RESUMO
The objective of this study was to compare the ratio of renal oxygen availability (RO2) to glomerular filtration rate (GFR), a measure of relative renal hypoxia, in adolescents with and without type 1 diabetes (T1D) and relate the ratio to albuminuria, renal plasma flow (RPF), fat mass, and insulin sensitivity (M/I). RO2 was estimated by blood oxygen level-dependent MRI; fat mass was estimated by DXA; GFR and RPF were estimated by iohexol and p-aminohippurate clearance; albuminuria was estimated by urine albumin-to-creatinine ratio (UACR); and M/I was estimated from steady-state glucose infusion rate/insulin (mg/kg/min) by hyperglycemic clamp in 50 adolescents with T1D (age 16.1 ± 3.0 years, HbA1c 8.6 ± 1.2%) and 20 control patients of similar BMI (age 16.1 ± 2.9 years, HbA1c 5.2 ± 0.2%). The RO2:GFR (ms/mL/min) was calculated as RO2 (T2*, ms) divided by GFR (mL/min). Whole-kidney RO2:GFR was 25% lower in adolescents with T1D versus control patients (P < 0.0001). In adolescents with T1D, lower whole-kidney RO2:GFR was associated with higher UACR (r = -0.31, P = 0.03), RPF (r = -0.52, P = 0.0009), and fat mass (r = -0.33, P = 0.02). Lower medullary RO2:GFR was associated with lower M/I (r = 0.31, P = 0.03). In conclusion, adolescents with T1D exhibited relative renal hypoxia that was associated with albuminuria and with increased RPF, fat mass, and insulin resistance. These data suggest a potential role of renal hypoxia in the development of diabetic kidney disease.
Assuntos
Diabetes Mellitus Tipo 1/complicações , Hipóxia , Oxigênio/metabolismo , Adiposidade , Adolescente , Composição Corporal , Criança , Diabetes Mellitus Tipo 1/metabolismo , Nefropatias Diabéticas , Feminino , Furosemida , Taxa de Filtração Glomerular , Técnica Clamp de Glucose , Hemoglobinas Glicadas/genética , Hemoglobinas Glicadas/metabolismo , Humanos , Insulina , Iohexol/farmacologia , Masculino , Adulto Jovem , Ácido p-Aminoipúrico/farmacologiaRESUMO
Lansoprazole, a proton pump inhibitor, potently inhibits human organic anion transporter, hOAT3 (SLC22A8). Lansoprazole has an asymmetric atom in its structure and is clinically administered as a racemic mixture of (R)-and (S)-enantiomers. However, little is known about the stereoselective inhibitory potencies of lansoprazole against hOAT3 and its homolog, hOAT1. In the present study, the stereoselective inhibitory effect of lansoprazole was evaluated using hOAT1-and hOAT3-expressing cultured cells. hOAT1 and hOAT3 transported [14C]p-aminohippurate and [3H]estrone-3-sulfate (ES) with Michaelis-Menten constants of 29.8 ± 4.0 and 30.1 ± 9.0 µmol/L respectively. Lansoprazole enantiomers inhibited hOAT1- and hOAT3-mediated transport of each substrate in a concentration-dependent manner. The IC50 value of (S)-lansoprazole against hOAT3-mediated transport of [3H]ES (0.61 ± 0.08 µmol/L) was significantly lower than that of (R)-lansoprazole (1.75 ± 0.31 µmol/L). In contrast, stereoselectivity was not demonstrated for the inhibition of hOAT1. Furthermore, (S)-lansoprazole inhibited hOAT3-mediated transport of pemetrexed and methotrexate (hOAT3 substrates) more strongly than the corresponding (R)-lansoprazole. This study is the first to demonstrate that the stereoselective inhibitory potency of (S)-lansoprazole against hOAT3 is greater than that of (R)-lansoprazole. The present findings provide novel information about the drug interactions associated with lansoprazole.
