Effects of Febuxostat on Oxidative Stress.

PURPOSE: We previously examined factors that affect the measured derivatives of reactive oxygen metabolites (d-ROMs), an indicator of reactive oxygen species production, and biological antioxidant potential (BAP), an indicator of antioxidant capacity, in typical health checkup examinees and reported the usefulness of measuring both indicators simultaneously. In addition, a positive correlation reportedly exists between d-ROMs and the visceral fat area measured by using computed tomography. A recent study of the relationship between uric acid levels and various obesity-related factors found that visceral fat was the factor most strongly related to uric acid levels. Uric acid is itself a potent endogenous antioxidant, but because reactive oxygen species are produced during uric acid generation, it is suggested that uric acid may have opposing effects. The objective of this study was to analyze the effect of febuxostat, a novel xanthine oxidase inhibitor, on oxidative stress. METHODS: Study subjects were 43 hyperuricemia outpatients receiving care in the internal medicine department of our institution. The subjects were divided into a new administration group (29 patients) and a switched administration group (14 patients); the latter were allopurinol-treated patients with hyperuricemia who were switched to febuxostat. In addition to measuring the patients' uric acid and creatinine levels and estimated glomerular filtration rate before and after treatment, their d-ROMs and BAP as well as the BAP/d-ROMs ratio were also measured. FINDINGS: Both groups exhibited significant decreases in uric acid levels, as well as significant decreases in d-ROMs and BAP. No significant changes were observed in the BAP/dROMs ratio or renal function, including creatinine levels and estimated glomerular filtration rate. IMPLICATIONS: Febuxostat could significantly reduce d-ROMs. However, BAP levels were also significantly reduced concurrently. No changes were observed in the BAP/d-ROMs ratios. This regulatory mechanism is believed to have counteracted changes in the in vivo oxidative stress balance caused by febuxostat administration.

Significance of measuring oxidative stress in lifestyle-related diseases from the viewpoint of correlation between d-ROMs and BAP in Japanese subjects.

In recent years, oxidative stress has been postulated to be an important factor in the pathogenesis and development of lifestyle-related diseases. In this study, we investigated the association between the derivatives of reactive oxygen metabolites (d-ROMs), as an index of products of reactive oxygen species (ROS), and biological antioxidant potential (BAP), as an index of antioxidant potential. We also investigated the associations between d-ROMs or BAP and the risk factors for lifestyle-related diseases or metabolic syndrome-associated factors to evaluate their usefulness in preventive medicine. There were 442 subjects who underwent health checkup examination in our facilities. In addition to standard medical checkup items, we analyzed d-ROMs, BAP, brachial-ankle pulse wave velocity, high-sensitivity C-reactive protein level and visceral fat area (VFA) visualized on a computed tomography scan. The mean d-ROM value in females was significantly higher than that in males. There was a positive correlation between the d-ROM and VFA levels. On correlation analysis, there was a negative correlation between the d-ROM and creatinine levels. As factors that influence d-ROMs, the level of VFA was selected, suggesting the significance of oxidative stress measurement with d-ROMs. In addition, there was a positive correlation between d-ROMs and BAP values. Further research is required to resolve whether increased production of ROS or the antioxidant potential that can compensate for such an increase of ROS is more important in vivo.

Brain pharmacokinetics of tetramethylpyrazine after intranasal and intravenous administration in awake rats.

Brain pharmacokinetic behaviors of tetramethylpyrazine (TMP) following intranasal (i.n.) and intravenous (i.v.) administration, have been investigated using brain microdialysis technique in free-moving rats. A cross-over design was employed in the present experiment. The same set of rats (n=5) received i.v. injection at a dose of 10 mg/kg TMP via tail vein. Equal dose was administered intranasally. After application, the dialysates sampled from the left striatum were measured by HPLC-UV detection. The results indicated that the mean corrected TMP concentration of 1.49 microg/ml was obtained at 5 min following i.n. dosing while no TMP in the dialysate sampled 5 min after i.v. injection was detected, in the range of our measurement limit. No compartment model was most suitable for analysis of the concentration vs. time results after i.n. dosing. Thus, a non-compartment model was used in the analysis of all experimental data. No significant differences in brain pharmacokinetic parameters, except Cmax, were found between both i.n. and i.v. administration routes. AUCi.n./AUCi.v. ratio was 92.42%. Finally, compared with i.v. application, intranasal administration of TMP could obtain significantly fast absorption from nasal to ipsilateral striatum and equal bioavailability. Therapeutically relevant nasal formulation is a potential alternative for intravenous administration approach for TMP.

