Abstract-Reactive oxygen species (ROS) participate in cardioprotection of ischemic reperfusion (I/R) injury via preconditioning mechanisms. Mitochondrial ROS have been shown to play a key role in this process. Angiotensin II (Ang II) exhibits pharmacological preconditioning; however, the involvement of NAD(P)H oxidase, known as an ROS-generating enzyme responsive to Ang II stimuli, in the preconditioning process remains unclear. We compared the effects of 5-hydroxydecanoate (5-HD; an inhibitor of mitochondrial ATP-sensitive potassium channels), apocynin (an NAD(P)H oxidase inhibitor), and 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol; a membrane permeable radical scavenger) on pharmacological preconditioning by Ang II in rat cardiac I/R injury in vivo. Treatment with a pressor dose of Ang II before a 30-minute coronary occlusion reduced infarct size as determined 24 hours after reperfusion. The protective effects of Ang II were eliminated by pretreatment with 5-HD or apocynin, similar to tempol. Both 5-HD and apocynin suppressed the enhanced cardiac lipid peroxidation and activation of the apoptosis signal-regulating kinase/p38, c-Jun NH 2 -terminal kinase (JNK) pathways, but not the Raf/MEK/extracellular signal-regulated kinase pathway, elicited by acutely administered Ang II. Apocynin but not 5-HD suppressed Ang II-induced augmentations of the NAD(P)H oxidase complex formation (p47 phox , p22 phox , and Rac-1) and its activity in the heart. Finally, 5-HD suppressed superoxide production by isolated cardiac mitochondria without any effect on their respiration. These results suggest that the preconditioning effects of Ang II for cardiac I/R injury may be mediated by cardiac mitochondriaderived ROS enhanced through NAD(P)H oxidase via JNK and p38 mitogen-activated protein kinase activation. Key Words: angiotensin Ⅲ antioxidants Ⅲ free radicals Ⅲ heart T he opening of cardiac mitochondrial ATP-sensitive potassium (mitoK ATP ) channels protects against subsequent ischemic reperfusion (I/R) tissue injury in the heart. 1,2 Receptor activation ligands, such as bradykinin, opioids, and acetylcholine, have been shown to trigger a preconditioning state 3 similar to mitoK ATP channel openers, which are inhibited by 5-hydroxydecanoate (5-HD), a specific mitoK ATP channel inhibitor. In the process of mitoK ATP channel-mediated preconditioning, the involvement of mitochondriaderived free radicals 4,5 and the posteffectors mitogen-activated protein (MAP) kinases, especially p38 MAP kinase 6 -8 has been strongly suggested. 1,9 Angiotensin II (Ang II) is well known as a powerful inducer of oxidative stress to cardiovascular tissues, and the reactive oxygen species (ROS) generated participate in Ang II-induced intracellular signaling pathways. 10 We demonstrated that acutely administered Ang II stimulates redoxsensitive cardiac MAP kinase activation, which was eliminated by tempol, a superoxide dismutase mimetic, in vivo. 11 To date, NAD(P)H oxidase has been considered a source of ROS corresponding to Ang II effects in c...

Abstract-Reactive oxygen species (ROS) are key mediators in signal transduction of angiotensin II (Ang II). However, roles of vascular mitochondria, a major intracellular ROS source, in response to Ang II stimuli have not been elucidated. This study aimed to examine the involvement of mitochondria-derived ROS in the signaling pathway and the vasoconstrictor mechanism of Ang II. Using 5-hydroxydecanoate (5-HD; a specific inhibitor of mitochondrial ATP-sensitive potassium [mitoK ATP ] channels) and tempol (a superoxide dismutase mimetic), the effects of Ang II and diazoxide (a mitoK ATP channel opener) were compared on redox-sensitive mitogen-activated protein (MAP) kinase activation in rat vascular smooth muscle cells (RVSMCs) in vitro and in rat aorta in vivo. Stimulation of RVSMCs by Ang II or diazoxide increased phosphorylated MAP kinases (ERK1/2, p38, and JNK), as well as superoxide production, which were then suppressed by 5-HD pretreatment in a dose-dependent manner, except for ERK1/2 activation by Ang II. The same events were reproduced in rat aorta in vivo. Ang II-like diazoxide depolarized the mitochondrial membrane potential (⌬⌿ M ) of RVSMCs determined by JC-1 fluorescence, which was inhibited by 5-HD. 5-HD did not modulate Ang II-induced calcium mobilization in RVSMCs and did not affect on the vasoconstrictor effect in either acute or chronic phases of Ang II-induced hypertension. These results reveal that Ang II stimulates mitochondrial ROS production through the opening of mitoK ATP channels in the vasculature-like diazoxide, leading to reduction of ⌬⌿ M and redox-sensitive activation of MAP kinase; however, generated ROS from mitochondria do not contribute to Ang II-induced vasoconstriction. Key Words: angiotensin Ⅲ oxidative stress R eactive oxygen species (ROS) are recognized as mediators of vascular signal transduction and are involved in activation of mitogen-activated protein (MAP) kinases. 1 Participation of ROS in blood pressure regulation has been shown in Ang II-induced hypertension 2-4 and other hypertensive models. [5][6][7] We recently demonstrated time-dependent transition of ROS sensitivity of Ang II hypertension in rats, in which in the early phase of Ang II infusion, high blood pressure does not depend on ROS production, but thereafter it shifts to being ROS-sensitive. 8 In this study, it was also found that in the acute phase of Ang II infusion, MAP kinases are stimulated through a ROS sensitive mechanism.Recent reports have supported the hypothesis that the enzyme NAD(P)H oxidase plays a major role as the most important source of superoxide anions in vascular cells and contributes significantly to the functional and structural alterations present in hypertension or atherosclerosis. 9,10 It has been further proposed that NAD(P)H oxidase is essential for production of superoxide in response to Ang II stimuli to vascular tissues. Mice deficient in the p47 phox gene showed significantly lower arterial blood pressure elevation during chronic Ang II infusion. 11 Moreover, endothelial ce...

