Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor symptoms and neuropathological features, such as loss of dopaminergic neurons in the substantia nigra pars compacta and accumulation of alpha-synuclein (α-Syn). Progranulin (PGRN) is a secreted growth factor that exhibits anti-inflammatory properties and regulates lysosomal function. Although autophagy-lysosome pathway is the main degradative pathway for α-Syn, the molecular mechanistic relationship between PD and PGRN remains unclear. In this study, we investigated the role of PGRN in PD pathology. PGRN protein expression in striatum was increased in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model mice. Intracerebroventricular (i.c.v.) administration of PGRN ameliorated the decrease in expression of tyrosine hydroxylase, a dopaminergic neuron marker, in MPTP-treated mice. Furthermore, i.c.v. administration of PGRN ameliorated 6-hydroxydopamine-induced motor deficits. In SH-SY5Y human neuroblastoma cells, 1-methyl-4-phenylpyridinium ion (MPP⁺), an active metabolite of MPTP, increased α-Syn expression. In contrast, PGRN ameliorated MPP⁺-induced increase in α-Syn expression. Although PGRN decreased the levels of autophagy-related proteins Sequestosome-1 (p62) and MAP1LC3 (LC3)-II, PGRN did not influence the phosphorylation of AMP-activated protein kinase and mechanistic target of rapamycin, which are also proteins that regulate autophagy. Immunostaining analysis showed that PGRN ameliorated MPP⁺-induced increase of LC3 puncta, indicator of autophagosome, and co-localization of LC3 and α-Syn. The DALGreen assay showed that PGRN ameliorated MPP⁺-induced decreasing trend of autolysosomes. These results suggest that PGRN participates in α-Syn degradation via acceleration of the autophagy-lysosome pathway and is a potential therapeutic target for PD.

Thyroid cancer (TC) is the most common malignant tumor of endocrine system and head and neck. Ononin is an isoflavone component, which exhibited great antioxidant and anti-inflammatory activities. This study was conducted to explore the functions of ononin in the TC progression. The cell counting kit-8 (CCK8) assay was applied for the cell viability determination. The cell death and apoptosis rate were analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and flow cytometry. The quantitative real-time PCR (qRT-PCR) and Western blot assays were performed for the relative expressions determination. Lactate dehydrogenase (LDH) release assay was used to assess cytotoxicity. Ononin treatment prominently inhibited the cell viability and induced the cell apoptosis of the TC cells. Besides, caspase 3 (CASP3) was down-regulated and CD274 was up-regulated in TC. Ononin treatment prominently decreased the CD274 levels and increased the CASP3 levels in the TC cells. Additionally, ononin treatment dramatically enhanced the LDH release of the cytotoxicity of T cells. What is more, CASP3 overexpression or CD274 knockdown promoted the role of ononin in TC cells. Ononin treatment induced the cell death of the TC cells through regulating the CASP3 and CD274 expressions.

Bortezomib, an anticancer drug for multiple myeloma and mantle cell lymphoma, causes severe adverse events and leads to peripheral neuropathy. The associated neuropathy limits the use of bortezomib and could lead to discontinuation of the treatment; therefore, effective intervention is crucial. In the present study, we statistically searched for a drug that could alleviate bortezomib-induced peripheral neuropathy using adverse event self-reports. We observed that specific inhibitors of the mechanistic target of rapamycin (mTOR) lowered the incidence of bortezomib-induced peripheral neuropathy. These findings were experimentally validated in mice, which exhibited long-lasting mechanical hypersensitivity after repeated bortezomib treatment. This effect was inhibited for hours after a systemic injection with rapamycin or everolimus in a dose-dependent manner. Bortezomib-induced allodynia was accompanied by the activation of spinal astrocytes, and intrathecal injection of mTOR inhibitors or an inhibitor of ribosomal protein S6 kinase 1, a downstream target of mTOR, exhibited considerable analgesic effects in a dose-dependent manner. These results suggest that mTOR inhibitors, which are readily available to patients prescribed bortezomib, are one of the most effective therapeutics for bortezomib-induced peripheral neuropathy.