Role of AT1 receptor in isoproterenol-induced cardiac hypertrophy and oxidative stress in mice.

Elevated activities of the sympathetic nerve and renin-angiotensin systems are common features of heart failure. This study was designed to investigate the roles of the AT1 receptor in cardiac hypertrophy and oxidative stress during excessive beta-adrenoceptor stimulation using an AT1 receptor antagonist (ARB) and AT1a receptor-deficient (AT1aR(-/-)) mice. Isoproterenol (ISO) was given to C57BL mice with or without ARB (olmesartan) treatment and to AT1aR(-/-) mice by a subcutaneously implanted osmotic mini-pump for 11 days at a rate of 15 mg/kg/day. Chronic ISO infusion to C57BL mice caused concentric cardiac hypertrophy (sham; 4.1+/-0.1, ISO; 5.2+/-0.2 mg/g heart to body weight ratio), accompanied by enhancement of cardiac collagen accumulation, lipid peroxidation, superoxide generation and NADPH oxidase activity. The AT1a and beta-1,2 receptor mRNA expressions were down-regulated in the heart of ISO-infused mice. Olmesartan markedly suppressed cardiac mass enlargement as well as increases of oxidative indicators without any effects on heart rate. Olmesartan did not affect the cardiac angiotensin and beta-adrenergic receptor mRNA expression patterns. The AT1a receptor contribution to ISO-induced cardiac hypertrophy was reproduced in AT1aR(-/-) mice. These data suggest that the AT1 receptor plays a crucial role in the development of cardiac hypertrophy and oxidative stress under excessive beta-adrenergic stimulation, and that ARB treatment is beneficial for sympatho-excitatory cardiac hypertrophy and failure in mice.

Effects of adrenomedullin 2 on regional hemodynamics in conscious rats.

The present study aimed to assess the effects of rat adrenomedullin 2 on systemic and regional hemodynamics in conscious Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Intravenous infusion of rat adrenomedullin 2 (0.25-5 micro g/kg/min) decreased blood pressure, and increased heart rate in a dose-dependent manner in both types of rats. Rat adrenomedullin 2 (5 micro g/kg/min) increased heart rate and cardiac output. As a result, total peripheral resistance significantly decreased. In SHRs, adrenomedullin 2 significantly increased regional blood flow in the heart, liver, spleen, kidneys, and adrenal glands. Especially, effects on heart, liver, and kidneys were remarkable. Regional hemodynamic changes were reproduced in WKY rats, and there was no qualitative difference in regional responses to rat adrenomedullin 2 between SHRs and WKY rats. Thus, rat adrenomedullin 2 predominantly increased flow rates in organs that were richly blood-supplied from cardiac output. Rat adrenomedullin 2 may contribute to the regulation of the cardiovascular system, by acting as a local vasodilatory hormone as well as a circulatory hormone.

Renoprotective effects of l-carnosine on ischemia/reperfusion-induced renal injury in rats.

We examined the renoprotective effects of l-carnosine (beta-alanyl-l-histidine) on ischemia/reperfusion (I/R)-induced acute renal failure (ARF) in rats. Ischemic ARF was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. In vehicle (0.9% saline)-treated rats, renal sympathetic nerve activity (RSNA) was significantly augmented during the renal ischemia, and renal function was markedly decreased at 24 h after reperfusion. Intracerebroventricular injection of l-carnosine (1.5 and 5 pmol/rat) to ischemic ARF rats dose-dependently suppressed the augmented RSNA during ischemia and the renal injury at 24 h after reperfusion. N-alpha-Acetyl-l-carnosine [N-acetyl-beta-alanyl-l-histidine; 5 pmol/rat intracerebroventricular (i.c.v.)], which is resistant to enzymatic hydrolysis by carnosinase, did not affect the renal injury, and l-histidine (5 pmol/rat i.c.v.), a metabolite cleaved from l-carnosine by carnosinase, ameliorated the I/R-induced renal injury. Furthermore, a selective histamine H(3) receptor antagonist, thioperamide (30 nmol/rat i.c.v.) eliminated the preventing effects by l-carnosine (15 nmol/rat intravenously) on ischemic ARF. In contrast, a selective H(3) receptor agonist, R-alpha-methylhistamine (5 pmol/rat i.c.v.), prevented the I/R-induced renal injury as well as l-carnosine (5 pmol/rat) did. These results indicate that l-carnosine prevents the development of I/R-induced renal injury, and the effect is accompanied by suppressing the enhanced RSNA during ischemia. In addition, the present findings suggest that the renoprotective effect of l-carnosine on ischemic ARF is induced by its conversion to l-histidine and l-histamine and is mediated through the activation of histamine H(3) receptors in the central nervous system.

