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1.
Drug Metab Dispos ; 52(2): 118-125, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38050024

RESUMO

The organic anion uptake and efflux transporters [organic anion-transporting polypeptide (OATP)1B1, OATP1B3 and multidrug resistance-associated protein (MRP)2 and MRP3] that mediate the transport of the hepatobiliary-specific contrast agent gadoxetate (Gd-EOB-DTPA) are direct or indirect targets of the farnesoid X receptor (FXR), a key regulator of bile acid and lipid homeostasis. In benign liver tumors, FXR expression and activation is not yet characterized. We investigated the expression and activation of FXR and its targets in hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH) and their correlation with Gd-EOB-DTPA-enhanced magnetic resonance imaging (MRI). Gd-EOB-DTPA MRI patterns were assessed by an expert radiologist. The intensity of the lesions on the hepatobiliary phase was correlated to mRNA expression levels of OATP1B1, OATP1B3, MRP2, MRP3, FXR, and small heterodimer partner (SHP) in fresh surgical specimens of patients with FNH or HCA subtypes. Normal and tumor sample pairs of 43 HCA and 14 FNH were included. All FNH (14/14) were hyperintense. Of the 34 HCA with available Gd-EOB-DTPA-enhanced MRI, 6 were hyperintense and 28 HCA were hypointense. OATP1B3 was downregulated in the hypointense tumors compared with normal surrounding liver tissue (2.77±3.59 vs. 12.9±15.6, P < 0.001). A significant positive correlation between FXR expression and activation and OATP1B3 expression level was found in the HCA cohort. SHP showed a trend toward downregulation in hypointense HCA. In conclusion, this study suggests that the MRI relative signal in HCA may reflect expression level and/or activity of SHP and FXR. Moreover, our data confirms the pivotal role of OATP1B3 in Gd-EOB-DTPA uptake in HCA. SIGNIFICANCE STATEMENT: FXR represents a valuable target for the treatment of liver disease and metabolic syndrome. Currently, two molecules, ursodeoxycholate and obeticholate, are approved for the treatment of primary biliary cirrhosis and cholestasis, with several compounds in clinical trials for the treatment of metabolic dysfunction-associated fatty liver disease. Because FXR expression and activation is associated with gadoxetate accumulation in HCA, an atypical gadoxetate-enhanced MRI pattern might arise in patients under FXR-targeted therapy, thereby complicating the differential diagnosis.


Assuntos
Adenoma de Células Hepáticas , Hiperplasia Nodular Focal do Fígado , Neoplasias Hepáticas , Transportadores de Ânions Orgânicos , Humanos , Transportadores de Ânions Orgânicos/metabolismo , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/metabolismo , Meios de Contraste/metabolismo , Hiperplasia Nodular Focal do Fígado/diagnóstico , Hiperplasia Nodular Focal do Fígado/metabolismo , Hiperplasia Nodular Focal do Fígado/patologia , Imageamento por Ressonância Magnética/métodos , Proteína 2 Associada à Farmacorresistência Múltipla , Ânions/metabolismo , Estudos Retrospectivos
2.
Circ Res ; 130(1): 80-95, 2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-34809444

RESUMO

BACKGROUND: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. METHODS: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. RESULTS: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25, overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. CONCLUSIONS: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.


Assuntos
Proteínas Nucleares/metabolismo , Splicing de RNA , Receptores de LDL/genética , Colesterol/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Lipoproteínas LDL/metabolismo , Fígado/metabolismo , Mutação , Proteínas Nucleares/genética , Receptores de LDL/metabolismo , Spliceossomos/metabolismo
3.
Int J Mol Sci ; 24(3)2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36768423

RESUMO

Tyrosine kinase inhibitors (TKIs) decisively contributed in revolutionizing the therapeutic approach to cancer, offering non-invasive, tolerable therapies for a better quality of life. Nonetheless, degree and duration of the response to TKI therapy vary depending on cancer molecular features, the ability of developing resistance to the drug, on pharmacokinetic alterations caused by germline variants and unwanted drug-drug interactions at the level of membrane transporters and metabolizing enzymes. A great deal of approved TKIs are inhibitors of the organic cation transporters (OCTs). A handful are also substrates of them. These transporters are polyspecific and highly expressed in normal epithelia, particularly the intestine, liver and kidney, and are, hence, arguably relevant sites of TKI interactions with other OCT substrates. Moreover, OCTs are often repressed in cancer cells and might contribute to the resistance of cancer cells to TKIs. This article reviews the OCT interactions with approved and in-development TKIs reported in vitro and in vivo and critically discusses the potential clinical ramifications thereof.