Glinus oppositifolius is an endemic herbaceous plant found in tropical Asian countries and is native in Vietnam. It is used in traditional folk medicine because of its flavor and antiseptic and laxative effects. In the current research, the effects of Tox-off, Biovip, and the purified compounds isolated from G. oppositifolius in the previous study were evaluated on the activation of adenosine 5′-monophosphate-activated protein kinase (AMPK)-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) in C2C12 myoblasts. In addition, the most potent active compounds, traphanoside-GO1 (TRA-GO1) and TRA-GO5 have validated the reduction of fatty acid synthase (FAS) and sterol regulatory element binding protein (SREBP)-1c in HepG2 cells. We found that Tox-off and Biovip significantly increased the phosphorylation of AMPK and ACC in C2C12 myoblasts. Furthermore, TRA-GO1 and TRA-GO5 significantly increased the AMPK activation and phosphorylation of its downstream substrate ACC in a concentration-dependent way compared to the dimethyl sulfoxide (DMSO) control. Besides, the protein level of FAS and SREBP-1c decreased by TRA-GO1 and TRA-GO5 in a concentration-dependent manner. Taken together, our results showed that the increased AMPK and ACC phosphorylation by active components of G. oppositifolius may activate the AMPK signaling pathways, which are useful for the anti-obesity and its related metabolic disorders.

Pazopanib is one of recommended treatment for metastatic renal cell carcinoma (RCC). Despite its effectiveness, patients often difficult to continue pazopanib treatment due to adverse events (AEs). We established an ambulatory care pharmacy practice that enables pharmacist–urologist collaboration to manage patients with RCC. This study evaluated the usefulness of this collaborative management. We retrospectively reviewed the medical records of 51 consecutive patients with metastatic RCC receiving pazopanib at the Kobe City Medical Center General Hospital between April 2014 and December 2020. Our collaborative management was implemented in October 2016. The time to pazopanib discontinuation was compared between patients who started pazopanib before (n = 30) and after (n = 21) the implementation of the collaborative management. A multivariate Cox regression analysis was performed to analyze the factors associated with pazopanib discontinuation. In the collaborative management, the oncology pharmacists had a total of 245 face-to-face patient consultations, and provided 286 suggestions [according to supportive care in pazopanib treatment (214 suggestions) were most frequent], and 236 (82.5%) were accepted by urologists. The median time to discontinuation (6.1 months vs. 2.4 months, p = 0.024) was significantly longer in the after group. Multivariate analysis showed that collaborative management (hazard ratio (HR) 0.49, 95% confidence interval (CI) 0.26–0.88, p = 0.017), and Eastern Cooperative Oncology Group performance status (ECOG PS) ≥2 at pazopanib initiation (HR 3.87, 95% CI 1.47–9.13, p = 0.008) were significantly associated with pazopanib discontinuation. These results suggested that, compared to conventional management, collaborative management is effective at prolonging the time to pazopanib discontinuation.

A cytokine known as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has the ability to precisely cause the death of cancer cells, while normal cells are left undisturbed. Recent studies show that certain cancer cells are sensitive to the apoptotic effect of TRAIL. In this study, HT29 colorectal adenocarcinoma cells exposed to TRAIL were treated with heptaphylline and 7-methoxyheptaphylline from Clausena harmandiana in an effort to comprehend the mechanisms involved behind this activity. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test was utilized to determine cell survival, and phase contrast microscopy was used to examine cell morphology. Through using real-time RT-PCR, Western blotting, and RT-PCR, the molecular mechanisms were investigated. According to the findings, whilst hepataphylline caused cytotoxicity in normal colon FHC cells, in comparison to healthy colon FHC cells, 7-methoxyheptaphylline inhibited cancer cells in a concentration-dependent manner. Heptaphylline alone or in conjunction with TRAIL showed no discernible effect on TRAIL-induced HT29 cell death, but 7-methoxyheptaphylline boosted caspase-3 cleavage. The study showed that the c-Jun N-terminal kinase (JNK) pathway was responsible for the 7-methoxyheptaphylline’s enhancement of the death receptor 5 (DR5) mRNA, TRAIL receptor, and protein. The results demonstrated that the 7-methoxyheptaphylline of Clausena harmandiana increased the expression of DR5 via the JNK pathway, intensifying TRAIL-induced HT29 cell death.

