Yoshihito Tashiro

Nicorandil prevents endothelial dysfunction due to antioxidative effects via normalisation of NADPH oxidase and nitric oxide synthase in streptozotocin diabetic rats.

BackgroundNicorandil, an anti-angina agent, reportedly improves outcomes even in angina patients with diabetes. However, the precise mechanism underlying the beneficial effect of nicorandil on diabetic patients has not been examined. We investigated the protective effect of nicorandil on endothelial function in diabetic rats because endothelial dysfunction is a major risk factor for cardiovascular disease in diabetes.MethodsMale Sprague-Dawley rats (6 weeks old) were intraperitoneally injected with streptozotocin (STZ, 40 mg/kg, once a day for 3 days) to induce diabetes. Nicorandil (15 mg/kg/day) and tempol (20 mg/kg/day, superoxide dismutase mimetic) were administered in drinking water for one week, starting 3 weeks after STZ injection. Endothelial function was evaluated by measuring flow-mediated dilation (FMD) in the femoral arteries of anaesthetised rats. Cultured human coronary artery endothelial cells (HCAECs) were treated with high glucose (35.6 mM, 24 h) and reactive oxygen species (ROS) production with or without L-NAME (300 µM), apocynin (100 µM) or nicorandil (100 µM) was measured using fluorescent probes.ResultsEndothelial function as evaluated by FMD was significantly reduced in diabetic as compared with normal rats (diabetes, 9.7 ± 1.4%; normal, 19.5 ± 1.7%; n = 6-7). There was a 2.4-fold increase in p47phox expression, a subunit of NADPH oxidase, and a 1.8-fold increase in total eNOS expression in diabetic rat femoral arteries. Nicorandil and tempol significantly improved FMD in diabetic rats (nicorandil, 17.7 ± 2.6%; tempol, 13.3 ± 1.4%; n = 6). Nicorandil significantly inhibited the increased expressions of p47phox and total eNOS in diabetic rat femoral arteries. Furthermore, nicorandil significantly inhibited the decreased expression of GTP cyclohydrolase I and the decreased dimer/monomer ratio of eNOS. ROS production in HCAECs was increased by high-glucose treatment, which was prevented by L-NAME and nicorandil suggesting that eNOS itself might serve as a superoxide source under high-glucose conditions and that nicorandil might prevent ROS production from eNOS.ConclusionsThese results suggest that nicorandil improved diabetes-induced endothelial dysfunction through antioxidative effects by inhibiting NADPH oxidase and eNOS uncoupling.

22-Oxacalcitriol prevents progression of endothelial dysfunction through antioxidative effects in rats with type 2 diabetes and early-stage nephropathy

BACKGROUND Vitamin D deficiency is associated with endothelial dysfunction in type 2 diabetes patients, but the effectiveness of vitamin D supplementation remains controversial. We assessed whether 22-oxacalcitriol (OCT) could prevent endothelial dysfunction in type 2 diabetes mellitus (DM) rats. METHODS DM rats with early-stage nephropathy were treated for 10 weeks with OCT (0.2 μg/kg) three times per week or by an implanted insulin pellet. Endothelial dysfunction was assessed by femoral flow-mediated dilation (FMD). RESULTS Insulin significantly improved FMD as blood glucose levels normalized. OCT also improved FMD without hypercalcemia or hyperphosphatemia and without affecting blood glucose or blood pressure. In femoral arteries, OCT significantly suppressed the elevated expression of p22(phox), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit, and improved the endothelial nitric oxide synthase (eNOS) dimer-to-monomer ratio. In cultured endothelial cells, OCT significantly inhibited high-glucose (HG)-induced reactive oxygen species (ROS) production. Simultaneously, OCT significantly suppressed HG-induced p22(phox) expression and improved eNOS uncoupling as was observed in the in vivo study. CONCLUSION In DM rats, OCT improved endothelial dysfunction, at least in part, by suppressing ROS generation through p22(phox) expression, which might contribute to improving eNOS uncoupling.

