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BACKGROUND: Residential exposure to pesticides may occur via inhalation of airborne pesticides, direct skin contacts with pesticide-contaminated surfaces, and consumption of food containing pesticide residues. The aim was to study the association of dermal exposure to pesticides between the use and non-use periods, between farmer and non-farmer families and between dermal exposure and the excretion of metabolites from urine in residents living close to treated agricultural fields. METHODS: In total, 112 hand wipes and 206 spot urine samples were collected from 16 farmer and 38 non-farmer participants living within 50 m from an agricultural field in the Netherlands. The study took place from May 2016 to December 2017 during the use as well as the non-use periods of pesticides. Hand wipes were analysed for the parent compound and urines samples for the corresponding urinary metabolite of five applied pesticides: asulam, carbendazim (applied as thiophanate-methyl), chlorpropham, prochloraz and tebuconazole. Questionnaire data was used to study potential determinants of occurrence and levels of pesticides in hand wipes according to univariate and multivariate analysis. RESULTS: Carbendazim and tebuconazole concentrations in hand wipes were statistically significantly higher in the pesticide-use period compared to the non-use period. In addition, especially during the use periods, concentrations were statistically significantly higher in farmer families compared to non-farmer families. For asulam, chlorpropham and prochloraz, the frequency of non-detects was too high (57-85%) to be included in this analysis. The carbendazim contents in urine samples and hand wipes were correlated on the first and second day after taking the hand wipe, whereas chlorpropham was only observed to be related on the second day following the spray event. CONCLUSIONS: Concentrations in hand wipes were overall higher in pesticide use periods compared to non-use periods and higher in farmer families compared to non-farmer families. Only for carbendazim a strong correlation between concentrations in hand wipes and its main metabolite in urine was observed, indicating dermal exposure via contaminated indoor surfaces. We expect this to be related to the lower vapour pressure and longer environmental lifetime of carbendazim compared to the other pesticides studies.
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Residuos de Plaguicidas , Plaguicidas , Biomarcadores , Exposición a Riesgos Ambientales/análisis , Monitoreo del Ambiente , Mano , Humanos , Países Bajos , Plaguicidas/análisisRESUMEN
INTRODUCTION: An increasing number of women becomes pregnant while suffering from chronic kidney disease (CKD). As a result of decreased renal function, uremic solutes circulate at high levels in the maternal circulation. This study aimed to acquire more knowledge about the placental transfer of uremic solutes across the human placenta. METHODS: Placental transfer was studied in healthy term placentas, via the ex vivo dual-side human cotyledon perfusion technique (closed-closed set-up for both maternal and fetal circulations). Uremic solute concentrations in maternal and fetal perfusates were measured via LC-MS/MS over 180 min of perfusion. RESULTS: We found that the studied compounds demonstrated different degrees of placental transfer. Fetal-to-maternal perfusate ratios at t = 180 min were for anthranilic acid 1.00 ± 0.02, indole-3-acetic acid 0.47 ± 0.08, hippuric acid 0.36 ± 0.18, l-arabinitol 0.33 ± 0.04, indoxyl sulfate 0.33 ± 0.11, neopterin 0.28 ± 0.14 and kynurenic acid 0.13 ± 0.03. All uremic solutes studied also emerged in the perfusates when cotyledons were perfused in the absence of uremic solute concentrations added to the maternal reservoir. For kynurenin these concentrations were so high, it complicated the calculation of a transfer ratio for the exogenously administered compound. DISCUSSION: After 180 min of exposure the extent of placental transfer differs substantially for the solutes studied, reflecting different transfer rates. Future studies should investigate to what extent specific uremic solutes reach the fetal circulation in vivo and how they may interfere with organ function and development of the unborn child.