Chronic hepatitis C virus (HCV) infection can lead to liver cirrhosis and hepatocellular carcinoma. Although current medications using direct-acting antivirals (DAAs) are highly effective and well-tolerated for treating patients with chronic HCV, high prices and the existence of DAA-resistant variants hamper treatment. There is thus a need for easily accessible antivirals with different mechanisms of action. During the screening of Indonesian medicinal plants for anti-HCV activity, we found that a crude extract of Dryobalanops aromatica leaves possessed strong antiviral activity against HCV. Bioassay-guided purification identified an oligostilbene, vaticanol B, as an active compound responsible for the anti-HCV activity. Vaticanol B inhibited HCV infection in a dose-dependent manner with 50% effective and cytotoxic concentrations of 3.6 and 559.5 µg/mL, respectively (Selectivity Index: 155.4). A time-of-addition study revealed that the infectivity of HCV virions was largely lost upon vaticanol B pretreatment. Also, the addition of vaticanol B following viral entry slightly but significantly suppressed HCV replication and HCV protein expression in HCV-infected and a subgenomic HCV replicon cells. Thus, the results clearly demonstrated that vaticanol B acted mainly on the viral entry step, while acting weakly on the post-entry step as well. Furthermore, co-treatment of the HCV NS5A inhibitor daclatasvir with vaticanol B increased the anti-HCV effect. Collectively, the present study has identified a plant-derived oligostilbene, vaticanol B, as a novel anti-HCV compound.

Clindamycin phosphate (CLP) is a broad-spectrum antibiotic that is used widely for different types of infections. It has a short half-life and hence it should be taken every six hours to ensure adequate antibiotic blood concentration. On the other hand, microsponges are extremely porous polymeric microspheres, offering the prolonged controlled release of the drug. The present study aims to develop and evaluate innovative CLP-loaded microsponges (named Clindasponges) to prolong and control the drug release and enhance its antimicrobial activity, consequently improving patient compliance. The clindasponges were fabricated successfully by quasi-emulsion solvent diffusion technique using Eudragit S100 (ES100) and ethyl cellulose (EC) as carriers at various drug-polymer ratios. Several variables were optimized for the preparation technique including the type of solvent, stirring time, and stirring speed. The clindasponges were then characterized in terms of particle size, production yield, encapsulation efficiency, scanning electron microscopy, Fourier Transform Infrared Spectroscopy analysis, in vitro drug release with kinetic modeling, and antimicrobial activity study. Moreover, in vivo, pharmacokinetics parameters of CLP from the candidate formula were simulated based on the convolution method and in vitro–in vivo correlation (IVIVC-Level A) was built up successfully. Uniform spherical microsponges with 82.3 µm mean particle size with a porous spongy structure were evident. ES2 batch exhibited the highest production yield and encapsulation efficiency (53.75 and 74.57%, respectively) and it was able to exhaust 94% of the drug at the end of 8 h of the dissolution test. The release profile data of ES2 was best fitted to Hopfenberg kinetic model. ES2 was significantly (p < 0.05) effective against Staphylococcus aureus and Escherichia coli compared to the control. Also, ES2 displayed a twofold increase in the simulated area under the curve (AUC) compared to the reference marketed product.

Since small extracellular vesicle (sEVs) are involved in cell-to-cell communication via transfer of certain bioactive molecules and have the capability to overcome biological barriers against drug transport, their use as a drug delivery system (DDS) has been demonstrated in treatment of a diverse range of diseases. However, some issues in drug encapsulation have been pointed out, including low encapsulation efficiency and poor reproducibility. It was previously reported that liposomes containing phosphatidylserine (PS) can fuse together in the presence of calcium ion, which allows for drug encapsulation into the resultant liposomes (i.e., calcium fusion method). On the other hand, PS is reportedly present in lipid membrane of sEVs as a distinct lipid composition. We therefore hypothesized that PS-mediated membrane fusion of sEVs with PS-liposomes encapsulating therapeutic agents via the calcium fusion method can be applied to convenient drug encapsulation into sEVs. Membrane fusion of PS-liposomes and sEVs derived from murine melanoma B16F1 cells (B16-sEVs) was firstly confirmed. The obtained nanoparticles, termed chimeric nanoparticles (CM-NP), showed comparable cellular uptake to B16-sEVs into B16F1 cells. Moreover, CM-NP encapsulating an anticancer drug doxorubicin (DOX) (CM-NP-DOX) could be prepared by membrane fusion of PS-liposomes encapsulating DOX (PS-Lipo-DOX) and B16-sEVs. CM-NP-DOX exhibited a superior anticancer effect on B16F1 cells in vitro compared with PS-Lipo-DOX. These findings suggest that the calcium fusion method could be applied for membrane fusion of sEVs and PS-liposomes, and that this approach would likely be useful for efficient drug encapsulation into sEVs, as well as increasing liposome functionality.

