RESUMO
Custom oligonucleotides (oligos) are widely used reagents in biomedical research. Some common applications of oligos include polymerase chain reaction (PCR), sequencing, hybridization, microarray, and library construction. The reliability of oligos in such applications depends on their purity and specificity. Here, we report that commercially available oligos are frequently contaminated with nonspecific sequences (i.e. other unrelated oligonucleotides). Most of the oligos that we designed to amplify clustered regularly interspersed palindromic repeats (CRISPR) guide sequences contained nonspecific CRISPR guides. These contaminants were detected in research-grade oligos procured from eight commercial oligo-suppliers located in three different geographic regions of the world. Deep sequencing of some of the oligos revealed a variety of contaminants. Given the wide range of applications of oligos, the impact of oligo cross-contamination varies greatly depending on the field and the experimental method. Incorporating appropriate control experiments in research design can help ensure that the quality of oligo reagents meets the intended purpose. This can also minimize risk depending on the purposes for which the oligos are used.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Contaminação de Medicamentos , Indicadores e Reagentes , Oligonucleotídeos , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Oligonucleotídeos/química , Oligonucleotídeos/normas , Técnicas Genéticas , Indicadores e Reagentes/análise , Indicadores e Reagentes/normas , Indústrias/normasRESUMO
ESCO1/2 acetyltransferases mediating SMC3 acetylation and sister chromatid cohesion (SCC) are differentially required for genome integrity and development. Here we established chicken DT40 cell lines with mutations in ESCO1/2, SMC3 acetylation, and the cohesin remover WAPL. Both ESCO1 and ESCO2 promoted SCC, while ESCO2 was additionally and specifically required for proliferation and centromere integrity. ESCO1 overexpression fully suppressed the slow proliferation and centromeric separation phenotypes of esco2 cells but only partly suppressed its chromosome arm SCC defects. Concomitant inactivation of ESCO1 and ESCO2 caused lethality owing to compromised mitotic chromosome segregation. Neither wapl nor acetyl-mimicking smc3-QQ mutations rescued esco1 esco2 lethality. Notably, esco1 esco2 wapl conditional mutants showed very severe proliferation defects associated with catastrophic mitoses and also abnormal interphase chromatin organization patterns. The results indicate that cohesion establishment by vertebrate ESCO1/2 is linked to interphase chromatin architecture formation, a newly identified function of cohesin acetyltransferases that is both fundamentally and medically relevant.
Assuntos
Acetiltransferases/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Estruturas Cromossômicas/genética , Instabilidade Genômica/genética , Acetilação , Acetiltransferases/genética , Animais , Linhagem Celular , Proliferação de Células/genética , Centrômero/genética , Galinhas , Proteínas Cromossômicas não Histona/genética , Técnicas de Inativação de Genes , Inativação Gênica , Interfase/genética , Proteínas Nucleares/genéticaRESUMO
BACKGROUND: Recent studies have identified that low levels of some tumour suppressor microRNAs (miRNAs) in the blood contribute to tumour progression and poor outcomes in various cancers. However, no study has proved these miRNAs are associated with cancer immune mechanisms. METHODS: From a systematic review of the NCBI and miRNA databases, four tumour suppressor miRNA candidates were selected (miR-5193, miR-4443, miR-520h, miR-496) that putatively target programmed cell death ligand 1 (PD-L1). RESULTS: Test-scale and large-scale analyses revealed that plasma levels of miR-5193 were significantly lower in gastric cancer (GC) patients than in healthy volunteers (HVs). Low plasma levels of miR-5193 were associated with advanced pathological stages and were an independent prognostic factor. Overexpression of miR-5193 in GC cells suppressed PD-L1 on the surface of GC cells, even with IFN-γ stimulation. In the coculture model of GC cells and T cells stimulated by anti-CD3/anti-CD28 beads, overexpression of miR-5193 increased anti-tumour activity of T cells by suppressing PD-L1 expression. Subcutaneous injection of miR-5193 also significantly enhanced the tumour-killing activity and trafficking of T cells in mice. CONCLUSIONS: Low blood levels of miR-5193 are associated with GC progression and poor outcomes and could be a target of nucleic acid immunotherapy in GC patients.
