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, nϭ6); aldosterone (0.75 g/H, SC, nϭ8); aldosterone plus a selective mineralocorticoid receptor antagonist; eplerenone (0.125% in chow, nϭ8); aldosterone plus an antioxidant; and tempol (3 mmol/L in drinking solution, nϭ8). The activities of MAPKs, including extracellular signal-regulated kinases (ERK)1/2, c-Jun-NH 2 -terminal kinases (JNK), p38MAPK, and big-MAPK-1 (BMK1) in renal cortical tissues were measured by Western blot analysis. Aldosteroneinfused rats showed higher systolic blood pressure (165Ϯ5 mm Hg) and urinary excretion of protein (106Ϯ24 mg/d) than vehicle-infused rats (118Ϯ3 mm Hg and 10Ϯ3 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 (0.23Ϯ0.02) than vehicle-infused rats (0.09Ϯ0.01 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 (127Ϯ2 and 125Ϯ5 mm Hg), and the elevations of urinary excretion of protein (10Ϯ2 and 9Ϯ2 mg/day) or TBARS contents (0.08Ϯ0.01 and 0.11Ϯ0.01 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.

Whether temporary angiotensin II (AngII) blockade at the prediabetic stage attenuates renal injury in type 2 diabetic OLETF rats later in life was investigated. OLETF rats were treated with an AT 1 receptor antagonist (olmesartan, 0.01% in food), angiotensin-converting enzyme inhibitor (temocapril, 0.01% in food), a combination of the two, or hydralazine (25 mg/kg per d) at the prediabetic stage (4 to 11 wk of age) and then monitored without further treatment until 50 wk of age. At 11 wk of age, blood glucose levels and urinary protein excretion (U protein V) were similar between OLETF and control LETO rats. However, OLETF rats showed higher kidney AngII contents and type IV collagen mRNA expression than LETO rats at this age. These decreased with olmesartan, temocapril, and a combination of these but not with hydralazine. At 50 wk of age, diabetic OLETF rats showed higher BP, U protein V, and intrarenal AngII levels than LETO rats. Temporary AngII blockade did not affect glucose metabolism or the development of hypertension in OLETF rats but significantly suppressed proteinuria and ameliorated glomerular injury. However, no parameters were affected by temporary hydralazine treatment. The present study demonstrated that intrarenal AngII and type IV collagen expression are already augmented long before diabetes becomes apparent in OLETF rats. Furthermore, temporary AngII blockade at the prediabetic stage attenuates the progression of renal injury in these animals. These data suggest that early AngII blockade could be an effective strategy for preventing the development of type 2 diabetic renal injury later in life. D iabetic nephropathy is a major complication in diabetes and a leading cause of end-stage renal failure, which causes disabilities and a high mortality rate in patients with this disease (1). The mechanisms underlying the development of diabetic nephropathy are extremely complex; however, the potential role of the renin-angiotensin system (RAS) has been suggested (2-13). Recent studies indicate that in diabetes, intrarenal generation of angiotensin II (AngII) is elevated despite suppressed circulating RAS (5,6). Furthermore, AT 1 receptor blockers (ARB) or angiotensin-converting enzyme inhibitors (ACEI) have been shown to attenuate the progression of diabetic nephropathy (3,4,7-13). Several clinical trials have shown that ARB are more effective than traditional antihypertensive therapies in reducing renal failure progression in patients with type 2 diabetes and that the renoprotective effects of ARB are independent of their antihypertensive actions (8 -11). Of interest, it has also been shown that ACEI treatment of normotensive patients with diabetes and little or no proteinuria (early stages of diabetic nephropathy) results in long-term stabilization of plasma creatinine levels and urinary protein excretion rates (U protein V) (12,13). These observations suggest that angiotensin blockade has clinical benefits for patients who have diabetes and have no or early signals of renal disease.It has been sho...

