Post-treatment with JP-1302 protects against renal ischemia/reperfusion-induced acute kidney injury in rats.

Ischemia/reperfusion injury is the most common cause of acute kidney injury. We previously revealed that pre-treatment with yohimbine or JP-1302 attenuated renal ischemia/reperfusion injury by inhibition of α2C-adrenoceptor antagonist. The aim of the present study is to investigate the effects of post-treatment with JP-1302 on renal ischemia/reperfusion injury in rats. Male Sprague Dawley rats were randomly divided into four groups: sham operation, ischemia/reperfusion, pre-treatment with JP-1302 (3.0 mg/kg) and post-treatment with JP-1302 groups. In ischemia/reperfusion injury, renal functional parameters, such as blood urea nitrogen, plasma creatinine and creatinine clearance, deteriorated after reperfusion. Renal venous norepinephrine concentrations, as well as inflammatory molecules in the kidney increased after reperfusion. Both pre- and post-treatment with JP-1302 improved renal dysfunction, tissue damage, renal venous norepinephrine concentrations and inflammatory molecules expression in the kidney. In conclusion, these results suggest that post-treatment with JP-1302 protects on ischemia/reperfusion-induced acute kidney injury by suppressing cytokine upregulation via α2C-adrenoceptors.

Inhibition of α2C-adrenoceptors ameliorates cisplatin-induced acute renal failure in rats.

Nephrotoxicity is a major adverse reaction of the anticancer drug, cisplatin. We investigated the renoprotective effects of the α2-adrenoceptor antagonist, yohimbine and selective α2C-adrenoceptor antagonist, JP-1302, in cisplatin-treated Sprague Dawley rats. Rats were given a single intravenous dose of 7.5 mg/kg cisplatin and then yohimbine or JP-1302 was administered intraperitoneally at 0.1 or 3 mg/kg/day, respectively, for four days. Renal functional parameters, such as blood urea nitrogen, plasma creatinine, creatinine clearance and renal venous norepinephrine concentrations were measured. Kidney tissue damage and tumour necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) mRNA levels were assessed after the animals were euthanized. Cisplatin treatment aggravated the kidney functional parameters of blood urea nitrogen, plasma creatinine and creatinine clearance. Renal venous norepinephrine concentrations were also elevated after cisplatin administration. Treatment with yohimbine or JP-1302 clearly ameliorated kidney function and cell apoptosis. These treatments suppressed elevated renal plasma norepinephrine, TNF-α, MCP-1 and cleaved caspase 3 expressions which occurred after administration of cisplatin. These results suggest that yohimbine can prevent cisplatin-induced renal toxicity associated with acute kidney injury by suppressing cytokine expression through α2C-adrenoceptors.

Effect of monoamine oxidase inhibitors on ischaemia/reperfusion-induced acute kidney injury in rats.

Increases in renal sympathetic nerve activity during ischaemia and renal venous norepinephrine levels after reperfusion play important roles in the development of ischaemia/reperfusion-induced acute kidney injury. In the present study, we examined the effect of isatin, an endogenous monoamine oxidase inhibitor, on renal venous norepinephrine levels, superoxide production after reperfusion, and ischaemia/reperfusion-induced acute kidney injury. Ischaemia/reperfusion-induced acute kidney injury was accomplished by clamping the left renal artery and vein for 45min, followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal superoxide production and norepinephrine overflow were elevated and significant renal tissue damage was observed following ischaemia/reperfusion injury. Intravenous injection of isatin (10mg/kg) at 5min before ischaemia increased the renal venous plasma norepinephrine level after reperfusion and aggravated ischaemia/reperfusion-induced renal dysfunction and histological damage. The excessive superoxide production after reperfusion was significantly suppressed by isatin administration, indicating that the inhibition of oxidative deamination effectively suppressed superoxide production. These data suggest that the exacerbation effect of isatin is associated, at least in part, with increased norepinephrine levels but not with superoxide production. To the best of our knowledge, this is the first report of isatin involvement in the pathogenesis and/or development of acute kidney injury.

Sex differences in ischaemia/reperfusion-induced acute kidney injury depends on the degradation of noradrenaline by monoamine oxidase.