Assuntos
MicroRNAs , Neoplasias Gástricas , Humanos , Animais , Camundongos , Neoplasias Gástricas/genética , Neoplasias Gástricas/terapia , Antígeno B7-H1 , MicroRNAs/metabolismo , Genes Supressores de Tumor , ImunoterapiaRESUMO
Metabolic abnormalities play a pivotal role in various pathological conditions, necessitating the quantification of specific metabolites for diagnosis. While mass spectrometry remains the primary method for metabolite measurement, its limited throughput underscores the need for biosensors capable of rapid detection. Previously, we reported that pillar[6]arene with 12 carboxylate groups (P6AC) forms host-guest complexes with 1-methylnicotinamide (1-MNA), which is produced in vivo by nicotinamide N-methyltransferase (NNMT). P6AC acts as a biosensor by measuring the fluorescence quenching caused by photoinduced electron transfer upon 1-MNA binding. However, the low sensitivity of P6AC makes it impractical for detecting 1-MNA in unpurified biological samples. In this study, we found that P6A with 12 sulfonate groups (P6AS) is a specific and potent supramolecular host for 1-MNA interactions even in biological samples. The 1-MNA binding affinity of P6AS in water was found to be (5.68 ± 1.02) × 106 M-1, which is approximately 700-fold higher than that of P6AC. Moreover, the 1-MNA detection limit of P6AS was determined to be 2.84 × 10-7 M, which is substantially lower than that of P6AC. Direct addition of P6AS to culture medium was sufficient to quantify 1-MNA produced by cancer cells. Furthermore, this sensor was able to specifically detect 1-MNA even in unpurified human urine. P6AS therefore enables rapid and high-throughput quantification of 1-MNA, and further improvement of our strategy will contribute to the establishment of high-throughput screening of NNMT inhibitors, diagnosis of liver diseases, and imaging of human cancer cells in vivo.
RESUMO
Dotinurad was developed as a uricosuric agent, inhibiting urate (UA) reabsorption through the UA transporter URAT1 in the kidneys. Due to its high selectivity for URAT1 among renal UA transporters, we investigated the mechanism underlying this selectivity by identifying dotinurad binding sites specific to URAT1. Dotinurad was docked to URAT1 using AutoDock4, utilizing the AlphaFold2-predicted structure. The inhibitory effects of dotinurad on wild-type and mutated URAT1 at the predicted binding sites were assessed through URAT1-mediated [14C]UA uptake in Xenopus oocytes. Nine amino acid residues in URAT1 were identified as dotinurad-binding sites. Sequence alignment with UA-transporting organic anion transporters (OATs) revealed that H142 and R487 were unique to URAT1 among renal UA-transporting OATs. For H142, IC50 values of dotinurad increased to 62, 55, and 76 nM for mutated URAT1 (H142A, H142E, and H142R, respectively) compared with 19 nM for the wild type, indicating that H142 contributes to URAT1-selective interaction with dotinurad. H142 was predicted to interact with the phenyl-hydroxyl group of dotinurad. The IC50 of the hydroxyl group methylated dotinurad (F13141) was 165 µM, 8420-fold higher than dotinurad, suggesting the interaction of H142 and the phenyl-hydroxyl group by forming a hydrogen bond. Regarding R487, URAT1-R487A exhibited a loss of activity. Interestingly, the URAT1-H142A/R487A double mutant restored UA transport activity, with the IC50 value of dotinurad for the mutant (388 nM) significantly higher than that for H142A (73.5 nM). These results demonstrate that H142 and R487 of URAT1 determine its selectivity for dotinurad, a uniqueness observed only in URAT1 among UA-transporting OATs. SIGNIFICANCE STATEMENT: Dotinurad selectively inhibits the urate reabsorption transporter URAT1 in renal urate-transporting organic ion transporters (OATs). This study demonstrates that dotinurad interacts with H142 and R487 of URAT1, located in the extracellular domain and unique among OATs when aligning amino acid sequences. Mutations in these residues reduce affinity of dotinurad for URAT1, confirming their role in conferring selective inhibition. Additionally, the interaction between dotinurad and URAT1 involving H142 is found to mediate hydrogen bonding.
