Ilex cornuta leaves extracts ameliorate hyperuricemia by modulating uric acid transporters.

ETHNOPHARMACOLOGICAL RELEVANCE: Ilex cornuta is a valuable species of the Holly genus (Aquifoliaceae), and mainly distributed in eastern China. It is not only made into tea, namely Kudingcha, but also used as traditional medicine to relieve cough, headache, gout, and nourish liver and kidney. AIM OF THE STUDY: The purpose of this study was to explore the exact efficacy of different extracts from Ilex cornuta in the treatment of hyperuricemia in vitro and in vivo, and to explore its pharmacological mechanism, so as to bring new ideas for the development of new drugs for reducing uric acid (UA) and anti-gout. MATERIALS AND METHODS: Five crude extracts from Ilex cornuta leaves were extracted by different methods. Then, the xanthine oxidase inhibitory activity and antioxidant capacity of 5 extracts in vitro were compared to screen the extract with the most UA regulating potential. In vivo experiment, hyperuricemia model of mice was established by intragastric administration of potassium oxonate and feeding high yeast diet. Biochemical indexes such as serum UA level, xanthine oxidase activity, liver and kidney index of mice in each group were detected. The pathological sections of kidney and liver tissues were also observed and compared. The mechanism of Ilex cornuta leaves (western blotting, and RT-qPCR) in the treatment of hyperuricemia was further explored by targeting UA transporters ABCG2, GLUT9, and URAT1. RESULTS: The in vitro results of inhibitory activity of xanthine oxidase showed that the crude saponin extract was the best, followed by crude flavonoids extract. Then, the in vivo results reflected that both crude saponins and crude flavonoids extracts could significantly reduce the serum UA level, inhibit the activity of xanthine oxidase in serum and liver, and maintain serum urea nitrogen and creatinine at normal level. Meanwhile, there was no liver and kidney injury in mice. Through the comparison of the mechanism results, it was found that both extracts could up-regulate the expression of ABCG2 protein and mRNA related to UA excretion, and down-regulate the expression of GLUT9 and URAT1 protein and mRNA. CONCLUSION: The crude flavonoids and saponins of Ilex cornuta leaves not only inhibited XOD activity in vitro, but also significantly controlled XOD activity and reduced UA level in hyperuricemia mice in vivo. One of the potential mechanisms was to regulate UA level in vivo by regulating ABCG2, GLUT9, and URAT1 transporters directly related to UA transport, thus achieving the effect of intervening hyperuricemia. This study provided a preliminary experimental basis for the development of new drugs of Ilex cornuta leaves for treating hyperuricemia.

Plasma and urinary CP I and CP III concentrations in chimeric mice with human hepatocytes after rifampicin administration.

The interest in transporter-mediated drug interactions has been increasing in the field of drug development. In this study, we measured the plasma and urinary concentrations of coproporphyrin (CP) I and CP III as endogenous substrates for organic anion-transporting polypeptide (OATP) using chimeric mice with human hepatocytes (PXB mice) and examined the influence of an OATP inhibitor, rifampicin (RIF). CP I and CP III were actively taken up intracellularly, and RIF inhibited the uptake in a concentration-dependent manner for both CP I and CP III in human hepatocytes (PXB-cells). Single doses of RIF at 10 and 30 mg/kg were orally or intravenously administered to PXB mice and wild-type ICR mice. Plasma concentrations (AUC0-8h) of CP I increased in both mice. However, a marked increase in CP III was only observed in ICR mice, after intravenous administration of RIF at 30 mg/kg. The IC50 values of RIF for intracellular CP I/III uptake and the unbound plasma concentrations of RIF suggested that the increase in plasma CP I is associated with the exposure of RIF to OATPs. The 24-h cumulative urinary excretions of CP I and CP III increased in both mice, but more markedly in PXB mice. Thus, RIF increased the plasma and urinary concentrations of CP I and CP III in the mice, as reported in humans, and CP I may be a more sensitive biomarker of OATP-mediated drug interactions in PXB mice.

