Prolonged Duration of Erythropoiesis-Stimulating Agents' Action Delays Disease Progression in Anti-Thy 1 Antibody-Induced Chronic Glomerulonephritis Rats.

BACKGROUND: Although erythropoiesis-stimulating agents (ESAs) exert renoprotective effects in renal disease models, it has not been revealed whether the prolonged duration of action of ESAs contributes to their renoprotective effects. OBJECTIVE: We examined whether the prolonged duration of ESAs' action contributes to their renoprotective effects by comparing a divided administration of a short-acting ESA, epoetin beta (EPO), or a single administration of a long-acting ESA, epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.), to a single administration of EPO in chronic glomerulonephritis (GN) rats. MATERIALS AND METHODS: Chronic GN was induced by intravenous injection of anti-Thy 1.1 antibody (0.6 mg/kg) into uninephrectomized rats (day 0). Chronic GN rats were intravenously injected once with vehicle (disease control; DC), EPO 5,000 IU/kg (single EPO), or C.E.R.A. 25 µg/kg (single C.E.R.A.) on day 1; or 3 times during the first week with EPO 1,667 IU/kg from day 1 (divided EPO; total 5,000 IU/kg). Hemoglobin (Hb) level and urinary total protein (U-TP) level which are the indexes of hematopoiesis and renoprotective effects, respectively, were measured several times over 8 weeks. RESULTS: Divided EPO and single C.E.R.A. increased Hb levels more greatly than did single EPO. In all chronic GN rats, elevated U-TP levels decreased transiently 2 weeks after chronic GN induction and then flared again. Single EPO significantly suppressed this exacerbation of U-TP levels compared to DC. Divided EPO and single C.E.R.A. each significantly suppressed the exacerbation of U-TP levels compared to single EPO. CONCLUSION: Prolonged duration of ESAs' action contributed significantly to their renoprotective effects.

Intermittent Ibandronate Maintains Bone Mass, Bone Structure, and Biomechanical Strength of Trabecular and Cortical Bone After Discontinuation of Parathyroid Hormone Treatment in Ovariectomized Rats.

Although parathyroid hormone (PTH) expresses an anabolic effect on bone mass, the increased bone mass disappears once PTH treatment is withdrawn. Therefore, sequential treatment with anti-bone-resorptive agents is required to maintain bone mass after PTH treatment. We examined the effect of sequential treatment with ibandronate (IBN), a nitrogen-containing bisphosphonate, following PTH in ovariectomized (OVX) rats. Wistar-Imamichi rats (27 weeks old) were ovariectomized and treated with PTH (10 µg/kg, s.c.; 5 times/week; PTH group) for 8 weeks from 8 weeks after OVX. Thereafter, PTH was withdrawn and rats were administered IBN (10 µg/kg, s.c.; every 4 weeks; PTH-IBN group) or vehicle (PTH-Veh group) for another 8 weeks. PTH increased bone mineral density (BMD) measured by dual-energy X-ray absorptiometry and biomechanical strength in the lumbar spine and femur as compared to the disease control rats. BMD and biomechanical strength in the PTH-Veh group were lower than in the PTH group, whereas in the PTH-IBN group they were maintained at the level of the PTH group. Microstructure of the trabecular and cortical bone in the PTH-IBN group was not significantly different from that in the PTH group. In histomorphometric analysis of the lumbar vertebra, eroded surface and osteoclast surface in the PTH-Veh group were no different from those in the PTH group, whereas they were lower in the PTH-IBN group. Osteoid surface, osteoblast surface, and mineralize surface decreased in both PTH-IBN and PTH-Veh groups compared to the PTH group, and these parameters in the PTH-IBN group were lower than in the PTH-Veh group. These results indicated that intermittent IBN after PTH treatment suppressed bone turnover and maintained BMD, biomechanical strength, and microstructure in the lumbar spine and femur of OVX rats.

Nicorandil prevents sirolimus-induced production of reactive oxygen species, endothelial dysfunction, and thrombus formation.