Assuntos
Transportadores de Ânions Orgânicos , Ácido Úrico , Uricosúricos , Animais , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/genética , Ácido Úrico/metabolismo , Ácido Úrico/farmacologia , Sítios de Ligação , Humanos , Uricosúricos/farmacologia , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Xenopus laevis , Rim/metabolismo , Rim/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/efeitos dos fármacos , Benzotiazóis/farmacologia , Simulação de Acoplamento MolecularRESUMO
The role of the kidney as an excretory organ for exogenous and endogenous compounds is well recognized, but there is a wealth of data demonstrating that the kidney has significant metabolizing capacity for a variety of exogenous and endogenous compounds that in some cases surpass the liver. The induction of drug-metabolizing enzymes by some chemicals can cause drug-drug interactions and intraindividual variability in drug clearance. In this study, we evaluated the expression and induction of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) isoforms in 3D-cultured primary human renal proximal tubule epithelial cells (RPTEC) to elucidate their utility as models of renal drug metabolism. CYP2B6, CYP2E1, CYP3A4, CYP3A5, and all detected UGTs (UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) mRNA levels in 3D-RPTEC were significantly higher than those in 2D-RPTEC and HK-2 cells and were close to the levels in the human kidney cortex. CYP1B1 and CYP2J2 mRNA levels in 3D-RPTEC were comparable to those in 2D-RPTEC, HK-2 cells, and the human kidney cortex. Midazolam 1'-hydroxylation, trifluoperazine N-glucuronidation, serotonin O-glucuronidation, propofol O-glucuronidation, and morphine 3-glucuronidation in the 3D-RPTEC were significantly higher than the 2D-RPTEC and comparable to those in the HepaRG cells, although bupropion, ebastine, and calcitriol hydroxylations were not different between the 2D- and 3D-RPTEC. Treatment with ligands of the aryl hydrocarbon receptor and farnesoid X receptor induced CYP1A1 and UGT2B4 expression, respectively, in 3D-RPTEC compared with 2D-RPTEC. We provided information on the expression, activity, and induction abilities of P450s and UGTs in 3D-RPTEC as an in vitro human renal metabolism model. SIGNIFICANCE STATEMENT: This study demonstrated that the expression of cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) in 3D-cultured primary human renal proximal tubule epithelial cells (3D-RPTEC) was higher than those in 2D-cultured primary human renal proximal tubule epithelial cells and HK-2 cells. The results were comparable to that in the human kidney cortex. 3D-RPTEC are useful for evaluating the induction of kidney P450s, UDP-glucuronosyltransferases, and human renal drug metabolism in cellulo.
Assuntos
Sistema Enzimático do Citocromo P-450 , Células Epiteliais , Glucuronosiltransferase , Túbulos Renais Proximais , Humanos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Glucuronosiltransferase/metabolismo , Glucuronosiltransferase/genética , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Células Cultivadas , Indução Enzimática/efeitos dos fármacos , Linhagem Celular , Técnicas de Cultura de Células/métodos , RNA Mensageiro/metabolismo , RNA Mensageiro/genéticaRESUMO
Hepatic physiology depends on the liver's complex structural composition which among others, provides high oxygen supply rates, locally differential oxygen tension, endothelial paracrine signaling, as well as residual hemodynamic shear stress to resident hepatocytes. While functional improvements were shown by implementing these factors into hepatic culture systems, direct cause-effect relationships are often not well characterized-obfuscating their individual contribution in more complex microphysiological systems. By comparing increasingly complex hepatic in vitro culture systems that gradually implement these parameters, we investigate the influence of the cellular microenvironment to overall hepatic functionality in pharmacological applications. Here, hepatocytes were modulated in terms of oxygen tension and supplementation, endothelial coculture, and exposure to fluid shear stress delineated from oxygen influx. Results from transcriptomic and metabolomic evaluation indicate that particularly oxygen supply rates are critical to enhance cellular functionality-with cellular drug metabolism remaining comparable to physiological conditions after prolonged static culture. Endothelial signaling was found to be a major contributor to differential phenotype formation known as metabolic zonation, indicated by WNT pathway activity. Lastly, oxygen-delineated shear stress was identified to direct cellular fate towards increased hepatic plasticity and regenerative phenotypes at the cost of drug metabolic functionality - in line with regenerative effects observed in vivo. With these results, we provide a systematic evaluation of critical parameters and their impact in hepatic systems. Given their adherence to physiological effects in vivo, this highlights the importance of their implementation in biomimetic devices, such as organ-on-a-chip systems. Considering recent advances in basic liver biology, direct translation of physiological structures into in vitro models is a promising strategy to expand the capabilities of pharmacological models.