Interaction of myricetin, ampelopsin (dihydromyricetin), and their sulfate metabolites with serum albumin, cytochrome P450 (CYP2C9, 2C19, and 3A4) enzymes, and organic anion-transporting polypeptides (OATP1B1 and OATP2B1).

Myricetin (MYR) and ampelopsin (AMP, or dihydromyricetin) are flavonoid aglycones found in certain plants and dietary supplements. During the presystemic biotransformation of flavonoids, mainly sulfate and glucuronide derivatives are produced, which are the dominant metabolites in the circulation. In this study, we tested the interactions of MYR, myricetin-3'-O-sulfate (M3'S), AMP, and ampelopsin-4'-O-sulfate (A4'S) with human serum albumin (HSA), cytochrome P450 enzymes (CYPs), and organic anion-transporting polypeptides (OATPs) using in vitro models, including the recently developed method for measuring flavonoid levels in living cells. M3'S and MYR bound to albumin with high affinity, and they showed moderate displacing effects versus the Site I marker warfarin. MYR, M3'S, AMP, and A4'S exerted no or only minor inhibitory effects on CYP2C9, CYP2C19, and CYP3A4 enzymes. M3'S and MYR caused considerable inhibitory actions on OATP1B1 at low micromolar concentrations (IC50 = 1.7 and 6.4 µM, respectively), while even their nanomolar levels resulted in strong inhibitory effects on OATP2B1 (IC50 = 0.3 and 0.4 µM, respectively). In addition, M3'S proved to be a substrate of OATP1B1 and OATP2B1. These results suggest that MYR-containing dietary supplements may affect the OATP-mediated transport of certain drugs, and OATPs are involved in the tissue uptake of M3'S.

Structural insight into the Arabidopsis vacuolar anion channel ALMT9 shows clade specificity.

The Arabidopsis thaliana aluminum-activated malate transporter 9 (AtALMT9) functions as a vacuolar chloride channel that regulates the stomatal aperture. Here, we present the cryoelectron microscopy (cryo-EM) structures of AtALMT9 in three distinct states. AtALMT9 forms a dimer, and the pore is lined with four positively charged rings. The apo-AtALMT9 state shows a putative endogenous citrate obstructing the pore, where two W120 constriction residues enclose a gate with a pore radius of approximately 1.8 Å, representing an open state. Interestingly, channel closure is solely controlled by W120. Compared to wild-type plants, the W120A mutant exhibits more sensitivity to drought stress and is unable to restore the visual phenotype on leaves upon water recovery, reflecting persistent stomatal opening. Furthermore, notable variations are noted in channel gating and substrate recognition of Glycine max ALMT12, AtALMT9, and AtALMT1. In summary, our investigation enhances comprehension of the interplay between structure and function within the ALMT family.

AtALMT5 mediates vacuolar fumarate import and regulates the malate/fumarate balance in Arabidopsis.

Malate and fumarate constitute a significant fraction of the carbon fixed by photosynthesis, and they are at the crossroad of central metabolic pathways. In Arabidopsis thaliana, they are transiently stored in the vacuole to keep cytosolic homeostasis. The malate and fumarate transport systems of the vacuolar membrane are key players in the control of cell metabolism. Notably, the molecular identity of these transport systems remains mostly unresolved. We used a combination of imaging, electrophysiology and molecular physiology to identify an important molecular actor of dicarboxylic acid transport across the tonoplast. Here, we report the function of the A. thaliana Aluminium-Activated Malate Transporter 5 (AtALMT5). We characterised its ionic transport properties, expression pattern, localisation and function in vivo. We show that AtALMT5 is expressed in photosynthetically active tissues and localised in the tonoplast. Patch-clamp and in planta analyses demonstrated that AtALMT5 is an ion channel-mediating fumarate loading of the vacuole. We found in almt5 plants a reduced accumulation of fumarate in the leaves, in parallel with increased malate concentrations. These results identified AtALMT5 as an ion channel-mediating fumarate transport in the vacuoles of mesophyll cells and regulating the malate/fumarate balance in Arabidopsis.