Sirolimus (SRL) is widely used to prevent restenosis after percutaneous coronary intervention. However, its beneficial effect is hampered by complications of thrombosis. Several studies imply that reactive oxygen species (ROS) play a critical role in endothelial dysfunction and thrombus formation. The present study investigated the protective effect of nicorandil (NIC), an anti-angina agent, on SRL-associated thrombosis. In human coronary artery endothelial cells (HCAECs), SRL stimulated ROS production, which was prevented by co-treatment with NIC. The preventive effect of NIC on ROS was abolished by 5-hydroxydecanoate but not by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. NIC also inhibited SRL-induced up-regulation of NADPH oxidase subunit p22(phox) mRNA. Co-treatment with NIC and SRL significantly up-regulated superoxide dismutase 2. NIC treatment significantly improved SRL-induced decrease in viability of HCAECs. The functional relevance of the preventive effects of NIC on SRL-induced ROS production and impairment of endothelial viability was investigated in a mouse model of thrombosis. Pretreatment with NIC inhibited the SRL-induced acceleration of FeCl3-initiated thrombus formation and ROS production in the testicular arteries of mice. In conclusion, NIC prevented SRL-induced thrombus formation, presumably due to the reduction of ROS and to endothelial protection. The therapeutic efficacy of NIC could represent an additional option in the prevention of SRL-related thrombosis.

Nicorandil suppresses urinary protein excretion and activates eNOS in Dahl salt-sensitive hypertensive rats.

BACKGROUND: Hypertension is a risk factor common to both chronic kidney disease and cardiovascular disease. Nicorandil is widely used for the treatment of angina. We investigated the benefits of nicorandil with respect to renal dysfunction in Dahl salt-sensitive hypertensive (DS) rats. METHOD: DS rats were fed a high-salt (HS) diet and nicorandil was administered via the drinking water. Blood pressure and renal function were measured for 4 weeks after starting the rats on the HS diet. RESULTS: In rats fed the HS diet, renal dysfunction was manifested by an increase in urinary protein and N-acetyl-ß-D-glucosaminidase excretion. Nicorandil ameliorated renal function with a concomitant reduction in urinary 8-hydroxy-2'-deoxyguanosine and an increase in urinary NOx. Significant upregulation of endothelial nitric oxide synthase (eNOS) expression and an increase in the eNOS dimer/monomer ratio (reduction of eNOS uncoupling) was demonstrated in glomeruli following nicorandil treatment. The blood pressure of DS rats was increased by salt loading; however, no significant change in blood pressure was observed with nicorandil treatment. CONCLUSION: In DS rats fed a HS diet, nicorandil prevented the development of renal dysfunction, which was accompanied by an increase in eNOS expression in the kidneys.

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.

IL-6 receptor antibody treatment improves muscle weakness in experimental autoimmune myasthenia gravis mouse model.

Myasthenia gravis (MG) is a chronic autoimmune disease characterized by muscle weakness and fatigue. It is caused by pathological autoantibodies against components expressed at neuromuscular junctions, such as acetylcholine receptor (AChR). Interleukin-6 (IL-6) has been suggested to play a role in the pathogenesis of MG, and IL-6 receptor (IL-6R) antibody treatment may provide a novel therapeutic option. In this study, we investigated the effects of IL-6R antibody treatment in an experimental autoimmune MG (EAMG) mouse model. We demonstrated that IL-6R antibody treatment improved muscle weakness, reduced IgG deposition at neuromuscular junctions, and the levels of AChR autoantibodies in serum. In addition, follicular helper T cells and Th17, plasma cells in lymph nodes were lower in IL-6R antibody treated mice. Our findings suggest that IL-6R blockade may be a novel and effective therapeutic strategy for the treatment of MG.

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.

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 ß(2)-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.

Intradermal AQP4 peptide immunization induces clinical features of neuromyelitis optica spectrum disorder in mice.

We challenged to create a mouse model of neuromyelitis optica spectrum disorder (NMOSD) induced by AQP4 peptide immunization. Intradermal immunization with AQP4 p201-220 peptide induced paralysis in C57BL/6J mice, but not in AQP4 KO mice. AQP4 peptide-immunized mice showed pathological features similar to NMOSD. Administration of anti-IL-6 receptor antibody (MR16-1) inhibited the induction of clinical signs and prevented the loss of GFAP/AQP4 and deposition of complement factors in AQP4 peptide-immunized mice. This novel experimental model may contribute to further understanding the pathogenesis of NMOSD, elucidating the mechanism of action of therapeutic agents, and developing new therapeutic approaches.

Paclitaxel-induced endothelial dysfunction in living rats is prevented by nicorandil via reduction of oxidative stress.

