Masanori Yoshizumi

Possible Contributions of Reactive Oxygen Species and Mitogen-Activated Protein Kinase to Renal Injury in Aldosterone/Salt-Induced Hypertensive Rats

Abstract—Studies were performed to test the hypothesis that reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) contribute to the pathogenesis of aldosterone/salt-induced renal injury. Rats were given 1% NaCl to drink and were treated with one of the following combinations for 6 weeks: vehicle (0.5% ethanol, SC, n=6); aldosterone (0.75 &mgr;g/H, SC, n=8); aldosterone plus a selective mineralocorticoid receptor antagonist; eplerenone (0.125% in chow, n=8); aldosterone plus an antioxidant; and tempol (3 mmol/L in drinking solution, n=8). The activities of MAPKs, including extracellular signal-regulated kinases (ERK)1/2, c-Jun-NH2-terminal kinases (JNK), p38MAPK, and big-MAPK-1 (BMK1) in renal cortical tissues were measured by Western blot analysis. Aldosterone-infused rats showed higher systolic blood pressure (165±5 mm Hg) and urinary excretion of protein (106±24 mg/d) than vehicle-infused rats (118±3 mm Hg and 10±3 mg/d). Renal cortical mRNA expression of p22phox, Nox-4, and gp91phox, measured by real-time polymerase chain reaction, was increased in aldosterone-infused rats by 2.3, 4.3, and 3.0-fold, respectively. Thiobarbituric acid-reactive substances (TBARS) content in renal cortex was also higher in aldosterone (0.23±0.02) than vehicle-infused rats (0.09±0.01 nmol/mg protein). ERK1/2, JNK, and BMK1 activities were significantly elevated in aldosterone-infused rats by 3.3, 2.3, and 3.0-fold, respectively, whereas p38MAPK activity was not changed. Concurrent administration of eplerenone or tempol to aldosterone-infused rats prevented the development of hypertension (127±2 and 125±5 mm Hg), and the elevations of urinary excretion of protein (10±2 and 9±2 mg/day) or TBARS contents (0.08±0.01 and 0.11±0.01 nmol/mg protein). Furthermore, eplerenone and tempol treatments normalized the activities of ERK1/2, JNK, and BMK1. These data suggest that ROS and MAPK play a role in the progression of renal injury induced by chronic elevations in aldosterone.

Fluid shear stress inhibits TNF-α activation of JNK but not ERK1/2 or p38 in human umbilical vein endothelial cells: Inhibitory crosstalk among MAPK family members

Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and IL-1 stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. TNF-α and IL-1 regulate gene expression in ECs, in part, by stimulating mitogen-activated protein kinases (MAPK), which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAPK in EC. To test this hypothesis, we determined the effects of flow (shear stress = 12 dynes/cm2) on TNF-α and IL-1-stimulated activity of three MAPK in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). Flow alone stimulated ERK1/2 and p38 activity but decreased JNK activity compared with static controls. TNF-α or IL-1 alone activated ERK1/2, p38, and JNK maximally at 15 min in HUVEC. Preexposing HUVEC for 10 min to flow inhibited TNF-α and IL-1 activation of JNK by 46% and 49%, respectively, but had no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, which inhibits flow-mediated ERK1/2 activation, prevented flow from inhibiting cytokine activation of JNK. Phorbol 12-myristate 13-acetate, which strongly activates ERK1/2, also inhibited TNF-α activation of JNK. These findings indicate that fluid shear stress inhibits TNF-α-mediated signaling events in HUVEC via the activation of the ERK1/2 signaling pathway. Inhibition of TNF-α signal transduction represents a mechanism by which steady laminar flow may exert atheroprotective effects on the endothelium.

Reactive Oxygen Species Activate p90 Ribosomal S6 Kinase via Fyn and Ras

Reactive oxygen species and growth factors stimulate similar intracellular signal transduction events including activation of Src kinase family members and extracellular signal-regulated kinases (ERK1/2). A potentially important downstream effector of Src and ERK1/2 is p90 ribosomal S6 kinase (p90RSK), which plays an important role in cell growth by activating several transcription factors as well as the Na+/H+ exchanger. In the present study, we determined whether H2O2 activates p90RSK to gain insight into signal transduction mechanisms activated by reactive oxygen species. H2O2 (200 μm) stimulated ERK1/2 and p90RSK activity in lymphocytes, endothelial cells, and fibroblasts. The MEK-1 inhibitor, PD98059 (30 μm), inhibited H2O2-mediated activation of ERK1/2 but not of p90RSK. An essential role for Fyn and Ras in p90RSK activation was suggested by five findings. 1) The tyrosine kinase inhibitor, herbimycin A, and the specific Src kinase family inhibitor, PP1, blocked p90RSK activation by H2O2 in a concentration-dependent manner. 2) p90RSK activation by H2O2 was significantly reduced in fibroblasts derived from transgenic mice deficient in Fyn, but not c-Src. 3) H2O2rapidly activated Ras (peak at 2–5 min), which preceded p90RSK activation (peak at 20 min). 4) Dominant negative Ras completely blocked H2O2-induced activation of p90RSK. 5) In Fyn−/− fibroblasts, activation of Ras by H2O2 was significantly attenuated. These results show essential roles for Fyn and Ras in H2O2-mediated activation of p90RSK and establish redox-sensitive regulation of Ras and p90RSK as a new function for Fyn.