Vitamin D Receptor Signaling Enhances Locomotive Ability in Mice

Bone fractures markedly reduce quality of life and life expectancy in elderly people. Although osteoporosis increases bone fragility, fractures frequently occur in patients with normal bone mineral density. Because most fractures occur on falling, preventing falls is another focus for reducing bone fractures. In this study, we investigated the role of vitamin D receptor (VDR) signaling in locomotive ability. In the rotarod test, physical exercise enhanced locomotive ability of wild‐type (WT) mice by 1.6‐fold, whereas exercise did not enhance locomotive ability of VDR knockout (KO) mice. Compared with WT mice, VDR KO mice had smaller peripheral nerve axonal diameter and disordered AChR morphology on the extensor digitorum longus muscle. Eldecalcitol (ED‐71, ELD), an analog of 1,25(OH)2D3, administered to rotarod‐trained C57BL/6 mice enhanced locomotor performance compared with vehicle‐treated nontrained mice. The area of AChR cluster on the extensor digitorum longus was greater in ELD‐treated mice than in vehicle‐treated mice. ELD and 1,25(OH)2D3 enhanced expression of IGF‐1, myelin basic protein, and VDR in rat primary Schwann cells. VDR signaling regulates neuromuscular maintenance and enhances locomotive ability after physical exercise. Further investigation is required, but Schwann cells and the neuromuscular junction are targets of vitamin D3 signaling in locomotive ability.

Renoprotection by continuous erythropoietin receptor activator in puromycin aminonucleoside-induced nephrotic syndrome.

Background/Aims: Recent studies have demonstrated that erythropoiesis-stimulating agents (ESAs) induce a tissue-protective effect in the kidney. In this study, we examined whether continuous erythropoietin receptor activator (CERA), a long-acting ESA, could prevent kidney injury, especially podocyte damage, in a rat model of nephrotic syndrome induced by puromycin aminonucleoside (PAN). Methods: Rats were injected with CERA (30 µg/kg) or vehicle 4 h before the injection of PAN (50 mg/kg). Renal function, kidney injury, and podocyte damage were assessed at 7 days. Results: The levels of proteinuria, BUN, and plasma creatinine significantly increased in rats with PAN-induced nephrosis. Treatment with CERA significantly prevented these deteriorations induced by PAN. Glomerular lesions, especially vacuolation of podocytes, and the increase of desmin expression in PAN-treated rats were significantly ameliorated by treatment with CERA. Treatment with CERA also significantly prevented the decrease in the protein productions of nephrin and podocin in the kidneys of PAN-treated rats. We found persistent activation of the Akt signaling pathway in the kidneys of CERA-treated rats. Conclusion: CERA could ameliorate renal dysfunction in PAN-induced nephrosis, which might be due to the amelioration of podocyte injury. CERA inhibited the depletion of nephrin and podocin, key components of the glomerular filtration barrier, and alleviated proteinuria. Activation of the Akt signaling pathway might be involved in the renoprotective effect of CERA

Epoetin beta pegol prevents endothelial dysfunction as evaluated by flow-mediated dilation in chronic kidney disease rats

Chronic kidney disease (CKD) patients have a poor prognosis due to cardiovascular disease. Anemia and endothelial dysfunction are important risk factors for cardiovascular events in CKD patients, and treatment with erythropoiesis-stimulating agent (ESA) has been reported to improve the quality of life in CKD patients. In this study, we evaluated the effect of anemia correcting dose of epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.) on endothelial function in 5/6 nephrectomized rats (Nx rats). C.E.R.A. was subcutaneously administered once a fortnight, 5 times in total, from 1 week after nephrectomy. Twenty-four hours after last administration, endothelial function was evaluated by measuring flow-mediated dilation (FMD) in the femoral arteries of anesthetized Nx rats by ultrasound system. Femoral arteries were harvested for western blot analysis. C.E.R.A. significantly increased FMD of Nx rats. Endothelium-independent vasodilation induced by nitroglycerin injection was not influenced by C.E.R.A treatment. Nox4 expression and nitrotyrosine accumulation were significantly decreased, and phosphorylation of eNOS was significantly enhanced in the femoral arteries of C.E.R.A.-treated rats. C.E.R.A. normalized hemoglobin levels but did not affect body weight, systolic blood pressure, heart rate, urinary protein excretion and plasma creatinine. These results indicate that C.E.R.A. prevented endothelial dysfunction in Nx rats, possibly through reduction of local oxidative stress and enhancement of eNOS phosphorylation in the arteries. This study provides the first evidence that C.E.R.A. prevented endothelial dysfunction in CKD model rats under conditions of amelioration of anemia.