Cancer Therapeutic Potential and Prognostic Value of the SLC25 Mitochondrial Carrier Family: A Review.

Transporters of the solute carrier family 25 (SLC25) regulate the intracellular distribution and concentration of nucleotides, amino acids, dicarboxylates, and vitamins within the mitochondrial and cytoplasmic matrices. This mechanism involves changes in mitochondrial function, regulation of cellular metabolism, and the ability to provide energy. In this review, important members of the SLC25 family and their pathways affecting tumorigenesis and progression are elucidated, highlighting the diversity and complexity of these pathways. Furthermore, the significant potential of the members of SLC25 as both cancer therapeutic targets and biomarkers will be emphasized.

Arachidonic acid enhances hepatocyte bile acid uptake and alleviates cholestatic liver disease by upregulating OATP1 expression.

Cholestatic liver disease is caused by disorders of bile synthesis, secretion, and excretion. Over the long term, progressive liver cell damage from the disease evolves into liver fibrosis and cirrhosis, ultimately leading to liver failure and even cancer. Notably, cholestatic liver disease has a complex pathogenesis that remains relatively unclear. In this study, we generated two mouse models of cholestatic liver disease using a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet and α-naphthyl isothiocyanate (ANIT) gavage. Quantitative proteomics using liquid chromatography-tandem mass spectrometry showed that arachidonic acid metabolism was a common pathway in both models. Additionally, serum arachidonic acid concentrations were lower in both models than in the control group. Arachidonic acid supplementation in the diet of DDC model mice significantly reduced the levels of serum markers of cholestasis (alanine aminotransferase, aspartate transaminase, alkaline phosphatase, total bile acid, and total bilirubin) and decreased the degree of bile duct hyperplasia and cholestasis. To elucidate the mechanisms by which arachidonic acid improved bile stasis, we analyzed gene expression after arachidonic acid administration and found that Oatp1 was upregulated in the liver tissue of cholestatic mice. Arachidonic acid also increased Oatp1 expression in AML12 cells, which promoted bile acid uptake. Conclusively, our research showed that arachidonic acid mitigates cholestatic liver disease by upregulating Oatp1, promoting bile acid uptake by hepatocytes and participating in intestinal-hepatic circulation. Overall, these results suggest that supplementing foods with arachidonic acid in the daily diet may be an effective treatment strategy for cholestatic liver disease.

Identification and Analysis of Aluminum-Activated Malate Transporter Gene Family Reveals Functional Diversification in Orchidaceae and the Expression Patterns of Dendrobium catenatum Aluminum-Activated Malate Transporters.

Aluminum-activated malate transporter (ALMT) genes play an important role in aluminum ion (Al3+) tolerance, fruit acidity, and stomatal movement. Although decades of research have been carried out in many plants, there is little knowledge about the roles of ALMT in Orchidaceae. In this study, 34 ALMT genes were identified in the genomes of four orchid species. Specifically, ten ALMT genes were found in Dendrobium chrysotoxum and D. catenatum, and seven were found in Apostasia shenzhenica and Phalaenopsis equestris. These ALMT genes were further categorized into four clades (clades 1-4) based on phylogenetic relationships. Sequence alignment and conserved motif analysis revealed that most orchid ALMT proteins contain conserved regions (TM1, GABA binding motif, and WEP motif). We also discovered a unique motif (19) belonging to clade 1, which can serve as a specifically identified characteristic. Comparison with the gene structure of AtALMT genes (Arabidopsis thaliana) showed that the gene structure of ALMT was conserved across species, but the introns were longer in orchids. The promoters of orchid ALMT genes contain many light-responsive and hormone-responsive elements, suggesting that their expression may be regulated by light and phytohormones. Chromosomal localization and collinear analysis of D. chrysotoxum indicated that tandem duplication (TD) is the main reason for the difference in the number of ALMT genes in these orchids. D. catenatum was chosen for the RT-qPCR experiment, and the results showed that the DcaALMT gene expression pattern varied in different tissues. The expression of DcaALMT1-9 was significantly changed after ABA treatment. Combining the circadian CO2 uptake rate, titratable total acid, and RT-qPCR data analysis, most DcaALMT genes were highly expressed at night and around dawn. The result revealed that DcaALMT genes might be involved in photosynthate accumulation. The above study provides more comprehensive information for the ALMT gene family in Orchidaceae and a basis for subsequent functional analysis.