Augmentation of intrarenal angiotensin II levels in uninephrectomized aldosterone/salt-treated hypertensive rats; renoprotective effects of an ultrahigh dose of olmesartan.

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.

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.

Effects of adrenomedullin on cardiac oxidative stress and collagen accumulation in aldosterone-dependent malignant hypertensive rats.

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 = 5], 1% NaCl in drinking water and vehicle (n = 8), 1% NaCl and aldosterone (0.75 microg/h s.c., n = 8), and 1% NaCl, aldosterone, and adrenomedullin (1.3 microg/kg/h s.c., n = 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.

Synergistic effect of mechanical stretch and angiotensin II on superoxide production via NADPH oxidase in vascular smooth muscle cells.

OBJECTIVE: Mechanical forces and angiotensin II influence the structure and function of vascular cells, and play an important role in reactive oxygen species production. In this study, we examined the effects of mechanical stretch and angiotensin II on the expression of p22-phox and Nox-1, essential membrane components of NADPH oxidase, and superoxide production in rat vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Neither a stretch force nor angiotensin II alone altered p22-phox and Nox-1 expression in VSMCs. Combined stimulation markedly increased p22-phox and Nox-1 mRNA, however, which was associated with increased NADPH oxidase activity, superoxide production and total 8-iso-prostaglandin F2alpha concentration. The increases in p22-phox mRNA levels induced by a stretch force in combination with angiotensin II were prevented by treatment with an angiotensin type I (AT1) receptor antagonist, RNH-6270 (100 nmol/l). Protein expression of the AT1 receptor was upregulated by a stretch force. CONCLUSIONS: 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.

Contribution of reactive oxygen species to the pathogenesis of left ventricular failure in Dahl salt-sensitive hypertensive rats: effects of angiotensin II blockade.

OBJECTIVE: We investigated the contribution of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species (ROS) generation to the pathogenesis of diastolic heart failure (DHF) in Dahl salt-sensitive (DS) hypertensive rats, with the aim of testing our hypothesis that the cardioprotective effects of angiotensin II (Ang II) blockade are provided by the suppression of this pathway. METHODS: DS rats were maintained on high (H: 8.0% NaCl) or low (L: 0.3% NaCl) salt diets from age 7 to 17 weeks. DS/H rats were also treated with candesartan cilexetil (10 mg/kg per day, orally) or a superoxide dismutase mimetic, tempol (3 mmol/l in drinking water) from age 7 to 17 weeks. RESULTS: DS/H rats represented hypertension, left ventricular (LV) relaxation abnormality and myocardial stiffening with preserved systolic heart function. As compared with DS/L rats, DS/H rats showed higher levels of transforming growth factor-beta (TGF-beta), connective tissue growth factor (CTGF), p22phox and gp91phox mRNA expression, NADPH oxidase activity and thiobarbituric acid-reactive substance (TBARS) contents in LV tissues. Gene expression of uncoupling protein-2 (UCP-2), an inner mitochondrial membrane proton transporter, was also 2.8 +/- 0.5-fold higher. In DS/H rats, treatment with candesartan did not alter blood pressure, but resulted in a marked improvement of the hemodynamic deterioration; these therapeutic effects were accompanied by decreases in myocardial NADPH oxidase activity, TBARS contents and the expression of TGF-beta, CTGF, p22phox, gp91phox and UCP-2. Similar therapeutic effects were provided by treatment with tempol in DS/H rats. CONCLUSIONS: Our data suggest that NADPH oxidase-mediated ROS production contributes to the pathogenesis of DHF in DS hypertensive rats, and that the cardioprotective effects of AngII blockade are, at least partially, mediated through the suppression of this pathway.