Assuntos
Fígado , Sistemas Microfisiológicos , Fígado/metabolismo , Hepatócitos/metabolismo , Perfilação da Expressão Gênica , Oxigênio/metabolismoRESUMO
Recent advancements in bioengineering have introduced potential alternatives to liver transplantation via the development of self-assembled liver organoids, derived from human-induced pluripotent stem cells (hiPSCs). However, the limited maturity of the tissue makes it challenging to implement this technology on a large scale in clinical settings. In this study, we developed a highly efficient method for generating functional liver organoids from hiPSC-derived carboxypeptidase M liver progenitor cells (CPM+ LPCs), using a microwell structure, and enhanced maturation through direct oxygenation in oxygen-permeable culture plates. We compared the morphology, gene expression profile, and function of the liver organoid with those of cells cultured under conventional conditions using either monolayer or spheroid culture systems. Our results revealed that liver organoids generated using polydimethylsiloxane-based honeycomb microwells significantly exhibited enhanced albumin secretion, hepatic marker expression, and cytochrome P450-mediated metabolism. Additionally, the oxygenated organoids consisted of both hepatocytes and cholangiocytes, which showed increased expression of bile transporter-related genes as well as enhanced bile transport function. Oxygen-permeable polydimethylsiloxane membranes may offer an efficient approach to generating highly mature liver organoids consisting of diverse cell populations.
Assuntos
Células-Tronco Pluripotentes Induzidas , Metaloendopeptidases , Humanos , Oxigênio/metabolismo , Diferenciação Celular , Fígado/metabolismo , Técnicas de Cultura de Células/métodos , Organoides/metabolismo , Dimetilpolisiloxanos , Proteínas Ligadas por GPIRESUMO
Controlling RAS mutant cancer progression remains a significant challenge in developing anticancer drugs. Whereas Ras G12C-covalent binders have received clinical approval, the emergence of further mutations, along with the activation of Ras-related proteins and signals, has led to resistance to Ras binders. To discover novel compounds to overcome this bottleneck, we focused on the concurrent and sustained blocking of two major signaling pathways downstream of Ras. To this end, we synthesized 25 drug-drug conjugates (DDCs) by combining the MEK inhibitor trametinib with Akt inhibitors using seven types of linkers with structural diversity. The DDCs were evaluated for their cell permeability/accumulation and ability to inhibit proliferation in RAS-mutant cell lines. A representative DDC was further evaluated for its effects on signaling proteins, induction of apoptosis-related proteins, and the stability of hepatic metabolic enzymes. These in vitro studies identified a series of DDCs, especially those containing a furan-based linker, with promising properties as agents for treating RAS-mutant cancers. Additionally, in vivo experiments in mice using the two selected DDCs revealed prolonged half-lives and anticancer efficacies comparable to those of trametinib. The PK profiles of trametinib and the Akt inhibitor were unified through the DDC formation. The DDCs developed in this study have potential as drug candidates for the broad inhibition of RAS-mutant cancers.
Assuntos
Antineoplásicos , Neoplasias , Animais , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Transdução de Sinais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Mutação , Inibidores da Angiogênese/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Linhagem Celular TumoralRESUMO
Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.