Aldosterone Stimulates Collagen Gene Expression and Synthesis Via Activation of ERK1/2 in Rat Renal Fibroblasts

Recently, we demonstrated that in rats treated chronically with aldosterone and salt, severe tubulointerstitial fibrosis is associated with the activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERK1/2). Here, we investigated whether aldosterone stimulates collagen synthesis via ERK1/2-dependent pathways in cultured rat renal fibroblasts. Gene expression of mineralocorticoid receptor (MR) and types I, II, III, and IV collagen was measured by real-time polymerase chain reaction (PCR). MR protein expression and ERK1/2 activity were evaluated by Western blotting analysis with anti-MR and anti–phospho-ERK1/2 antibodies, respectively. Collagen synthesis was determined by [3H]-proline incorporation. Significant levels of MR mRNA and protein expression were observed in rat renal fibroblasts. Treatment with aldosterone (0.1 to 10 nmol/L) increased ERK1/2 phosphorylation in a concentration-dependent manner with a peak at 5 minutes. Aldosterone (10 nmol/L) also increased the mRNA levels of types I, III, and IV collagen at 36 hours but had no effect on the type II collagen mRNA level. [3H]-proline incorporation was significantly increased by aldosterone in both the medium and cell layer at 48 hours. Aldosterone-induced ERK1/2 phosphorylation was markedly attenuated by pretreatment with eplerenone (10 &mgr;mol/L), a selective MR antagonist, or PD98059 (10 &mgr;mol/L), a specific inhibitor of MAPK kinase/ERK kinase, which is the upstream activator of ERK1/2. In addition, both eplerenone and PD98059 prevented the aldosterone-induced increases in types I, III, and IV collagen mRNA and [3H]-proline incorporation. These results suggest that aldosterone stimulates collagen gene expression and synthesis via MR-mediated ERK1/2 activation in renal fibroblasts, which may contribute to the progression of aldosterone-induced tubulointerstitial fibrosis.

Involvement of Aldosterone and Mineralocorticoid Receptors in Rat Mesangial Cell Proliferation and Deformability

We demonstrated recently that chronic administration of aldosterone to rats induces glomerular mesangial injury and activates mitogen-activated protein kinases including extracellular signal-regulated kinases 1/2 (ERK1/2). We also observed that the aldosterone-induced mesangial injury and ERK1/2 activation were prevented by treatment with a selective mineralocorticoid receptor (MR) antagonist, eplerenone, suggesting that the glomerular mesangium is a potential target for injuries induced by aldosterone via activation of MR. In the present study, we investigated whether MR is expressed in cultured rat mesangial cells (RMCs) and involved in aldosterone-induced RMC injury. MR expression and localization were evaluated by Western blotting analysis and fluorolabeling methods. Cell proliferation and micromechanical properties were determined by [3H]-thymidine uptake measurements and a nanoindentation technique using an atomic force microscope cantilever, respectively. ERK1/2 activity was measured by Western blotting analysis with an anti-phospho–ERK1/2 antibody. Protein expression and immunostaining revealed that MR was abundant in the cytoplasm of RMCs. Aldosterone (1 to 100 nmol/L) dose-dependently activated ERK1/2 in RMCs with a peak at 10 minutes. Pretreatment with eplerenone (10 &mgr;mol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased [3H]-thymidine incorporation and decreased the elastic modulus, indicating cellular proliferative and deforming effects of aldosterone, respectively. These aldosterone-induced changes in cellular characteristics were prevented by pretreatment with eplerenone or an ERK (MEK) inhibitor, PD988059 (100 &mgr;mol/L). The results indicate that aldosterone directly induces RMC proliferation and deformability through MR and ERK1/2 activation, which may contribute to the pathogenesis of glomerular mesangial injury.