Assuntos
Neoplasias , Humanos , Qualidade de Vida , Interações Medicamentosas , Proteínas de Membrana Transportadoras , Neoplasias/tratamento farmacológico , Cátions , Proteínas de Transporte de Cátions Orgânicos/genética
4.
Int J Mol Sci ; 24(3)2023 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-36769329

RESUMO

Sphingolipids are exceptionally diverse, comprising hundreds of unique species. The bulk of circulating sphingolipids are synthesized in the liver, thereby plasma sphingolipid profiles represent reliable surrogates of hepatic sphingolipid metabolism and content. As changes in plasma sphingolipid content have been associated to exposure to drugs inducing hepatotoxicity both in vitro and in rodents, in the present study the translatability of the preclinical data was assessed by analyzing the plasma of patients with suspected drug-induced liver injury (DILI) and control subjects. DILI patients, whether intrinsic or idiosyncratic cases, had no alterations in total sphingoid base levels and profile composition compared to controls, whereby cardiovascular disease (CVD) was a confounding factor. Upon exclusion of CVD individuals, elevation of 1-deoxysphingosine (1-deoxySO) in the DILI group emerged. Notably, 1-deoxySO values did not correlate with ALT values. While 1-deoxySO was elevated in all DILI cases, only intrinsic DILI cases concomitantly displayed reduction of select shorter chain sphingoid bases. Significant perturbation of the sphingolipid metabolism observed in this small exploratory clinical study is discussed and put into context, in the consideration that sphingolipids might contribute to the onset and progression of DILI, and that circulating sphingoid bases may function as mechanistic markers to study DILI pathophysiology.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Esfingolipídeos/metabolismo , Fígado/metabolismo
5.
J Biol Chem ; 296: 100204, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33334877

RESUMO

The carnitine/organic cation transporter novel 2 (OCTN2) is responsible for the cellular uptake of carnitine in most tissues. Being a transmembrane protein OCTN2 must interact with the surrounding lipid microenvironment to function. Among the main lipid species that constitute eukaryotic cells, cholesterol has highly dynamic levels under a number of physiopathological conditions. This work describes how plasma membrane cholesterol modulates OCTN2 transport of L-carnitine in human embryonic kidney 293 cells overexpressing OCTN2 (OCTN2-HEK293) and in proteoliposomes harboring human OCTN2. We manipulated the cholesterol content of intact cells, assessed by thin layer chromatography, through short exposures to empty and/or cholesterol-saturated methyl-ß-cyclodextrin (mßcd), whereas free cholesterol was used to enrich reconstituted proteoliposomes. We measured OCTN2 transport using [3H]L-carnitine, and expression levels and localization by surface biotinylation and Western blotting. A 20-min preincubation with mßcd reduced the cellular cholesterol content and inhibited L-carnitine influx by 50% in comparison with controls. Analogously, the insertion of cholesterol in OCTN2-proteoliposomes stimulated L-carnitine uptake in a dose-dependent manner. Carnitine uptake in cells incubated with empty mßcd and cholesterol-saturated mßcd to preserve the cholesterol content was comparable with controls, suggesting that the mßcd effect on OCTN2 was cholesterol dependent. Cholesterol stimulated L-carnitine influx in cells by markedly increasing the affinity for L-carnitine and in proteoliposomes by significantly enhancing the affinity for Na+ and, in turn, the L-carnitine maximal transport capacity. Because of the antilipogenic and antioxidant features of L-carnitine, the stimulatory effect of cholesterol on L-carnitine uptake might represent a novel protective effect against lipid-induced toxicity and oxidative stress.


Assuntos
Carnitina/metabolismo , Colesterol/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto/metabolismo , Transporte Biológico , Células HEK293 , Humanos , Proteolipídeos/metabolismo
6.
Int J Mol Sci ; 23(22)2022 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-36430444

RESUMO

The nuclear receptor farnesoid X receptor (FXR, NR1H4) is a bile acid (BA) sensor that links the enterohepatic circuit that regulates BA metabolism and elimination to systemic lipid homeostasis. Furthermore, FXR represents a real guardian of the hepatic function, preserving, in a multifactorial fashion, the integrity and function of hepatocytes from chronic and acute insults. This review summarizes how FXR modulates the expression of pathway-specific as well as polyspecific transporters and enzymes, thereby acting at the interface of BA, lipid and drug metabolism, and influencing the onset and progression of hepatotoxicity of varying etiopathogeneses. Furthermore, this review article provides an overview of the advances and the clinical development of FXR agonists in the treatment of liver diseases.