Hyperuricemia and its related diseases: mechanisms and advances in therapy.

Hyperuricemia, characterized by elevated levels of serum uric acid (SUA), is linked to a spectrum of commodities such as gout, cardiovascular diseases, renal disorders, metabolic syndrome, and diabetes, etc. Significantly impairing the quality of life for those affected, the prevalence of hyperuricemia is an upward trend globally, especially in most developed countries. UA possesses a multifaceted role, such as antioxidant, pro-oxidative, pro-inflammatory, nitric oxide modulating, anti-aging, and immune effects, which are significant in both physiological and pathological contexts. The equilibrium of circulating urate levels hinges on the interplay between production and excretion, a delicate balance orchestrated by urate transporter functions across various epithelial tissues and cell types. While existing research has identified hyperuricemia involvement in numerous biological processes and signaling pathways, the precise mechanisms connecting elevated UA levels to disease etiology remain to be fully elucidated. In addition, the influence of genetic susceptibilities and environmental determinants on hyperuricemia calls for a detailed and nuanced examination. This review compiles data from global epidemiological studies and clinical practices, exploring the physiological processes and the genetic foundations of urate transporters in depth. Furthermore, we uncover the complex mechanisms by which the UA induced inflammation influences metabolic processes in individuals with hyperuricemia and the association with its relative disease, offering a foundation for innovative therapeutic approaches and advanced pharmacological strategies.

Structural basis for the transport and substrate selection of human urate transporter 1.

High serum urate levels are the major risk factor for gout. URAT1, the primary transporter for urate absorption in the kidneys, is well known as an anti-hyperuricemia drug target. However, the clinical application of URAT1-targeted drugs is limited because of their low specificity and severe side effects. The lack of structural information impedes elucidation of the transport mechanism and the development of new drugs. Here, we present the cryoelectron microscopy (cryo-EM) structures of human URAT1(R477S), its complex with urate, and its closely related homolog OAT4. URAT1(R477S) and OAT4 exhibit major facilitator superfamily (MFS) folds with outward- and inward-open conformations, respectively. Structural comparison reveals a 30° rotation between the N-terminal and C-terminal domains, supporting an alternating access mechanism. A conserved arginine (OAT4-Arg473/URAT1-Arg477) is found to be essential for chloride-mediated inhibition. The URAT1(R477S)-urate complex reveals the specificity of urate recognition. Taken together, our study promotes our understanding of the transport mechanism and substrate selection of URAT1.

Design, synthesis and bioactivity evaluation of isobavachin derivatives as hURAT1 inhibitors for hyperuricemia agents.

