Essential Role of NADPH Oxidase-Dependent Production of Reactive Oxygen Species in Maintenance of Sustained B Cell Receptor Signaling and B Cell Proliferation.

Reactive oxygen species (ROS) are not only toxic substances inducing oxidative stress but also play a role as a second messenger in signal transduction through various receptors. Previously, B cell activation was shown to involve prolonged ROS production induced by ligation of BCR. However, the mechanisms for ROS production and ROS-mediated activation in B cells are still poorly understood. In this study, we demonstrate that BCR ligation induces biphasic ROS production in both mouse spleen B cells and the mouse B cell line BAL17; transient and modest ROS production is followed by sustained and robust ROS production at 2-6 h after BCR ligation. ROS production in the late phase but not in the early phase augments activation of signaling pathways, such as the NF-κB and PI3K pathways, and is essential for B cell proliferation. ROS production in the late phase appears to be mediated by NADPH oxidases (NOXes) because prolonged ROS production is inhibited by various NOX inhibitors, including the specific inhibitor VAS2870. BCR ligation-induced ROS production is also inhibited by CRISPR/Cas9-mediated deletion of either the Cyba gene encoding p22phox, the regulator of NOX1-4 required for their activation, or NOX3, whereas ROS production is not affected by double deficiency of the DUOXA1 and DUOXA2 genes essential for the activation of the NOX isoforms DUOX1 and DUOX2. These results indicate that NOXes play a crucial role in sustained but not early BCR signaling and suggest an essential role of NOX-dependent sustained BCR signaling in B cell activation.

Effects of Agmatine on Contrast-Induced Nephropathy in Rats and Rabbits.

Renal insufficiency secondary to contrast administration remains a prevalent and debilitating complication of angiographic procedures. Contrast-induced nephropathy (CIN) is a common clinical problem for which there is no effective medical treatment. However, agmatine has been shown to be effective against ischemia/reperfusion-induced acute kidney injury in rats, a similar condition to CIN. Our aim was to examine the protective effects of agmatine in a rat model of CIN and, based on those results, in a rabbit model of CIN. CIN in the rat model was induced by intravenous administration of indomethacin (10 mg/kg), Nω-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) and iopamidol (OYPALOMIN, 7.4 g iodine/kg) at 2 weeks after a unilateral nephrectomy. CIN in the rabbit model was induced by intrarenal arterial injection of only iopamidol (BYSTAGE, 4.8 g iodine/kg). Intravenous injection of agmatine (0.1 and 0.3 mmol/kg) did not attenuate the CIN-induced renal insufficiency in the rat model. Intravenous injection of agmatine (0.3 mmol/kg) attenuated the CIN-induced renal insufficiency in the rabbit model such as increases in blood urea nitrogen and plasma creatinine levels. Renal histological damage was also improved by the agmatine administration. The difference in effects of agmatine injection between CIN rats and CIN rabbits was caused by indomethacin and L-NAME administrations. These results indicate that agmatine prevents the development of CIN-induced renal insufficiency in rabbits, and the effect is accompanied by activation of nitric oxide synthase and subsequent increase of blood flow.

Grape Extract from Chardonnay Seeds Restores Deoxycorticosterone Acetate-Salt-Induced Endothelial Dysfunction and Hypertension in Rats.

Grape extract (GE), which contains various polyphenolic compounds, exerts protective effects against lifestyle-related diseases, such as diabetes and hypertension. We pharmacologically investigated whether dietary supplements with an extract from Chardonnay exerted antihypertensive effects in deoxycorticosterone acetate (DOCA)-salt-induced hypertensive rats. GE increased nitric oxide (NO) production by activating the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in cultured endothelial cells and induced vasorelaxation in the aorta and mesenteric artery via the same pathway. The development and progression of hypertension by the DOCA-salt treatment was significantly inhibited in GE-fed rats. Reduced vasoreactive responses to acetylcholine in the aorta of DOCA-salt rats were significantly ameliorated by the GE diet. Dietary GE supplements slightly diminished vascular superoxide anion production induced by the DOCA-salt treatment. On the other hand, dietary GE supplements had no effect on the progression of hypertension in rats in which NO synthase was pharmacologically and chronically suppressed. In addition, the oral administration of GE for 5 d in healthy rats enhanced endothelial NO synthase (eNOS) gene expression and vascular reactivity to acetylcholine in the aorta. Thus, GE has endothelium-dependent vasorelaxant properties that are mediated by the activation of endothelial NO synthase via the PI3K/Akt pathway, and this mechanism is conducive to the antihypertensive effects of GE observed in DOCA-salt-treated rats.