Assuntos
Ácidos e Sais Biliares , Doença Hepática Induzida por Substâncias e Drogas , Humanos , Receptores Citoplasmáticos e Nucleares , Homeostase , Lipídeos
7.
Int J Mol Sci ; 23(13)2022 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-35806196

RESUMO

Vascular and lymphatic vessels drive breast cancer (BC) growth and metastasis. We assessed the cell growth (proliferation, migration, and capillary formation), gene-, and protein-expression profiles of Vascular Endothelial Cells (VECs) and Lymphatic Endothelial Cells (LECs) exposed to a conditioned medium (CM) from estrogen receptor-positive BC cells (MCF-7) in the presence or absence of Estradiol. We demonstrated that MCF-7-CM stimulated growth and capillary formation in VECs but inhibited LEC growth. Consistently, MCF-7-CM induced ERK1/2 and Akt phosphorylation in VECs and inhibited them in LECs. Gene expression analysis revealed that the LECs were overall (≈10-fold) more sensitive to MCF-7-CM exposure than VECs. Growth/angiogenesis and cell cycle pathways were upregulated in VECs but downregulated in LECs. An angiogenesis proteome array confirmed the upregulation of 23 pro-angiogenesis proteins in VECs. In LECs, the expression of genes related to ATP synthesis and the ATP content were reduced by MCF-7-CM, whereas MTHFD2 gene, involved in folate metabolism and immune evasion, was upregulated. The contrasting effect of MCF-7-CM on the growth of VECs and LECs was reversed by inhibiting the TGF-ß signaling pathway. The effect of MCF-7-CM on VEC growth was also reversed by inhibiting the VEGF signaling pathway. In conclusion, BC secretome may facilitate cancer cell survival and tumor growth by simultaneously promoting vascular angiogenesis and inhibiting lymphatic growth. The differential effects of BC secretome on LECs and VECs may be of pathophysiological relevance in BC.


Assuntos
Neoplasias da Mama , Células Endoteliais , Trifosfato de Adenosina/metabolismo , Neoplasias da Mama/metabolismo , Células Endoteliais/metabolismo , Feminino , Humanos , Linfangiogênese/genética , Células MCF-7 , Neovascularização Patológica/metabolismo , Secretoma , Transcriptoma
8.
Mol Pharmacol ; 97(5): 314-323, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32098797

RESUMO

Farnesoid X receptor (FXR), or NR1H4, protects the liver from insults of various etiologies. A role of FXR in drug-induced liver injury has also been hypothesized yet only marginally investigated. The aim of this study was to assess the effect of FXR activation on gene expression and phenotype of the liver of mice treated with valproic acid (VPA), or 2-propylpentanoic acid, a prototypical hepatotoxic drug. Obeticholic acid (OCA) was used to activate FXR both in mice and in human hepatocellular carcinoma (Huh-7) cells. Next-generation sequencing of mouse liver tissues was performed from control, VPA, and VPA + OCA-treated mice. Pathway analysis validation was performed using real-time reverse-transcription polymerase chain reaction, Western blotting, immunohistochemistry, and fluorometric assays. FXR activation induced antioxidative pathways, which was confirmed by a marked reduction in VPA-induced lipid peroxidation and endoplasmic reticulum stress. In vitro, VPA-induced oxidative stress was independent of lipid accumulation, stemmed from the cytoplasm, and was mitigated by OCA. In the liver of the mice treated with OCA, the levels of cytochrome P450 potentially involved in VPA metabolism were increased. The hepatic lipid-lowering effect observed in animals cotreated with VPA and OCA in comparison with that of animals treated with VPA was associated with regulation of the genes involved in the steatogenic nuclear receptor peroxisome proliferator-activated γ (PPARγ) pathway. In conclusion, pronounced antioxidant activity, repression of the PPARγ pathway, and higher expression of P450 enzymes involved in VPA metabolism may underlie the hepatoprotective of FXR activation during VPA treatment. SIGNIFICANCE STATEMENT: Valproic acid-induced oxidative stress occurs in absence of lipid accumulation and is not of mitochondrial origin. Valproic acid exposure induces the expression of the steatogenic nuclear receptor peroxisome proliferator-activated γ (PPARγ) and its downstream target genes. Constitutive activation of the farnesoid X receptor (FXR) reduces PPARγ hepatic expression and induces hepatic antioxidant activity. The variability in FXR expression level/activity, for instance in individuals carrying loss-of-function genetic variants of the FXR gene, could contribute to valproic acid pharmacokinetic and toxicokinetic profile.