Previously, we reported a novel natural scaffold compound, isobavachin (4',7-dihydroxy-8-prenylflavanone), as a highly potent hURAT1 inhibitor with anti-hyperuricemia effect. However, the structure-activity relationship remains unknown and the poor pharmacokinetic (PK) parameters may limit further clinical use. Herein, a series of isobavachin derivatives were rationally designed and synthesized to explore the structure-activity relationship of isobavachin target hURAT1, and to improve their PK properties. Among them, compounds 15d, 15f, 15g, 27b and 27d showed promising hURAT1 inhibitory activities, which could comparable to that of isobavachin (IC50 = 0.24 µM). In addition, 27b also inhibited another urate reabsorption transporter GLUT9 with an IC50 of 4.47 µM. Compound 27b displayed greater urate-lowering activity in a hyperuricemia mouse model at a dose of 10 mg/kg compared to isobavachin and lesinurad. Overall, our results suggest that compound 27b represents a novel, safe hURAT1 and GLUT9 dual-target inhibitor with excellent drug availability and is worthy of further investigation as an anti-hyperuricemia agent.

Significant increases in detection capability for assessment of organic anion transporting polypeptide-mediated drug-drug interaction in cynomolgus monkeys by modifying pretreatment of Coproporphyrin I and III, and glycochenodeoxycholate-3-sulfate in plasma.

BACKGROUND: Coproporphyrin I (CP-I), Coproporphyrin III (CP-III), and glycochenodeoxycholate-3-sulfate (GCDCA-S) act as endogenous substrates of Organic Anion Transporting Polypeptide (OATP) 1B and have been considered for application in OATP1B-mediated drug‒drug interaction (DDI) risk assessments. Prior assays of the endogenous OATP substrates might exhibit reduced DDI detection capability and possibly overlook low DDI risk. We pioneered a simultaneous assay of the three substrates in monkey plasma using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) and applied it to monkey studies to identify lower DDI risk. RESULTS: The methodology development indicated that precursors of CP-I/III were oxidized to form CP-I/III, diminishing the detection capability in DDI risk assessments. A precursor eliminated analytical (PEA) method was developed to eliminate the precursors through solid-phase extraction. This method aimed to prevent the oxidation of CP-I/III precursors by incorporating edaravone. For comparison, a precursor oxidized analytical (POA) method was also developed, wherein the precursors of CP-I/III were fully oxidized to CP-I/III. The PEA method achieved high sensitivity for CP-I/III and GCDCA-S, with lower quantification limits of 0.01 ng mL-1 and 0.5 ng mL-1, respectively. Both methods ensured that the validation parameters met the acceptance criteria. The two methods were applied to a monkey study, with CP-I/III showcasing notably enhanced DDI detection capabilities through the novel PEA method in comparison to the POA method. SIGNIFICANCE: This study's methodology has future implications for OATP-mediated DDI risk assessment using endogenous substrates. The novel PEA method can identify lower OATP-mediated DDI risks for drugs that the current methods cannot detect. Our method is likely applicable in clinical settings, and its utility should be assessed in clinical trials.

Plantaginis Semen Ameliorates Hyperuricemia Induced by Potassium Oxonate.

Plantaginis semen is the dried ripe seed of Plantago asiatica L. or Plantago depressa Willd., which has a long history in alleviating hyperuricemia (HUA) and chronic kidney diseases. While the major chemical ingredients and mechanism remained to be illustrated. Therefore, this work aimed to elucidate the chemicals and working mechanisms of PS for HUA. UPLC-QE-Orbitrap-MS was applied to identify the main components of PS in vitro and in vivo. RNA sequencing (RNA-seq) was conducted to explore the gene expression profile, and the genes involved were further confirmed by real-time quantitative PCR (RT-qPCR). A total of 39 components were identified from PS, and 13 of them were detected in the rat serum after treating the rat with PS. The kidney tissue injury and serum uric acid (UA), xanthine oxidase (XOD), and cytokine levels were reversed by PS. Meanwhile, renal urate anion transporter 1 (Urat1) and glucose transporter 9 (Glut9) levels were reversed with PS treatment. RNA-seq analysis showed that the PPAR signaling pathway; glycine, serine, and threonine metabolism signaling pathway; and fatty acid metabolism signaling pathway were significantly modified by PS treatment. Further, the gene expression of Slc7a8, Pck1, Mgll, and Bhmt were significantly elevated, and Fkbp5 was downregulated, consistent with RNA-seq results. The PPAR signaling pathway involved Pparα, Pparγ, Lpl, Plin5, Atgl, and Hsl were elevated by PS treatment. URAT1 and PPARα proteins levels were confirmed by Western blotting. In conclusion, this study elucidates the chemical profile and working mechanisms of PS for prevention and therapy of HUA and provides a promising traditional Chinese medicine agency for HUA prophylaxis.