Assuntos
Lansoprazol/química , Lansoprazol/farmacologia , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Inibidores da Bomba de Prótons/química , Inibidores da Bomba de Prótons/farmacologia , Interações Medicamentosas , Estrona/análogos & derivados , Estrona/farmacologia , Células HEK293 , Humanos , Rim/metabolismo , Metotrexato/farmacologia , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Pemetrexede/farmacologia , Probenecid/farmacologia , Estereoisomerismo , Ácido p-Aminoipúrico/farmacologiaRESUMO
Probe substrates for, and inhibitors of, specific transporters are desired to evaluate quantitatively the in vivo functions of transporters in humans. Based on published data, adefovir and benzylpenicillin were selected as organic anion transporter (OAT) 1- and OAT3-selective probe substrates, respectively. In human kidney slices, probenecid potently inhibited the uptake of both adefovir and benzylpenicillin with inhibition constant (Ki) values of 18.6±5.1 and 12.6±4.2µM, respectively, whereas p-aminohippurate (PAH) preferentially inhibited adefovir uptake. A clinical drug-interaction study involving healthy subjects was performed to investigate the dose-dependent inhibition potencies of probenecid and PAH on the renal clearance of the probe substrates. Adefovir or benzylpenicillin was coadministered with different oral doses of probenecid (500, 750, or 1500mg) or intravenous PAH infusion rates (70, 120, or 210mg/min/person) to the same subject using a crossover design. The renal clearance of adefovir was reduced by 45% and 46% in the subjects treated with the maximum dose of probenecid and PAH, respectively, which was in accordance with the results of in vitro inhibition study. On the other hand, renal clearance of benzylpenicillin was reduced by 78% in the subjects treated with the maximum dose of probenecid (1500mg), which could be explained by its in vitro Ki values. However, PAH unexpectedly increased the renal clearance of benzylpenicillin by 47%. These results suggest that adefovir and benzylpenicillin can be used as probe drugs for OAT1 and OAT3, respectively, and that PAH can be used to investigate the role of OAT1 in the urinary excretion of drugs in humans, whereas it may modulate other transport processes in the kidney.
Assuntos
Adenina/análogos & derivados , Rim/efeitos dos fármacos , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Organofosfonatos/farmacocinética , Penicilina G/farmacocinética , Probenecid/farmacologia , Ácido p-Aminoipúrico/farmacologia , Adenina/sangue , Adenina/farmacocinética , Adulto , Interações Medicamentosas , Células HEK293 , Humanos , Técnicas In Vitro , Rim/metabolismo , Masculino , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Organofosfonatos/sangue , Penicilina G/sangue , Adulto JovemRESUMO
Aging and abnormal aldosterone regulation are both associated with vascular disease. We hypothesized that aldosterone dysregulation influences the age-related risk of renal vascular and cardiovascular disease. We conducted an analysis of 562 subjects who underwent detailed investigations under conditions of liberal and restricted dietary sodium intake (1124 visits) in the General Clinical Research Center. Aldosterone regulation was characterized by the ratio of maximal suppression to stimulation (supine serum aldosterone on a liberal sodium diet divided by the same measure on a restricted sodium diet). We previously demonstrated that higher levels of this Sodium-modulated Aldosterone Suppression-Stimulation Index (SASSI) indicate greater aldosterone dysregulation. Renal plasma flow (RPF) was determined via p-aminohippurate clearance to assess basal renal hemodynamics and the renal vascular responses to dietary sodium manipulation and angiotensin II infusion. Cardiovascular risk was calculated using the Framingham Risk Score. In univariate linear regression, older age (ß=-4.60; P<0.0001) and higher SASSI (ß=-58.63; P=0.001) predicted lower RPF and a blunted RPF response to sodium loading and angiotensin II infusion. We observed a continuous, independent, multivariate-adjusted interaction between age and SASSI, where the inverse relationship between SASSI and RPF was most apparent with older age (P<0.05). Higher SASSI and lower RPF independently predicted higher Framingham Risk Score (P<0.0001) and together displayed an additive effect. Aldosterone regulation and age may interact to mediate renal vascular disease. Our findings suggest that the combination of aldosterone dysregulation and renal vascular dysfunction could additively increase the risk of future cardiovascular outcomes; therefore, aldosterone dysregulation may represent a modifiable mechanism of age-related vascular disease.