It has recently been shown that glomerular mesangial injury is associated with increases in renal cortical reactive oxygen species (ROS) levels in rats treated chronically with aldosterone and salt. This study was conducted to determine the mechanisms responsible for aldosterone-induced ROS production in cultured rat mesangial cells (RMC). Oxidative fluorescent dihydroethidium was used to evaluate intracellular production of superoxide anion (O 2 ؊ ) in intact cells. The lucigeninderived chemiluminescence assay was used to determine NADPH oxidase activity. The staining of dihydroethidium was increased in a dose-dependent manner by aldosterone (1 to 100 nmol/L) with a peak at 3 h in RMC. Aldosterone A growing body of evidence supports a role for aldosterone in the progression of renal injury (1-4). In rats, chronic administrations of aldosterone and salt led to severe proteinuria and glomerular injury (5,6). Similarly, exogenous infusion of aldosterone reversed the renoprotective effects of angiotensin-converting enzyme (ACE) inhibitors in remnant kidney hypertensive rats (7) and stroke-prone, spontaneously hypertensive rats (SHRSP) (8). In addition, treatment with the mineralocorticoid receptor (MR) antagonists ameliorated glomerular injury in SHRSP (9) and in rats treated with angiotensin II (AngII) and nitric oxide synthase inhibitor (10), cyclosporine A (11) or radiation (12), independent of BP reduction. In patients with chronic renal failure (13) and early diabetic nephropathy (14), addition of a nonselective MR antagonist, spironolactone, to ACE inhibitors did not exert hemodynamic effects, but markedly reduced urinary excretion rate of protein (U protein V). These observations suggest that aldosterone has direct deleterious effects on the kidney via activation of the MR. However, the mechanisms responsible for the aldosterone/MR-induced renal injury remain undetermined.Recent studies have indicated the potential participation of reactive oxygen species (ROS) in the pathophysiology of aldosterone-induced cardiovascular tissue injury (5,(15)(16)(17)(18)(19)(20)(21)(22). In aldosterone/salt-treated hypertensive rats, vascular NADPH oxidase activity and ROS production were markedly increased (15,16). In these animals, treatment with an NADPH oxidase inhibitor, apocynin, prevented BP elevation and cardiovascular hypertrophy (17). It was also shown that treatment with a selective MR antagonist, eplerenone, improved endothelial dysfunction and reduced vascular superoxide anion (O 2 Ϫ ) generation in diet-induced atherosclerosis (18). Similarly, eplerenone reduced aortic atherosclerotic lesions and O 2 Ϫ generation in peritoneal macrophages of apolipoprotein E-deficient mice (19,20). Mazak et al. (21) showed that aldosterone potentiates AngII-induced signaling in vascular smooth muscle cells, and that these effects of aldosterone were blocked by antioxidants. The authors also showed that spironolactone decreased NADPH

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...

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Abstract-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 [ 3 H]-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.[3 H]-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 mol/L), a selective MR antagonist, or PD98059 (10 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 [ Key Words: aldosterone Ⅲ collagen Ⅲ fibroblasts Ⅲ mineralocorticoids R ecent studies have indicated the usefulness of mineralocorticoid receptor (MR) antagonists in ameliorating renal injury. [1][2][3][4][5][6][7][8][9][10][11][12][13] In stroke-prone spontaneously hypertensive rats 4 and rats treated with angiotensin II and a nitric oxide synthase inhibitor, 5 cyclosporine A, 6 or radiation, 7 MR antagonists had no effect on systemic blood pressure but markedly ameliorated glomerular and tubulointerstitial fibrosis. In clinical studies, the addition of a nonselective MR antagonist, spironolactone, to angiotensin-converting enzyme inhibitors had no hemodynamic effects but markedly reduced proteinuria in patients with chronic renal failure 8 and early diabetic nephropathy. 9 It has also been shown that monotherapy with spironolactone 10 or eplerenone, 11 a selective MR antagonist, is more effective than angiotensin-converting enzyme inhibitors in reducing proteinuria in hypertensive patients. Furthermore, White et al 12 showed that in hypertensive patients, eplerenone had a similar blood pressurelowering effect to that of a calcium antagonist, amlodipine, but reduced the urinary albumin-to-creatinine ratio to a greater extent. These observations suggest that MR blockade has renoprotective effects through mechanisms that cannot be simply explained by blood pressure and hemodynamic changes.R...

