RESUMEN
Proximal tubules in the kidney play a crucial role in reabsorbing and eliminating substrates from the body into the urine, leading to high local concentrations of xenobiotics. This makes the proximal tubule a major target for drug toxicity that needs to be evaluated during the drug development process. Here, we describe an advanced in vitro model consisting of fully polarized renal proximal tubular epithelial cells cultured in a microfluidic system. Up to 40 leak-tight tubules were cultured on this platform that provides access to the basolateral as well as the apical side of the epithelial cells. Exposure to the nephrotoxicant cisplatin caused a dose-dependent disruption of the epithelial barrier, a decrease in viability, an increase in effluent LDH activity, and changes in expression of tight-junction marker zona-occludence 1, actin, and DNA-damage marker H2A.X, as detected by immunostaining. Activity and inhibition of the efflux pumps P-glycoprotein (P-gp) and multidrug resistance protein (MRP) were demonstrated using fluorescence-based transporter assays. In addition, the transepithelial transport function from the basolateral to the apical side of the proximal tubule was studied. The apparent permeability of the fluorescent P-gp substrate rhodamine 123 was decreased by 35% by co-incubation with cyclosporin A. Furthermore, the activity of the glucose transporter SGLT2 was demonstrated using the fluorescent glucose analog 6-NBDG which was sensitive to inhibition by phlorizin. Our results demonstrate that we developed a functional 3D perfused proximal tubule model with advanced renal epithelial characteristics that can be used for drug screening studies.
Asunto(s)
Técnicas de Cultivo de Célula , Células Epiteliales/efectos de los fármacos , Enfermedades Renales/inducido químicamente , Túbulos Renales Proximales/efectos de los fármacos , Moduladores del Transporte de Membrana/toxicidad , Proteínas de Transporte de Membrana/efectos de los fármacos , Perfusión , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transporte Biológico , Línea Celular , Polaridad Celular , Cisplatino/toxicidad , Ciclosporina/toxicidad , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Dispositivos Laboratorio en un Chip , Proteínas de Transporte de Membrana/metabolismo , Técnicas Analíticas Microfluídicas , Florizina/toxicidad , Transportador 2 de Sodio-Glucosa/efectos de los fármacos , Transportador 2 de Sodio-Glucosa/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2/toxicidad , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/patologíaRESUMEN
In this study, we aimed to investigate modulation of glucose uptake by the HTR-8/SVneo human first-trimester extravillous trophoblast cell line by a series of compounds and to study its consequences upon cell proliferation, viability and migration. We observed that uptake of (3)H-deoxy-d-glucose ((3)H-DG; 10 nM) was time-dependent, saturable, inhibited by cytochalasin B (50 and 100 µM), phloretin (0.5 mM) and phloridzin (1 mM), insulin-insensitive and sodium-independent. In the short term (30 min), neither 5-HT (100-1000 µM), melatonin (10 nM) nor the drugs of abuse ethanol (100 mM), nicotine (100 µM), cocaine (25 µM), amphetamine (10-25 µM) and 3,4-methylenedioxy-N-methamphetamine (10 µM) affected (3)H-DG uptake, while dexamethasone (100-1000 µM), fluoxetine (100-300 µM), quercetin, epigallocatechin-3-gallate (30-1000 µM), xanthohumol (XH) and resveratrol (1-500 µM) decreased it. XH was the most potent inhibitor [IC50 = 3.55 (1.37-9.20) µM] of (3)H-DG uptake, behaving as a non-competitive inhibitor of (3)H-DG uptake, both after short- and long-term (24 h) treatment. The effect of XH (5 µM; 24 h) upon (3)H-DG uptake involved mammalian target of rapamycin, tyrosine kinases and c-Jun N-terminal kinases intracellular pathways. Moreover, XH appeared to decrease cellular uptake of lactate due to inhibition of the monocarboxylate transporter 1. Additionally, XH (24 h; 5 µM) decreased cell viability, proliferation, culture growth and migration. The effects of XH upon cell viability and culture growth, but not the antimigratory effect, were mimicked by low extracellular glucose conditions and reversed by high extracellular glucose conditions. We thus suggest that XH, by inhibiting glucose cellular uptake and impairing HTR-8/SVneo cell viability and proliferation, may have a deleterious impact in the process of placentation.