Discovery of a Potent and Orally Bioavailable Xanthine Oxidase/Urate Transporter 1 Dual Inhibitor as a Potential Treatment for Hyperuricemia and Gout.

The main uric acid-lowering agents in clinical use for hyperuricemia and gout are xanthine oxidase (XO) inhibitors or urate transporter 1 (URAT1) inhibitors. While these therapies can partially control the disease, they have various limitations. The development of XO/URAT1 dual inhibitors offers the potential to enhance therapeutic potency and reduce toxicity compared with single-target inhibitors. Through scaffold hopping from the XO inhibitor febuxostat (2) and the URAT1 inhibitor probenecid (3), followed by structure-activity relationship (SAR) studies, we identified compound 27 as a potent dual inhibitor of XO and URAT1. Compound 27 demonstrated significant dual inhibition in vitro (XO IC50 = 35 nM; URAT1 IC50 = 31 nM) and exhibited favorable pharmacology and pharmacokinetic (PK) profiles in multiple species including monkeys. Furthermore, toxicity studies in rats and monkeys revealed general safety profiles, supporting that compound 27 emerges as a promising novel drug candidate with potent XO/URAT1 dual inhibition for the treatment of gout.

The toxicity of cisplatin derives from effects on renal organic ion transporters expression and serum endogenous substance levels.

Acute kidney injury (AKI) is a worldwide public health problem with high morbidity and mortality. Cisplatin is a widely used chemotherapeutic agent for treating solid tumors, but the induction of AKI restricts its clinical application. In this study, the effect of cisplatin on the expression of organic ion transporters was investigated through in vivo and in vitro experiments. Targeted metabolomics techniques were used to measure the levels of selected endogenous substances in serum. Transmission electron microscopy was used to observe the microstructure of renal tubular epithelial cells. Our results show that the toxicity of cisplatin on HK-2 cells or HEK-293 cells was time- and dose-dependent. Administration of cisplatin decreased the expression of OAT1/3 and OCT2 and increased the expression of MRP2/4. Mitochondrial damage induced by cisplatin lead to renal tubular epithelial cell injury. In addition, administration of cisplatin resulted in significant changes in endogenous substance levels in serum, including amino acids, carnitine, and fatty acids. These serum amino acids and metabolites (α-aminobutyric acid, proline, and alanine), carnitines (tradecanoylcarnitine, hexanylcarnitine, octanoylcarnitine, 2-methylbutyroylcarnitine, palmitoylcarnitine, and linoleylcarnitine) and fatty acids (9E-tetradecenoic acid) represent endogenous substances with diagnostic potential for cisplatin-induced AKI.

The role of drug efflux and uptake transporters in the plasma pharmacokinetics and tissue disposition of morphine and its main metabolites.

