Sodium-dependent phosphate cotransporter type 1 sequence polymorphisms in male patients with gout.

OBJECTIVES: Molecular biological approaches have recently identified urate transporters in renal proximal tubular cells. Human sodium-dependent phosphate cotransporter type 1 encoded by SLC17A1 is a urate transporter localised to the renal proximal tubular cells and candidate molecule to secret urate from renal tubular cells to urine. This study investigated the roles of SLC17A1 in the development of gout. PATIENTS AND METHODS: Single nucleotide polymorphisms in the human SLC17A1 gene (rs1165176, rs1165151, rs1165153, rs1165196, rs1165209, rs1165215, rs1179086, rs3799344 and rs3757131) were selected, and an association study was conducted using male patients with gout (n=175) and male controls (n=595). RESULTS: There were significant differences between gout and control groups in the distribution of genotypes at rs1165196 (T806C; Ile269Thr, odds ratio (OR) 0.55, p=0.0035), rs1179086 (OR 0.57, p=0.0018) and rs3757131 (OR 0.54, p=0.0026). In controls, T806C alone had no effect on serum uric acid (sUA) levels. However, T806C showed significant interaction with a reduction of sUA in obese individuals (body mass index > or = 25) using multiple regression analysis. CONCLUSIONS: Our data suggest that SLC17A1 polymorphisms are associated with the development of gout.

Detection of six single-nucleotide polymorphisms associated with rheumatoid arthritis by a loop-mediated isothermal amplification method and an electrochemical DNA chip.

An electrochemical DNA chip using an electrochemically active intercalator and DNA probe immobilized on a gold electrode has been developed for genetic analysis. In this study, the six polymorphisms associated with rheumatoid arthritis (RA), N-acetyltransferase2 (NAT2) gene polymorphisms T341C, G590A, and G857A, methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms C677T and A1298C, and serum amyloid A1 (SAA1) gene promoter polymorphism C-13T were simultaneously detected by the electrochemical DNA chip and the loop-mediated isothermal amplification (LAMP) method, which is a novel technique for DNA amplification. Human genomic DNAs were extracted from blood, and the targets containing the six polymorphisms were amplified by the LAMP method. A sample containing the six LAMP products was reacted with the electrochemical DNA chip using a DNA detection system that controls hybridization reaction, washing, electrochemical detection, and data analysis automatically. A total of 31 samples were genotyped by this method, and the results were completely consistent with those determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis or the PCR direct sequence analysis. The time required for this method was only 2 h, and operations were very simple. Therefore, this method is expected to contribute to personalized medicine based on genotype.

Validation of the associations between single nucleotide polymorphisms or haplotypes and responses to disease-modifying antirheumatic drugs in patients with rheumatoid arthritis: a proposal for prospective pharmacogenomic study in clinical practice.

BACKGROUND: For prevention of joint destruction in rheumatoid arthritis, optimal management of therapy with disease-modifying antirheumatic drugs is essential. Pharmacogenomic evidence, if reliable, may be incorporated in the treatment of rheumatoid arthritis to achieve a more efficient activity control with minimized adverse events. METHODS: We conducted retrospective studies to validate our previous three different results about the association between adverse events or efficacy of two different disease-modifying antirheumatic drugs and genomic variations. Association between single nucleotide polymorphisms in N-acetyltransferase 2 gene (NAT2) and adverse events by sulfasalazine and association between C677T or A1298C in 5,10-methylenetetrahydrofolate reductase gene (MTHFR) and responses to methotrexate were examined. RESULTS: Patients without the wild-type haplotype at NAT2 were more likely to suffer from overall adverse events [n=186, P=0.001, relative risk (RR) 3.31, 95% confidence interval (CI) 1.76-6.22] and severe adverse events (P=0.015, RR 24.6, 95% CI 2.37-254.53) by sulfasalazine. Patients with the T allele at C677T in MTHFR were more susceptible to overall adverse events (n=156, P=0.003; RR 2.4, 95% CI 1.29-4.55) while patients with the C allele at A1298C were less likely to be treated with a higher dose (>6 mg/week) of methotrexate in one year of treatment (n=159, P=0.008, RR 1.84, 95% CI 1.12-3.01). In all three association studies, the results were essentially the same as previously reported. CONCLUSION: As three studies on the associations between genomic variations and adverse events or efficacy of two different disease-modifying antirheumatic drugs were replicated, the usefulness of the tests is worth being tested in clinical practice.

Proliferation of donor mitochondrial DNA in nuclear transfer calves (Bos taurus) derived from cumulus cells.

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.

Potassium-induced increase in renal kallikrein secretion is attenuated in dissected renal connecting tubules of young spontaneously hypertensive rats.