Eldecalcitol prevents endothelial dysfunction in postmenopausal osteoporosis model rats.

Postmenopausal women have high incidence of cardiovascular events as estrogen deficiency can cause endothelial dysfunction. Vitamin D is reported to be beneficial on endothelial function, but it remains controversial whether vitamin D is effective for endothelial dysfunction under the treatment for osteoporosis in postmenopausal women. The aim of this study was to evaluate the endothelial protective effect of eldecalcitol (ELD) in ovariectomized (OVX) rats. ELD (20  ng/kg) was orally administrated five times a week for 4 weeks from 1 day after surgery. After that, flow-mediated dilation (FMD) as an indicator of endothelial function was measured by high-resolution ultrasound in the femoral artery of living rats. ELD ameliorated the reduction of FMD in OVX rats. ELD inhibited the increase in NOX4, nitrotyrosine, and p65 and the decrease in dimer/monomer ratio of nitric oxide synthase in OVX rat femoral arteries. ELD also prevented the decrease in peroxisome proliferator-activated receptor gamma (PPARγ) in femoral arteries and cultured endothelial cells. Although PPARγ is known to inhibit osteoblastogenesis, ELD understandably increased bone mineral density of OVX rats without increase in PPARγ in bone marrow. These results suggest that ELD prevented the deterioration of endothelial function under condition of preventing bone loss in OVX rats. This endothelial protective effect of ELD might be exerted through improvement of endothelial nitric oxide synthase uncoupling, which is mediated by an antioxidative effect through normalization of vascular PPARγ/NF-κB signaling.

Epoetin beta pegol alleviates oxidative stress and exacerbation of renal damage from iron deposition, thereby delaying CKD progression in progressive glomerulonephritis rats

The increased deposition of iron in the kidneys that occurs with glomerulopathy hinders the functional and structural recovery of the tubules and promotes progression of chronic kidney disease (CKD). Here, we evaluated whether epoetin beta pegol (continuous erythropoietin receptor activator: CERA), which has a long half‐life in blood and strongly suppresses hepcidin‐25, exerts renoprotection in a rat model of chronic progressive glomerulonephritis (cGN). cGN rats showed elevated urinary total protein excretion (uTP) and plasma urea nitrogen (UN) from day 14 after the induction of kidney disease (day 0) and finally declined into end‐stage kidney disease (ESKD), showing reduced creatinine clearance with glomerulosclerosis, tubular dilation, and tubulointerstitial fibrosis. A single dose of CERA given on day 1, but not on day 16, alleviated increasing uTP and UN, thereby delaying ESKD. In the initial disease phase, CERA significantly suppressed urinary 8‐OHdG and liver‐type fatty acid–binding protein (L‐FABP), a tubular damage marker. CERA also inhibited elevated plasma hepcidin‐25 levels and alleviated subsequent iron accumulation in kidneys in association with elevated urinary iron excretion and resulted in alleviation of growth of Ki67‐positive tubular and glomerular cells. In addition, at day 28 when the exacerbation of uTP occurs, a significant correlation was observed between iron deposition in the kidney and urinary L‐FABP. In our study, CERA mitigated increasing kidney damage, thereby delaying CKD progression in this glomerulonephritis rat model. Alleviation by CERA of the exacerbation of kidney damage could be attributable to mitigation of tubular damage that might occur with lowered iron deposition in tubules.

Nicorandil ameliorated hypertensive renal injury without lowering blood pressure in spontaneously hypertensive rats.