Paclitaxel-eluting stents dramatically reduce rates of in-stent restenosis; however, paclitaxel is known to lead to endothelial dysfunction. Protective effects of nicorandil on paclitaxel-induced endothelial dysfunction by examining flow-mediated dilation (FMD) were investigated in anesthetized rats. After 7-day osmotic infusion of paclitaxel (5 mg/kg per day), FMD was measured by high-resolution ultrasound in the femoral artery of living rats. Paclitaxel significantly reduced FMD (21.6% ± 3.2% to 7.1% ± 1.7%); this reduction was prevented by co-treatment with nicorandil (15 mg/kg per day), while paclitaxel did not affect nitroglycerin-induced vasodilation. Diazoxide and tempol, but not isosorbide dinitrate, had an effect similar to nicorandil in preventing paclitaxel-induced decrease in FMD. Nicorandil significantly prevented paclitaxel-induced reduction in acetylcholine-induced vasodilation. On the underling mechanisms, paclitaxel increased reactive oxygen species (ROS) production (dihydrorhodamine 123, DCF fluorescence intensity) and NADPH oxidase (p47(phox), gp91(phox) mRNA) in arteries and human coronary artery endothelial cells (HCAECs), while paclitaxel reduced nitric oxide (NO) release (DAF-2 fluorescence intensity), but not endothelial NO synthase (eNOS) phosphorylation in HCAECs. Nicorandil prevented the increased ROS production in arteries and HCAECs, which was 5-hydroxydecanoate (5-HD)-sensitive but 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)-resistant, without significant effect on the reduced NO release. In conclusion, nicorandil prevents paclitaxel-induced endothelial dysfunction, which may be brought by improved NO bioavailability due to the reduction of oxidative stress via K(ATP) channel activation.

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

BACKGROUND: Nicorandil, 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. METHODS: Male 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. RESULTS: Endothelial 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. CONCLUSIONS: These results suggest that nicorandil improved diabetes-induced endothelial dysfunction through antioxidative effects by inhibiting NADPH oxidase and eNOS uncoupling.

Interleukin-6: evolving role in the management of neuropathic pain in neuroimmunological disorders.

BACKGROUND: Neuropathic pain in neuroimmunological disorders refers to pain caused by a lesion or disease of the somatosensory system such as multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). MS and NMOSD are autoimmune disorders of the central nervous system, and ≥ 50% of patients with these disorders experience chronic neuropathic pain. The currently available medications for the management of neuropathic pain have limited effectiveness in patients with MS and NMOSD, and there is an unmet medical need to identify novel therapies for the management of chronic neuropathic pain in these patients. In this review article, we summarize the role of interleukin-6 (IL-6) in the pathogenesis of MS and NMOSD and the ameliorative effects of anti-IL-6 therapies in mouse models of experimental autoimmune encephalomyelitis (EAE). MAIN BODY: Intraperitoneal injection of MR16-1, an anti-IL-6 receptor (IL-6R) antibody, reduced mechanical allodynia and spontaneous pain in EAE mice, which was attributed to a reduction in microglial activation and inhibition of the descending pain inhibitory system. The effect of anti-IL-6 therapies in ameliorating neuropathic pain in the clinical setting is controversial; a reduction in pain intensity has been reported with an anti-IL-6 antibody in four studies, namely a case report, a pilot study, a retrospective observational study, and a case series. Pain intensity was evaluated using a numerical rating scale (NRS), with a lower score indicating lesser pain. A reduction in the NRS score was reported in all four studies. However, in two randomized controlled trials of another anti-IL-6R antibody, the change in the visual analog scale pain score was not statistically significantly different when compared with placebo. This was attributed to the low mean pain score at baseline in both the trials and the concomitant use of medications for pain in one of the trials, which may have masked the effects of the anti-IL-6R antibody on neuropathic pain. CONCLUSION: Thus, anti-IL-6 therapies might have a potential to reduce neuropathic pain, but further investigations are warranted to clarify the effect of inhibition of IL-6 signaling on neuropathic pain associated with MS and NMOSD.

New BBB Model Reveals That IL-6 Blockade Suppressed the BBB Disorder, Preventing Onset of NMOSD.