Assuntos
Cisplatino , Testículo , Humanos , Camundongos , Animais , Criança , Recém-Nascido , Masculino , Testículo/metabolismo , Técnicas de Cultura de Órgãos/métodos , Cisplatino/toxicidade , Espermatogênese , Proteínas de Fluorescência Verde/genéticaRESUMO
BACKGROUND: Hyposalivation is treated using oral cholinergic drugs; however, systemic side effects occasionally lead to discontinuation of treatment. We aimed to investigate the effects of transdermal pilocarpine on the salivary gland skin on saliva secretion and safety in rats. METHODS: Pilocarpine was administered to rats orally (0.5 mg/kg) or topically on the salivary gland skin (5 mg/body). Saliva volume, the number of sweat dots, and fecal weight were measured along with pilocarpine concentration in plasma and submandibular gland tissues. RESULTS: Saliva volume significantly increased 0.5 h after oral administration and 0.5, 3, and 12 h after topical administration. Fecal weight and sweat dots increased significantly 1 h after oral administration; however, no changes were observed after topical application. The pilocarpine concentration in the submandibular gland tissues of the topical group was higher than that in the oral group at 0.5, 3, and 12 h of administration. CONCLUSIONS: Pilocarpine application to salivary gland skin persistently increased salivary volume in rats without inducing sweating or diarrhea. Transdermal pilocarpine applied to the skin over the salivary glands may be an effective and safe treatment option for hyposalivation.
Assuntos
Administração Cutânea , Pilocarpina , Glândulas Salivares , Salivação , Xerostomia , Pilocarpina/administração & dosagem , Pilocarpina/farmacologia , Animais , Salivação/efeitos dos fármacos , Ratos , Masculino , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/metabolismo , Xerostomia/induzido quimicamente , Xerostomia/tratamento farmacológico , Agonistas Muscarínicos/administração & dosagem , Agonistas Muscarínicos/farmacologia , Saliva/metabolismo , Saliva/química , Administração Oral , Glândula Submandibular/efeitos dos fármacos , Glândula Submandibular/metabolismo , Ratos Sprague-DawleyRESUMO
This study investigated novel tumor suppressor microRNAs (miRNAs) that decrease in plasma and predict chemosensitivity to neoadjuvant chemotherapy (NAC) for esophageal squamous cell carcinoma (ESCC) and revealed their usefulness as novel therapeutic agents. We selected four miRNA candidates (miR-323, 345, 409, and 1254) based on the microRNA microarray comparing pre-treatment plasma levels in ESCC patients with high and low histopathological responses to NAC and an NCBI database review. Among these miRNA candidates, miR-1254 was more highly elevated in pre-treatment plasma of ESCC patients with a high histopathological response than in those with a low histopathological response (P = 0.0021, area under the receiver-operating characteristic curve 0.7621). High plasma miR-1254 levels tended to correlate with the absence of venous invasion (P = 0.0710) and were an independent factor predicting a higher response to chemotherapy (P = 0.0022, odds ratio 7.86) and better prognosis (P = 0.0235, hazard ratio 0.23). Overexpressing miR-1254 in ESCC cells significantly enhanced chemosensitivity to cisplatin through the transcriptional regulation of ABCC1 in vitro. Moreover, increased plasma miR-1254 levels by subcutaneous injection significantly improved responses to cisplatin in mice. Plasma miR-1254 might be a useful biomarker for predicting responses to NAC, and the restoration of plasma miR-1254 levels might improve chemosensitivity in ESCC.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , MicroRNAs , Animais , Camundongos , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Cisplatino , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , PrognósticoRESUMO
Patients with kidney dysfunction exhibit distinct pharmacokinetic profiles compared to those with normal kidney function. Hence, it is desirable to monitor the drug efficacy and toxicity caused by fluctuations in plasma drug concentrations associated with kidney dysfunction. Recently, pharmacokinetic information of drugs excreted mainly through the urine of patients with kidney dysfunction has been reported via drug-labeling information. Pharmacokinetic changes in drugs mainly eliminated by the liver cannot be overlooked as drug metabolism and/or transport activity in the liver may also be altered in patients with kidney dysfunction; however, the underlying mechanisms remain unclear. To plan an appropriate dosage regimen, it is necessary to clarify the underlying processes of functional changes in pharmacokinetic proteins. In recent years, uremic toxins have been shown to reduce the activity and/or expression of renal and hepatic transporters. This inhibitory effect has been reported to be time-dependent. In addition, inflammatory cytokines, such as interleukin-6, released from immune cells activated by uremic toxins and/or kidney injury can reduce the expression levels of drug-metabolizing enzymes and transporters in human hepatocytes. In this mini-review, we have summarized the renal and hepatic pharmacokinetic changes as well as the potential underlying mechanisms in kidney dysfunction, such as the chronic kidney disease and acute kidney injury. SIGNIFICANCE STATEMENT: Patients with kidney dysfunction exhibit distinct pharmacokinetic profiles compared to those with normal kidney function. Increased plasma concentrations of uremic toxins and inflammatory cytokines during kidney disease may potentially affect the activities and/or expression levels of drug-metabolizing enzymes and transporters in the liver and kidneys.