Ischaemic acute kidney injury (AKI) is a leading killer of both sexes; however, resistance to this injury is higher among women than men. We found that renal venous noradrenaline (NAd) overflow after reperfusion played important roles in the development of ischaemic AKI, and that the attenuation of AKI observed in female rats may be dependent on depressing the renal sympathetic nervous system with endogenous oestrogen. In the present study, we used male and female Sprague-Dawley rats to investigate whether sex differences in the pathogenesis of ischaemic AKI are related to the degradation of NAd by monoamine oxidase (MAO) in the kidney. Ischaemic AKI was achieved by clamping the left renal artery and vein for 45 minutes followed by reperfusion 2 weeks after contralateral nephrectomy. Renal injury was more severe in male rats than in female rats and renal venous plasma NAd levels after reperfusion were markedly elevated in males, but not in females. These sex differences were eliminated by a treatment with isatin, a non-selective MAO inhibitor, and moclobemide, a selective MAOA inhibitor, but not by selegiline, a selective MAOB inhibitor. Ischaemia decreased the mRNA expression levels of both MAOs in the kidney 1 day after reperfusion; however, MAOA mRNA expression levels were higher in female rats than in male rats. These results suggest that the degradation of NAd by MAOA in the kidney contributes to sex differences in the pathogenesis of ischaemia/reperfusion-induced AKI.

Renoprotective effect of yohimbine on ischaemia/reperfusion-induced acute kidney injury through α2C-adrenoceptors in rats.

Excitation of renal sympathetic nervous activity and the resulting increased levels of renal venous norepinephrine play important roles in renal ischaemia/reperfusion injury in rats. This study examined the effects of yohimbine, a non-selective α2-adrenoceptor antagonist, on renal venous norepinephrine levels and kidney function in acute kidney injury. Acute ischaemia/reperfusion-induced kidney injury was induced in rats by clamping the left renal artery and vein for 45min, followed by reperfusion, 2 weeks after a contralateral nephrectomy. Intravenous injection of yohimbine (0.1mg/kg) 5min prior to ischaemia significantly attenuated kidney injury and decreased the renal venous norepinephrine levels, as compared with vehicle-treated rats. To investigate the involvement of α2-adrenoceptor subtypes, we pre-treated with JP-1302, a selective α2C-adrenoceptor antagonist (1mg/kg). This suppressed renal venous norepinephrine levels and tumour necrosis factor-α and monocyte chemoattractant protein-1 mRNA levels after reperfusion and improved kidney function. Pre-treatment with BRL44408, a selective α2A-adrenoceptor antagonist (1mg/kg), or imiloxan, a selective α2B-adrenoceptor antagonist (1mg/kg) had no effect on renal function or tissue injury. These results suggest that yohimbine prevented ischaemia/reperfusion-induced kidney injury by inhibiting α2C-adrenoceptors and suppressing pro-inflammatory cytokine expression.

Hsc70 contributes to cancer cell survival by preventing Rab1A degradation under stress conditions.

Heat shock cognate protein 70 (Hsc70) acts as a molecular chaperone for the maintenance of intracellular proteins, which allows cancer cells to survive under proteotoxic stress. We attempted to use Hsc70 to identify key molecules in cancer cell survival. Here, we performed mass-spectrometry-based proteomics analysis utilizing affinity purification with anti-Hsc70 antibodies; as a result, 83 differentially expressed proteins were identified under stress conditions. This result implies that there was a change in the proteins with which Hsc70 interacted in response to stress. Among the proteins identified under both serum-depleted and 5-fluorouracil-treated conditions, Rab1A was identified as an essential molecule for cancer cell survival. Hsc70 interacted with Rab1A in a chaperone-dependent manner. In addition, Hsc70 knockdown decreased the level of Rab1A and increased the level of its ubiquitination under stress conditions, suggesting that Hsc70 prevented the degradation of Rab1A denatured by stress exposure. We also found that Rab1A knockdown induced cell death by inhibition of autophagosome formation. Rab1A may therefore contribute to overcoming proteotoxic insults, which allows cancer cells to survive under stress conditions. Analysis of Hsc70 interactors provided insight into changes of intracellular status. We expect further study of the Hsc70 interactome to provide a more comprehensive understanding of cancer cell physiology.

Protective effect of moxonidine on ischemia/reperfusion-induced acute kidney injury through α2/imidazoline I1 receptor.