Preventive Effects of Grape Extract on Ischemia/Reperfusion-Induced Acute Kidney Injury in Mice.

Since grape extract (GE) contains oligomeric proanthocyanidins and numerous polyphenols, dietary GE supplements may exert protective effects against various diseases. The present study investigated the pharmacological effects of GE derived from Chardonnay in vitro and in vivo. GE (100 µg/mL) completely inhibited tumor necrosis factor-α-induced endothelin-1, monocyte chemoattractant protein-1, interleukin-1ß, and intercellular adhesion molecule-1 gene expression in cultured endothelial cells. GE also strongly stimulated the phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) pathway. In the in vivo study, the effects of GE on ischemic acute kidney injury (AKI) were examined using male C57bl/6J wild-type and eNOS-/- mice. Right nephrectomized mice were exposed to 45 min of ischemia in the left kidney and this was followed by reperfusion. Although renal functional parameters in AKI mice significantly increased 48 h after reperfusion, the administration of GE (0.1 and 1 mg/kg, intravenous (i.v.)) 5 min before ischemia dose-dependently improved post-ischemic renal dysfunction in wild-type mice. Renal histopathological studies on AKI mice revealed tubular necrosis, proteinaceous casts in tubuli, and medullary congestion. The administration of GE ameliorated this damage in wild-type mice, but not in eNOS-/- mice. Furthermore, GE significantly restored decreases in the renal nitric oxide metabolite content due to ischemia in wild-type mice, but not in eNOS-/- mice. Thus, eNOS is closely involved in the renoprotective effects of GE, strongly suggesting that GE supplements are useful as a prophylactic treatment for the development of ischemic AKI.

Involvement of γ-Glutamyl Transpeptidase in Ischemia/Reperfusion-Induced Cardiac Dysfunction in Isolated Rat Hearts.

GGsTop is a highly potent and specific, and irreversible γ-glutamyl transpeptidase (GGT) inhibitor without any influence on glutamine amidotransferases. The aim of the present study was to investigate the involvement of GGT in ischemia/reperfusion-induced cardiac dysfunction by assessing the effects of a treatment with GGsTop. Using a Langendorff apparatus, excised rat hearts underwent 40 min of global ischemia without irrigation and then 30 min of reperfusion. GGT activity was markedly increased in cardiac tissues exposed to ischemia, and was inhibited by the treatment with GGsTop. Exacerbation of cardiac functional parameters caused by ischemia and reperfusion, namely the reduction of left ventricular (LV) developed pressure and the maximum and negative minimum values of the first derivative of LV pressure, and the increment in LV end-diastolic pressure was significantly attenuated by GGsTop treatment. The treatment with GGsTop suppressed excessive norepinephrine release in the coronary perfusate, a marker for myocardial dysfunction, after ischemia/reperfusion. In addition, oxidative stress indicators in myocardium, including superoxide and malondialdehyde, after ischemia/reperfusion were significantly low in the presence of GGsTop. These observations demonstrate that enhanced GGT activity contributes to cardiac damage after myocardial ischemia/reperfusion, possibly via increased oxidative stress and subsequent norepinephrine overflow. GGT inhibitors have potential as a therapeutic strategy to prevent myocardial ischemia/reperfusion injury in vivo.

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.

Mechanisms underlying the renoprotective effect of GABA against ischaemia/reperfusion-induced renal injury in rats.

Excitation of the renal sympathetic nervous system is important for the development of ischaemic acute kidney injury (AKI) in rats. We reported that intravenous treatment with GABA has preventive effects against ischaemia/reperfusion (I/R)-induced renal dysfunction with histological damage in rats; however, the mechanisms underlying these effects on renal injury remain unknown. Thus, the aim of the present study was to clarify how GABA mechanistically affects ischaemic AKI in rats. Ischaemic AKI was induced in rats by clamping the left renal artery and vein for 45 min and then reperfusing the kidney to produce I/R-induced injury. Treatment with the GABAB receptor antagonist CGP52432 (100 nmol/kg, i.v., or 1 nmol/kg, i.c.v.) abolished the suppressive effects of 50 µmol/kg, i.v., GABA on enhanced renal sympathetic nerve activity (RSNA) during ischaemia, leading to elimination of the renoprotective effects of GABA. Intracerebroventricular treatment with 0.5 µmol/kg GABA or i.v. treatment with 1 µmol/kg baclofen, a selective GABAB receptor agonist, prevented the I/R-induced renal injury equivalent to i.v. treatment with GABA. Conversely, i.v. treatment with 10 µmol/kg bicuculline, a GABAA receptor antagonist, failed to affect the preventive effects of GABA against ischaemic AKI. We therefore concluded that GABAB receptor stimulation in the central nervous system, rather than peripheral GABAB receptor stimulation, mediates the preventive effect of GABA against ischaemic AKI by suppressing the enhanced RSNA induced by renal ischaemia.