Assuntos
Injúria Renal Aguda , Insuficiência Renal Crônica , Humanos , Toxinas Urêmicas , Citocinas/metabolismo , Rim/metabolismo , Fígado/metabolismo , Insuficiência Renal Crônica/metabolismoRESUMO
Urate transporter 1 (URAT1) is a transporter responsible for uric acid (UA) reabsorption by renal proximal tubules and a pharmacological target of uricosuric agents. Probenecid and benzbromarone have been used as uricosuric agents, while dotinurad was recently approved in Japan. Notably, the in vitro IC 50 of dotinurad on URAT1 is not strong enough to explain its in vivo uricosuric effect estimated based on clinical unbound plasma concentrations, suggesting the presence of mechanisms other than competition with UA uptake at the extracellular domain of URAT1 (cis-inhibition). In this study, trans-inhibition was hypothesized as the mechanism underlying URAT1 inhibition by dotinurad, wherein intracellularly accumulated dotinurad inactivates URAT1. In URAT1-expressing Madin-Darby Canine Kidney-II cells and Xenopus oocytes, pre-incubation with dotinurad potentiated the inhibitory effect more than co-incubation alone, but this effect was not observed with benzbromarone or probenecid. Under co-incubation, dotinurad inhibited UA uptake in a competitive manner (cis-inhibition). When we pre-injected dotinurad directly into oocytes and immediately measured [14C]UA uptake without coincubation (only trans-inhibition), dotinurad noncompetitively inhibited UA uptake. URAT1 is an exchange transporter for UA and monocarboxylates such as nicotinic acid (NA). Pre-injected dotinurad and extracellular UA attenuated and facilitated efflux of [3H]NA, respectively, whereas pre-injection of benzbromarone or probenecid did not affect it, suggesting that dotinurad exhibits trans-inhibition by attenuating URAT1-mediated efflux of monocarboxylates, which is a driving force for UA uptake by URAT1. Accordingly, dotinurad ameliorates URAT1-mediated UA reabsorption by both cis- and trans-inhibition, explaining its clinically stronger uricosuric effect than that estimated by the in vitro IC50 value. SIGNIFICANCE STATEMENT: The uricosuric agent dotinurad inhibits uric acid reabsorptive transporter (URAT) 1 with a clinical potency stronger than that estimated from IC 50 obtained by in vitro URAT1 inhibition. This in vivo-in vitro discrepancy was explained by the trans-inhibition effect of dotinurad on URAT1. Trans-inhibition was due to the attenuation of monocarboxylates efflux via URAT1, which is a driving force for URAT1-mediated exchange transport of uric acid. Overall, this is the first study to experimentally demonstrate trans-inhibition mechanism of URAT1.
RESUMO
The proximal tubule plays an important role in the kidney and is a major site of drug interaction and toxicity. Analysis of kidney toxicity via in vitro assays is challenging, because only a few assays that reflect functions of drug transporters in renal proximal tubular epithelial cells (RPTECs) are available. In this study, we aimed to develop a simple and reproducible method for culturing RPTECs by monitoring organic anion transporter 1 (OAT1) as a selection marker. Culturing RPTECs in spherical cellular aggregates increased OAT1 protein expression, which was low in the conventional two-dimensional (2D) culture, to a level similar to that in human renal cortices. By proteome analysis, it was revealed that the expression of representative two proximal tubule markers was maintained and 3D spheroid culture improved the protein expression of approximately 7% of the 139 transporter proteins detected, and the expression of 2.3% of the 4,800 proteins detected increased by approximately fivefold that in human renal cortices. Furthermore, the expression levels of approximately 4,800 proteins in three-dimensional (3D) RPTEC spheroids (for 12 days) were maintained for over 20 days. Cisplatin and adefovir exhibited transporter-dependent ATP decreases in 3D RPTEC spheroids. These results indicate that the 3D RPTEC spheroids developed by monitoring OAT1 gene expression are a simple and reproducible in vitro experimental system with improved gene and protein expressions compared with 2D RPTECs and were more similar to that in human kidney cortices. Therefore, it can potentially be used for evaluating human renal proximal tubular toxicity and drug disposition. SIGNIFICANCE STATEMENT: This study developed a simple and reproducible spheroidal culture method with acceptable throughput using commercially available RPTECs by monitoring OAT1 gene expression. RPTECs cultured using this new method showed improved mRNA/protein expression profiles to those in 2D RPTECs and were more similar to those of human kidney cortices. This study provides a potential in vitro proximal tubule system for pharmacokinetic and toxicological evaluations during drug development.