Enhancement of renal sympathetic nerve activity during renal ischemia and norepinephrine overflow from the kidney after reperfusion play important roles in the development of ischemic acute kidney injury. Recently, we have found that moxonidine, an α2/imidazoline Ι1-receptor agonist, has preventive effects on ischemic acute kidney injury by suppressing the excitation of renal sympathetic nervous system after reperfusion. In the present study, to clarify the renoprotective mechanisms of moxonidine (360 nmol/kg, i.v.) against ischemic acute kidney injury, we investigated the effect of intravenous (i.v.) and intracerebroventricular (i.c.v.) injection of efaroxan, an α2/Ι1 receptor antagonist, on the moxonidine-exhibited actions. Ischemic acute kidney injury was induced by clamping the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. The suppressive effect of moxonidine on enhanced renal sympathetic nerve activity during renal ischemia was not observed in the rat treated with either i.v. (360 nmol/kg) or i.c.v. (36 nmol/kg) of efaroxan. Furthermore, i.v. injection of efaroxan eliminated the preventive effect of moxonidine on ischemia/reperfusion-induced kidney injury and norepinephrine overflow, and i.c.v. injection of efaroxan did not completely inhibit the moxonidine's effects. These results indicate that moxonidine prevents the ischemic kidney injury by sympathoinhibitory effect probably via α2/Ι1 receptors in central nervous system and by suppressing the norepinephrine overflow through α2/Ι1 receptors on sympathetic nerve endings.

Protective effect of ischemic preconditioning on ischemia/reperfusion-induced acute kidney injury through sympathetic nervous system in rats.

We have found that a series of brief renal ischemia and reperfusion (preconditioning), before the time of ischemia significantly attenuated the ischemia/reperfusion-induced acute kidney injury through endothelial nitric oxide synthase. In this study, we examined the effects of ischemic preconditioning on renal sympathetic nervous system and kidney function in ischemia/reperfusion-induced acute kidney injury with or without nitric oxide synthase inhibitor. Ischemia/reperfusion-induced acute kidney injury was made by clamping the left renal artery and vein for 45-min followed by reperfusion, 2 weeks after the contralateral nephrectomy. Ischemic preconditioning, consisting of three cycles of 2-min ischemia followed by 5-min reperfusion, was performed before the 45-min ischemia. Ischemic preconditioning suppressed the enhanced renal sympathetic nerve activity during ischemia and the elevated renal venous plasma norepinephrine level after reperfusion, and attenuated renal dysfunction and histological damage. The renoprotective effect of ischemic preconditioning was diminished by N(G)-nitro-L-arginine methyl ester (0.3 mg/kg, i.v.), a nonselective nitric oxide synthase inhibitor, 5 min before the start of ischemic preconditioning. Thus, ischemic preconditioning decreased renal sympathetic nerve activity and norepinephrine release probably through activating nitric oxide production, thereby improving ischemia/reperfusion-induced acute kidney injury.

Sex differences in ischemia/reperfusion-induced acute kidney injury are dependent on the renal sympathetic nervous system.

Resistance to ischemic acute kidney injury has been shown to be higher in female rats than in male rats. We found that renal venous norepinephrine overflow after reperfusion played important roles in the development of ischemic acute kidney injury. In the present study, we investigated whether sex differences in the pathogenesis of ischemic acute kidney injury were derived from the renal sympathetic nervous system using male and female Sprague-Dawley rats. Ischemia/reperfusion-induced acute kidney injury was achieved by clamping the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function was impaired after reperfusion in both male and female rats; however, renal dysfunction and histological damage were more severe in male rats than in female rats. Renal venous plasma norepinephrine levels after reperfusion were markedly elevated in male rats, but were not in female rats. These sex differences were eliminated by ovariectomy or treatment with tamoxifen, an estrogen receptor antagonist, in female rats. Furthermore, an intravenous injection of hexamethonium (25mg/kg), a ganglionic blocker, 5 min before ischemia suppressed the elevation in renal venous plasma norepinephrine levels after reperfusion, and attenuated renal dysfunction and histological damage in male rats, and ovariectomized and tamoxifen-treated female rats, but not in intact females. Thus, the present findings confirmed sex differences in the pathogenesis of ischemic acute kidney injury, and showed that the attenuation of ischemia/reperfusion-induced acute kidney injury observed in intact female rats may be dependent on depressing the renal sympathetic nervous system with endogenous estrogen.

Protective effect of 17ß-estradiol on ischemic acute kidney injury through the renal sympathetic nervous system.