Molecular mechanisms and therapeutic strategies of chronic renal injury: renoprotective effects of aldosterone blockade.

Recent clinical and pre-clinical studies have indicated the utility of mineralocorticoid receptor (MR) antagonists in renal injury. We have demonstrated in rats that chronic treatment with aldosterone results in severe proteinuria and renal injury, characterized by glomerular changes, tubulointerstitial fibrosis, and collagen accumulation. We also observed increased reactive oxygen species (ROS) generation and mitogen-activated protein kinases (MAPKs) activity in renal cortical tissues. Treatment with a selective MR antagonist, eplerenone, prevented elevation of ROS levels and MAPK activity, as well as ameliorating renal injury. In vitro studies revealed that MRs are highly expressed in rat glomerular mesangial cells (RMC) and rat renal fibroblasts. In RMC, aldosterone induces cellular injuries through NADPH oxidase-dependent ROS production and/or MAPK activation. Aldosterone-induced renal cellular injuries were markedly attenuated by treatment with eplerenone. These data suggest that aldosterone induces renal injury through activation of MRs and support the notion that MR blockade has beneficial effects on aldosterone-dependent renal injury through mechanisms that cannot be simply explained by hemodynamic changes. In this review, we summarized our recent findings pertaining to the roles of aldosterone and MRs in the pathogenesis of renal injury. Potential molecular mechanisms responsible for aldosterone/MR-induced renal injury were also discussed.

Effects of calcium channel blockade on angiotensin II-induced peritubular ischemia in rats.

Recent studies have indicated that derangement of peritubular capillary (PTC) circulation with consequent tubulointerstitial hypoxia plays a pivotal role in the pathogenesis of renal injury. The present study was performed to determine whether azelnidipine, a new dihydropyridine calcium channel blocker, attenuates angiotensin II (AngII)-induced peritubular ischemia in anesthetized rats. The superficial PTCs were visualized directly using an intravital fluorescence videomicroscope system, and the PTC blood flow was evaluated by analyzing the velocity of fluorescein isothiocyanate-labeled erythrocytes. Intravenous infusion of AngII (50 ng/kg/min, 10 min) significantly increased mean arterial pressure (MAP) and renal vascular resistance (RVR) (by 35 +/- 3% and 110 +/- 32%, respectively), and decreased total renal blood flow (RBF) and PTC erythrocyte velocity (by -34 +/- 4 and -37 +/- 1%, respectively). Treatment with azelnidipine (5 microg/kg/min i.v., 10 min) had no effect on basal MAP, RBF, RVR, or PTC erythrocyte velocity. However, azelnidipine markedly attenuated the AngII-induced increases in MAP (7 +/- 3%) and RVR (40 +/- 4%) and decreases in RBF (-24 +/- 1%) and PTC erythrocyte velocity (-22 +/- 1%). Similar attenuation in the AngII-induced responses of MAP, RBF, RVR, and PTC erythrocyte velocity were observed in rats treated with a higher dose of azelnidipine (20 microg/kg/min i.v., 10 min), which significantly decreased basal MAP and RVR and increased RBF and PTC erythrocyte velocity. These data suggest that calcium channel blockade attenuates AngII-induced peritubular ischemia, which may be involved in its beneficial effects on renal injury.

Roles of adrenomedullin 2 in regulating the cardiovascular and sympathetic nervous systems in conscious rats.

Recently, a new member of the calcitonin gene-related peptide (CGRP) family, adrenomedullin 2 (AM2) or intermedin (IMD), was identified. AM2/IMD has been shown to have a vasodilator effect in mice and rats and an effect on urine formation in rats. In the present study, we investigated the effects of intravenously infused rat AM2 (rAM2) on blood pressure (BP), heart rate (HR), renal sympathetic nerve activity (RSNA), and renal blood flow (RBF) in conscious unrestrained rats relative to the effects of rat adrenomedullin (rAM) and proadrenomedullin NH2-terminal 20 peptide (rPAMP). Intravenous infusion of rAM2 (5 nmol/kg) significantly decreased BP and increased HR, RSNA, and RBF. These hypotensive and sympathoexcitatory effects diminished after 20 min, and HR returned to control levels 30 min after cessation of the infusion. In contrast, a significant increase in RBF was still evident 60 min after cessation of the peptide infusion. The duration of BP, HR, and RSNA responses was longer with rAM (5 nmol/kg) than with rAM2 infusion, whereas the increases in RBF induced by rAM2 and rAM were similar in their amplitude and duration. Infusion of rPAMP (200 nmol/kg) increased HR and RSNA but had no effect on RBF. Baroreceptor denervation suppressed, but did not diminish, the increases in HR and RSNA to rAM2. These findings indicate that the physiological roles of rAM2 and rAM are similar and that rAM2 also has a long-lasting vasodilator action on the renal vascular bed.

