Inhibitory effect of Citrus flavonoids on the in vitro transport activity of human urate transporter 1 (URAT1/SLC22A12), a renal re-absorber of urate

Toyoda, Yu; Takada, Tappei; Saito, Hiroki; Hirata, Hiroshi; Ota-Kontani, Ami; Kobayashi, Naoyuki; Tsuchiya, Youichi; Suzuki, Hiroshi

doi:10.1038/s41538-020-0063-7

Cited by 25 publications

(37 citation statements)

References 14 publications

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“…Whole-cell lysate samples were separated by SDS-PAGE and transferred to an Immobilon-P PVDF membrane (Millipore, Bedford, MA, USA) by electroblotting at 15 V for 60 min, as described previously [15]. Blots were probed with appropriate antibodies (Table 1), and the signals were visualized by chemiluminescence and detected using a multi-imaging Analyzer Fusion Solo 4 TM system (Vilber Lourmat, Eberhardzell, Germany).…”

Section: Preparation Of Protein Lysates and Immunoblottingmentioning

confidence: 99%

“…For confocal laser scanning microscopic observation, 48 h after the transfection, 293A cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and further processed according to previous studies [15,20]. In brief, the cells were treated with a fluorescent wheat germ agglutinin conjugate (WGA, Alexa Fluor ® 594 conjugate; Thermo Fisher Scientific) to visualize plasma membranes, followed by nuclear staining using TO-PRO-3 Iodide (Molecular Probes, Eugene, OR, USA).…”

Section: Confocal Microscopymentioning

confidence: 99%

“…The urate uptake assay using URAT1-expressing 293A cells was conducted according to our previous studies [15,18] with minor modifications. In brief, 48 h after plasmid transfection, cells were washed twice with Cl − -free transport buffer (Buffer T2: 125 mM Na-gluconate, 4.8 mM K-gluconate, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 1.3 mM Ca-gluconate, 25 mM HEPES, 5.6 mM D-glucose, and pH 7.4) and pre-incubated in Buffer T2 for 15 min at 37 • C. The buffer was then exchanged with pre-warmed fresh Buffer T2 containing 5 µM [8-14 C]-urate with or without test compound at the indicated concentrations (0, 0.1, 0.3, 1, 3, 10, 30, 100, or 300 µM), and the cells were further incubated for 20 sec; 1% DMSO was used as a vehicle control.…”

Section: Urate Uptake Assay Using Urat1-expressing 293a Cellsmentioning

confidence: 99%

“…where E max is the maximum effect, EC 50 is the half maximal effective concentration, C is the concentration of the test compound, and n is the sigmoid-fit factor. Finally, based on the results, the IC 50 was calculated as described previously [15].…”

Section: Urate Uptake Assay Using Urat1-expressing 293a Cellsmentioning

confidence: 99%

“…In this context, daily consumption of nutrients with URAT1-inhibitory activity may have a beneficial effect on SUA management in subjects with high SUA levels. Actually, food ingredients that inhibit URAT1 function have attracted great interest; we and other groups identified some such natural ingredients from fruit flavonoids [15], coumarins [16], and wood pigments [17]. Nevertheless, despite the nutritional significance of FAs, their effects on URAT1 activity remain to be elucidated.…”

Section: Introductionmentioning

confidence: 99%

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Omega-3 Polyunsaturated Fatty Acids Inhibit the Function of Human URAT1, a Renal Urate Re-Absorber

et al. 2020

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The beneficial effects of fatty acids (FAs) on human health have attracted widespread interest. However, little is known about the impact of FAs on the handling of urate, the end-product of human purine metabolism, in the body. Increased serum urate levels occur in hyperuricemia, a disease that can lead to gout. In humans, urate filtered by the glomerulus of the kidney is majorly re-absorbed from primary urine into the blood via the urate transporter 1 (URAT1)-mediated pathway. URAT1 inhibition, thus, contributes to decreasing serum urate concentration by increasing net renal urate excretion. Here, we investigated the URAT1-inhibitory effects of 25 FAs that are commonly contained in foods or produced in the body. For this purpose, we conducted an in vitro transport assay using cells transiently expressing URAT1. Our results showed that unsaturated FAs, especially long-chain unsaturated FAs, inhibited URAT1 more strongly than saturated FAs. Among the tested unsaturated FAs, eicosapentaenoic acid, α-linolenic acid, and docosahexaenoic acid exhibited substantial URAT1-inhibitory activities, with half maximal inhibitory concentration values of 6.0, 14.2, and 15.2 μM, respectively. Although further studies are required to investigate whether the ω-3 polyunsaturated FAs can be employed as uricosuric agents, our findings further confirm FAs as nutritionally important substances influencing human health.