Enhanced renal sympathetic nerve activity during an ischemic period and renal venous norepinephrine overflow after reperfusion play important roles in the development of ischemic acute kidney injury. In this study, we examined the effect of 17ß-estradiol on the renal sympathetic nervous system and kidney function in ischemia/reperfusion-induced acute kidney injury in anesthetized rats. Ischemic acute kidney injury was induced by clamping the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after a contralateral nephrectomy. Intravenous injection of 17ß-estradiol (100 µg/kg) 15 min before reperfusion suppressed enhanced renal sympathetic nerve activity during renal ischemia, also suppressed renal venous norepinephrine overflow after reperfusion, and attenuated ischemia/reperfusion-induced renal dysfunction with histological damage. The above renoprotective effects of 17ß-estradiol were reversed by pretreatment with tamoxifen (5 mg/kg), an estrogen receptor antagonist, or N(G)-nitro-L-arginine methyl ester (0.3 mg/kg), a non-selective nitric oxide synthase inhibitor. These results indicate that 17ß-estradiol can suppress enhanced renal sympathetic nerve activity during renal ischemia, and its consequent effect on norepinephrine overflow from nerve endings, by nitric oxide production via estrogen receptors. These effects appear to contribute to renoprotection against ischemia/reperfusion-induced renal injury.

Effects of liposome clodronate on renal leukocyte populations and renal fibrosis in murine obstructive nephropathy.

Although liposome-encapsulated clodronate has been used as a means to deplete macrophages from certain tissues, target leukocyte subtypes within the kidney are not clearly known under normal and pathologic conditions. The present study was therefore conducted to examine the effects of liposome clodronate on renal infiltrating cell type following unilateral ureteral obstruction (UUO) and tried to correlate these changes to the mechanisms of early development of renal fibrosis. Renal infiltrating leukocyte subtypes and counts were determined by using multicolor flow cytometric analysis of cell suspensions from obstructed kidneys. UUO for 5 days elicited renal tubular apoptosis and renal fibrosis and showed 4-fold increase in renal leukocytes including monocytes/macrophages, dendritic cells, and T-cells. Repeated administration of liposome clodronate selectively depleted F4/80+ monocytes/macrophages and F4/80+ dendritic cells but not F4/80(-) dendritic cells or other cell types in both obstructed and non-obstructed kidneys. Tubular apoptosis and renal fibrosis were also significantly attenuated by liposome clodronate. Increased gene expression of TNF-alpha and TGF-beta observed in obstructed kidneys were markedly attenuated by depletion of renal mononuclear phagocytes. These findings suggest that F4/80+ monocytes/macrophages and/or F4/80+ dendritic cells play a pivotal role in the development of obstruction-induced tubular apoptosis and renal fibrosis, possibly through TNF-alpha and TGF-beta dependent mechanisms.

Moxonidine prevents ischemia/reperfusion-induced renal injury in rats.

Enhancement of renal sympathetic nerve activity during renal ischemia and its consequent effect on norepinephrine overflow from nerve endings after reperfusion play important roles in the development of ischemic acute kidney injury. In the present study, we evaluated whether moxonidine, an alpha(2)-adrenaline/I(1)-imidazoline receptor agonist which is known to elicit sympathoinhibitory action, would prevent the post-ischemic renal injury. Ischemic acute kidney injury was induced by clamping the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Intravenous (i.v.) injection of moxonidine at a dose of 360 nmol/kg to ischemic acute kidney injury rats suppressed the enhanced renal sympathetic nerve activity during the ischemic period, to a degree similar to findings with intracerebroventricular (i.c.v.) injection of moxonidine at a dose of 36 nmol/kg. On the other hand, suppressive effects of the i.v. treatment on renal venous norepinephrine overflow, renal dysfunction and tissue injury in the post-ischemic kidney were significantly greater than those elicited by the i.c.v. treatment. These results suggest that renoprotective effects of moxonidine on ischemic acute kidney injury probably result from its suppressive action on the ischemia-enhanced renal sympathetic nerve activity followed by norepinephrine spillover from the nerve endings of the post-ischemic kidney.

Investigation of the role of the amino acid residue at position 230 for catalysis in monomeric carbonyl reductase 3.