Abstract-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 [ 3 H]-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 mol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased Key Words: mineralocorticoids Ⅲ aldosterone T he utility of mineralocorticoid receptor (MR) antagonists in renal injury has been suggested in preclinical and clinical studies. 1-12 MR blockade had no effect on systemic blood pressure but markedly ameliorated glomerular injury in stroke-prone spontaneously hypertensive rats 3 and rats treated with angiotensin II (Ang II) and an NO synthase inhibitor, 4 cyclosporine A 5 or radiation. 6 In patients with chronic renal failure 7 and early diabetic nephropathy, 8 addition of a nonselective MR antagonist, spironolactone, to angiotensinconverting enzyme (ACE) inhibitors had no hemodynamic effects but markedly reduced the urinary protein excretion rate (U protein V). For hypertensive patients, it has also been indicated that monotherapy with spironolactone 9 or a selective MR antagonist, eplerenone, 10 is more effective than ACE inhibitors in reducing U protein V. Furthermore, White et al 11 showed that in hypertensive patients, eplerenone has a similar blood pressure-lowering effect to a calcium antagonist, amlodipine, but reduced the urinary albumin-to-creatinine ratio to a greater extent than amlodipine. Thus, these observations support the notion that MR blockade has renoprotective effects through mechanisms that cannot be simply explained by hemodynamic changes.We demonstrated recently that chronic administration of aldosterone to rats induced glomerular injury characterized by mesangial matrix expansion and cell overgrowth. 12 We also observed that the aldosterone-induced glomerular injury was prevented by treatment with eplerenone. These results in...

Recent studies demonstrated a possible role of aldosterone in mediating cell senescence. Thus, the aim of this study was to investigate whether aldosterone induces cell senescence in the kidney and whether aldosterone-induced renal senescence affects the development of renal injury. Aldosterone infusion (0.75 μg/h) into rats for 5 weeks caused hypertension and increased urinary excretion rates of proteins and N-acetyl-β-D-glucosaminidase. Aldosterone induced senescence-like changes in the kidney, exhibited by increased expression of the senescence-associated β-galactosidase, overexpression of p53 and cyclin-dependent kinase inhibitor (p21), and decreased expression of SIRT1. These changes were abolished by eplerenone (100 mg/kg/d), a mineralocorticoid receptor (MR) antagonist, but unaffected by hydralazine (80 mg/liter in drinking water). Furthermore, aldosterone induced similar changes in senescence-associated β-galactosidase, p21, and SIRT1 expression in cultured human proximal tubular cells, which were normalized by an antioxidant, N-acetyl L-cysteine, or gene silencing of MR. Aldosterone significantly delayed wound healing and reduced the number of proliferating human proximal tubular cells, while gene silencing of p21 diminished the effects, suggesting impaired recovery from tubular damage. These findings indicate that aldosterone induces renal senescence in proximal tubular cells via the MR and p21-dependent pathway, which may be involved in aldosterone-induced renal injury.