Androgen Receptor Gene Knockout Male Mice Exhibit Impaired Cardiac Growth and Exacerbation of Angiotensin II-induced Cardiac Fibrosis

Androgen has anabolic effects on cardiac myocytes and has been shown to enhance left ventricular enlargement and function. However, the physiological and patho-physiological roles of androgen in cardiac growth and cardiac stress-induced remodeling remains unclear. We aimed to clarify whether the androgen-nuclear androgen receptor (AR) system contributes to the cardiac growth and angiotensin II (Ang II)-stimulated cardiac remodeling by using systemic AR-null male mice. AR knock-out (ARKO) male mice, at 25 weeks of age, and age-matched wild-type (WT) male mice were treated with or without Ang II stimulation (2.0 mg/kg/day) for 2 weeks. ARKO mice with or without Ang II stimulation showed a significant reduction in the heart-to-body weight ratio compared with those of WT mice. In addition, echocardiographic analysis demonstrated impairments of both the concentric hypertrophic response and left ventricular function in Ang II-stimulated ARKO mice. Western blot analysis of the myocardium revealed that activation of extracellular signal-regulated kinases (ERK) 1/2 and ERK5 by Ang II stimulation were lower in ARKO mice than those of WT mice. Ang II stimulation caused more prominent cardiac fibrosis in ARKO mice than in WT mice with enhanced expression of types I and III collagen and transforming growth factor-β1 genes and with increased Smad2 activation. These results suggest that, in male mice, the androgen-AR system participates in normal cardiac growth and modulates cardiac adaptive hypertrophy and fibrosis during the process of cardiac remodeling under hypertrophic stress.

Quercetin glucuronide prevents VSMC hypertrophy by angiotensin II via the inhibition of JNK and AP-1 signaling pathway

We previously reported that quercetin, a bioflavonoid belonging to polyphenols, inhibited Angiotensin II (Ang II)-induced vascular smooth muscle cell (VSMC) hypertrophy through the inhibition of c-Jun N-terminal kinase (JNK) activation. However, we recently found that orally administered quercetin appeared in plasma as glucuronide-conjugated forms in rats and humans. Therefore we examined the effect of chemically synthesized quercetin glucuronide on Ang II-induced mitogen-activated protein (MAP) kinase activation and hypertrophy of cultured rat aortic smooth muscle cells (RASMC). Ang II activated extracellular signal-regulated kinase (ERK)1/2, JNK, and p38 in RASMC. Ang II-induced JNK activation was inhibited by quercetin 3-O-beta-d-glucuronide (Q3GA) whereas ERK1/2 and p38 activations were not affected. Q3GA scavenged 1,1-diphenyl-2-picrylhydrazyl radical measured by a method of electron paramagnetic resonance. Q3GA also inhibited Ang II-induced increases in activator protein-1 (AP-1) DNA binding, a downstream transcription factor of JNK. Finally, Ang II-induced [3H]leucine incorporation into RASMC was abolished by Q3GA. These findings suggest that the preventing effect of Q3GA on Ang II-induced VSMC hypertrophy is attributable in part to its inhibitory effect on JNK and the AP-1 signaling pathway. Q3GA would be an active metabolite of quercetin in plasma and may possess a preventing effect for cardiovascular diseases relevant to VSMC growth.

Ebselen attenuates oxidative stress-induced apoptosis via the inhibition of the c-Jun N-terminal kinase and activator protein-1 signalling pathway in PC12 cells.

Ebselen (2‐phenyl‐1,2‐benzisoselenazol‐3[2H]‐one) is a selenoorganic compound exhibiting both glutathione peroxidase activity and antioxidant activity. Although it has been reported that ebselen is effective for oxidative stress‐induced neuronal damage both in vivo and clinically, the precise mechanisms of the efficacy have not yet been elucidated. Thus, we hypothesized that ebselen may affect reactive oxygen species‐induced mitogen‐activated protein (MAP) kinase activation in cultured PC12 cells. Our findings showed that hydrogen peroxide (H2O2) stimulated rapid and significant activation of extracellular signal‐regulated kinase (ERK)1/2, c‐Jun N‐terminal kinase (JNK) and p38 in PC12 cells, which is a model of catecholamine‐containing neurons. H2O2‐induced JNK activation was inhibited by ebselen, whereas ERK1/2 and p38 activation by H2O2 were not affected by ebselen. Inhibition by ebselen of H2O2‐induced hydroxyl radical generation in PC12 cells was observed using electron paramagnetic resonance measurements. Ebselen also inhibited H2O2‐induced increases in DNA binding activity of activator protein‐1 (AP‐1), a downstream transcription factor of JNK, composed of the c‐Jun homo/heterodimer. Finally, pretreatment of cells with ebselen resulted in a significant recovery from cell death including apoptosis by H2O2 in PC12 cells. These findings suggest that ebselen attenuates oxidative stress‐induced neuronal cell death through the inhibition of the JNK and AP‐1 signalling pathway. Thus, inhibition of JNK by ebselen may imply its usefulness for treatment of ischaemic cerebral diseases relevant to neuronal cell death.