Assuntos
Ácido Quenodesoxicólico/análogos & derivados , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/patologia , Estresse Oxidativo , Ácido Valproico/efeitos adversos , Animais , Antioxidantes/farmacologia , Peso Corporal/efeitos dos fármacos , Linhagem Celular Tumoral , Ácido Quenodesoxicólico/farmacologia , Ácido Quenodesoxicólico/uso terapêutico , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/fisiopatologia , Feminino , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Testes de Função Hepática , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcriptoma/genética
9.
J Pharmacol Exp Ther ; 372(1): 46-53, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31624079

RESUMO

The human organic cation transporter 2 (OCT2) mediates the first step of tubular secretion of most positively charged substances. We describe the role of plasma membrane cholesterol in OCT2 activity. Human embryonic kidney 293 cells overexpressing OCT2 (OCT2-HEK293) and wild-type HEK293 cells (WT-HEK293) were employed. Cellular cholesterol content, assessed by thin layer chromatography, was manipulated using empty methyl-ß-cyclodextrin (mßcd) and cholesterol-presaturated mßcd (RAMEB). The effect of mßcd on OCT2 protein stability and oligomerization state was evaluated by immunofluorescence and Western blotting. Transport activity of OCT2 was measured using [3H]1-methyl-4-phenylpyridinium (MPP+). A 20-minute incubation with mßcd reduced the total cellular cholesterol content by 40% to 60% as compared with that in untreated cells, without altering the content of the other main lipid species. In this condition, OCT2-mediated uptake of MPP+ was reduced by ∼50%. When cells were coincubated with empty mßcd and RAMEB, the cholesterol content and OCT2-mediated uptake of MPP+ were comparable to those in untreated cells, suggesting that the mßcd effect on OCT2 activity was cholesterol dependent. In untreated cells, the MPP+ influx kinetics was allosteric, whereas in cells treated with mßcd, one binding site was observed. Our findings suggest that changes in cellular cholesterol content can dramatically alter OCT2-mediated transport, potentially resulting in abnormal tubular secretion and unexpected drug toxicity and drug-drug interactions. SIGNIFICANCE STATEMENT: Plasma membrane cholesterol is important for the allosteric properties of OCT2. From a pharmacologic standpoint, the variability in cholesterol content stemming from certain pathophysiologic conditions such as aging and acute kidney injury should be taken into account as additional source of interpatient pharmacokinetic/pharmacodynamic variability and unexpected toxicity profile of OCT2 substrates, which can escape preclinical and clinical development.


Assuntos
1-Metil-4-fenilpiridínio/farmacologia , Membrana Celular/metabolismo , Colesterol/metabolismo , Herbicidas/farmacologia , Transportador 2 de Cátion Orgânico/metabolismo , Regulação Alostérica , Sítio Alostérico , Membrana Celular/efeitos dos fármacos , Células HEK293 , Humanos , Transportador 2 de Cátion Orgânico/química , Ligação Proteica , beta-Ciclodextrinas/farmacologia
10.
J Pharmacol Exp Ther ; 375(2): 239-246, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32848075

RESUMO

Mitochondrial damage is considered a hallmark of drug-induced liver injury (DILI). However, despite the common molecular etiology, the evolution of the injury is usually unpredictable, with some cases that are mild and reversible upon discontinuation of the treatment and others characterized by irreversible acute liver failure. This suggests that additional mechanisms of damage play a role in determining the progression of the initial insult. To uncover novel pathways potentially involved in DILI, we investigated in vitro the metabolic perturbations associated with nefazodone, an antidepressant associated with acute liver failure. Several pathways associated with ATP production, including gluconeogenesis, anaerobic glycolysis, and oxidative phosphorylation, were altered in human hepatocellular carcinoma-derived (Huh7) cells after 2-hour exposure to a 50 µM extracellular concentration of nefazodone. In the presence or absence of glucose, ATP production of Huh7 cells was glycolysis- and oxidative phosphorylation-dependent, respectively. In glucose-containing medium, nefazodone-induced ATP depletion from Huh7 cells was biphasic. Huh7 cells in glucose-free medium were more sensitive to nefazodone than those in glucose-containing medium, losing the biphasic inhibition. Nefazodone-induced ATP depletion in primary cultured mouse hepatocytes, mainly dependent on oxidative phosphorylation, was monophasic. At lower extracellular concentrations, nefazodone inhibited the oxygen consumption of Huh7 cells, whereas at higher extracellular concentrations, it also inhibited the extracellular acidification. ATP content was rescued by increasing the extracellular concentration of glucose. In conclusion, nefazodone has a dual inhibitory effect on mitochondrial-dependent and mitochondrial-independent ATP production. SIGNIFICANCE STATEMENT: Mitochondrial damage is a hallmark of drug-induced liver injury, yet other collateral alterations might contribute to the severity and evolution of the injury. Our in vitro study supports previous results arguing that a deficit in hepatic glucose metabolism, concomitant to the mitochondrial injury, might be cardinal in the prognosis of the initial insult to the liver. From a drug development standpoint, coupling anaerobic glycolysis and mitochondrial function assessment might increase the drug-induced liver injury preclinical screening performance.