Morphine is a widely used opioid for the treatment of pain. Differences in drug transporter expression and activity may contribute to variability in morphine pharmacokinetics and response. Using appropriate mouse models, we investigated the impact of the efflux transporters ABCB1 and ABCG2 and the OATP uptake transporters on the pharmacokinetics of morphine, morphine-3-glucuronide (M3G), and M6G. Upon subcutaneous administration of morphine, its plasma exposure in Abcb1a/1b-/-;Abcg2-/--, Abcb1a/1b-/-;Abcg2-/-;Oatp1a/1b-/-;Oatp2b1-/- (Bab12), and Oatp1a/1b-/-;Oatp2b1-/- mice was similar to that found in wild-type mice. Forty minutes after dosing, morphine brain accumulation increased by 2-fold when mouse (m)Abcb1 and mAbcg2 were ablated. Relative recovery of morphine in small intestinal content was significantly reduced in all the knockout strains. In the absence of mOatp1a/1b and mOatp2b1, plasma levels of M3G were markedly increased, suggesting a lower elimination rate. Moreover, Oatp-deficient mice displayed reduced hepatic and intestinal M3G accumulation. Mouse Oatps similarly affected plasma and tissue disposition of subcutaneously administered M6G. Human OATP1B1/1B3 transporters modestly contribute to the liver accumulation of M6G. In summary, mAbcb1, in combination with mAbcg2, limits morphine brain penetration and its net intestinal absorption. Variation in ABCB1 activity due to genetic polymorphisms/mutations and/or environmental factors might, therefore, partially affect morphine tissue exposure in patients. The ablation of mOatp1a/1b increases plasma exposure and decreases the liver and small intestinal disposition of M3G and M6G. Since the contribution of human OATP1B1/1B3 to M6G liver uptake was quite modest, the risks of undesirable drug interactions or interindividual variation related to OATP activity are likely negligible.

Functional knockout of the Oatp1d1 membrane transporter affects toxicity of diclofenac in zebrafish embryos.

Organic anion transporting polypeptides (OATPs) facilitate the cellular uptake of a large number of compounds. Zebrafish Oatp1d1 matches the functional capabilities of human OATP orthologs, particularly in hormone and drug transport. It is highly expressed in the liver and later stages of embryonic development, indicating its critical role in zebrafish physiology and development. Data from previous in vitro analyses have shown a high affinity of zebrafish Oatp1d1 for pharmaceuticals and xenobiotics, providing the basis for further in vivo studies on its defence and developmental functions. Using CRISPR-Cas9 technology, we have generated an Oatp1d1 zebrafish mutant that has highly reduced Oatp1d1 expression in embryos and adult tissues compared to wild type (WT). The absence of Oatp1d1 was confirmed using custom-made antibodies. To evaluate its ecotoxicological relevance, mutant and WT embryos were exposed to increasing concentrations of diclofenac, an NSAID known for its wide and frequent use, environmental pseudo-persistence and ecological implications. WT embryos showed developmental delays and malformations such as spinal curvature, cardiac edema and blood pooling at higher diclofenac concentrations, whereas the Oatp1d1 mutant embryos showed marked resilience, with milder developmental defects and delayed toxic effects. These observations suggest that the absence of Oatp1d1 impedes the efficient entry of diclofenac into hepatocytes, thereby slowing its biotransformation into potentially more toxic metabolites. In addition, the changes in transcript expression of other uptake transporters revealed a highly probable and complex network of compensatory mechanisms. Therefore, the results of this study point to the importance of Oatp1d1-mediated transport of diclofenac, as demonstrated for the first time in vivo using an Oatp1 deficient zebrafish line. Finally, our data indicates that the compensatory role of other transporters with overlapping substrate preferences needs to be considered for a reliable understanding of the physiological and/or defensive role(s) of membrane transporters.

The Competitive Counterflow Assay for Identifying Drugs Transported by Solute Carriers: Principle, Applications, Challenges/Limits, and Perspectives.