Therapy for patients of metastatic breast cancer based on palbociclib, a cyclin-dependent kinase 4/6 inhibitor, has been approved in Japan. However, the risk factors for palbociclib-induced severe neutropenia in Japanese patients are rarely reported. Hence, the present study is aimed to identify the risk factors for adverse events requiring palbociclib dose reduction or discontinuation, and to identify the factors necessary to identify a more stable strategy for treatment continuation. This retrospective cohort analysis included patients with advanced breast cancer treated with 125 mg/d palbociclib. We demonstrated that severe neutropenia required significant dose reduction or therapy cessation. Most (77%) of the patients had severe neutropenia within the three courses. Risk factors for grade 3 or higher included low neutrophil counts (< 3250 /µL) before treatment [odds ratio (OR) = 9.10, 95% confidence interval (CI) (2.80–29.41), p < 0.001] and high age-adjusted Charlson comorbidity index (> 9) [OR = 1.64, 95% CI (1.09–2.48), p = 0.018]. Thus, low baseline neutrophil counts and high values for Age-adjusted Charlson comorbidity index are prospective predictive markers for palbociclib-induced severe neutropenia.

Secondary non-response to infliximab (IFX) occurs in some patients with rheumatoid arthritis (RA). Although therapeutic drug monitoring (TDM) is a useful tool to optimize IFX therapy, it is unclear whether it can help to identify the risk of secondary non-response. This study aimed to explore the utility of serum levels of IFX or other biomarkers to predict IFX discontinuation owing to secondary non-response. A single-center, retrospective study was conducted using the Kyoto University Rheumatoid Arthritis Management Alliance cohort database between 2011 and 2020. Serum IFX levels were measured using liquid chromatography-tandem mass spectrometry. An electrochemiluminescence assay was used to quantify serum levels of tumor necrosis factor-α and interleukin-6 and detect anti-drug antibodies. Eighty-four out of 310 patients were eligible for this study. The cutoff levels of biomarkers were determined by receiver operating characteristic analysis. IFX persistence was similar between groups stratified using IFX levels, tumor necrosis factor-α levels, interleukin-6 levels, and anti-drug antibodies positivity. The group with lower IFX and higher interleukin-6 levels had the worst therapy persistence (p = 0.017) and the most frequent disease worsening (90.0%, p < 0.001). Evaluating both interleukin-6 and IFX levels, not just IFX alone, enabled us to identify patients at risk of discontinuing IFX treatment. These findings support the utility of measuring IFX and interleukin-6 levels for successful maintenance therapy for RA.

To clarify the pharmacological properties of the Na⁺/Ca²⁺ exchanger (NCX) inhibitor SEA0400 as an antiarrhythmic agent, we assessed its effects on rapid component of delayed rectifier K⁺ current (I_Kr) blocker-induced torsade de pointes (TdP) in isoflurane-anesthetized rabbits. Atrioventricular block was induced in rabbits using a catheter ablation technique, and the monophasic action potential (MAP) of the right ventricle was measured under electrical pacing at 60 beats/min. In non-treated control animals, intravenous administration of low-dose (0.3 mg/kg) or high-dose nifekalant (3 mg/kg) prolonged the MAP duration (MAP₉₀) by 113 ± 11 ms (n = 5) and 237 ± 39 ms (n = 5), respectively, where TdP was induced in 1/5 animals treated with a low dose and in 3/5 animals treated with a high dose of nifekalant. In SEA0400-treated animals, low- and high-dose nifekalant prolonged the MAP₉₀ by 65 ± 13 ms (n = 5) and 230 ± 20 ms (n = 5), respectively. No TdP was induced by the low dose but 1/5 animals treated with a high dose of nifekalant developed TdP. In verapamil-treated animals, low-dose and high-dose nifekalant prolonged MAP₉₀ by 50 ± 12 ms (n = 5) and 147 ± 30 ms (n = 5), respectively, without inducing TdP. These results suggest that SEA0400 has the potential to inhibit low-dose nifekalant-induced TdP by suppressing the MAP-prolonging action of nifekalant, whereas the drug inhibited high-dose nifekalant-induced TdP without affecting the MAP-prolonging action of nifekalant. This may reveal that, in contrast to verapamil, the antiarrhythmic effects of SEA0400 on I_Kr blocker-induced TdP may be multifaceted, depending on the severity of the proarrhythmogenic conditions present.