Assuntos
Aldosterona/sangue , Doenças Cardiovasculares/fisiopatologia , Rim/fisiopatologia , Fluxo Plasmático Renal/fisiologia , Fatores Etários , Angiotensina II/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/diagnóstico , Estudos Transversais , Dieta Hipossódica , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Humanos , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Fluxo Plasmático Renal/efeitos dos fármacos , Fatores de Risco , Sódio na Dieta/administração & dosagem , Ácido p-Aminoipúrico/farmacologiaRESUMO
BACKGROUND: A good in vitro model should approximate an in vivo-like behavior as closely as possible in order to reflect most likely the in vivo situation. Regarding renal physiology of different species, humans are more closely related to pigs than to rodents, therefore primary porcine kidney cells (PKC) and their subsequent cell strain could be a valid alternative to primary human cells for renal in vitro toxicology. For this PKC must display inherent characteristics (e.g. structural organization) and functions (e.g. transepithelial transport) as observed under in vivo conditions within the respective part of the kidney. RESULTS: We carried out a comprehensive characterization of PKC and their subsequent cell strain, including morphology and growth as well as transporter expression and functionality. The data presented here demonstrate that PKC express various transporters including pMrp1 (abcc1), pMrp2 (abcc2), pOat1 (slc22a6) and pOat3 (slc22a8), whereas pMdr1 (abcb1) and pOatp1a2 (slco1a2) mRNA could not be detected in either the PKCs or in the porcine cortical tissue. Functionality of the transporters was demonstrated by determining the specific PAH transport kinetics. CONCLUSIONS: On the basis of the presented results it can be concluded that PKC and to some extent their subsequent cell strain represent a valuable model for in vitro toxicology, which might be used as an alternative to human primary cells.
Assuntos
Citotoxinas/farmacologia , Células Epiteliais/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Ácido p-Aminoipúrico/farmacologia , Animais , Transporte Biológico , Biomarcadores/metabolismo , Radioisótopos de Carbono , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Expressão Gênica , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Cinética , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Proteína 2 Associada à Farmacorresistência Múltipla , Cultura Primária de Células , SuínosRESUMO
STUDY DESIGN: Laboratory investigation in rats submitted to experimental spinal cord injury (SCI). OBJECTIVE: To characterize changes in renal function during acute SCI. METHODS: Sprague Dawley rats were subjected to severe spinal cord contusion at T8 level or to laminectomy as control. Twenty-four hours after spine surgery, clearance assessments of a single dose of iohexol (120 mg kg(-1)) or of p-aminohippuric acid (PAH, 100 mg kg(-1)) were used to evaluate glomerular filtration rate (GFR) and tubular secretion (TS), respectively. Blood sampling was used to determine concentrations of both compounds by high-performance liquid chromatography for pharmacokinetic measurements. RESULTS: Iohexol clearance decreased significantly after injury, which resulted in increased concentrations and half-life of iohexol in blood; PAH clearance remained unchanged. CONCLUSION: GFR but not TS is altered during spinal shock. These observations should be of interest to professionals caring for early cord-injured patients, in order to prevent toxicity and therapeutic failure when administering drugs eliminated by the kidney.
Assuntos
Taxa de Filtração Glomerular , Iohexol/farmacocinética , Rim/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Ácido p-Aminoipúrico/farmacologia , Doença Aguda , Animais , Feminino , Taxa de Depuração Metabólica , Ratos , Ratos Sprague-DawleyRESUMO
INTRODUCTION: Renal uptake of Tc-99m-MG3 involves organic anion transporter (OAT). Treatment with drugs showing OAT affinity might interfere with renal uptake of Tc-99m-MAG3, leading to misinterpretation in Tc-99m-MAG3. This study was conducted to discuss a possible drug interference with Tc-99m-MAG3 diagnosis on OAT sites. METHODS: Renal uptake and plasma clearance of Tc-99m-MAG3 were analyzed in healthy volunteers under control and OAT1 and OAT3 related drug treatment conditions. An in vitro uptake study using OAT1 or OAT3 expressing cells was also conducted. RESULTS: Both PAH and probenecid treatment induced delays in Tc-99m-MAG3 clearance from blood, and reductions in the renal uptake clearance. As a result, the normalized effective renal plasma flow estimated from Tc-99m-MAG3 clearance was significantly underestimated, whereas the glomerular filtration rate estimated from plasma creatinine levels was unchanged. The transport activity of Tc-99m-MAG3 was higher in OAT1-expressing cells than in OAT3-expressing cells. CONCLUSION: Drugs with OAT1 affinity affect the renal uptake of Tc-99m-MAG3 and blood clearance. This might cause misinterpretation of functional diagnosis of the kidney using Tc-99m-MAG3.