Assuntos
Antidepressivos/efeitos adversos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metabolômica , Piperazinas/efeitos adversos , Triazóis/efeitos adversos , Trifosfato de Adenosina/metabolismo , Anaerobiose/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Camundongos
11.
Liver Int ; 40(4): 844-859, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31883408

RESUMO

BACKGROUND & AIMS: Patients with non-alcoholic fatty liver disease (NAFLD) exhibit higher levels of plasma 1-deoxysphingolipids than healthy individuals. The aim of this study was to investigate the role of farnesoid X receptor (FXR) in 1-deoxysphingolipid de novo synthesis and degradation. METHODS: Mice were fed with a high-fat diet (HFD) to induce obesity and NAFLD, and then treated with the FXR ligand obeticholic acid (OCA). Histology and gene expression analysis were performed on liver tissue. Sphingolipid patterns from NAFLD patients and mouse models were assessed by liquid chromatography-mass spectrometry. The molecular mechanism underlying the effect of FXR activation on sphingolipid metabolism was studied in Huh7 cells and primary cultured hepatocytes, as well as in a 1-deoxysphinganine-treated mouse model. RESULTS: 1-deoxysphingolipids were increased in both NAFLD patients and mouse models. FXR activation by OCA protected the liver against oxidative stress, apoptosis, and reduced 1-deoxysphingolipid levels, both in a HFD-induced mouse model of obesity and in 1-deoxysphinganine-treated mice. In vitro, FXR activation lowered intracellular 1-deoxysphingolipid levels by inducing Cyp4f-mediated degradation, but not by inhibiting de novo synthesis, thereby protecting hepatocytes against doxSA-induced cytotoxicity, mitochondrial damage, and apoptosis. Overexpression of Cyp4f13 in cells was sufficient to ameliorate doxSA-induced cytotoxicity. Treatment with the Cyp4f pan-inhibitor HET0016 or FXR knock-down fully abolished the protective effect of OCA, indicating that OCA-mediated 1-deoxysphingolipid degradation is FXR and Cyp4f dependent. CONCLUSIONS: Our study identifies FXR-Cyp4f as a novel regulatory pathway for 1-deoxysphingolipid metabolism. FXR activation represents a promising therapeutic strategy for patients with metabolic syndrome and NAFLD.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Amidinas , Animais , Humanos , Fígado , Camundongos , Camundongos Endogâmicos C57BL , Receptores Citoplasmáticos e Nucleares , Esfingolipídeos
12.
Int J Mol Sci ; 21(21)2020 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-33114309

RESUMO

Individual cells and epithelia control the chemical exchange with the surrounding environment by the fine-tuned expression, localization, and function of an array of transmembrane proteins that dictate the selective permeability of the lipid bilayer to small molecules, as actual gatekeepers to the interface with the extracellular space. Among the variety of channels, transporters, and pumps that localize to cell membrane, organic cation transporters (OCTs) are considered to be extremely relevant in the transport across the plasma membrane of the majority of the endogenous substances and drugs that are positively charged near or at physiological pH. In humans, the following six organic cation transporters have been characterized in regards to their respective substrates, all belonging to the solute carrier 22 (SLC22) family: the organic cation transporters 1, 2, and 3 (OCT1-3); the organic cation/carnitine transporter novel 1 and 2 (OCTN1 and N2); and the organic cation transporter 6 (OCT6). OCTs are highly expressed on the plasma membrane of polarized epithelia, thus, playing a key role in intestinal absorption and renal reabsorption of nutrients (e.g., choline and carnitine), in the elimination of waste products (e.g., trimethylamine and trimethylamine N-oxide), and in the kinetic profile and therapeutic index of several drugs (e.g., metformin and platinum derivatives). As part of the Special Issue Physiology, Biochemistry, and Pharmacology of Transporters for Organic Cations, this article critically presents the physio-pathological, pharmacological, and toxicological roles of OCTs in the tissues in which they are primarily expressed.