Monomeric carbonyl reductase 3 (CBR3) is a member of the short-chain dehydrogenase/reductase family. CBR3 exhibits much lower activity than monomeric carbonyl reductase 1 (CBR1) in humans and Chinese hamsters although they are highly homologous to each other in amino acid sequence levels. In the present study, we first cloned the CBR3 gene of rat origin (rCBR3), and characterized its enzymatic activity. rCBR3 also exhibited a limited catalytic efficiency similarly to the other CBR3 orthologues of humans and Chinese hamsters. Among the CBR3 orthologues, the human enzyme showed considerably lower activity. Compared with the amino acid sequences of CBR1 and CBR3 among humans, rats, Chinese hamsters, and mice, the tryptophan residue at position 230 is highly conserved while human CBR3 possesses rigid amino acid, proline, at that position instead. Thus, the Trp-230 was expected to be one of the important residues for catalysis since it locates in the hinge region at the substrate-binding loop. The substitution of tryptophan for proline in hCBR3 failed to affect the enzymatic characteristics. Similarly, the substitution of proline for tryptophan in either Chinese hamster CBR3 (CHCR3) or rCBR3 showed no significant change in the catalytic properties. These results suggest that limited catalytic efficiency of carbonyl reductase activity of CBR3 is a common property among animal species, and the substitution of the amino acid residue at position 230 alone has no apparent impact on their enzymatic activities.

Cobalt protoporphyrin attenuates rat obstructive nephropathy: role of cellular infiltration.

OBJECTIVES: Renal interstitial inflammation is closely related to the progressive renal fibrosis. It has been reported that heme oxygenase-1 (HO-1) induction attenuated renal fibrosis in obstructive nephropathy. To elucidate the antifibrogenic mechanisms of HO-1, we examined the effect of HO-1 induction on renal interstitial inflammation. METHODS: Adult male rats underwent unilateral ureteral obstruction (UUO). The rats were pretreated with cobalt protoporphyrin (CoPP, a potent HO-1 inducer; 15 or 50 mg/kg) subcutaneously on the day -6 and -1 before UUO. Sham-operated rats served as controls. Renal interstitial fibrosis, macrophage and T cell infiltration were immunohistochemically assessed on the day 5 after UUO. Gene expressions of HO-1 and profibrogenic molecules were determined by real-time reverse transcriptase-polymerase chain reaction. RESULTS: CoPP dose-dependently induced HO-1 activity, protein, and messenger RNA (mRNA) expression in the renal cortices. CoPP significantly attenuated the renal fibrosis in a dose-dependent manner. Gene expressions of transforming growth factor-beta and extracellular matrix proteins were upregulated in UUO and were attenuated by CoPP. CoPP markedly inhibited T cell infiltration. Unexpectedly, it enhanced macrophage influx dose dependently. Double immunostaining of macrophage and HO-1 showed that CoPP elicited HO-1 overexpression in infiltrating macrophages, whereas UUO alone did not. CONCLUSIONS: HO-1 induction protected against the renal interstitial fibrosis in rat obstructive nephropathy. It is suggested that inhibition of T cell influx is, at least in part, involved in the protection. Increased macrophages that overexpress HO-1 may play an important role in attenuating renal fibrosis.

Attenuation of renal fibrosis by curcumin in rat obstructive nephropathy.

OBJECTIVES: To test whether curcumin has a protective action against interstitial inflammation and the development of renal fibrosis in obstructive nephropathy. We also tested whether inhibition of nuclear factor kappa-B (NF-kappaB) and activator protein-1 (AP-1) by curcumin is involved in these mechanisms. METHODS: Adult male rats underwent unilateral ureteral obstruction. The rats were treated with curcumin (200 mg/kg/day or 800 mg/kg/day), NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC; 200 mg/kg/day), or vehicle by gavage. Sham-operated rats served as controls. Seven days after unilateral ureteral obstruction, the activity of NF-kappaB and AP-1 was examined by electrophoretic mobility shift assay using nuclear protein extracts from the renal cortex. Gene expression of chemokines and pro-fibrotic molecules was determined by real-time reverse transcriptase-polymerase chain reaction. Macrophage infiltration and collagen III accumulation in the cortical interstitium was examined immunohistochemically. RESULTS: Both curcumin and PDTC significantly attenuated interstitial macrophage influx and renal fibrosis. Ureteral occlusion activated both NF-kappaB and AP-1-DNA binding. Curcumin and PDTC significantly inhibited NF-kappaB activity, but not AP-1. Gene expression of chemokines and pro-fibrotic molecules was upregulated in unilateral ureteral obstruction that was attenuated by either curcumin or PDTC. CONCLUSIONS: Curcumin protected against the renal interstitial inflammation and fibrosis elicited by ureteral occlusion. Inhibition of the NF-kappaB-dependent pathway is at least in part involved in the mechanisms, but AP-1 inhibition is unlikely to be involved in the beneficial effects of curcumin.