The identification of substrates for solute carriers (SLCs) handling drugs is an important challenge, owing to the major implication of these plasma membrane transporters in pharmacokinetics and drug-drug interactions. In this context, the competitive counterflow (CCF) assay has been proposed as a practical and less expensive approach than the reference functional uptake assays for discriminating SLC substrates and non-substrates. The present article was designed to summarize and discuss key-findings about the CCF assay, including its principle, applications, challenges and limits, and perspectives. The CCF assay is based on the decrease of the steady-state accumulation of a tracer substrate in SLC-positive cells, caused by candidate substrates. Reviewed data highlight the fact that the CCF assay has been used to identify substrates and non-substrates for organic cation transporters (OCTs), organic anion transporters (OATs), and organic anion transporting polypeptides (OATPs). The performance values of the CCF assay, calculated from available CCF study data compared with reference functional uptake assay data, are, however, rather mitigated, indicating that the predictability of the CCF method for assessing SLC-mediated transportability of drugs is currently not optimal. Further studies, notably aimed at standardizing the CCF assay and developing CCF-based high-throughput approaches, are therefore required in order to fully precise the interest and relevance of the CCF assay for identifying substrates and non-substrates of SLCs.

9-Hydroxy-8-oxypalmatine, a novel liver-mediated oxymetabolite of palmatine, alleviates hyperuricemia and kidney inflammation in hyperuricemic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Palmatine is a main bioactive alkaloid of Cortex Phellodendri, which has been commonly prescribed for the treatment of hyperuricemia (HUA) in China. The metabolites of palmatine were crucial to its prominent biological activity. 9-Hydroxy-8-oxypalmatine (9-OPAL) is a novel liver-mediated secondary oxymetabolite of palmatine. AIM OF THE STUDY: The current study was to assess the efficacy of 9-OPAL, a novel liver-mediated secondary oxymetabolite of palmatine derived from Cortex Phellodendri, in experimental HUA mouse model and further explore its underlying mechanism. MATERIALS AND METHODS: An in vitro metabolic experiment with oxypalmatine was carried out using liver samples. We separated and identified a novel liver metabolite, and investigated its anti-HUA effect in mice. HUA mice were induced by potassium oxonate and hypoxanthine daily for one week. After 1 h of modeling, mice were orally administered with different doses of 9-OPAL (5, 10 and 20 mg/kg). The pathological changes of the kidneys were evaluated using hematoxylin-eosin staining (H&E). The acute toxicity of 9-OPAL was assessed. The effects of 9-OPAL on serum levels of uric acid (UA), adenosine deaminase (ADA), xanthine oxidase (XOD), creatinine (CRE), blood urea nitrogen (BUN) and inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA) or biochemical method. Furthermore, Western blot, quantitative real-time PCR (qRT-PCR) and molecular docking were used to investigate the effect of 9-OPAL on the expression of renal urate transporters and NLRP3 signaling pathway in HUA mice. RESULTS: 9-OPAL had been discovered to be a novel liver-mediated oxymetabolite of palmatine for the first time. Treatment with 9-OPAL significantly reduced the UA, CRE as well as BUN levels, and also effectively attenuated abnormal renal histopathological deterioration with favorable safety profile. Besides, 9-OPAL significantly decreased the serum and hepatic activities of XOD and ADA, dramatically inhibited the up-regulation of UA transporter protein 1 (URAT1) and glucose transporter protein 9 (GLUT9), and reversed the down-regulation of organic anion transporter protein 1 (OAT1). Additionally, 9-OPAL effectively mitigated the renal inflammatory markers (TNF-α, IL-1ß, IL-6 and IL-18), and downregulated the transcriptional and translational expressions of renal Nod-like receptor family pyrin domain containing 3 (NLRP3), caspase-1, apoptosis-associated speck-like (ASC) and IL-1ß in HUA mice. Molecular docking results revealed 9-OPAL bound firmly with XOD, OAT1, GLUT9, URAT1, NLRP3, caspase-1, ASC and IL-1ß. CONCLUSIONS: 9-OPAL was found to be a novel liver-mediated secondary metabolite of palmatine with favorable safety profile. 9-OPAL had eminent anti-hyperuricemic and renal-protective effects, and the mechanisms might be intimately associated with repressing XOD activities, modulating renal urate transporter expression and suppressing the NLRP3 inflammasome activation. Our investigation might also provide further experimental evidence for the traditional application of Cortex Phellodendri in the treatment of HUA.