Assuntos
Bicamadas Lipídicas/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Preparações Farmacêuticas/metabolismo , Toxinas Biológicas/farmacocinética , Transporte Biológico , Humanos , Concentração de Íons de Hidrogênio , Absorção Intestinal , Proteínas de Transporte de Cátions Orgânicos/genética , Farmacocinética , Reabsorção Renal
13.
Antimicrob Agents Chemother ; 63(12)2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31591120

RESUMO

The polymixin colistin represents a last resort antibiotic for multidrug resistant infections, but its use is limited by the frequent onset of acute drug-induced kidney injury (DIKI). It is essential to closely monitor kidney function prior to and during colistin treatment in order to pinpoint early signs of injury and minimise long-term renal dysfunction. To facilitate this, a mouse model of colistin-induced nephrotoxicity was used to uncover novel early markers of colistin-induced DIKI. Increased urinary levels of kidney injury molecule 1 (Kim-1) as well as glycosuria were observed in colistin-treated mice, where alterations of established clinical markers of acute kidney injury (serum creatinine and albuminuria) and emerging markers such as cystatin C were inaccurate in flagging renal damage as confirmed by histology. A direct interaction of colistin with renal glucose reabsorption was ruled out by a cis-inhibition assay in mouse brush border membrane vesicles (BBMV). Immunohistochemical examination and protein quantification by western blotting showed a marked reduction in the protein amount of sodium-glucose transporter 2 (Sglt2), the main kidney glucose transporter, in renal tissue from colistin-treated mice in comparison to control animals. Consistently, BBMV isolated from treated mouse kidneys also showed a reduction in ex vivo glucose uptake when compared to BBMV isolated from control kidneys. These findings support pathology observations of colistin-induced proximal tubule damage at the site of the brush border membrane, where Sglt2 is expressed, and open avenues for the study of glycosuria as a sensitive, specific, and accessible marker of DIKI during colistin therapy.

14.
Liver Int ; 39(12): 2350-2359, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31408569

RESUMO

BACKGROUND & AIMS: The organic anion-transporting polypeptide 1B1 (OATP1B1) is an anion exchanger expressed at the hepatocyte sinusoidal membrane, which mediates the uptake of several endogenous metabolites and drugs. OATP1B1 expression level and activity are major sources of inter-patient variability of pharmacokinetics and pharmacodynamics of several drugs. Besides the genotype, factors that contribute to the inter-individual variability in OATP1B1 expression level are practically unknown. The aim of this work was to uncover novel epigenetic mechanisms of OATP1B1 regulation. METHODS: A functional screening strategy to assess the effect of microRNAs on the uptake of estrone-3-sulphate, an OATP1B1 substrate, into human hepatocellular carcinoma (Huh-7) cells was used. microRNA-206 (miR-206) expression in human liver tissues was measured by real-time RT-PCR. OATP1B1 expression in Huh-7 and in human liver tissues was assessed by real-time RT-PCR, Western blotting and immunostaining. The mRNA-miRNA interaction was assessed by reporter assay. RESULTS: miR-206 mimic repressed mRNA and protein expression of OATP1B1 in Huh-7 cells. The intracellular accumulation of estrone-3-sulphate was reduced by 30% in cells overexpressing miR-206. The repressive effect of miR-206 on the activity of the firefly luciferase gene 2 under the control of the OATP1B1 3' untranslated region was lost upon deletion of the predicted miR-206 binding site. Hepatic miR-206 level negatively correlated with OATP1B1 mRNA and protein levels extracted from normal human liver tissues. CONCLUSIONS: miR-206 exerts a suppressive effect on OATP1B1 expression by an epigenetic mechanism. Individuals with high hepatic levels of miR-206 appear to display lower level of OATP1B1.


Assuntos
Estrona/análogos & derivados , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Fígado/metabolismo , MicroRNAs/metabolismo , Animais , Células CHO , Linhagem Celular Tumoral , Simulação por Computador , Cricetulus , Estrona/metabolismo , Feminino , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Masculino , Pessoa de Meia-Idade
15.
J Pathol ; 246(3): 352-365, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30058725

RESUMO

Pancreatic ductal adenocarcinoma (PDAC), which is the primary cause of pancreatic cancer mortality, is poorly responsive to currently available interventions. Identifying new targets that drive PDAC formation and progression is critical for developing alternative therapeutic strategies to treat this lethal malignancy. Using genetic and pharmacological approaches, we investigated in vivo and in vitro whether uptake of the monoamine serotonin [5-hydroxytryptamine (5-HT)] is required for PDAC development. We demonstrated that pancreatic acinar cells have the ability to readily take up 5-HT in a transport-mediated manner. 5-HT uptake promoted activation of the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for transdifferentiation of acinar cells into acinar-to-ductal metaplasia (ADM), a key determinant in PDAC development. Consistent with the central role played by Rac1 in ADM formation, inhibition of the 5-HT transporter Sert (Slc6a4) with fluoxetine reduced ADM formation both in vitro and in vivo in a cell-autonomous manner. In addition, fluoxetine treatment profoundly compromised the stromal reaction and affected the proliferation and lipid metabolism of malignant PDAC cells. We propose that Sert is a promising therapeutic target to counteract the early event of ADM, with the potential to stall the initiation and progression of pancreatic carcinogenesis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Carcinoma Ductal Pancreático/enzimologia , Proliferação de Células , Genes ras , Neuropeptídeos/metabolismo , Pâncreas/enzimologia , Neoplasias Pancreáticas/enzimologia , Serotonina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/prevenção & controle , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transdiferenciação Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Embrião de Galinha , Modelos Animais de Doenças , Ativação Enzimática , Fluoxetina/farmacologia , Predisposição Genética para Doença , Humanos , Metaplasia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/prevenção & controle , Fenótipo , Ratos , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Transdução de Sinais , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo
16.
Molecules ; 24(3)2019 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-30759858