Asunto(s)
Desoxiglucosa/metabolismo , Flavonoides/farmacología , Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Placentación/efectos de los fármacos , Propiofenonas/farmacología , Trofoblastos/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Transformada , Movimiento Celular/efectos de los fármacos , Citocalasina B/farmacología , Citocalasina B/toxicidad , Dexametasona/farmacología , Dexametasona/toxicidad , Femenino , Flavonoides/toxicidad , Glucosa/farmacología , Proteínas Facilitadoras del Transporte de la Glucosa/fisiología , Humanos , Drogas Ilícitas/farmacología , Drogas Ilícitas/toxicidad , Melatonina/farmacología , Melatonina/toxicidad , Floretina/farmacología , Floretina/toxicidad , Florizina/farmacología , Florizina/toxicidad , Polifenoles/farmacología , Polifenoles/toxicidad , Embarazo , Primer Trimestre del Embarazo , Propiofenonas/toxicidad , Proteínas Tirosina Quinasas/fisiología , Resveratrol , Transducción de Señal/efectos de los fármacos , Estilbenos/farmacología , Estilbenos/toxicidad , Serina-Treonina Quinasas TOR/fisiología , Trofoblastos/citologíaRESUMEN
The dihydrochalcone phlorizin is a natural product and dietary constituent found in a number of fruit trees. It has been used as a pharmaceutical and tool for physiology research for over 150 years. Phlorizin's principal pharmacological action is to produce renal glycosuria and block intestinal glucose absorption through inhibition of the sodium-glucose symporters located in the proximal renal tubule and mucosa of the small intestine. This review covers the role phlorizin has played in the history of diabetes mellitus and its use as an agent to understand fundamental concepts in renal physiology as well as summarizes the physiology of cellular glucose transport and the pathophysiology of renal glycosuria. It reviews the biology and pathobiology of glucose transporters and discusses the medical botany of phlorizin and the potential effects of plant flavonoids, such as phlorizin, on human metabolism. Lastly, it describes the clinical pharmacology and toxicology of phlorizin, including investigational uses of phlorizin and phlorizin analogs in the treatment of diabetes, obesity, and stress hyperglycemia.
Asunto(s)
Diabetes Mellitus/fisiopatología , Florizina/fisiología , Animales , Transporte Biológico , Glucosa/metabolismo , Glucosuria , Humanos , Florizina/farmacología , Florizina/toxicidadRESUMEN
Four Dorset wethers were studied in a Latin square design with 72-h periods to determine the metabolic adaptations that occur in support of increased glucose demand in ruminants. Wethers injected at 8-h intervals with excipient or a total of .5, 1.0, or 2.0 g/d of phlorizin excreted an average of 0, 72.7, 97.9, and 98.5 g/d of glucose into the urine, respectively. Both acute (2 to 24 h after the first injection) and chronic (8-h intervals from 8 to 72 h after the first injection) adaptations of plasma variables to phlorizin treatment were assessed. Concentrations of plasma glucose decreased linearly with increasing phlorizin dose during the 1st 24 h of treatment and tended to decrease linearly with phlorizin dose during 8 to 72 h of treatment. Urea N tended to increase linearly during 2 to 24 h and increased linearly during 8 to 72 h. Nonesterified fatty acids increased linearly with phlorizin injection during the entire treatment period. beta-Hydroxybutyrate increased quadratically with phlorizin injection during 2 to 24 h and tended to increase quadratically during 8 to 72 h. The ratio of insulin to glucagon tended to decrease linearly with phlorizin injection during the 1st 24 h but was unaffected from 8 to 72 h. Triiodothyronine, but not thyroxine, tended to decrease linearly with phlorizin injection during 8 to 72 h. Cortisol was not affected by treatment. Digestibilities of energy and N were not affected by treatment. Urinary energy excretion increased with phlorizin injection in proportion to the amounts of glucose excreted into the urine. These data indicate that phlorizin-treated wethers largely adapted to phlorizin treatment by 24 h after the first injection and are a suitable model for further investigations of hepatic adaptation to increased glucose demand in ruminants.
Asunto(s)
Glucosa/metabolismo , Glucosuria/veterinaria , Florizina/toxicidad , Ovinos/metabolismo , Ácido 3-Hidroxibutírico/sangre , Animales , Glucemia/metabolismo , Nitrógeno de la Urea Sanguínea , Modelos Animales de Enfermedad , Ácidos Grasos no Esterificados/sangre , Glucagón/sangre , Gluconeogénesis/efectos de los fármacos , Glucosuria/inducido químicamente , Glucosuria/metabolismo , Hidrocortisona/sangre , Insulina/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Florizina/administración & dosificación , Ovinos/orina , Enfermedades de las Ovejas/inducido químicamente , Enfermedades de las Ovejas/metabolismo , Tiroxina/sangre , Triyodotironina/sangreRESUMEN
This study utilized phloridzin (P1) and its aglucone phloretin (P2), two known inhibitors of glucose transmembrane transport, to inhibit tumor cell growth in vivo. The efficacy of hydrazine sulfate as an anticachexic agent was also evaluated. Utilizing the rat mammary adenocarcinoma and Fischer bladder cell carcinoma cell lines, it has been shown that the i.p. administration of P1 and P2 can produce significant differences in mean tumor diameters as compared to the untreated controls.