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Section: Preparation Of Protein Lysates and Immunoblottingmentioning

confidence: 99%

Section: Confocal Microscopymentioning

confidence: 99%

Section: Urate Uptake Assay Using Urat1-expressing 293a Cellsmentioning

confidence: 99%

Section: Urate Uptake Assay Using Urat1-expressing 293a Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Omega-3 Polyunsaturated Fatty Acids Inhibit the Function of Human URAT1, a Renal Urate Re-Absorber

et al. 2020

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Mechanism of flavonoids inhibiting xanthine oxidase and alleviating hyperuricemia from structure–activity relationship and animal experiments: A review

Xue

Gong

et al. 2023

Food Frontiers

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Hyperuricemia (HUA) is a metabolic disease caused by excessive uric acid in patients. Flavonoids have attracted extensive attention due to their safety, effectiveness, and little side effects in the treatment of HUA and inhibition of xanthine oxidase (XO) activity. In this paper, the studies on the treatment of HUA and the inhibition of XO activity by flavonoids (including flavones, flavonols, flavanones, flavanols, isoflavonoids, anthocyanins, and chalcones) were comprehensively reviewed from the aspects of structure–activity relationship and animal experiments. Results showed that hydrogen bonds and hydrophobic interactions were the two most important forces involved in the interaction between flavonoids and XO. Flavonoids could combine with the hydrophobic cavity of XO to cause changes in XO structure and influence the action of enzyme and substrate, thereby decreasing the catalytic activity of XO. Moreover, flavonoids undergo a series of metabolic reactions in the organism, which plays a role in alleviating HUA by affecting the expression of related genes and proteins (e.g., glucose transporter 9, urate transporter 1, and organic anion transporter 1) in the kidneys, liver, and intestinal tract of the organism and changing the internal environment in organs. In addition, this paper provides new perspectives on the shortcomings of the current research, to promote the in‐depth development and application of flavonoids.

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Vitamin C transporter SVCT1 serves a physiological role as a urate importer: functional analyses and in vivo investigations

Toyoda

Miyata

Uchida

et al. 2023

Pflugers Arch - Eur J Physiol

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Uric acid, the end product of purine metabolism in humans, is crucial because of its anti-oxidant activity and a causal relationship with hyperuricemia and gout. Several physiologically important urate transporters regulate this water-soluble metabolite in the human body; however, the existence of latent transporters has been suggested in the literature. We focused on the Escherichia coli urate transporter YgfU, a nucleobase-ascorbate transporter (NAT) family member, to address this issue. Only SLC23A proteins are members of the NAT family in humans. Based on the amino acid sequence similarity to YgfU, we hypothesized that SLC23A1, also known as sodium-dependent vitamin C transporter 1 (SVCT1), might be a urate transporter. First, we identified human SVCT1 and mouse Svct1 as sodium-dependent low-affinity/high-capacity urate transporters using mammalian cell-based transport assays. Next, using the CRISPR-Cas9 system followed by the crossing of mice, we generated Svct1 knockout mice lacking both urate transporter 1 and uricase. In the hyperuricemic mice model, serum urate levels were lower than controls, suggesting that Svct1 disruption could reduce serum urate. Given that Svct1 physiologically functions as a renal vitamin C re-absorber, it could also be involved in urate re-uptake from urine, though additional studies are required to obtain deeper insights into the underlying mechanisms. Our findings regarding the dual-substrate specificity of SVCT1 expand the understanding of urate handling systems and functional evolutionary changes in NAT family proteins.

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Inhibitory effect of Citrus flavonoids on the in vitro transport activity of human urate transporter 1 (URAT1/SLC22A12), a renal re-absorber of urate

Cited by 25 publications

References 14 publications

Omega-3 Polyunsaturated Fatty Acids Inhibit the Function of Human URAT1, a Renal Urate Re-Absorber

Omega-3 Polyunsaturated Fatty Acids Inhibit the Function of Human URAT1, a Renal Urate Re-Absorber

Mechanism of flavonoids inhibiting xanthine oxidase and alleviating hyperuricemia from structure–activity relationship and animal experiments: A review

Vitamin C transporter SVCT1 serves a physiological role as a urate importer: functional analyses and in vivo investigations

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