RESUMO

The emergence of multidrug resistant (MDR) infections and the shortage of new therapeutic options have made colistin, a polymyxin antibiotic, the main option for the treatment of MDR Gram-negative bacterial infections in the last decade. However, the rapid onset of renal damage often prevents the achievement of optimal therapeutic doses and/or forces the physicians to interrupt the therapy, increasing the risk of drug resistance. The proper management of colistin-induced nephrotoxicity remains challenging, mostly because the investigation of the cellular and molecular pharmacology of this drug, off the market for decades, has been largely neglected. For years, the renal damage induced by colistin was considered a mere consequence of the detergent activity of this drug on the cell membrane of proximal tubule cells. Lately, it has been proposed that the intracellular accumulation is a precondition for colistin-mediated renal damage, and that mitochondria might be a primary site of damage. Antioxidant approaches (e.g., ascorbic acid) have shown promising results in protecting the kidney of rodents exposed to colistin, yet none of these strategies have yet reached the bedside. Here we provide a critical overview of the possible mechanisms that may contribute to colistin-induced renal damage and the potential protective strategies under investigation.


Assuntos
Injúria Renal Aguda/induzido quimicamente , Antibacterianos/farmacologia , Colistina/farmacologia , Rim/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Membrana Celular/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Humanos
17.
Mol Pharmacol ; 94(2): 802-811, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29743187

RESUMO

Inflammation has a recognized role in nonalcoholic fatty liver disease (NAFLD) progression. In the present work, we studied the effect of high-fat diet (HFD) on arachidonic acid metabolism in the liver and investigated the role of the farnesoid X receptor (FXR, NR1H4) in eicosanoid biosynthetic pathways and nuclear factor κ light-chain enhancer of activated B cells (NF-kB) signaling, major modulators of the inflammatory cascade. Mice were fed an HFD to induce NAFLD and then treated with the FXR ligand obeticholic acid (OCA). Histology and gene expression analyses were performed on liver tissue. Eicosanoid levels were measured from serum and urine samples. The molecular mechanism underlying the effect of FXR activation on arachidonic acid metabolism and NF-kB signaling was studied in human liver Huh7 cells and primary cultured hepatocytes. NAFLD was characterized by higher (∼25%) proinflammatory [leukotrienes (LTB4)] and lower (∼3-fold) anti-inflammatory [epoxyeicosatrienoic acids (EETs)] eicosanoid levels than in chow mice. OCA induced the expression of several hepatic cytochrome P450 (P450) epoxygenases, the enzymes responsible for EET synthesis, and mitigated HFD-induced hepatic injury. In vitro, induction of CYP450 epoxygenases was sufficient to inhibit NF-kB signaling and cell migration. The CYP450 epoxygenase pan-inhibitor gemfibrozil fully abolished the protective effect of OCA, indicating that OCA-mediated inhibition of NF-kB signaling was EET-dependent. In summary, NAFLD was characterized by an imbalance in arachidonate metabolism. FXR activation reprogramed arachidonate metabolism by inducing P450 epoxygenase expression and EET production. In vitro, FXR-mediated NF-kB inhibition required active P450 epoxygenases.


Assuntos
Ácido Araquidônico/metabolismo , Ácido Quenodesoxicólico/análogos & derivados , Dieta Hiperlipídica/efeitos adversos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Ácido Araquidônico/sangue , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Ácido Quenodesoxicólico/administração & dosagem , Ácido Quenodesoxicólico/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/genética , Receptores Citoplasmáticos e Nucleares/genética , Células THP-1
18.
Biochim Biophys Acta Mol Basis Dis ; 1864(4 Pt B): 1498-1506, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28882625

RESUMO

Drug-induced liver injury includes a spectrum of pathologies, some related to the mode of injury, some to the cell type primarily damaged. Among these, drug-induced bile duct injury is characterized by the destruction of the biliary epithelium following exposure to a drug. Most of the drugs associated with bile duct injury cause immune-mediated lesions to the epithelium of interlobular ducts. These share common histopathological features with primary biliary cholangitis, such as inflammation and necrosis at the expense of cholangiocytes and, if the insult persists, bile duct loss and biliary cirrhosis. Some drugs selectively target larger ducts. Such injury is often dose-dependent and thought to be the result of intrinsic drug toxicity. The histological changes resemble those seen in primary sclerosing cholangitis. This overview focuses on the clinical and pathological features of bile duct injury associated with drug treatment and on the immunological and biochemical effects that drugs exert on the biliary epithelium. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.


