Kayoko Miyata

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.

Aldosterone Stimulates Reactive Oxygen Species Production through Activation of NADPH Oxidase in Rat Mesangial Cells

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 lucigenin-derived 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 (100 nmol/L for 3 h) also significantly increased NADPH oxidase activity from 232 +/- 18 to 346 +/- 30 cpm/5 x 10(4) cells. Immunoblotting data showed that aldosterone (100 nmol/L for 3 h) increased p47phox and p67phox protein levels in the membrane fraction by approximately 2.1- and 2.3-fold, respectively. On the other hand, mRNA expression of NADPH oxidase membrane components, p22phox, Nox-1, and Nox-4, were not altered by aldosterone (for 3 to 12 h) in RMC. Pre-incubation with the selective mineralocorticoid receptor (MR) antagonist, eplerenone (10 micromol/L), significantly attenuated aldosterone-induced O(2)(-) production, NADPH oxidase activation and membranous translocation of p47phox and p67phox. These results suggest that aldosterone-induced ROS generation is associated with NAPDH oxidase activation through MR-mediated membranous translocation of p47phox and p67phox in RMC. These cellular actions of aldosterone may play a role in the pathogenesis of glomerular mesangial injury.

Urinary Angiotensinogen as a Novel Biomarker of the Intrarenal Renin-Angiotensin System Status in Hypertensive Patients

We reported previously that urinary angiotensinogen (UAGT) levels provide a specific index of the intrarenal renin-angiotensin system (RAS) status in angiotensin II–dependent hypertensive rats. To study this system in humans, we recently developed a human angiotensinogen ELISA. To test the hypothesis that UAGT is increased in hypertensive patients, we recruited 110 adults. Four subjects with estimated glomerular filtration levels <30 mL/min per 1.73 m2 were excluded because previous studies have already shown that UAGT is highly correlated with estimated glomerular filtration in this stage of chronic kidney disease. Consequently, 106 paired samples of urine and plasma were analyzed from 70 hypertensive patients (39 treated with RAS blockers [angiotensin-converting enzyme inhibitors or angiotensin II type 1 receptor blockers; systolic blood pressure: 139±3 mm Hg] and 31 not treated with RAS blockers [systolic blood pressure: 151±4 mm Hg]) and 36 normotensive subjects (systolic blood pressure: 122±2 mm Hg). UAGT, normalized by urinary concentrations of creatinine, were not correlated with race, gender, age, height, body weight, body mass index, fractional excretion of sodium, plasma angiotensinogen levels, or estimated glomerular filtration. However, UAGT/urinary concentration of creatinine was significantly positively correlated with systolic blood pressure, diastolic blood pressure, urinary albumin:creatinine ratio (r=0.5994), and urinary protein:creatinine ratio (r=0.4597). UAGT/urinary concentration of creatinine was significantly greater in hypertensive patients not treated with RAS blockers (25.00±4.96 &mgr;g/g) compared with normotensive subjects (13.70±2.33 &mgr;g/g). Importantly, patients treated with RAS blockers exhibited a marked attenuation of this augmentation (13.26±2.60 &mgr;g/g). These data indicate that UAGT is increased in hypertensive patients, and treatment with RAS blockers suppresses UAGT, suggesting that the efficacy of RAS blockade to reduce the intrarenal RAS activity can be assessed by measurements of UAGT.

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.

Temporary angiotensin II blockade at the prediabetic stage attenuates the development of renal injury in type 2 diabetic rats.

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.

Urinary angiotensinogen as a potential biomarker of severity of chronic kidney diseases

We previously reported that urinary excretion rates of angiotensinogen (AGT) provide a specific index of the activity of the intrarenal renin-angiotensin system in angiotensin II-dependent hypertensive rats. Meanwhile, we have recently developed direct enzyme-linked immunosorbent assays (ELISAs) to measure plasma and urinary AGT in humans. This study was performed to test a hypothesis that urinary AGT levels are enhanced in chronic kidney disease (CKD) patients and correlated with some clinical parameters. Eighty patients with CKD (37 women and 43 men, from 18 to 94 years old) and seven healthy volunteers (two women and five men, from 27 to 43 years old) were included. Plasma AGT levels showed a normal distribution; however, urinary AGT-creatinine ratios (UAGT/UCre) deviated from the normal distribution. When a logarithmic transformation was executed, Log(UAGT/UCre) levels showed a normal distribution. Therefore, Log(UAGT/UCre) levels were used for further analyses. Log(UAGT/UCre) levels were not correlated with age, gender, height, body weight, body mass index, systolic blood pressure, diastolic blood pressure, serum sodium levels, serum potassium levels, urinary sodium-creatinine ratios, plasma renin activity, or plasma AGT levels. However, Log(UAGT/UCre) levels were significantly correlated positively with urinary albumin-creatinine ratios, fractional excretion of sodium, urinary protein-creatinine ratios, and serum creatinine, and correlated negatively with estimated glomerular filtration rate. Log(UAGT/UCre) levels were significantly increased in CKD patients compared with control subjects (1.8801 +/- 0.0885 vs. 0.9417 +/- 0.1048; P = .0024). These data confirmed our earlier report and showed that a new ELISA assay is a valid approach for measuring urinary AGT.