Assuntos
Testes de Função Renal , Rim/efeitos dos fármacos , Rim/fisiologia , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Probenecid/farmacologia , Tecnécio Tc 99m Mertiatida/metabolismo , Ácido p-Aminoipúrico/farmacologia , Adulto , Transporte Biológico/efeitos dos fármacos , Estudos Cross-Over , Descoberta de Drogas , Taxa de Filtração Glomerular/efeitos dos fármacos , Voluntários Saudáveis , Humanos , Rim/metabolismo , Masculino , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Probenecid/metabolismo , Probenecid/uso terapêutico , Ligação Proteica , Adulto Jovem , Ácido p-Aminoipúrico/metabolismo , Ácido p-Aminoipúrico/uso terapêuticoRESUMO
We investigated transport of the organic anion Chlorophenol Red (CPR) in the tardigrade Halobiotus crispae using a new method for quantifying non-fluorescent dyes. We compared the results acquired from the tardigrade with CPR transport data obtained from Malpighian tubules of the desert locust Schistocerca gregaria. CPR accumulated in the midgut lumen of H. crispae, indicating that organic anion transport takes place here. Our results show that CPR transport is inhibited by the mitochondrial un-coupler DNP (1 mmol l(-1); 81% reduction), the Na(+)/K(+)-ATPase inhibitor ouabain (10 mmol l(-1); 21% reduction) and the vacuolar H(+)-ATPase inhibitor bafilomycin (5 µmol l(-1); 21% reduction), and by the organic anions PAH (10 mmol l(-1); 44% reduction) and probenecid (10 mmol l(-1); 61% reduction, concentration-dependent inhibition). Transport by locust Malpighian tubules exhibits a similar pharmacological profile, albeit with markedly higher concentrations of CPR being reached in S. gregaria. Immunolocalization of the Na(+)/K(+)-ATPase α-subunit in S. gregaria revealed that this transporter is abundantly expressed and localized to the basal cell membranes. Immunolocalization data could not be obtained from H. crispae. Our results indicate that organic anion secretion by the tardigrade midgut is transporter mediated with likely candidates for the basolateral entry step being members of the Oat and/or Oatp transporter families. From our results, we cautiously suggest that apical H(+) and possibly basal Na(+)/K(+) pumps provide the driving force for the transport; the exact coupling between electrochemical gradients generated by the pumps and transport of ions, as well as the nature of the apical exit step, are unknown. This study is, to our knowledge, the first to show active epithelial transport in tardigrades.
Assuntos
Transporte Biológico Ativo/fisiologia , Gafanhotos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Tardígrados/metabolismo , 2,4-Dinitrofenol/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Transporte de Íons , Macrolídeos/farmacologia , Túbulos de Malpighi/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/metabolismo , Fenolsulfonaftaleína/análogos & derivados , Fenolsulfonaftaleína/metabolismo , Probenecid/farmacologia , Ácido p-Aminoipúrico/farmacologiaRESUMO
Mechanisms of transcellular transport of 4-chloro-2-methylphenoxyacetic acid (MCPA) across the small intestine were investigated using Caco-2 cells cultured on permeable membranes. The cell monolayers were incubated with MCPA, either from apical side at pH 6.0 or 7.4, or basolateral side at pH 7.4. The accumulation and apical-to-basolateral transport of MCPA were markedly stimulated by the acidic pH on the apical side (inwardly directed H(+) gradient), dependent on metabolic energy and inhibited by co-incubation with acetic acid or benzoic acid. Without the H(+) gradient, on the other hand, the basolateral-to-apical transport of MCPA (secretory transport) was higher than the apical-to-basolateral transport (absorptive transport), although the secretory transport of MCPA was markedly lower than the absorptive transport under the H(+) gradient. Co-incubation of MCPA with probenecid from the basolateral side significantly inhibited the accumulation and transport of MCPA, whereas co-incubation with p-aminohippuric acid did not. These results suggest that the absorptive transport of MCPA is mediated by H(+)-linked monocarboxylic acid transporters expressed on the apical membranes, while secretory transport is mediated by a probenecid-sensitive transporter expressed on the basolateral membranes of Caco-2 cell monolayers.