The SOD mimetic tempol ameliorates glomerular injury and reduces mitogen-activated protein kinase activity in Dahl salt-sensitive rats.

It was shown recently that renal injury in Dahl salt-sensitive (DS) hypertensive rats is accompanied by mitogen-activated protein kinase (MAPK) activation. The present study was conducted to elucidate the contribution of reactive oxygen species to MAPK activities and renal injury in DS rats. DS rats were maintained on high salt (H; 8.0% NaCl; n = 7) or low salt (L; 0.3% NaCl; n = 6) diets; H + a superoxide dismutase mimetic, tempol (3 mmol/L in drinking water; n = 8); or H + hydralazine (0.5 mmol/L in drinking water; n = 8) for 4 wk. Mean BP (MBP) in DS/H and DS/L rats was 185 +/- 7 and 113 +/- 3 mmHg, respectively. DS/H rats showed a higher ratio of urinary protein excretion and creatinine (U(protein)V/U(cr)V; 20.3 +/- 1.1) and a higher cortical collagen content (22 +/- 1 micro g/mg) than in DS/L rats (2.4 +/- 0.1 and 13 +/- 1 micro g/mg, respectively). The expression of p22-phox and Nox-1, essential components of NAD(P)H oxidase, in renal cortical tissue was approximately threefold higher in DS/H rats than in DS/L rats. Increased activities of renal cortical MAPK, including extracellular signal-regulated kinases (ERK) 1/ERK2 and c-Jun NH(2)-terminal kinases (JNK) were also observed in DS/H rats by 7.0 +/- 0.7- and 4.3 +/- 0.2-fold, respectively. Tempol treatment significantly decreased MBP (128 +/- 3 mmHg), U(protein)V/U(cr)V (4.8 +/- 0.4), and cortical collagen content (14 +/- 1 micro g/mg) and normalized ERK1/ERK2 and JNK activities in DS/H rats. Histologically, tempol markedly ameliorated progressive sclerotic and proliferative glomerular changes in DS/H rats. Hydralazine-treated DS/H rats showed similar MBP (127 +/- 5 mmHg) to tempol-treated DS/H rats. Hydralazine also decreased U(protein)V/U(cr)V (16.2 +/- 1.5) and cortical collagen content (19 +/- 1 micro g/mg) in DS/H rats. However, these values were significantly higher than those of tempol-treated rats. Furthermore, although hydralazine significantly reduced JNK activity (-56 +/- 3%), ERK1/ERK2 activities were unaffected. These data suggest that reactive oxygen species, generated by NAD(P)H oxidase, contribute to the progression of renal injury through ERK1/ERK2 activation in DS/H hypertensive rats.

Aldosterone Stimulates Vascular Smooth Muscle Cell Proliferation Via Big Mitogen-Activated Protein Kinase 1 Activation

The nongenomic effects of aldosterone have been implicated in the pathogenesis of various cardiovascular diseases. Aldosterone-induced nongenomic effects are attributable in part to the activation of extracellular signal-regulated kinase 1/2 (ERK1/2), a classical mitogen-activated protein (MAP) kinase. Big MAP kinase 1 (BMK1), a newly identified MAP kinase, has been shown to be involved in cell proliferation, differentiation, and survival. We examined whether aldosterone stimulates BMK1-mediated proliferation of cultured rat aortic smooth muscle cells (RASMCs). Mineralocorticoid receptor (MR) expression and localization were evaluated by Western blotting analysis and fluorolabeling methods. ERK1/2 and BMK1 activities were measured by Western blotting analysis with the respective phosphospecific antibodies. Cell proliferation was determined by Alamar Blue colorimetric assay. Aldosterone (0.1 to 100 nmol/L) dose-dependently activated BMK1 in RASMCs, with a peak at 30 minutes. To clarify whether aldosterone-induced BMK1 activation is an MR-mediated phenomenon, we examined the effect of eplerenone, a selective MR antagonist, on aldosterone-induced BMK1 activation. Eplerenone (0.1 to 10 &mgr;mol/L) dose-dependently inhibited aldosterone-induced BMK1 activation in RASMCs. Aldosterone also stimulated RASMC proliferation, which was inhibited by eplerenone. Aldosterone-mediated phenomena were concluded to be attributable to a nongenomic effect because cycloheximide failed to inhibit aldosterone-induced BMK1 activation. Transfection of dominant-negative MAP kinase/ERK kinase 5 (MEK5), which is an upstream regulator of BMK1, partially inhibited aldosterone-induced RASMC proliferation, which was almost completely inhibited by MEK inhibitor PD98059. In addition to the classical steroid activity, rapid nongenomic effects induced by aldosterone may represent an alternative etiology for vascular diseases such as hypertension.