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Background: Monocarboxylate transporter 9 (MCT9), an orphan transporter member of the solute carrier family 16 (SLC16), possibly reabsorbs uric acid in the renal tubule and has been suggested by genome-wide association studies to be involved in the development of hyperuricemia and gout. In this study we investigated the mechanisms regulating the expression of human (h) MCT9, its degradation, and physiological functions. Methods and Results: hMCT9-FLAG was stably expressed in HEK293 cells and its degradation, intracellular localization, and urate uptake activities were assessed by pulse-chase analysis, immunofluorescence, and [14C]-urate uptake experiments, respectively. hMCT9-FLAG was localized on the plasma membrane as well as in the endoplasmic reticulum and Golgi apparatus. The proteasome inhibitors MG132 and lactacystine increased levels of hMCT9-FLAG protein expression with enhanced ubiquitination, prolonged their half-life, and decreased [14C]-urate uptake. [14C]-urate uptake was increased by both heat shock (HS) and the HS protein inducer geranylgeranylacetone (GGA). Both HS and GGA restored the [14C]-urate uptake impaired by MG132. Conclusions: hMCT9 does transport urate and is degraded by a proteasome, inhibition of which reduces hMCT9 expression on the cell membrane and urate uptake. HS enhanced urate uptake through hMCT9.

Inflammation induced by urate deposition in joints causes gout. Healthy individuals maintain serum levels of urate by balancing urate production/excretion, whereas a production/excretion imbalance increases urate levels. Hyperuricemia is diagnosed when the serum urate level is continuously above 7 mg/dl as the solubility limit, and urate accumulates in the kidneys and joints. Because hyperuricemia increases the risk of gout, therapies aim to eliminate urate deposition to prevent gouty arthritis and kidney injury.This review discusses the mechanism underlying hyperuricemia with respect to urate production and urate transport, along with urate-lowering therapeutics, including urate synthesis inhibitors, uricolytic enzymes, and uricosuric agents. The authors asses published data on relevant commercial therapy development projects and clinical trials.Available treatment options for hyperuricemia are limited. Allopurinol, a urate synthesis inhibitor, is generally administered at a reduced dosage to patients with renal impairment. Some URAT1 inhibitors have an unfavorable side effect profile. A promising strategy for treatment is the use of uricosuric agents that inhibit transporters (e.g. URAT1, URATv1/GLUT9, OAT10) which reabsorb urate from the urine.

Aims In addition to potentially progressing to either cirrhosis or hepatocellular carcinoma, non-alcoholic steatohepatitis (NASH) is currently the leading indication for liver transplantation. Nintedanib has been clinically used to treat idiopathic pulmonary fibrosis for many years, but its effects in an animal model of NASH have not been tested. The purpose of this study was to evaluate the effects of nintendanib on NASH in choline-deficient, l -amino acid-defined, high-fat diet (CDAHFD)-fed mice. Main methods Male C57BL/6 mice were fed a CDAHFD for 6 weeks to induce NASH with liver fibrosis, and they were administered nintedanib (60 mg/kg/day) or distilled water orally in the last 2 weeks of the feeding period. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglyceride, and non-esterified fatty acids concentrations were measured. Serum cytokeratin 18 fragment (CK18) was detected using ELISA. Liver tissue sections from mice were stained with hematoxylin-eosin and Masson's trichrome to assess the level of steatohepatitis and fibrosis. Key findings CDAHFD-fed mice exhibited higher serum ALT, AST, and ALP levels compared with Control mice. A significant increase in the serum CK18 level was observed in the NASH group compared with the Control group. CDAHFD feeding also enhanced steatohepatitis and hepatic fibrosis pathological features, which were reduced after nintedanib treatment. Significance Nintedanib exerted anti-inflammatory and anti-fibrotic effects in CDAHFD-induced NASH mice.

Background Intracellular uric acid is known to increase the protein level and channel current of atrial Kv1.5; however, mechanisms of the uric acid-induced enhancement of Kv1.5 expression remain unclear. Methods and Results: The effects of uric acid on mRNA and protein levels of Kv1.5, as well as those of Akt, HSF1 and Hsp70, in HL-1 cardiomyocytes were studied by using qRT-PCR and Western blotting. The uptake of uric acid was measured using radio-labeled uric acid. The Kv1.5-mediated channel current was also measured by using patch clamp techniques. Uric acid up-taken by HL-1 cells significantly increased the level of Kv1.5 proteins in a concentration-dependent manner, with this increase abolished by an uric acid transporter inhibitor. Uric acid slowed degradation of Kv1.5 proteins without altering its mRNA level. Uric acid enhanced phosphorylation of Akt and HSF1, and thereby increased both transcription and translation of Hsp70; these effects were abolished by a PI3K inhibitor. Hsp70 knockdown abolished the uric acid-induced increases of Kv1.5 proteins and channel currents. Conclusions Intracellular uric acid could stabilize Kv1.5 proteins through phosphorylation of Akt and HSF1 leading to enhanced expression of Hsp70.