BACKGROUND AND OBJECTIVES: To evaluate the pathophysiology of neuromyelitis optica spectrum disorder (NMOSD) and the therapeutic mechanism and levels of interleukin-6 (IL-6) blockade (satralizumab), especially with respect to blood-brain barrier (BBB) disruption with the new in vitro and ex vivo human BBB models and in vivo model. METHODS: We constructed new static in vitro and flow-based ex vivo models for evaluating continued barrier function, leukocyte transmigration, and intracerebral transferability of neuromyelitis optica-immunoglobulin G (NMO-IgG) and satralizumab across the BBB using the newly established triple coculture system that are specialized to closely mimic endothelial cell contact of pericytes and endfeet of astrocytes. In the in vivo study, we assessed the effects of an anti-IL-6 receptor antibody for mice (MR16-1) on in vivo BBB disruption in mice with experimental autoimmune encephalomyelitis in which IL-6 concentration in the spinal cord dramatically increases. RESULTS: In vitro and ex vivo experiments demonstrated that NMO-IgG increased intracerebral transferability of satralizumab and NMO-IgG and that satralizumab suppressed the NMO-IgG-induced transmigration of T cells and barrier dysfunction. In the in vivo study, the blockade of IL-6 signaling suppressed the migration of T cells into the spinal cord and prevented the increased BBB permeability. DISCUSSION: These results suggest that (1) our triple-cultured in vitro and in ex vivo BBB models are ideal for evaluating barrier function, leukocyte transmigration, and intracerebral transferability; (2) NMO-IgG increased the intracerebral transferability of NMO-IgG via decreasing barrier function and induced secretion of IL-6 from astrocytes causing more dysfunction of the barrier and disrupting controlled cellular infiltration; and (3) satralizumab, which can pass through the BBB in the presence of NMO-IgG, suppresses the BBB dysfunction and the infiltration of inflammatory cells, leading to prevention of onset of NMOSD.

Anti-IL-6 Receptor Antibody Inhibits Spontaneous Pain at the Pre-onset of Experimental Autoimmune Encephalomyelitis in Mice.

Chronic pain is a significant symptom in patients with autoimmune encephalomyelitis, such as multiple sclerosis and neuromyelitis optica. The most commonly used animal model of these diseases is experimental autoimmune encephalomyelitis (EAE). We previously reported that evoked pain, such as mechanical allodynia, was improved by an anti-IL-6 receptor antibody in EAE mice. However, few reports have evaluated spontaneous pain in EAE mice. Here, we assessed spontaneous pain in EAE mice by utilizing the Mouse Grimace Scale (MGS, a standardized murine facial expression-based coding system) and evaluated the influence of an anti-IL-6 receptor antibody (MR16-1). EAE was induced in female C57BL/6J mice by subcutaneous immunization with myelin oligodendrocyte glycoprotein 35-55 emulsified in adjuvant and administration of pertussis toxin. Mice were placed individually in cubicles and filmed for about 10 min. Ten clear head shots per mouse from the video recording were given a score of 0, 1, or 2 for each of three facial action units: orbital tightening, nose bulge, and ear position. Clinical symptoms of EAE were also scored. Measurement of 5-HT in the spinal cord and functional imaging of the periaqueductal gray (PAG) were also performed. Compared with control mice, MGS score was significantly higher in EAE mice. MR16-1 prevented this increase, especially in pre-onset EAE mice. Promotion of spinal 5-HT turnover and reduction of PAG activity were observed in pre-onset EAE mice. These results suggest that MR16-1 prevented spontaneous pain developed before EAE onset.

Anti-IL-6 receptor antibody improves pain symptoms in mice with experimental autoimmune encephalomyelitis.

BACKGROUND AND AIMS: Chronic pain is a prevalent symptom in patients with autoimmune encephalomyelitis such as multiple sclerosis and neuromyelitis optica. Although IL-6 is involved in various inflammatory and immune diseases, the roles of IL-6 in autoimmune-related pain have not been clarified. Therefore, we examined the effect of anti-IL-6 receptor antibody (MR16-1) on the pain sensitivity of experimental autoimmune encephalomyelitis (EAE) mice. MATERIALS AND METHODS: EAE was induced in female C57BL/6J mice by subcutaneous immunization with myelin oligodendrocyte glycoprotein 35-55 emulsified in adjuvant (Day 0). Pertussis toxin was intravenously administered at Days 0 and 2. Mice were sequentially scored for clinical symptoms of EAE. [Exp. 1] MR16-1 was intraperitoneally administered on Days 0 or 3. Sensitivity to pain was measured by the von Frey test (Days 7, 14, 20). The spinal cord was isolated and assessed by immunohistochemistry. [Exp. 2] MR16-1 was intraperitoneally administered on Day 12 when significant pain had already occurred. Pain assessment was conducted before the immunization, on Day 12 and after EAE onset. And then, spinal cord was isolated and flow cytometry was performed. RESULTS: [Exp. 1] MR16-1 prevented the increase in clinical score and sensitivity to pain in EAE mice. Immunohistochemical analysis showed that Iba1+ microglia were increased in the spinal cord of EAE mice, and were reduced by MR16-1. [Exp. 2] Administration of MR16-1 on Day 12 also reduced sensitivity to pain under EAE onset. Flow cytometry showed that CD45lowCD11b+ microglia were increased in the spinal cord of EAE mice, and that this increase was inhibited by MR16-1. CONCLUSION: These findings suggest that MR16-1 can decrease mechanical allodynia in EAE mice through inhibition of microglial activation and proliferation in the spinal cord.