Assuntos
Ácido 2-Metil-4-clorofenoxiacético/metabolismo , Absorção Intestinal , Intestino Delgado/metabolismo , Transporte Biológico , Células CACO-2 , Humanos , Concentração de Íons de Hidrogênio , Absorção Intestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Transportadores de Ácidos Monocarboxílicos/efeitos dos fármacos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Permeabilidade , Probenecid/farmacologia , Temperatura , Fatores de Tempo , Ácido p-Aminoipúrico/farmacologiaRESUMO
SCOPE: Hydroxycinnamic acids are abundant antioxidants in our diet. In humans, hydroxycinnamic acids are metabolized to form sulfates and glucuronides, with the majority recovered in urine. METHODS AND RESULTS: We assessed the potential roles of organic anion transporters (OATs) and ATP-binding cassette (ABC) transporters in the renal uptake and efflux of hydroxycinnamic acid conjugates. Uptake studies using OAT1 (SLC22A6)-, OAT2 (SLC22A7)-, and OAT3 (SLC22A8)-expressing 293H embryonic kidney cells showed that OAT1 and OAT3, but not OAT2, accepted hydroxycinnamic acid conjugates as substrates. OAT1 and OAT3 mediated the basolateral uptake of hydroxycinnamic acid sulfates and glucuronide conjugates, respectively. Hydroxycinnamic acid sulfates are substrates of OAT4 and were capable of trans-stimulating 5-carboxyfluorescein uptake mediated by OAT4. On the other hand, hydroxycinnamic acid conjugates are not substrates for the ABC transporters, multidrug resistance protein 2 (MRP2/ABCC2) or breast cancer resistance protein (BCRP/ABCG2), demonstrated by the inability to alter ATPase activity. Cis-inhibition studies with OATs and MRPs revealed that hydroxycinnamic acid conjugates have limited impact on the transport of model substrates significantly at physiological concentrations. CONCLUSION: Concerted action of OAT1, OAT3, and OAT4 is involved in the elimination of hydroxycinnamic acid conjugates into urine, whereas MRP2 and breast cancer resistance protein are not involved in the disposition of these conjugates.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácidos Cumáricos/metabolismo , Ácidos Cumáricos/farmacocinética , Rim/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Fluoresceínas/farmacocinética , Glucuronídeos/farmacocinética , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Sulfatos/farmacocinética , Ácido p-Aminoipúrico/farmacologiaRESUMO
Vitamin D regulates the renin-angiotensin system (RAS) in experimental animals, but corresponding human data are limited. We examined the relation between plasma 25-hydroxyvitamin D and elements of the RAS in 184 normotensive individuals in high sodium balance; these included circulating levels of plasma renin activity and angiotensin II (Ang II) and the renal plasma flow response to infused Ang II, which is an indirect measure of the intrinsic RAS activity in the kidney. Compared with individuals with sufficient 25-hydroxyvitamin D levels (> or = 30.0 ng/mL), those with insufficiency (15.0 to 29.9 ng/mL) and deficiency (<15.0 ng/mL) had higher circulating Ang II levels (P for trend=0.03). Moreover, those with vitamin D deficiency had significantly blunted renal plasma flow responses to infused Ang II (mean decrease of 115 mL/min per 1.73 m(2) in renal plasma flow versus 145 mL/min per 1.73 m(2) among those with sufficient vitamin D levels; P for trend=0.009). Although plasma renin activity was higher among individuals with insufficient levels of vitamin D, the result was not statistically significant. These data suggest that low plasma 25-hydroxyvitamin D levels may result in upregulation of the RAS in otherwise healthy humans.