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.

D-allose, an all-cis aldo-hexose, suppresses development of salt-induced hypertension in Dahl rats.

OBJECTIVE: D-allose, an all-cis aldo-hexose, is non-caloric and possesses antioxidant properties. We investigated the effects of oral D-allose supplementation on the development of high blood pressure and the oxidative status in two genetically hypertensive animal models: Dahl salt-sensitive hypertensive (DS) rats and spontaneously hypertensive rats. METHODS AND RESULTS: The systolic blood pressure of DS rats fed a 4% salt diet for 4 weeks significantly increased from 122+/-8 to 161+/-5 mmHg as compared with DS rats fed a normal salt diet (138+/-5 mmHg at 4 weeks), whereas concordant supplementation of D-allose, but not D-glucose, with a dose of 2 g/kg daily to salt-loaded DS rats suppressed the development of high blood pressure (135+/-7 mmHg at 4 weeks), accompanied with decreases in superoxide production in the aorta that was determined by the lucigenin chemiluminescence and dihydroethidium staining. The increases of urinary protein secretion of salt-loaded DS rats were prevented by D-allose supplementation (DS rats fed 0.5% salt, 18.2+/-6.3 mg/day; DS rats fed 4% salt alone, 81.8+/-16.5 mg/day; DS rats fed 4% salt+D-allose, 31.3+/-11.8 mg/day; DS rats fed 4% salt+D-glucose, 85.3+/-20.5 mg/day). On the other hand, D-allose supplementation in spontaneously hypertensive rats had no significant effect on the blood pressure or the aortic superoxide production during the early developing stage of hypertension. CONCLUSIONS: These results underscore the role of enhanced oxidative stress in the pathogenesis of high blood pressure development in DS rats, and suggest the possibility of D-allose supplementation for prevention of salt-sensitive hypertension.

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 micromol/L), a selective MR antagonist, or PD98059 (10 micromol/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.

Ischemic preconditioning protects post-ischemic renal function in anesthetized dogs: role of adenosine and adenine nucleotides.

AIM: To investigate the effects of renal ischemic preconditioning (IPC) on both renal hemodynamics and the renal interstitial concentrations of adenosine and adenine nucleotides induced by ischemia-reperfusion injury. METHODS: Renal hemodynamics responses to ischemia-reperfusion injury in mongrel dog models were determined with or without multiple brief renal ischemic preconditioning treatments, as well as the adenosine A1 receptor antagonist (KW-3902), respectively. The renal interstitial concentrations of adenosine and adenine nucleotides in response to ischemia-reperfusion injury, either following 1-3 cycles of IPC or not, were measured simultaneously using microdialysis sampling technology. RESULTS: One 10-min IPC, adenosine A1 receptor antagonist (KW-3902) also shortened the recovery time of renal blood flow (RBF) and urine flow (UF), as well as mean blood pressure (BP). Advanced renal IPC attenuated the increment of adenosine and adenine nucleotides, as well as recovery time during the 60-min reperfusion which followed the 60-min renal ischemia. All of these recovery times were dependent on the cycles of 10-min IPC. The renal interstitial concentrations of adenosine and adenine nucleotides increased and decreased during renal ischemia and reperfusion, respectively. CONCLUSION: A significant relativity in dog models exists between the cycles of 10-min renal IPC and the recovery time of BP, UF, and RBF during the 60-min renal reperfusion following 60-min renal ischemia, respectively. Renal IPC can protect against ischemia-reperfusion injury and the predominant effect of endogenous adenosine induced by prolonged renal ischemia; renal adenosine A1 receptor activation during the renal ischemia-reperfusion injury is detrimental to renal function.