Epoetin beta pegol ameliorates flow-mediated dilation with improving endothelial nitric oxide synthase coupling state in nonobese diabetic rats.

BACKGROUND/AIMS: Patients with diabetic nephropathy have a high cardiovascular mortality. Epoetin beta pegol (continuous erythropoietin receptor activator, C.E.R.A.) is a drug for the treatment of renal anemia. In this study, we investigated the effect of C.E.R.A. on vascular endothelial function as evaluated by flow-mediated dilation (FMD) and the relationship between hematopoiesis and FMD in diabetic nephropathy rats. METHODS: Male Spontaneously Diabetic Torii rats (SDT, 22 weeks old) were used. C.E.R.A. (0.6, 1.2 µg/kg) was administered subcutaneously once every 2 weeks for 8 weeks. At 1 week after last administration (31 weeks old), we assessed FMD in the femoral arteries of anesthetized rats using a high-resolution ultrasound system. FMD was also measured 1 week after single C.E.R.A. treatment (5.0 µg/kg) to examine the influence of hematopoiesis. RESULTS: Flow-mediated dilation was significantly decreased in SDT rats before the start of C.E.R.A. treatment (22 weeks old). Repeated administration of C.E.R.A. dose-dependently improved FMD in SDT rats (31 weeks old) without changing blood glucose, nitroglycerin-induced vasodilation, or kidney function. Long-term administration of C.E.R.A. improved the state of endothelial nitric oxide synthase uncoupling in the femoral arteries of SDT rats, which showed a positive correlation with FMD. On the other hand, there was no correlation between FMD and Hb or Hct in SDT rats. Furthermore, at 1 week after single administration of C.E.R.A., FMD was not significantly improved although hemoglobin levels were comparable with levels following long-term C.E.R.A. CONCLUSION: Long-term treatment with C.E.R.A. improved FMD in SDT rats even after onset of endothelial dysfunction.

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 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.

Contribution of sensory C-fiber neuron injury to mechanical dynamic allodynia in a murine model of postherpetic neuralgia.

Mechanical dynamic allodynia is a hallmark symptom of postherpetic neuralgia, but the mechanisms are unclear. This study examined the participation of injury to sensory C-fiber and A-fiber neurons in postherpetic dynamic allodynia. Percutaneous inoculation of mice with herpes simplex virus type-1 caused zoster-like skin lesions and dynamic allodynia, which persisted after lesion healed. In postherpetic mice, the intensity of dynamic allodynia was positively and negatively correlated with the withdrawal latency of nociceptive response to heat and the intensity of aversive response to capsaicin, respectively. Calcitonin gene-related peptide immunoreactivity (a C-fiber marker) was markedly reduced, but neurofilament 200 immunoreactivity (an A-fiber neuron marker) was unchanged in the scarred skin of postherpetic mice. In the affected dorsal root ganglion of postherpetic mice, peripherin-immunoreactive (a C-fiber neuron marker) neurons reduced significantly, whereas neurofilament 200-immunoreactive neurons did not. These results suggest that postherpetic dynamic allodynia is associated with injury to sensory C-fiber neurons and little damage to A-fiber neurons.

Left ventricular hypertrophy is associated with inflammation in sodium loaded subtotal nephrectomized rats.

The pathological influences of inflammation on left ventricular hypertrophy (LVH) were studied in subtotal nephrectomized (SNx) rats after 0.3% NaCl loading for 5 weeks. We found that mild hypertension, increased plasma levels of creatinine, inorganic phosphate, asymmetric dimethylarginine (ADMA), and parathyroid hormone (PTH) were observed in the present SNx rats without LVH. In the present study, the NaCl-loaded SNx (SNx + NaCl) rats were characterized by significant LVH and hypertension with aggravated values of all the parameters. We further confirmed that glomerular sclerosis, tubulointerstitial fibrosis, and inflammatory cell infiltration into the tubulointerstitial area, observed in the SNx rats, were more severely caused in the SNx + NaCl rats. In addition, plasma interleukin-6 (IL-6) levels in the SNx + NaCl rats were significantly increased compared to those in the SNx rats. These findings indicated that NaCl-loaded SNx rats developed LVH and hypertension, which were accompanied with increased plasma levels of PTH, creatinine, inorganic phosphorus, ADMA, and IL-6. Thus, these results suggest that inflammation as well as endothelial dysfunction would be correlated with LVH as non-traditional risk factors at the early stage in the present renal failure model.