lisinopril-, and taurine-treated animals showed a statistically significant decrease in ECM protein expression at both ages. Moreover, treatment with taurine reduced the TGF-beta1 mRNA levels in 24- and 30-mo-old rats by 40%. Taurine also completely blocked increases in type I and type IV collagen expression in mesangial cells in response to TGF-beta1. Our results demonstrate a protective role from both converting enzyme inhibitors and taurine in the age-related progressive renal sclerosis. In addition, taking into account that taurine is considered as an antioxidant amino acid, present data suggest a role for ROS in age-related progressive renal fibrosis, perhaps through interactions with the TGF-beta1 pathway.

Oxidant/antioxidant balance in isolated glomeruli and cultured mesangial cells.

The mechanisms responsible for age-related glomerular sclerosis (GS) have not been clearly identified. The present experiments were aimed at assessing the importance of the oxidant/antioxidant balance in the early stages of this process. For this purpose, the renal function (biochemical and clearance studies), some characteristics of isolated glomeruli, and reactive oxygen production (superoxide anion, hydrogen peroxide) as well as the antioxidant ability (superoxide dismutase, catalase, glutathione peroxidase) of glomeruli and cultured mesangial cells were studied in 3- and 18-month-old Fischer 344 rats (YOUNG and OLD rats, respectively). OLD animals show a normal renal function, increased urine protein excretion, and augmented protein glomerular content, an indirect index of GS. Isolated glomeruli from these rats produced increased amounts of superoxide anion and hydrogen peroxide, and catalase activity was increased. The glomerular thiobarbituric acid-reactive substances (TBARS) content was higher in OLD than in YOUNG animals. Similar results were obtained in cultured mesangial cells. In summary, the present results demonstrate, at an early stage of rat GS development, an association between the functional and structural changes of this process and an increased TBARS content (likely indicative of lipid oxidative damage) at the glomerular structures as well as in cultured mesangial cells. More extensive studies are needed to confirm the nature of this association.

Reactive oxygen species and platelet-activating factor synthesis in age-related glomerulosclerosis.

This work deals with the hypothesis that an increased synthesis of reactive oxygen species (ROS) or platelet-activating factor (PAF) (or both) may be implied in the genesis of age-related glomerulosclerosis. Plasma concentration and urinary excretion of both thiobarbituric acid-reactive substances (TBARS) and PAF were measured in young and old human beings and rats. Moreover, these same parameters as well as H2O2 synthesis and reduced glutathione (GSH) content were measured in isolated glomeruli of young (3 months) and old (18 months) Wistar rats. H2O2 synthesis and GSH content were also measured in cultured rat mesangial cells from young and old animals. Both human beings and rats showed a decreased glomerular filtration rate and an increased urinary protein excretion with respect to young individuals. Isolated glomeruli from old animals showed a higher protein content and a lower number of cell nuclei than those from young rats. No changes were detected in plasma concentration and urinary excretion of TBARS and PAF in either human beings or rats. Glomeruli from 18-month-old rats exhibited a higher content of TBARS and GSH and an increased synthesis of H2O2 and PAF than did those from 3-month-old rats. GSH content and H2O2 synthesis were higher in cultured cells from old rats than in those from young rats. These results point to the possibility that ROS or PAF could mediate some of the changes that characterize age-related glomerulosclerosis.

A dual effect of somatostatin on the proliferation of cultured rat mesangial cells.

The present experiments were designed to analyze the ability of somatostatin to modulate the proliferation of cultured rat mesangial cells. In the absence of fetal calf serum, somatostatin stimulated cell proliferation in a dose-dependent manner. In contrast, in proliferating cells, somatostatin inhibited cell proliferation, also in a dose-dependent fashion. Zaprinast, a rather specific cyclic GMP phosphodiesterase blocker, inhibited the somatostatin-dependent proliferation in the absence of growth factors. However, it potentiated the inhibitory effect on proliferating cells. These results support a dual role for somatostatin in the regulation of mesangial cell proliferation. The inhibitory effect of the peptide may be mediated by cyclic GMP.