Assuntos
Rim , Proteína 1 Transportadora de Ânions Orgânicos , Humanos , Rim/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Expressão Gênica , Células Epiteliais/metabolismoRESUMO
Transdermal scopolamine applied to the postauricular area is used to treat drooling. We investigated the duration of action of scopolamine ointment and the effect of the application site on drug efficacy and concentration in the salivary glands of rats. Scopolamine ointment was applied to the skin over the salivary glands (SSG) and back (SB). Saliva volume was measured after intraperitoneal administration of pilocarpine. Blood and salivary glands were collected after scopolamine ointment application, and scopolamine concentrations in the plasma and salivary glands were measured. Saliva volume after application in the SSG group was significantly lower at all time points than in the non-treated group, and the change in saliva volume in the SSG group was greater than that in the SB group at all time points. This suggests that applying scopolamine ointment to the SSG strongly suppresses salivary secretion. Scopolamine concentration in the salivary glands of the SSG group was significantly higher at 9 h. The change in the efficacy of scopolamine ointment depending on the application site was due to the difference in transfer to the salivary glands. Transdermal administration of scopolamine to the skin over the salivary glands may have high efficiency in treating drooling.
Assuntos
Escopolamina , Sialorreia , Ratos , Animais , Administração Cutânea , Sialorreia/tratamento farmacológico , Pomadas/uso terapêutico , Glândulas SalivaresRESUMO
ABSTRACT: Arakawa, H, Mori, M, and Tanimoto, M. Greater hip moments in rear-foot-elevated split squats than in conventional back squats with the same relative intensity of loads. J Strength Cond Res 37(5): 1009-1016, 2023-Rear-foot-elevated split squat (RFESS) is often performed as an alternative to conventional double-leg back squat (DLBS). This study aimed to compare 3-dimensional joint kinetics of DLBS and RFESS using the same relative intensity of loads. Eight male college rugby players performed 3 repetitions of DLBS and RFESS at 10-repetition-maximum (RM) loading. Before testing, both exercises were incorporated into the subjects' training program with a progressive increase in loads for 4 months. A 3-dimensional optical motion capture system and force platform were used for data collection. The 3-dimensional moments at the knee and hip joints in each of the 3 axes were calculated based on the inverse dynamic procedure. p values < 0.05 were considered statistically significant. The hip extension moment was 44% greater in the RFESS than in the DLBS at the bottom position ( p < 0.01) and 47% greater for the peak value ( p < 0.01) on harmonic averages. The hip abduction and external rotation moments at the bottom position were also greater in the RFESS than in the DLBS. The findings suggest that the magnitude of hip extension moment per leg in DLBS tends to be restricted to less than that expected from the given strength level. In conclusion, the mechanical contribution of hip extensors per leg can be greater in RFESS than in DLBS when using respective 10RM loads, even if the absolute load is smaller and the trunk is more upright in RFESS.