Involvement of renal sympathetic nerve overactivation in the progression of chronic kidney disease in rats.

Time-dependent changes in the renal sympathetic nerve activity (RSNA) in the progression of chronic kidney disease (CKD) have not been investigated, despite the fact that renal sympathetic nervous system is augmented in the condition of CKD. In the present study, we examined time-dependent changes in RSNA and renal venous norepinephrine concentrations for 12 weeks using 5 of 6 nephrectomized CKD rats. Both RSNA and norepinephrine concentrations were increased during the early phase in the progression of CKD. Urinary protein excretion and systolic blood pressure (SBP) were gradually increased during 12 weeks after 5 of 6 nephrectomy. Treatment with γ-aminobutyric acid or the combination of prazosin and propranolol in the early phase (0-4 weeks) after 5 of 6 nephrectomy significantly attenuated the increases in urinary protein excretion and SBP in 5 of 6 nephrectomized rats. On the other hand, the same treatment in the late phase (8-12 weeks) after 5 of 6 nephrectomy failed to suppress the proteinuria and increase in SBP. Treatment with hydralazine at hypotensive dose for 12 weeks also failed to affect the proteinuria in 5 of 6 nephrectomized CKD rats. In conclusion, the augmentation of renal sympathetic nervous system in early phase after 5 of 6 nephrectomy is closely related to the development of partial ablation-induced CKD in rats.

Oligomycin, an F1Fo-ATPase inhibitor, protects against ischemic acute kidney injury in male but not in female rats.

We investigated the effects of oligomycin, an F1Fo-ATPase inhibitor, on ischemic acute kidney injury in male and female rats. Ischemic acute kidney injury was induced by clamping the left renal artery and vein for 45 or 60 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal dysfunction and histological renal damage were observed 1 day after reperfusion in both male and female rats, although these renal injuries were more marked in male rats than in female rats. Intravenous bolus injection of oligomycin (0.5 mg/kg) 5 min before ischemia markedly attenuated the ischemia/reperfusion-induced renal injury in male rats. However, oligomycin did not show the protective effect in female rats subjected to ischemia/reperfusion-induced renal injury. Pre-ischemic treatment with oligomycin suppressed partly but significantly ischemia-induced renal ATP depletion only in male rats. These results indicate that oligomycin prevents the onset of ischemic acute kidney injury in male but not in female rats, and the effect is accompanied by suppression of the ATP depletion only in the male rat kidney during ischemia, thereby suggesting that the ATP hydrolysis pathway by mitochondrial F1Fo-ATPase induces a sex difference in ischemic acute kidney injury.

Effects of angiotensin II AT1-receptor blockade on high fat diet-induced vascular oxidative stress and endothelial dysfunction in Dahl salt-sensitive rats.

We examined the effects of angiotensin II AT1-receptor blockade with olmesartan on high fat (HF) diet-induced vascular oxidative stress and endothelial dysfunction in normal salt (NS) diet-fed Dahl salt-sensitive (DSS) rats. Treatment with NS + HF diet (32% crude fat, 0.3% NaCl) for 20 weeks significantly increased blood pressure in DSS rats. NS + HF diet-fed DSS rats also showed higher plasma levels of thiobarbituric acid-reactive substances, aortic superoxide production, and mRNA levels of p22(phox) and gp91(phox) in aortic tissues than NS diet-fed DSS rats. Furthermore, acetylcholine-induced vasorelaxation of aorta from NS + HF diet-fed DSS rats was significantly reduced. In NS + HF diet-fed DSS rats, treatment with olmesartan medoxomil (10 mg/kg per day, p.o.) and hydralazine (25 mg/kg per day, p.o.) similarly decreased blood pressure. However, in these animals, only olmesartan normalized plasma levels of thiobarbituric acid-reactive substances, vascular superoxide in aortic tissues, and acetylcholine-induced vasorelaxation. These data indicate that HF diet-induced hypertension is associated with vascular oxidative stress and endothelial dysfunction in NS diet-treated DSS rats. Inhibition of angiotensin II AT1 receptors may elicit beneficial effects on HF-induced hypertension and vascular injury in subjects that have genetically enhanced sodium-sensitive blood pressure.