Proteinuria, a symptom of hypertensive renal injury, is a powerful predictor of mortality in chronic kidney disease patients with hypertension. The present study investigated whether a nonhypotensive dose of nicorandil could decrease hypertensive renal injury in male spontaneously hypertensive rats (SHR). Nicorandil (15 mg/kg/day, for 20 weeks) was administered in the drinking water to rats from 11 weeks old. Heart size, kidney size, and β2-microglobulin occurring with tubular histopathological damage were each significantly greater in SHR than in Wistar-Kyoto (WKY) rats, as was 24-hour excretion of urinary protein (SHR: 33.1 ± 3.5 mg/day, WKY: 5.4 ± 0.3 mg/day). Nicorandil significantly decreased urinary protein (21.7 ± 2.8 mg/day), glomerular cell density, and histopathological score without affecting systolic blood pressure. Nicorandil increased expression of endothelial nitric oxide synthase (eNOS) protein in the renal cortex in SHR without affecting expressions of mRNA for endothelin or genes involved in tissue damage or fibrosis. eNOS expression was negatively correlated with glomerular cell density. In addition, nicorandil increased urinary excretion of NOx, but did not change the eNOS dimer-to-monomer ratio or the decreased level of renal heparan sulfate in SHR. In conclusion, in SHR, long-term administration of nicorandil can ameliorate hypertensive proteinuria, without lowering blood pressure, possibly through an increase in eNOS expression.

GATA-4 Transcription Factor Regulates Cardiac COX-2 Expression Induced by Nicorandil in Left Ventricle of Rats

Background and Aims: Cardioprotective effects induced by delayed ischemic preconditioning and by nicorandil are mediated via expression of cardioprotective factors such as COX-2. The present study was undertaken to evaluate whether nicorandil could induce COX-2 in rats and to elucidate its mode of induction pharmacologically. Methods and Results: Three hours after administration of nicorandil (10 mg/kg, p.o.), COX-2 mRNA and protein were significantly increased in the left ventricle, although other cardioprotective factors (Bcl-2, eNOS, hexokinase, HSP, and iNOS) were not increased. This COX-2 induction in the left ventricle was preceded by induction of GATA-4, which was significant from 1 h after administration. Ventricular levels of 6-keto-prostaglandin F1α were increased 6 h after administration. Although pinacidil or isosorbide dinitrate alone did not increase COX-2 mRNA, their combined application significantly increased COX-2 mRNA. Moreover, although glibenclamide or ODQ each partly inhibited the induction of COX-2 mRNA by nicorandil, their combined application significantly inhibited it. These results suggest that nicorandil induces COX-2 protein through both the activation of KATP channels and guanylate cyclase. Conclusion: The present study demonstrated that nicorandil induces COX-2 via GATA-4 induction in the heart through both KATP channel activation and its nitrate-like properties.

Epoetin beta pegol, but not recombinant erythropoietin, retains its hematopoietic effect in vivo in the presence of the sialic acid-metabolizing enzyme sialidase

Erythropoiesis-stimulating agents (ESAs) are widely used for treating chronic kidney disease (CKD)-associated anemia. The biological activity of ESAs is mainly regulated by the number of sialic acid-containing carbohydrates on the erythropoietin (EPO) peptide. Sialidase, a sialic acid-metabolizing enzyme that accumulates in CKD patients, is suspected of contributing to shortening the circulation half-life of ESAs. Epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.), is an EPO integrated with methoxypolyethylene glycol (PEG). It has been suggested that C.E.R.A. may exert a favorable therapeutic effect, even under conditions of elevated sialidase; however, no detailed investigation of the pharmacological profile of C.E.R.A. in the presence of sialidase has been reported. In the present study, we injected C.E.R.A. or EPO pre-incubated with sialidase into rats, and assessed the hematopoietic effect by reticulocyte count. The hematopoietic effect of C.E.R.A., but not EPO, was preserved after sialidase treatment, despite the removal of sialic acid. Proliferation of EPO-dependent leukemia cells (AS-E2) was significantly increased by desialylated C.E.R.A. and EPO compared to non-treated C.E.R.A. or EPO. In conclusion, we show that C.E.R.A. exerts a favorable hematopoietic effect even under conditions of elevated sialidase. Our findings may contribute to a better understanding of CKD and more effective therapeutic approaches based on a patient’s profile of anemia.