Assuntos
Angiotensina II/farmacologia , Sistema Renina-Angiotensina/fisiologia , Sódio na Dieta/farmacologia , Vitamina D/análogos & derivados , Adulto , Angiotensina II/sangue , Angiotensina II/efeitos dos fármacos , População Negra , Feminino , Homeostase , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Valores de Referência , Circulação Renal/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Sódio/sangue , Vitamina D/sangue , Vitamina D/farmacologia , População Branca , Ácido p-Aminoipúrico/administração & dosagem , Ácido p-Aminoipúrico/farmacologiaRESUMO
The purpose of this study was to thoroughly characterize the efflux transporters involved in the intestinal permeability of the oral microtubule polymerization inhibitor colchicine and to evaluate the role of these transporters in limiting its oral absorption. The effects of P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) inhibitors on colchicine bidirectional permeability were studied across Caco-2 cell monolayers, inhibiting one versus multiple transporters simultaneously. Colchicine permeability was then investigated in different regions of the rat small intestine by in situ single-pass perfusion. Correlation with the P-gp/MRP2 expression level throughout different intestinal segments was investigated by immunoblotting. P-gp inhibitors [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), verapamil, and quinidine], and MRP2 inhibitors [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), indomethacin, and p-aminohippuric acid (p-AH)] significantly increased apical (AP)-basolateral (BL) and decreased BL-AP Caco-2 transport in a concentration-dependent manner. No effect was obtained by the BCRP inhibitors fumitremorgin C (FTC) and pantoprazole. P-gp/MRP2 inhibitors combinations greatly reduced colchicine mucosal secretion, including complete abolishment of efflux (GF120918/MK571). Colchicine displayed low (versus metoprolol) and constant permeability along the rat small-intestine. GF120918 significantly increased colchicine permeability in the ileum with no effect in the jejunum, whereas MK571 augmented jejunal permeability without changing the ileal transport. The GF120918/MK571 combination caused an effect similar to that of MK571 alone in the jejunum and to that of GF120918 alone in the ileum. P-gp expression followed a gradient increasing from proximal to distal segments, whereas MRP2 decreased from proximal to distal small intestinal regions. Overall, it was revealed that the combined effect of P-gp and MRP2, but not BCRP, dominates colchicine transepithelial transport, leading to complete coverage of the entire small intestine, and makes the efflux transport dominate the intestinal permeability process.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Transporte Biológico/fisiologia , Colchicina/farmacocinética , Absorção Intestinal/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Acridinas/farmacologia , Administração Oral , Animais , Células CACO-2 , Interações Medicamentosas , Epitélio/efeitos dos fármacos , Epitélio/fisiologia , Interações Alimento-Droga , Humanos , Íleo/efeitos dos fármacos , Íleo/patologia , Indóis/administração & dosagem , Indóis/farmacologia , Absorção Intestinal/fisiologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Intestino Delgado/fisiologia , Jejuno/efeitos dos fármacos , Jejuno/patologia , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Oxotremorina/análogos & derivados , Oxotremorina/farmacocinética , Oxotremorina/farmacologia , Permeabilidade , Ratos , Ratos Wistar , Tetra-Hidroisoquinolinas/farmacologia , Verapamil/administração & dosagem , Verapamil/farmacologia , Ácido p-Aminoipúrico/farmacocinética , Ácido p-Aminoipúrico/farmacologiaRESUMO
The effects of folic acid-induced acute renal failure on the renal excretion of belotecan were investigated in rats after intravenous administration. Both glomeruli and renal tubules were seriously damaged by folic acid-induced acute renal failure. The renal excretion clearance, CLr, of belotecan was significantly decreased by folic acid-induced acute renal failure. Furthermore, glomerular filtration rate and secretion clearance of the drug were dramatically decreased by folic acid-induced acute renal failure. In vivo renal uptake of belotecan was inhibited by p-aminohippurate, whereas renal excretion was inhibited by GF120918, but not by verapamil and bromosulphalein. This indicates that Oat1/3 and Bcrp are involved in the renal uptake and urinary excretion of belotecan, respectively. Both mRNA and protein levels of Oat1, Oat3 and Bcrp were significantly decreased in folic acid-induced acute renal failure rats. Based on the finding that belotecan is a substrate of OAT1 but not of OAT3, the decrease in CLr of belotecan in folic acid-induced acute renal failure could, therefore, mainly be attributed to the down-regulation of Oat1 and Bcrp, in addition to the decrease in glomerular filtration rate.