This study was designed to investigate the effects of uric acid on vascular endothelial function in measurements carried out either at the bedside or the laboratory bench. First, we performed reactive hyperemia peripheral arterial tonometry using an EndoPAT 2000 device and measured serum uric acid levels in 92 outpatients with hypertension. The reactive hyperemia index (RHI) showed no correlation with serum uric acid level (R = -0.125, P = 0.235) in either overall patients or in a high-risk group of 51 patients with complications such as cardiovascular and cerebrovascular diseases, chronic kidney disease, and/or diabetes (R = -0.025, P = 0.860). However, in the remaining 41 patients in the low-risk group, RHI correlated negatively with serum uric acid level (R = -0.335, P = 0.032). Multiple regression analysis showed that serum uric acid level predicted RHI (R = -0.321, P = 0.043) in the low-risk group independent of age, body mass index, systolic blood pressure, and low density lipoprotein-cholesterol level. We then performed an in-vitro study using the WST-8 assay in human umbilical vein endothelial cells, which showed that hypoxic conditions reduced cell viability. Treatment with uric acid caused a further reduction in cell viability, while ascorbic acid improved viability. Using Western blot analysis, we observed that uric acid reduced endothelial nitric oxide synthase phosphorylation during hypoxic conditions. Serum uric acid level is associated with peripheral vascular endothelial function in patients with low-risk hypertension and uric acid could directly impair endothelial function under hypoxic conditions. These results are relevant to the interventional studies examining the cardiovascular protective effect of hypouricemic agents.

We evaluated the effects of anticholinergic drugs principally used for the therapy of overactive bladder (OAB) on the activity of P-glycoprotein, an efflux transport protein, in Caco-2 cells. The time-dependent changes in the fluorescence of residual rhodamine 123, a P-glycoprotein activity marker, in the apical region of Caco-2 cells were measured in the presence of anticholinergic drugs using time-lapse confocal laser scanning microscopy. The effect of anticholinergic drugs on human P-glycoprotein ATPase activity was also measured. The fluorescence of residual rhodamine 123 in untreated Caco-2 cells decreased over time. The gradual decrease in the fluorescence was significantly inhibited by treatment with cyclosporine A, darifenacin, and trospium. In contrast, oxybutynin, N-desethyl-oxybutynin (DEOB), propiverine, and its active metabolites (M-1, M-2), imidafenacin, solifenacin, or tolterodine had little effect on the efflux of rhodamine 123. P-Glycoprotein ATPase activity was increased by darifenacin. Darifenacin and trospium reduced the rhodamine 123 transfer across the apical cell membrane. These data suggest that darifenacin and trospium interact with P-glycoprotein. Additionally, darifenacin influenced P-glycoprotein ATPase activity. These results suggest that darifenacin may be a substrate of P-glycoprotein. This study is the first paper to test simultaneously the effects of 10 anticholinergic drugs used currently for the therapy of OAB, on the P-glycoprotein.

The purpose of this study was to create novel urate under-excretion animal models using pyrazinamide and to evaluate whether dihydropyridine calcium channel blockers (CCBs) have uricosuric effects in vivo.Adult male ICR mice were treated with pyrazinamide, vehicle (dimethyl sulfoxide: DMSO), or tap water. Thirty minutes later, pyrazinamide-treated mice were given benzbromarone, losartan, nilvadipine, nitrendipine, nifedipine or azelnidipine. Six hours after the second administration, urine (by urinary bladder puncture) and plasma were collected to measure uric acid and creatinine levels, and fractional excretion of uric acid (FEUA) and creatinine clearance (Ccr) were calculated and evaluated. There was no significant difference in the levels of plasma uric acid, plasma creatinine, Ccr, urinary N-acetyl-β-d-glucosaminidase (NAG) and urinary NAG-creatinine ratio between water, DMSO, and pyrazinamide-treated mice. But the FEUA of pyrazinamide-treated mice was significantly lower than water mice. The FEUA was significantly higher in mice taking the dihydropyridine CCBs (nilvadipine, nitrendipine, nifedipine, and high-dose azelnidipine) than in pyrazinamide-treated mice. There was no significant difference in Ccr.Thus, a novel animal model created with PZA administration was useful as a urate under-excretion animal model that was probably URAT1-mediated, and the uricosuric effects of dihydropyridine CCBs were confirmed in vivo. Keywords: Uric acid, Urate, Fractional excretion of uric acid, Urate under-excretion animal model, Calcium channel blocker

An integrated analysis was performed with data from 4 phase 2 and phase 3 studies of tofogliflozin in which patients with type 2 diabetes mellitus received the sodium‐glucose cotransporter 2 inhibitor tofogliflozin for up to 24 weeks. Sex differences, baseline haemoglobin A1c (HbA1c) and serum uric acid (UA) levels, and log10‐transformed urinary N‐acetyl‐β‐D‐glucosaminidase ratio were significantly correlated with the reduction in serum UA levels at both 4 and 24 weeks in multivariate analysis (respectively, P 8.6%) and lowest in the group with the lowest baseline HbA1c level (quartile 1; HbA1c ≤ 7.4%). The decrease in serum UA levels was greatest in the quartile 1 group and lowest in the quartile 4 group. In most groups, the maximum decrease in serum UA levels was seen in the first 4 weeks, while the maximum decrease in HbA1c was seen at week 24. Thus, serum UA levels were significantly decreased in patients with moderate HbA1c levels.