Abstract-Clinical reports indicate that patients with primary aldosteronism commonly have impaired glucose tolerance; however, the relationship between aldosterone and insulin signaling pathway has not been clarified. In this study, we examined the effects of aldosterone treatment on insulin receptor substrate-1 expression and insulin signaling pathway including Akt phosphorylation and glucose uptake in rat vascular smooth muscle cells. Insulin receptor substrate-1 protein expression and Akt phosphorylation were determined by Western blot analysis with anti-insulin receptor substrate-1 and phosphorylated-Akt antibodies, respectively. Glucose metabolism was evaluated using 3 H-labeled 2-deoxy-D-glucose uptake. Aldosterone (1-100 nmol/L) dose-dependently decreased insulin receptor substrate-1 protein expression with a peak at 18 hours (nϭ4). Aldosterone-induced degradation of insulin receptor substrate-1 was markedly attenuated by treatment with the selective mineralocorticoid receptor antagonist eplerenone (10 mol/L; nϭ4). Furthermore, degradation was blocked by the Src inhibitor PP1 (20 mol/L; nϭ4). Treatment with antioxidants, N-acetylcysteine (10 mmol/L), or ebselen (40 mol/L) also attenuated aldosterone-induced insulin receptor substrate-1 degradation (nϭ4). In addition, proteasome inhibitor MG132 (1 mol/L) prevented insulin receptor substrate-1 degradation (nϭ4). Aldosterone treatment abolished insulin-induced Akt phosphorylation (100 nmol/L; 5 minutes; nϭ4). Furthermore, aldosterone pretreatment decreased insulin-stimulated (100 nmol/L; 60 minutes; nϭ4) glucose uptake by 50%, which was reversed by eplerenone (10 mol/L; nϭ4). These data indicate that aldosterone decreases insulin receptor substrate-1 expression via Src and reactive oxygen species stimulation by proteasome-dependent degradation in vascular smooth muscle cells; thus, aldosterone may be involved in the pathogenesis of vascular insulin resistance via oxidative stress. Key Words: aldosterone Ⅲ oxidative stress Ⅲ insulin receptor substrate-1 Ⅲ insulin resistance Ⅲ type 2 diabetes mellitus Ⅲ metabolic syndrome Ⅲ eplerenone I nsulin resistance is a key attribute of type 2 diabetes and the metabolic syndrome. 1,2 Systemic glucose metabolism is maintained in the liver and skeletal muscle, and in the insulin resistant state, insulin-stimulated glucose uptake is attenuated and accompanied with subsequent increases in blood glucose concentration. High blood glucose concentration induces secretion of insulin from the pancreas and results in hyperinsulinemia. Both hyperglycemia and hyperinsulinemia affect the vasculature and are associated with microangiopathy, including retinopathy and nephropathy, and macroangiopathy, including cardiovascular disease and atherosclerosis. 3 Insulin resistance in the vasculature might also affect systemic glucose metabolism. However, the involvement of normal insulin signaling contributes to arteriosclerosis. 4,5 On the other hand, serine phosphorylation and degradation of insulin receptor substrate-1 (IRS-1) is a possible...

Background-Because the mechanism of the angiogenic property of nitric oxide (NO) was not fully understood in vivo, we focused on the role of vascular endothelial growth factor (VEGF) in angiogenesis induced by endothelial NO synthase (eNOS) gene transfer. Methods and Results-After intramuscular injection of eNOS DNA into a rat ischemic hindlimb, transfection of eNOS vector resulted in a significant increase in eNOS protein 1 week after transfection. In addition, tissue concentrations of nitrite and nitrate were significantly increased in rats transfected with the eNOS gene up to 2 weeks after transfection. The increase in tissue nitrite and nitrate concentrations was completely inhibited by N G -nitro-L-arginine methyl ester (L-NAME). In contrast, serum concentrations of nitrite and nitrate and blood pressure were not changed by eNOS gene transfer. Importantly, overexpression of the eNOS gene resulted in a significant increase in peripheral blood flow, whereas L-NAME inhibited the increase in blood flow. Interestingly, basal blood flow was significantly lower in rats treated with L-NAME than in control rats. A significant increase in capillary number was consistently detected in rats transfected with the eNOS gene at 4 weeks after transfection, accompanied by a significant increase in VEGF. Moreover, administration of neutralizing anti-VEGF antibody abolished the increase in blood flow and capillary density induced by eNOS plasmid injection. Conclusions-Overall, intramuscular injection of bovine eNOS plasmid induced therapeutic angiogenesis in a rat ischemic hindlimb model, a potential therapy for peripheral arterial disease.