It is suggested that attenuation of the renal kallikrein-kinin system (KKS) involved the development of hypertension in young spontaneously hypertensive rats (SHR). In the present study, a comparison was made between young SHR and Wistar Kyoto rats (WKY) to examine the ability to secrete renal kallikrein from the microdissected connecting tubules (CNT) by potassium or an ATP-sensitive potassium channel blocker, 4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexylhydrochloride (PNU-37883A), both of which are renal kallikrein secretagogues. Maximum effect of potassium on kallikrein secretion was observed 10 min after placing the tubules at concentration of 20 mM. Kallikrein secretion was also increased concentration-dependently by PNU-37883A (0.1, 1, 10, and 100 microM). In the presence of EDTA, NiCl2, verapamil, xestspongin C (an inositol 1,4,5-trisphosphate (IP3) receptor-selective antagonist), or ruthenium red (a ryanodine-sensitive receptor blocker), potassium-induced increase in renal kallikrein secretion was inhibited. Augmentation of renal kallikrein secretion by potassium or PNU-37883A was diminished in SHR compared to WKY. These results indicate that the ability to secrete renal kallikrein by potassium was attenuated in young SHR compared with WKY. Furthermore, it is suggested that the potassium-induced renal kallikrein secretion requires an extracellular Ca2+ entry through Ca2+ channels including L-type Ca2+ channels and Ca2+ release from intracellular Ca2+ stores through IP3 receptor and ryanodine receptor.

Role of kinin and prostaglandin in cutaneous thermal nociception.

Involvements of kinin and prostaglandin and their interaction in noxious thermal stimuli were investigated in noninflamed and inflamed rats. The nociceptive response was evaluated from the escape latency of foot withdrawal to the thermal stimuli with a beam of light. The escape latency in kininogens-deficient Brown Norway (B/N-) Katholiek rats was significantly longer than that in the normal strain, B/N-Kitasato rats. The latency in B/N-Kitasato rat was prolonged by administration of a bradykinin (BK) B2 receptor antagonist, FR173657 (30 mg/kg, p.o.), whereas it was shortened by pretreatment with a kininase II inhibitor, captopril (10 mg/kg, i.p.). Both agents did not affect the latency in B/N-Katholiek rats. In normal Sprague-Dawley (SD) rat, administration of indomethacin did not change the escape latency against the thermal stimuli. In contrast, administration of indomethacin or a relatively cyclooxygenase-1-selective inhibitor, mofezolac (10 mg/kg, p.o.) significantly reduced numbers of writhing reaction in mice induced by acetic acid solution. Injection of lipopolysaccharide (1 mg/kg, i.v.) resulted in shortening escape latency at 8 h after the injection in B/N-Kitasato rats. This hyperalgesia could be reversed by pretreatment of the rats with indomethacin, a cyclooxygenase-2-selective inhibitor JTE-522 (10 mg/kg, p.o.), or FR173657, but not with mofezolac. The hyperalgesia was not seen in B/N-Katholiek rats. These results indicate that kinin has major participation in peripheral skin thermal nociception under noninflamed condition, although cyclooxygenases may have little participation. Prostaglandins produced by cyclooxygenase-2 could coordinate with BK to elicit hyperalgesia during inflammation induced by lipopolysaccharide.

Cyclooxygenase-2 inhibitors attenuate increased blood pressure in renovascular hypertensive models, but not in deoxycorticosterone-salt hypertension.

COX-2 is an inducible cyclooxygenase (COX) that has been reported to be expressed in the macula densa and surrounding cortical thick ascending limb in normotensive rats. The present study assessed the contribution of COX-2 in three different rat models of hypertension, each characterized by a different activation of the renal renin-angiotensin system. Mean blood pressure (MBP) in the rat 2 kidney-1 clip (2K1C) model was significantly reduced with a COX-2 selective inhibitor, NS-398 (10 mg/kg, p.o., twice a day) (vehicle-administered rats (n = 8): 154 +/- 6 mmHg; NS-398-administered rats (n = 5): 128 +/- 10 mmHg). By contrast, a COX-1 selective inhibitor, mofezolac, did not lower MBP. Increased plasma renin activity (23 +/- 8 ng/kg/h (n = 6) vs. sham operation, 2.4 +/- 0.9 ng/kg/h (n = 4)) was markedly reduced to 6.8 +/- 2.7 ng/ml/h (n = 5) by NS-398, but not by mofezolac. The development of 1 kidney-1 clip (1K1C) hypertension was also inhibited by NS-398 (vehicle (n = 12): 133 +/- 1 mmHg; NS-398 (n = 7): 122 +/- 3 mmHg) accompanied by a reduction in plasma renin activity (3.0 +/- 0.3 ng/ml/h, n = 4) to 1.0 +/- 0.2 ng/ml/h (n = 5). The COX-2 inhibitor increased urinary excretions in the 1K1C model, but not in the 2K1C model. In a deoxycorticosterone acetate (DOCA)-salt model, plasma renin activity was markedly suppressed to less than 0.3 ng/ml/h. The COX-2 inhibitor caused no significant changes in MBP, plasma renin activity, or urinary excretion, suggesting that COX-2 made a lesser contribution in this model. Increased expression of COX-2 mRNA and protein was observed in the kidneys of 1K1C and 2K1C rats, but not in DOCA-salt rats. These results suggest that COX-2 plays a significant role in the development of 2K1C and 1K1C renovascular hypertension, in addition to making a substantial contribution to the diuretic effect in the 1K1C model.