Assuntos
Injúria Renal Aguda/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/urina , Camptotecina/análogos & derivados , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Acridinas/farmacologia , Injúria Renal Aguda/induzido quimicamente , Animais , Antineoplásicos/química , Bloqueadores dos Canais de Cálcio/farmacologia , Camptotecina/química , Camptotecina/farmacocinética , Camptotecina/urina , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Ácido Fólico/farmacologia , Taxa de Filtração Glomerular/efeitos dos fármacos , Indicadores e Reagentes/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Ratos , Ratos Sprague-Dawley , Tetra-Hidroisoquinolinas/farmacologia , Verapamil/farmacologia , Complexo Vitamínico B/farmacologia , Ácido p-Aminoipúrico/farmacologiaRESUMO
Because citrulline plasma concentration is elevated in kidney failure, citrulline could be a biomarker of renal insufficiency, although the mechanism regulating its disposition in the kidney has not been clarified. In rat kidney slices, citrulline uptake was apparently Na(+) dependent, saturable with K(m) 556 microM, and significantly inhibited by anionic (PAH) and cationic (TEA) compounds, but not by probenecid at 1 mM. Preincubation of kidney slices with glutarate increased citrulline uptake, while such an increase was not observed after preincubation of the slices in Na(+)-free buffer. This result suggested that a sodium-dependent dicarboxylate cotransporter is involved in citrulline uptake by rat kidney slices. In studies using transporter-overexpressing cells, human organic anion transporter 1 (OAT1) and rat Oat1 exhibited citrulline transport activity with K(m) values of 238 and 373 microM, respectively, while other OATs and organic cation transporters (OCTs) did not transport citrulline. Based on the relative activity factor method, the contribution of rat Oat1 to the overall uptake of citrulline in rat kidney slices was approximately 70%. Moreover, the interaction among citrulline, PAH, and probenecid uptakes via rat Oat1 suggested that there are multiple functional sites on Oat1 and that the citrulline site may be distinct from the PAH and probenecid site. Thus OAT1/Oat1 appears to be one of the major contributors to renal basolateral uptake of citrulline, and impaired activities of these transporters may contribute substantially to the increase in plasma citrulline in renal failure. Accordingly, citrulline may be useful for diagnosis of kidney function as is creatinine.
Assuntos
Citrulina/metabolismo , Rim/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/metabolismo , Cefazolina/farmacologia , Linhagem Celular , Glutaratos/farmacologia , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Masculino , Modelos Animais , Fator 1 de Transcrição de Octâmero/metabolismo , Fator 2 de Transcrição de Octâmero/metabolismo , Probenecid/farmacologia , Ratos , Ratos Wistar , Ácido p-Aminoipúrico/farmacologiaRESUMO
1. The purpose of this study was to investigate the involvement of rat Mrp2 and human MRP2 in benzylpenicillin transport using canalicular liver plasma membrane (cLPM) vesicles isolated from Sprague-Dawley or Easai hyperbilirubinemic (EHBR) rats, and MDCKII cells overexpressing MRP2. 2. The adenosine triphosphate (ATP)-dependent uptake of benzylpenicillin and oestradiol-17beta-D-glucuronide (E(2)17betaG), a representative substrate for Mrp2, into EHBR-cLPM vesicles was decreased relative to that seen with control-cLPM vesicles, which may reflect the absence of Mrp2 in the EHBR. The ATP-dependent uptake of taurocholate, which is not a substrate for Mrp2, was similar in both control and EHBR-cLPM vesicles. The concentration dependence of ATP-dependent benzylpenicillin uptake was reflected in a K(m) of 44.0 microM and a V(max) of 508.4 pmol mg(-1) min(-1). Additional inhibition studies using E(2)17betaG and methotrexate as representative substrates for Mrp2/MRP2 demonstrated the involvement of rat Mrp2, but not human MRP2, in benzylpenicillin efflux. Benzylpenicillin appears not to be a substrate for or inhibitor of other human efflux transporters such as MDR1, MRP1, MRP3, or BCRP. 3. In conclusion, rat Mrp2, but not human MRP2, plays an important role in ATP-dependent benzylpenicillin uptake in the bile canalicular membrane, which may explain why biliary excretion of benzylpenicillin is high in the rat but negligible in humans.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Antibacterianos/farmacocinética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Penicilina G/farmacocinética , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Ciclosporina/farmacologia , Cães , Estradiol/análogos & derivados , Estradiol/farmacocinética , Citometria de Fluxo , Humanos , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Ratos , Ratos Endogâmicos , Ratos Sprague-Dawley , Rodaminas/metabolismo , Especificidade da Espécie , Especificidade por Substrato , Ácido Taurocólico/farmacocinética , Vesículas Transportadoras/metabolismo , Ácido p-Aminoipúrico/farmacologiaRESUMO
Choline is essential for synthesis of the major membrane phospholipid phosphatidylcholine. Moreover, it serves as a precursor for synthesis of the neurotransmitter acetylcholine (ACh). Keratinocytes of the epidermis synthesize and release ACh. The uptake of choline is the rate-limiting step in both ACh synthesis and choline phospholipid metabolism, and it is a prerequisite for keratinocyte proliferation. However, the nature of the choline transport system in keratinocytes is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake into cultured human keratinocytes. Choline uptake into keratinocytes was independent of extracellular Na(+), saturable, and mediated by a single transport system with an apparent Michaelis-Menten constant of 12.3 muM. Choline uptake was reduced when the keratinocyte membrane potential was depolarized by high K(+). These results provide evidence that the choline transport activity is potential-sensitive. Various organic cations inhibit the choline transport system. RT-PCR demonstrated that keratinocytes expressed mRNA for choline transporter-like protein 1 (CTL1), mainly the CTL1a subtype. The present biochemical and pharmacological data suggest that CTL1a is functionally expressed in human keratinocytes and is responsible for the uptake of choline and organic cations in these cells.