Background Ischemia/reperfusion (I/R) injury triggers cardiac dysfunctions via creating reactive oxygen species (ROS). Because xanthine oxidase (XO) is one of the major enzymes that generate ROS, inhibition of XO is expected to suppress ROS-induced I/R injury. However, it remains unclear whether XO inhibition really yields cardioprotection during I/R. The protective effects of the XO inhibitors, topiroxostat and allopurinol, on cardiac I/R injury were evaluated.Methods and Results:Using isolated rat hearts, ventricular functions, occurrence of arrhythmias, XO activities and thiobarbituric acid reactive substances (TBARS) productions and myocardial levels of adenine nucleotides before and after I/R, and cardiomyocyte death markers during reperfusion, were evaluated. Topiroxostat prevented left ventricular dysfunctions and facilitated recovery from arrhythmias during I/R. Allopurinol and the antioxidant, N-acetylcysteine (NAC), exhibited similar effects at higher concentrations. Topiroxostat inhibited myocardial XO activities and TBARS productions after I/R. I/R decreased myocardial levels of ATP, ADP and AMP, but increased that of xanthine. While topiroxostat, allopurinol or NAC did not change myocardial levels of ATP, ADP or AMP after I/R, all of the agents decreased the level of xanthine. They also decreased releases of CPK and LDH during reperfusion. Conclusions Topiroxostat showed protective effects against I/R injury with higher potency than allopurinol or NAC. It dramatically inhibited XO activity and TBARS production, suggesting suppression of ROS generation.

Diabetes mellitus is a lifestyle-related disease that is characterized by inappropriate or diminished insulin secretion. Ex vivo pharmacological studies of hypoglycemic agents are often conducted using perfused pancreatic preparations. Pancreas preparations for organ bath experiments do not require cannulation and are therefore less complex than isolated perfused pancreas preparations. However, previous research has generated almost no data on insulin secretion from pancreas preparations using organ bath preparations. The purpose of this study was to investigate the applicability of isolated rat pancreas preparations using the organ bath technique in the quantitative analysis of insulin secretion from β-cells. We found that insulin secretion significantly declined during incubation in the organ bath, whereas it was maintained in the presence of 1 µM GLP-1. Conversely, amylase secretion exhibited a modest increase during incubation and was not altered in the presence of GLP-1. These results demonstrate that the pancreatic organ bath preparation is a sensitive and reproducible method for the ex vivo assessment of the pharmacological properties of hypoglycemic agents.

Background: Although urate impaired the endothelial function, its underlying mechanism remains unknown. We hypothesized that urate impaired nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) via activation of uric acid transporters (UATs). Purpose and method: In the present study, we studied effects of urate on NO production and eNOS protein expression in HUVEC cells in the presence and absence of urate lowering agents using molecular biological and biochemical assays. Results: HUVECs expressed the 4 kinds of UATs, URATv1, ABCG2, MRP4 and MCT9. Exposure to urate at 7 mg/dl for 24 h significantly reduced production of NO. Pretreatment with benzbromarone, losartan or irbesartan normalized NO production. The same exposure resulted in dephosphorylation of endothelial NO synthase (eNOS) in HUVECs. Again pretreatment with benzbromarone, losartan or irbesartan abolished this effect. Conclusion: Urate reduced NO production by impaired phosphorylation of eNOS in HUVEC via activation of UATs, which could be normalized by urate lowering agents.

Boron neutron capture therapy (BNCT) is a biochemically-targeted radiotherapy based on the nuclear capture and fission reactions that occur when boron-10 is irradiated with low energy thermal neutrons to yield high linear energy transfer alpha particles and recoiling lithium-7 nuclei with path-length less than 10 μm.It has been focused to use on the treatment of brain, skin, head and neck cancers. 4-Boronophenylalanine (BPA), a BNCT delivery agent, has been used in clinical field but its transport mechanism into the tumor lesion was not clear. It is known that L-type amino acid transporters, LAT1 and LAT3 are highly expressed in various types of cancer whereas LAT2 and LAT4 are expressed in normal tissues. The LAT1-selective compound(s) might be the ideal delivery agents for BNCT to obtain the specific accumulation in cancer tissue and reduce adverse effect on normal tissue. In this study we identify the transport of BPA by L-type amino acid transporters using Xenopus oocyte expression system and LC/MS-MS...