Molecular mechanisms and therapeutic strategies of chronic renal injury: the role of nuclear factor kappaB activation in the development of renal fibrosis.

Tubulointerstitial fibrosis is a common feature of many progressive renal diseases and is a main determinant that leads to an irreversible loss of renal function. In chronic cyclosporin A nephrotoxicity, we previously reported that inflammatory responses such as macrophage infiltration preceded interstitial fibrosis. This inflammation was accompanied by an elevation in renal nuclear factor kappaB (NF-kappaB) activity. Similar findings were obtained in chronic tacrolimus nephrotoxicity and obstructive nephropathy. Inhibition of NF-kappaB markedly attenuated renal inflammation and interstitial fibrosis in these models. Furthermore, administration of oral adsorbent (Kremezin) significantly attenuated the increase in renal NF-kappaB activity and concomitantly reduced interstitial inflammation and renal fibrosis in chronic renal failure rats. Elimination of indoxyl sulfate by this adsorbent is likely involved in this mechanism since it is known that indoxyl sulfate activates NF-kappaB in renal tubular cells. It is suggested that strategy aiming at NF-kappaB inhibition is important to prevent the progression of renal fibrosis.

Excess aldosterone under normal salt diet induces cardiac hypertrophy and infiltration via oxidative stress.

Aldosterone is known to play a role in the pathophysiology of some cardiovascular diseases. However, previous studies on aldosterone infusion have been mostly performed in animals receiving sodium loading and uninephrectomy, and thus the cardiac action of aldosterone alone remains to be fully clarified. The present study was undertaken to investigate the direct cardiac action of aldosterone infusion alone in rats not subjected to salt loading and uninephrectomy. Aldosterone (0.75 microg/h) was subcutaneously infused into rats via an osmotic minipump for 14 days. Aldosterone infusion, under a normal salt diet, induced only a slight increase in the blood pressure of normal rats throughout the infusion. However, aldosterone significantly induced cardiac hypertrophy, as shown by echocardiography and measurement of cardiomyocyte cross-sectional area. Furthermore, aldosterone caused not only cardiac interstitial macrophage infiltration but also cardiac focal inflammatory lesions, which were associated with an increase in cardiac monocyte chemoattractant protein-1 (MCP-1) and osteopontin mRNA. The slight elevation of blood pressure by aldosterone infusion was completely prevented by tempol, the superoxide dismutase mimetic. However, tempol failed to suppress cardiac hypertrophy, the formation of inflammatory lesions, and upregulation of cardiac MCP-1 and osteopontin by aldosterone, while N-acetylcysteine could inhibit all of them. Our data provide evidence that aldosterone alone can induce cardiac hypertrophy and severe inflammatory response in the heart, independently of blood pressure, even in the absence of salt loading or nephrectomy. Aldosterone seems to induce cardiac inflammation and gene expression via oxidative stress that is inhibited by N-acetylcysteine but not by tempol.

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.

Beneficial effect of candesartan on rat diastolic heart failure.

In this study, we examined whether an angiotensin II type 1 (AT1)-receptor blocker improves diastolic heart failure (DHF) in Dahl salt-sensitive (DS) rats. DHF was prepared by feeding DS rats on 8% NaCl diet from 7 weeks of age. DHF was estimated with echocardiography by measuring E velocity / A velocity (E/A) of left ventricular inflow. DS rats with established DHF were orally given candesartan (1 mg/kg per day) or vehicle. After 13 days of treatment, candesartan significantly improved DHF, as shown by the reduction of E/A from 4.49 +/- 1.04 to 1.98 +/- 0.54 (P<0.05) and prolonged survival rate more than the vehicle. Cardiac fibrosis, apoptosis, and gene expression were estimated by Sirius Red-staining, TUNEL-staining, and Northern blot analysis, respectively. The improvement of DHF by candesartan was accompanied by the decrease in cardiac hypertrophy, fibrosis, and apoptosis, and the reduction of gene expression of brain natriuretic peptide, collagen I, and monocyte chemoattractant protein-1. Moreover, candesartan decreased cardiac inflammatory cells and reactive oxygen species, estimated by counting ED-1-positive cells and the measurement of 4-hydroxy-2-nonenal staining, respectively. These results indicate that candesartan can improve diastolic dysfunction and may slow the progression of cardiac remodelling in DS rats with established DHF.