Assuntos
Ductos Biliares/patologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colangite Esclerosante/induzido quimicamente , Colestase/induzido quimicamente , Células Epiteliais/efeitos dos fármacos , Combinação Amoxicilina e Clavulanato de Potássio/farmacocinética , Combinação Amoxicilina e Clavulanato de Potássio/toxicidade , Ácidos e Sais Biliares/toxicidade , Ductos Biliares/citologia , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/metabolismo , Biomarcadores/análise , Biotransformação , Carbamazepina/farmacocinética , Carbamazepina/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Colagogos e Coleréticos/uso terapêutico , Colangite Esclerosante/diagnóstico , Colangite Esclerosante/tratamento farmacológico , Colangite Esclerosante/patologia , Colestase/diagnóstico , Colestase/tratamento farmacológico , Colestase/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia
19.
Drug Metab Dispos ; 46(8): 1129-1136, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29794161

RESUMO

[18F]fluorocholine is the fluorinated analog of [11C]choline and is used in positron emission tomography to monitor tumor metabolic activity. Although important to optimize its use and expand the clinical indications, the molecular determinants of fluorocholine cellular uptake are poorly characterized. In this work, we described the influx kinetics of fluorocholine mediated by the organic cation transporter 2 (OCT2, SLC22A2) and compared with that of choline. Then we characterized the expression pattern of OCT2 in renal cell carcinoma (RCC). In HEK293 cells stably transfected with OCT2 fluorocholine influx, kinetics was biphasic, suggesting two independent binding sites: a high-affinity (Km = 14 ± 8 µM, Vmax = 1.3 ± 0.5 nmol mg-1 min-1) and a low-affinity component (Km = 1.8 ± 0.3 mM, Vmax = 104 ± 4.5 nmol mg-1 min-1). Notably, choline was found to be transported with sigmoidal kinetics typical of homotropic positive cooperativity (h = 1.2, 95% confidence interval 1.1-1.3). OCT2 mRNA expression level was found significantly decreased in primary but not in metastatic RCC. Tissue microarray immunostaining of 216 RCC biopsies confirmed that the OCT2 protein level was consistent with that of the mRNA. The kinetic properties described in this work suggest that OCT2 is likely to play a dominant role in [18F]fluorocholine uptake in vivo. OCT2-altered expression in primary and metastatic cancer cells, as compared with the surrounding tissues, could be exploited in RCC imaging, especially to increase the detection sensitivity for small metastatic lesions, a major clinical challenge during the initial staging of RCC.


Assuntos
Transporte Biológico/fisiologia , Colina/análogos & derivados , Neoplasias Renais/metabolismo , Rim/metabolismo , Transportador 2 de Cátion Orgânico/metabolismo , Carcinoma de Células Renais/metabolismo , Linhagem Celular , Colina/metabolismo , Células HEK293 , Humanos , Cinética , RNA Mensageiro/metabolismo
20.
Annu Rev Physiol ; 76: 251-74, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24512081

RESUMO

The properties of intestinal folate absorption were documented decades ago. However, it was only recently that the proton-coupled folate transporter (PCFT) was identified and its critical role in folate transport across the apical brush-border membrane of the proximal small intestine established by the loss-of-function mutations identified in the PCFT gene in subjects with hereditary folate malabsorption and, more recently, by the Pcft-null mouse. This article reviews the current understanding of the properties of PCFT-mediated transport and how they differ from those of the reduced folate carrier. Other processes that contribute to the transport of folates across the enterocyte, along with the contribution of the enterohepatic circulation, are considered. Important unresolved issues are addressed, including the mechanism of intestinal folate absorption in the absence of PCFT and regulation of PCFT gene expression. The impact of a variety of ions, organic molecules, and drugs on PCFT-mediated folate transport is described.


Assuntos
Ácido Fólico/metabolismo , Absorção Intestinal/fisiologia , Animais , Circulação Êntero-Hepática/fisiologia , Deficiência de Ácido Fólico/genética , Deficiência de Ácido Fólico/metabolismo , Humanos , Absorção Intestinal/genética , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Síndromes de Malabsorção/genética , Síndromes de Malabsorção/metabolismo , Camundongos , Transportador de Folato Acoplado a Próton/genética , Transportador de Folato Acoplado a Próton/metabolismo
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