The anticancer drug oxaliplatin is associated with peripheral neuropathy as a side effect accompanied by mechanical and cold allodynia. Although the superficial layer of the spinal cord dorsal horn is known to receive information primarily from peripheral pain nerves, to our knowledge, no in vivo electrophysiological analyses have been conducted to determine whether oxaliplatin administration increases the excitability of superficial layer neurons. Therefore, in vivo extracellular recordings were performed to measure action potentials in the deep and superficial layers of the spinal cord dorsal horn in rats treated with a single dose (6 mg/kg) of oxaliplatin. Action potentials were produced by mechanical stimulation with von Frey filaments to the hindlimb receptive fields. The results revealed that the firing frequency of action potentials increased relative to the intensity of mechanical stimulation, and that both deep and superficial layer neurons in the spinal cord dorsal horn increased significantly in oxaliplatin-treated compared with vehicle-treated rats, especially in the superficial layer. Several superficial layer neurons showed spontaneous firing that was not seen in vehicle-treated rats. In addition, a clear increase was seen in the firing frequency of neurons in the superficial layer of oxaliplatin-treated rats in response to a cold stimulus (here, the addition of acetone to the hindlimb receptive field). This study suggests that the superficial spinal cord dorsal horn strongly reflects the pain pathophysiology in peripheral neuropathy induced by oxaliplatin administration, and that the superficial layer neurons are useful for in vivo electrophysiological analysis using this pathological model.

The estimation of health risks of chemical substances was historically investigated using animal studies; however, current research focuses on reducing the number of animal experiments. The toxicity of chemicals in fish screening systems is reportedly correlated with their hydrophobicity. The inverse relationship between absorption rates (intestinal cell permeability) and virtual hepatic/plasma pharmacokinetics of diverse chemicals has been previously evaluated by modeling oral administration in rats. In the current study, internal exposures, i.e., virtual maximum plasma concentrations (C_max) and areas under the concentration–time curves (AUC), of 56 food chemicals with reported hepatic lowest-observed-effect levels (LOELs) of ≤1000 mg/kg/d in rats were pharmacokinetically modeled using in silico estimated input pharmacokinetic parameters. After a virtual single oral dose of 1.0 mg/kg of 56 food chemicals, the output C_max and AUC values in rat plasma generated by modeling using the corresponding in silico estimated input parameters were not significantly correlated with the reported hepatic LOEL values. However, significant inverse relationships between hepatic/plasma concentrations of selected lipophilic food chemicals (i.e., octanol–water partition coefficient log P > 1) using forward dosimetry and reported LOEL values (≤300 mg/kg/d) were observed (n = 14, r=−0.52–0.66, p ≤ 0.05). This simple modeling approach, which uses no experimental pharmacokinetic data, has the potential to play a significant role in reducing the use of animals to estimate toxicokinetics or internal exposures of lipophilic food components after oral doses. Therefore, these methods are valuable for estimating hepatic toxicity by using forward dosimetry in animal toxicity experiments.