Assuntos
Perfilação da Expressão Gênica , Queratinócitos/metabolismo , Simportadores/genética , Simportadores/fisiologia , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Anisóis/farmacologia , Antígenos CD/genética , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Células Cultivadas , Colina/metabolismo , Colina/farmacocinética , Clonidina/farmacologia , Desipramina/farmacologia , Difenidramina/farmacologia , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Fator 3 de Transcrição de Octâmero/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Quinidina/farmacologia , Quinina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraetilamônio/farmacologia , Fatores de Tempo , Trítio , Ácido p-Aminoipúrico/farmacologiaRESUMO
Confocal microscopy and image analysis were used to compare driving forces, specificity, and regulation of transport of the fluorescent organic anion, Texas Red (sulforhodamine 101 free acid; TR), in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark (Squalus acanthias). CP from both species exhibited concentrative, specific, and metabolism-dependent TR transport from bath to subepithelial/vascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady-state TR accumulation in subepithelial/vascular spaces was reduced by Na(+)-replacement, but was not affected by a 10-fold increase in buffer K(+). In shark CP, Na(+)-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high-K(+) medium. In both species, steady-state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 was not involved. Several organic anions reduced cellular and subepithelial/vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate, and the Mrp1 inhibitor MK571. In rat CP, TR accumulation in subepithelial/vascular spaces increased with PKA activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR but do so using different basic mechanisms that respond to different regulatory signals.
Assuntos
Plexo Corióideo/metabolismo , Transportadores de Ânions Orgânicos/fisiologia , Squalus acanthias/metabolismo , Xantenos/farmacocinética , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Estrona/análogos & derivados , Estrona/farmacologia , Feminino , Técnicas In Vitro , Cinética , Leucotrieno C4/farmacologia , Masculino , Meglumina/farmacologia , Metotrexato/farmacologia , Modelos Biológicos , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Potássio/farmacologia , Propionatos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Cianeto de Sódio/farmacologia , Ácido Taurocólico/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Xantenos/metabolismo , Ácido p-Aminoipúrico/farmacologiaRESUMO
This study aimed to establish the relationship between ME intake and energy and nutrient absorption across the portal-drained viscera (PDV) of forage-fed beef steers. Eight Angus (328 +/- 40 kg of BW) steers were surgically fitted with portal, mesenteric arterial, and mesenteric venous catheters, and were fed alfalfa cubes in a replicated 4 x 4 Latin square design with 4 levels of energy intake between 1 and 2 times maintenance energy requirements. On d 28 of each experimental period, p-aminohippuric acid was infused to measure blood and plasma flow across the PDV, and blood samples (1 every hour, for 6 h) were collected simultaneously from arterial and venous catheters for net absorption measurements. Oxygen utilization, and therefore energy utilization, increased (P < 0.05) linearly in relation to ME intake. Glucose net uptake was unaffected, but lactate net release increased linearly in response to ME intake (P < 0.05). Net absorption of all AA except tryptophan, glutamate, and glutamine increased linearly with ME intake (P < 0.05). The constant net absorption of glutamate and glutamine indicated increased net utilization of these AA when dietary supply was increased. These data provide quantitative measures of the PDV effects on energy and AA availability for productive tissues, and suggest that the greater net utilization of some AA when ME intake is increased could relate to their catabolism for energy production. Prediction estimates of small intestinal AA absorption, based on the Cornell Net Carbohydrate and Protein System (CNCPS), exceeded observed net AA PDV absorption. Mean bias represented the greatest proportion (87 to 96%) of the deviation between individual AA absorption and observed net AA PDV absorption, suggesting that the CNCPS model may be used to predict AA net absorption when factors describing AA utilization by the PDV are applied to model predictions.