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Cotiledón/metabolismo , Placenta/metabolismo , Tóxinas Urémicas/metabolismo , Transporte Biológico , Cromatografía Liquida , Femenino , Humanos , Embarazo , Espectrometría de Masas en TándemRESUMEN
When drugs exert their effects in the brain, linear extrapolation of doses from adults could be harmful for children as the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) function is still immature. More specifically, age-related variation in membrane transporters may impact brain disposition. As human data on brain transporter expression is scarce, age dependent [gestational age (GA), postnatal age (PNA), and postmenstrual age (PMA)] variation in immunohistochemical localization and staining intensity of the ABC transporters P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins 1, 2, 4, and 5 (MRP1/2/4/5) was investigated. Post mortem brain cortical and ventricular tissue was derived from 23 fetuses (GA range 12.9-39 weeks), 17 neonates (GA range 24.6-41.3 weeks, PNA range 0.004-3.5 weeks), 8 children (PNA range 0.1-3 years), and 4 adults who died from a wide variety of underlying conditions. In brain cortical BBB, immunostaining increased with age for Pgp and BCRP, while in contrast, MRP1 and MRP2 staining intensity appeared higher in fetuses, neonates, and children, as compared to adults. BCSFB was positively stained for Pgp, MRP1, and MRP2 and appeared stable across age, while BCRP was not detected. MRP4 and MRP5 were not detected in BBB or BCSFB. In conclusion, human BBB and BCSFB ABC membrane transporters show brain location and transporter-specific maturation.
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Transportadoras de Casetes de Unión a ATP/biosíntesis , Barrera Hematoencefálica/metabolismo , Transportadoras de Casetes de Unión a ATP/análisis , Transportadoras de Casetes de Unión a ATP/líquido cefalorraquídeo , Adulto , Preescolar , Humanos , Inmunohistoquímica , LactanteRESUMEN
Tebuconazole (TEB) is a widely used triazole fungicide, but the toxicokinetics of its human metabolites are not fully described. For proper interpretation of biological monitoring data, knowledge on the metabolism and elimination of the compound is required. A human volunteer study was performed with the aim to describe the time courses of urinary excretion after controlled oral and dermal administration of TEB. Six healthy volunteers (three males and three females) received on separate occasions a single oral dose of 1.5 mg of TEB and a single dermal dose of 2.5 mg during 1 h. In addition to a pre-exposure urine sample, complete urine voids were collected over 48 h post-administration. The main metabolite hydroxy-tebuconazole (TEB-OH) was quantified in each urine sample. Peak excretion rates after oral and dermal administration were reached after 1.4 and 21 h, mean elimination half-lives were 7.8 and 16 h, and recoveries within 48 h were 38% and 1%, respectively. The time courses of excretion were compared to simulations with an established physiologically based toxicokinetic model for TEB that was extended with a parallel model for TEB-OH. Overall, TEB-OH was rapidly excreted into urine after oral exposure, and renal elimination was considerably slower after dermal exposure. Urinary time courses between individuals were similar. The model predictions were in good agreement with the observed time courses of excretion.
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Fungicidas Industriales , Modelos Biológicos , Triazoles , Administración Cutánea , Administración Oral , Adulto , Femenino , Fungicidas Industriales/administración & dosificación , Fungicidas Industriales/toxicidad , Fungicidas Industriales/orina , Voluntarios Sanos , Humanos , Masculino , Toxicocinética , Triazoles/administración & dosificación , Triazoles/toxicidad , Triazoles/orina , Adulto JovenRESUMEN
Young children differ from adults in their exposure and susceptibility to environmental chemicals (e.g. pesticides) because of various factors such as behavior, diet and physiology. Their heightened vulnerability to environmental stressors makes it important to obtain appropriate urine samples for exposure characterization. However, collecting urine from non-toilet-trained children has been shown to be methodologically and practically challenging. Four urine collection approaches were tested: a disposable diaper, a urine bag, a collection pad and the clean catch. The success rate and the user rating of each method was evaluated. The success rates were 67%, 21%, 17% and 4% for the disposable diaper, urine bag, collection pad and clean catch, respectively. The average user ratings on a 0-10 (0 = inconvenient, 10 = convenient) scale were 9.0, 4.7, 7.3 and 2.5, respectively. Subsequently, the best rated method, the disposable polyacrylate diaper was tested with hydroxy-tebuconazole as an exposure biomarker for the fungicide tebuconazole and creatinine for urine density adjustment. After LC-MS/MS analysis, the recoveries of hydroxy-tebuconazole in the range of 0.05-25 ng/mL were on average 106%, and for creatinine 87%. Precisions (relative standard deviation) were for both 3%. The overall procedure including collection and extraction was assessed, resulting in three out of seven positive samples. Based on this study, the disposable diaper is a suitable method for urine collection of non-toilet-trained children for biomonitoring of tebuconazole. This method can serve as a basis for extension to other substances of interest.
