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-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...

Recent studies have suggested a role for aldosterone in the pathogenesis of renal injury. This study investigated the potential contributions of Rho-kinase and TGF-␤ pathways to aldosterone-induced renal injury. Rats were uninephrectomized and then treated for 5 wk with 1% NaCl in a drinking solution and one of the following: Vehicle (2% ethanol, subcutaneously; n ‫؍‬ 9); aldosterone (0.75 g/h, subcutaneously; n ‫؍‬ 9); or aldosterone ؉ fasudil, a specific Rho-kinase inhibitor (10 mg/kg per d, subcutaneously; n ‫؍‬ 8). Phosphorylation of myosin phosphate target subunit-1 (MYPT1) and Smad2/3 in renal cortical tissue was measured by Western blotting with anti-phospho MYPT1 and Smad2/3 antibodies, respectively. Rats that received aldosterone infusion exhibited hypertension and severe renal injury characterized by proteinuria, glomerular sclerosis, and tubulointerstitial fibrosis with increases in ␣-smooth muscle actin staining and numbers of monocytes/macrophages in the interstitium. Renal cortical mRNA levels of types I and III collagen, TGF-␤, connective tissue growth factor, and monocyte chemoattractant protein-1 as well as Smad2/3 phosphorylation were significantly increased in rats that received aldosterone infusion. All of these changes were associated with an increase in renal tissue MYPT1 phosphorylation. Treatment with fasudil did not alter BP but significantly ameliorated proteinuria and renal injury in rats that received aldosterone infusion. Severe glomerular injury and interstitial fibrosis were observed in rats that were treated long term with aldosterone/ salt (3,4). Moreover, exogenous infusion of aldosterone reversed the renoprotective effects of angiotensin II blockade in remnant kidney hypertensive rats (5). In stroke-prone spontaneously hypertensive rats (6); Dahl salt-sensitive hypertensive rats (7); and rats that were treated with angiotensin II and a nitric oxide synthase inhibitor (8), cyclosporine A (9), or radiation (10), treatment with mineralocorticoid receptor (MR) antagonists had no effect on systemic BP (SBP) but significantly ameliorated renal injury. In patients with chronic renal failure (11) and early diabetic nephropathy (12), the addition of MR antagonists to angiotensin-converting enzyme (ACE) inhibitors had no hemodynamic effects but markedly reduced proteinuria. Further studies also showed that monotherapy with MR antagonists was more effective than ACE inhibitors in reducing proteinuria in hypertensive patients (13,14). We recently found that aldosterone stimulates reactive oxygen species production (15) and induces cell proliferation (16) or deformation (16) in cultured glomerular mesangial cells. In addition, aldosterone stimulated collagen synthesis in cultured renal fibroblasts (17). Collectively, these data suggest that aldosterone has deleterious effects on the kidney that cannot be explained simply by BP changes. However, the precise mechanisms that are responsible for aldosterone-induced renal injury remain undetermined.Rho-kinase, an effector of the small G protein ...

These data indicate that mechanical stretch and angiotensin II synergistically increase NADPH oxidase expression in VSMCs, and suggest that part of this mechanism is mediated through an upregulation of the AT1 receptor induced by mechanical stretch. The combined effects of mechanical strain and angiotensin II might promote vascular damage through the production of superoxide in a hypertensive state.

Antihypertensive treatment with dihydropyridine calcium channel blockers elicits sympathetic nerve activation, which may contribute to cardiovascular events. However, recent clinical studies showed that treatment with azelnidipine, a new dihydropyridine calcium channel blocker, significantly reduced blood pressure in hypertensive patients while either maintaining or actually decreasing heart rate (HR). In this study, we exam- 1023)Key Words: azelnidipine, amlodipine, renal sympathetic nerve activity (RSNA), spontaneously hypertensive rats (SHR), heart rate (HR)