Assuntos
Extremidade Inferior , Postura , Humanos , Masculino , Joelho , Articulação do Joelho , Perna (Membro) , Fenômenos BiomecânicosRESUMO
AIM: Identification of blood-brain barrier (BBB) uptake transporters is a major challenge in the research and development of central nervous system (CNS) drugs. However, conventional methods that consider known drug uptake characteristics have failed at identifying the responsible transporter molecule. The present study aimed at identifying aripiprazole uptake transporters in BBB model hCMEC/D3 cells using a knockdown screening study targeting various transporters, including uncharacterized ones. METHODS: We evaluated the effect of 214 types of siRNA targeting transporters on the uptake of aripiprazole, an atypical antipsychotic drug, in hCMEC/D3 cells. Aripiprazole uptake was determined using Xenopus oocytes expressing the candidate genes extracted from the siRNA screening assay. RESULTS: The estimated unbound brain to plasma concentration ratio (Kp,uu,brain) of aripiprazole was estimated as 0.67 in wild-type mice and 1.94 in abcb1a/1b/abcg2 knockout mice, suggesting the involvement of both uptake and efflux transporters in BBB permeation. According to siRNA knockdown screening studies, organic cation/carnitine transporter 2 (OCTN2) and long-chain fatty acid transporter 1 (FATP1) were identified as candidate genes. The uptake of aripiprazole by hCMEC/D3 cells was decreased by OCTN2 inhibitors, but not by FATP1 inhibitors. A partially increased uptake of aripiprazole was observed in OCTN2-expressing Xenopus oocytes. Finally, to evaluate transporter-mediated BBB permeation of drugs, the reported and estimated Kp,uu,brain values were summarized. CONCLUSIONS: A knockdown screening study in combination with Kp,uu,brain values showed that aripiprazole was a potential substrate of OCTN2. The technique described in this study can be applied to identifying novel BBB transporters for CNS drugs.
Assuntos
Barreira Hematoencefálica , Proteínas de Membrana Transportadoras , Animais , Aripiprazol/farmacologia , Transporte Biológico , Encéfalo , Camundongos , RNA Interferente Pequeno/genéticaRESUMO
The type of method adopted for the evaluation of drug-induced kidney injury (DIKI) plays an important role during the drug discovery process. In the present study, the usefulness of cultured rat kidney tissue slices maintained on gas-permeable poly(dimethylsiloxane) (PDMS) plates for DIKI was assessed by monitoring the ATP content as a marker of cell viability. The amount of ATP in the kidney slices cultured on the PDMS plates was higher than that in the slices cultured on gas-impermeable polystyrene plates. The protein expression of organic cation transporter-2 (Oct2) was maintained for 3 d. Cisplatin showed a time- and concentration-dependent reduction in ATP in the slices with a half-effective concentration value of 24 µM, which was alleviated by cimetidine, an Oct2 inhibitor, suggesting that cisplatin-induced kidney injury in the cultured slices was regulated by the basolateral uptake transporter Oct2. Furthermore, the intensity of platinum anticancer drug-induced nephrotoxicity in the cultured slices was consistent with that of the in vivo study. In conclusion, the primary culture of rat kidney tissue slices on gas-permeable plates is expected to aid in the prediction of the extent of nephrotoxicity of drugs, even when transporters are responsible for the accumulation of drugs in kidney tissues.
Assuntos
Antineoplásicos , Platina , Animais , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Cisplatino/efeitos adversos , Rim , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Platina/metabolismo , RatosRESUMO
Cluster of differentiation (CD) 38, a major enzyme for nicotinamide adenine dinucleotide (NAD+) degradation, plays a key role in inflammation. Meanwhile, intracellular NAD+ decline is also associated with inflammatory responses. However, whether CD38 activation is involved in gouty inflammation has not been elucidated. The present study aimed to clarify the role of CD38 in monosodium urate crystals (MSU)-triggered inflammatory responses. The results showed that MSU crystals increased the protein expression of CD38 in time- and concentration-dependent manner in THP-1 macrophages and mouse bone marrow-derived macrophages (BMDMs). Moreover, intracellular NAD+ levels were reduced by MSU crystals along with the increased IL-1ß release. However, CD38 inhibition by 78c elevated intracellular NAD+ levels and suppressed IL-1ß release in MSU crystals-treated THP-1 macrophages and BMDMs. Interestingly, CD38 inhibition without significant elevation of intracellular NAD+ also decreased IL-1ß release driven by MSU crystals in THP-1 macrophages. In conclusion, the present study revealed that MSU crystals could activate CD38 with the ensuing intracellular NAD+ decline to promote inflammatory responses in THP-1 macrophages and BMDMs, while CD38 inhibition could suppress MSU crystals-triggered inflammatory responses, indicating that CD38 is a potential therapeutic target for gout.