Megakaryoblastic leukemia 2 (MKL2)/myocardin-related transcription factor-B (MRTFB) is a serum response factor (SRF) cofactor that is enriched in the brain and controls SRF target genes and neuronal morphology. There are at least four isoforms of MKL2/MRTFB. Among these, MKL2/MRTFB isoform 1 and spliced neuronal long isoform of SRF transcriptional coactivator (SOLOIST)/MRTFB isoform 4 (MRTFB i4) are highly expressed in neurons. Although, when overexpressed in neurons, isoform 1 and SOLOIST/MRTFB i4 have opposing effects on dendritic morphology and differentially regulate SRF target genes, it is unknown how endogenous SOLOIST/MRTFB i4 regulates gene expression. Using isoform-specific knockdown, we investigated the role of endogenous SOLOST/MRTFB i4 in regulating the expression of other MKL2/MRTFB isoforms and SRF-target genes in Neuro-2a cells. Knockdown of SOLOIST/MRTFB i4 downregulated SOLOIST/MRTFB i4, while it upregulated isoform 1 without affecting isoform 3. Knockdown of SOLOIST/MRTFB i4 downregulated the SRF target immediate early genes egr1 and Arc, while it upregulated c-fos. Double knockdown of isoform 1 and SOLOIST/MRTFB i4 inhibited c-fos expression. Taken together, our findings in Neuro-2a cells suggest that endogenous SOLOIST/MRTFB i4 positively regulates egr1 and Arc expression. In addition, endogenous SOLOIST/MRTFB i4 may negatively regulate c-fos expression, possibly by downregulating isoform 1 in Neuro-2a cells.

Inositol hexaphosphate (IP6), a widely found natural bioactive substance in grains, effectively inhibits the progression of colorectal cancer (CRC) when used in combination with inositol (INS). We previously showed that supplementation of IP6 and INS upregulated the claudin 7 gene in orthotropic CRC xenografts in mice. The aim of this study was to elucidate the role of claudin 7 in the inhibition of CRC metastasis by IP6 and INS, and explore the underlying mechanisms. We found that IP6, INS and their combination inhibited the epithelial–mesenchymal transition (EMT) of colon cancer cell lines (SW480 and SW620), as indicated by upregulation of claudin 7 and E-cadherin, and downregulation of N-cadherin. The effect of IP6 and INS was stronger compared to either agent alone (combination index < 1). Furthermore, the silencing of the claudin 7 gene diminished the anti-metastatic effects of IP6 and INS on SW480 and SW620 cells. Consistent with in vitro findings, the combination of IP6 and INS suppressed CRC xenograft growth in a mouse model, which was neutralized by claudin 7. Taken together, the combination of IP6 and INS can inhibit CRC metastasis by blocking EMT of tumor cells through upregulation of claudin 7.

To examine the effects of blindness on sleep/wakefulness states, we compared locomotor activity and delayed recovery from isoflurane anesthesia induced by hypnotics during light and dark periods in sighted CBA/N and blind CBA/J mice. Locomotor activity around the switch from the dark to light period significantly differed in both mice. Delayed recovery induced by brotizolam was attenuated in both periods in CBA/J mice. In addition, the period specificity of delayed recovery caused by suvorexant or diphenhydramine in CBA/N mice was abolished in CBA/J mice. These results suggest that blindness impairs sleep quality.

Endothelial dysfunction—a hallmark of chronic kidney disease (CKD)—is one of the major risk factors for cardiovascular diseases (CVD). Imbalances in endothelium-derived relaxing factors (EDRFs) and contracting factors (EDCFs) specific to endothelial dysfunction in CKD are yet to be studied. Therefore, using adenine-treated rats—a CKD rat model—we investigated the responsiveness of superior mesenteric artery (SMA) endothelium to acetylcholine (ACh) stimulation under different experimental conditions. Nine-week-old male Wistar rats were treated daily with adenine (200 and 600 mg/kg body weight) by oral gavage, for 10 d; the two groups were named adenine-200 (200 mg/kg body weight) and adenine-600 (600 mg/kg body weight). The systolic blood pressure (measured 1-, 8-, and 15 d post-treatment) was significantly increased in the adenine-600 group compared with that in the control group; whereas that in the adenine-200 group showed only a slight increase. Moreover, in the adenine-600 group the serum creatinine and blood urea nitrogen (BUN) levels (measured at 18 d post-treatment) were significantly elevated when compared with those in control or adenine-200 groups. The ACh-mediated relaxation was slightly reduced in the adenine-200 group. The ACh- and sodium nitroprusside (SNP)-mediated relaxations were impaired in the adenine-600 group. Although no ACh-mediated contraction was observed in the presence of a nitric oxide (NO) synthase inhibitor, ACh-induced endothelium-derived hyperpolarizing factor-mediated relaxation was largely impaired in the adenine-600 mg/kg group. This study revealed that in the SMA of adenine-induced CKD model rats, EDCF signaling remained unaltered while the NO and EDHF signaling were impaired.