Objectives-Clinical studies have indicated the beneficial effect of an L/N-type calcium channel blocker (CCB), cilnidipine, on the progression of proteinuria in hypertensive patients compared with an L-type CCB, amlodipine. In the present study, we examined the effects of cilnidipine and amlodipine on the renal injury in spontaneously hypertensive rat/ND mcr-cp (SHR/ND) and their underlying mechanism.Methods and results-SHR/ND were treated with vehicle (n = 10), cilnidipine [33 mg/kg per day, orally (p.o.); n = 11] or amlodipine (20 mg/kg per day, p.o.; n = 9) for 20 weeks. SHR/ND developed proteinuria in an age-dependent manner. Cilnidipine suppressed the proteinuria greater than amlodipine did. The immunohistochemical analysis showed that N-type calcium channel and Wilm's tumor factor, a marker of podocyte, were co-expressed. SHR/ND had significantly greater desmin staining, an indicator of podocyte injury, with lower podocin and nephrin expression in the glomeruli than Wistar-Kyoto rat or SHR. Cilnidipine significantly prevented the increase in desmin staining and restored the glomerular podocin and nephrin expression compared with amlodipine. Cilnidipine also prevented the increase in renal angiotensin II content, the expression and membrane translocation of NADPH oxidase subunits and dihydroethidium staining in SHR/ND. In contrast, amlodipine failed to change these renal parameters.Conclusion-These data suggest that cilnidipine suppressed the development of proteinuria greater than amlodipine possibly through inhibiting N-type calcium channel-dependent podocyte injury in SHR/ND.

Recent studies have indicated that both endothelin (ET) and angiotensin (Ang

Recent studies have suggested that aldosterone plays a role in the pathogenesis of renal injury. In this study, we investigated whether local angiotensin II (Ang II) activity contributes to the progression of renal injury in aldosterone/salt-induced hypertensive rats. Uninephrectomized rats were treated with 1% NaCl in a drinking solution and one of the following combinations for 6 weeks: vehicle (2% ethanol, s.c.; n=9), aldosterone (0.75 mug/h, s.c.; n=8), aldosterone+Ang II type 1 receptor blocker olmesartan (10 mg/kg/day, p.o.; n=8), or aldosterone+olmesartan (100 mg/kg/day, p.o.; n=9). Aldosterone/salt-treated hypertensive rats exhibited severe proteinuria and renal injury characterized by glomerular sclerosis and tubulointerstitial fibrosis. Aldosterone/salt-induced renal injury was associated with augmented expression of angiotensin converting enzyme and Ang II levels in the renal cortex and medullary tissues. Renal cortical and medullary mRNA expression of transforming growth factor-beta (TGF-beta) and connective tissue growth factor (CTGF) as well as the collagen contents were increased in aldosterone/salt-treated hypertensive rats. Treatment with olmesartan (10 or 100 mg/kg/day) had no effect on blood pressure but attenuated proteinuria in a dose-dependent manner. Olmesartan at 10 mg/kg/day tended to decrease renal cortical and medullary Ang II levels, TGF-beta and CTGF expression, and collagen contents; however, these changes were not significant. On the other hand, an ultrahigh dose of olmesartan (100 mg/kg/day) significantly decreased these values and ameliorated renal injury. These data suggest that augmented local Ang II activity contributes, at least partially, to the progression of aldosterone/salt-dependent renal injury.

We examined the effects of adrenomedullin on cardiac oxidative stress and collagen accumulation in aldosterone-dependent malignant hypertensive rats. Spontaneously hypertensive rats (SHRs) were treated with one of the following combinations for 4 weeks: tap water and vehicle [0.5% ethanol, subcutaneously (s.c.), n ϭ 5], 1% NaCl in drinking water and vehicle (n ϭ 8), 1% NaCl and aldosterone (0.75 g/h s.c., n ϭ 8), and 1% NaCl, aldosterone, and adrenomedullin (1.3 g/kg/h s.c., n ϭ 8). Systolic blood pressure (SBP) and left ventricular (LV) weight were higher in aldosterone-treated SHRs than vehicle-or vehicle/1% NaCl-treated SHRs. Thiobarbituric acid reactive substances (TBARS) levels and NADPH oxidase activity in LV tissues of aldosterone-treated SHRs were also higher than those of vehicle-or vehicle/1% NaCl-treated SHRs, and these changes were associated with increases in LV mRNA levels of p22phox, gp91phox, fibronectin, collagen types I and III, as well as collagen content. Treatment with adrenomedullin did not alter SBP or LV weight but attenuated aldosterone-induced increases in TBARS levels, NADPH oxidase activity, and mRNA levels of p22phox, gp91phox, fibronectin, collagen types I and III, as well as collagen content in LV tissues. These data suggest that NADPH oxidase-mediated reactive oxygen species production is involved in the pathogenesis of cardiac collagen accumulation in aldosterone-dependent malignant hypertensive rats and that the cardioprotective effects of adrenomedullin are mediated through the suppression of this pathway.