Involvements of Rho-kinase and TGF-beta pathways in aldosterone-induced renal injury.

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-beta 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 microg/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 alpha-smooth muscle actin staining and numbers of monocytes/macrophages in the interstitium. Renal cortical mRNA levels of types I and III collagen, TGF-beta, 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. Furthermore, fasudil prevented MYPT1 phosphorylation and markedly decreased alpha-smooth muscle actin staining, numbers of monocytes/macrophages, mRNA levels of types I and III collagen, TGF-beta, connective tissue growth factor and monocyte chemoattractant protein-1, and Smad2/3 activity in renal cortical tissues. These results provide evidence, for the first time, that Rho-kinase is substantially involved in aldosterone-induced renal injury through activation of a TGF-beta-dependent pathway.

Intrarenal angiotensin II and angiotensinogen augmentation in chronic angiotensin II-infused mice

The objectives of this study were to determine the effects of chronic angiotensin II (ANG II) infusions on ANG II content and angiotensinogen expression in the mouse kidney and the role of the angiotensin II type 1 receptor (AT(1)R) in mediating these changes. C57BL/6J male mice were subjected to ANG II infusions at doses of 400 or 1,000 ng.kg(-1).min(-1) either alone or with an AT(1)R blocker (olmesartan; 3 mg.kg(-1).day(-1)) for 12 days. Systolic and mean arterial pressures were determined by tail-cuff plethysmography and radiotelemetry. On day 13, blood and kidneys were collected for ANG II determinations by radioimmunoanalysis and intrarenal angiotensinogen expression studies by quantitative RT-PCR, Western blotting, and immunohistochemistry. ANG II infusions at the low dose elicited progressive increases in systolic blood pressure (135 +/- 2.5 mmHg). In contrast, the high dose induced a rapid increase (152 +/- 2.5, P < 0.05 vs. controls, 109 +/- 2.8). Renal ANG II content was increased by ANG II infusions at the low dose (1,203 +/- 253 fmol/g) and the high dose (1,258 +/- 173) vs. controls (499 +/- 40, P < 0.05). Kidney angiotensinogen mRNA and protein were increased only by the low dose to 1.13 +/- 0.02 and 1.26 +/- 0.10, respectively, over controls (1.00, P < 0.05). These effects were not observed in mice infused at the high dose and those receiving olmesartan. The results indicate that chronic ANG II infusions augment mouse intrarenal ANG II content with AT(1)R-dependent uptake occurring at both doses, but only the low dose of infusion, which elicited a slow progressive response, causes an AT(1)R-dependent increase in intrarenal angiotensinogen expression.

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.

Determination of plasma and urinary angiotensinogen levels in rodents by newly developed ELISA

We recently reported that urinary excretion rates of angiotensinogen provide a specific index of the intrarenal renin-angiotensin system status in angiotensin II-dependent hypertensive rats. Angiotensinogen concentrations in mouse plasma are thought to be much lower than those in rat plasma; however, detailed information is deficient due to lack of direct quantitative measurements of rodent angiotensinogen. To elucidate this issue, we have developed a quantitative method for measurement of rodent angiotensinogen using a sandwich-type ELISA. The standard curve for mouse and rat angiotensinogen exhibited a high linearity at 0.16-10 and 0.08-5 ng/ml, respectively, with correlation coefficients >0.99. While plasma angiotensinogen concentrations of male high serum IgA (HIGA) mice (IgA nephritis model animals, 1,308 +/- 47 ng/ml; n = 10) were lower than those of control BALB/c mice (1,620 +/- 384; n = 12), urinary angiotensinogen concentrations of HIGA mice (14.6 +/- 1.5 ng/ml; n = 34) were higher than those of BALB/c mice (4.6 +/- 0.1; n = 2). In a similar manner, while plasma angiotensinogen concentrations of Zucker diabetic fatty (ZDF) obese rats (type 2 diabetic model animals, 1,789 +/- 50 ng/ml; n = 5) were lower than those of control ZDF lean rats (2,296 +/- 47; n = 5), urinary angiotensinogen concentrations of ZDF obese rats (88.2 +/- 11.4 ng/ml; n = 15) were higher than those of ZDF lean rats (31.3 +/- 1.9; n = 15). These data indicate that plasma and urinary angiotensinogen concentrations are less in mice than rats. However, these data suggest that urinary angiotensinogen levels are different from plasma angiotensinogen levels in rodents. The development of rodent angiotensinogen ELISA allows quantitative comparisons in mouse and rat angiotensinogen levels in models of hypertension and cardiovascular and kidney diseases.