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Pañales Infantiles , Monitoreo del Ambiente/métodos , Contaminantes Ambientales/orina , Fungicidas Industriales/orina , Eliminación Renal , Control de Esfínteres , Triazoles/orina , Toma de Muestras de Orina , Biotransformación , Preescolar , Biomarcadores Ambientales , Exposición a Riesgos Ambientales/efectos adversos , Contaminantes Ambientales/efectos adversos , Femenino , Fungicidas Industriales/efectos adversos , Humanos , Hidroxilación , Lactante , Recién Nacido , Masculino , Proyectos Piloto , Reproducibilidad de los Resultados , Medición de Riesgo , Triazoles/efectos adversos , UrinálisisRESUMEN
BACKGROUND: Diclofenac increases the risk of anastomotic leakage, but the underlying mechanism is unknown. As diclofenac is excreted largely as biliary metabolites, the aim of this study was to determine the effect of these metabolites on intestinal anastomoses. METHODS: This was a randomized controlled blinded experiment using 210 male Wistar rats to assess the effect of 'diclofenac bile' on the anastomotic complication score, leak rate and anastomotic strength following oral and parenteral administration of diclofenac. Bile duct and duodenal catheterization techniques were used for diversion and replacement of bile, and biliary diclofenac metabolites were determined. RESULTS: Replacement of control bile with diclofenac bile resulted in higher anastomotic complication scores (P = 0·006) and leakage in five of 18 animals, compared with one of 18 controls (P = 0·089). In turn, following oral diclofenac administration, replacement of diclofenac bile with control bile reduced anastomotic complications (P = 0·016). The leak rate was seven of 15 versus 13 of 17 without replacement (P = 0·127). After intramuscular administration of diclofenac, the reduction in anastomotic complications was not significant when bile was replaced with control bile (P = 0·283), but it was significant when bile was drained without replacement (P = 0·025). Diclofenac metabolites in bile peaked within 2 h after administration. Administration of diclofenac bile resulted in nearly undetectable plasma levels of diclofenac (mean(s.d.) 0·01(0·01) µg/ml) after 120 min. Following oral diclofenac, bile replacement with control bile did not affect the plasma concentration of diclofenac (0·12(0·08) µg/ml versus 0·10(0·05) µg/ml with diclofenac bile; P = 0·869). CONCLUSION: Altered bile composition as a result of diclofenac administration increases the ileal anastomotic complication rate in rats.
RESUMEN
Currently, tacrolimus is the most potent immunosuppressive agent for renal transplant recipients and is commonly prescribed during pregnancy. As data on placental exposure and transfer are limited, we studied tacrolimus placental handling in samples obtained from renal transplant recipients. We found transfer to venous umbilical cord blood, but particularly noted a strong placental accumulation. In patient samples, tissue concentrations in a range of 55-82â¯ng/g were found. More detailed ex vivo dual-side perfusions of term placentas from healthy women revealed a tissue-to-maternal perfusate concentration ratio of 113⯱â¯49 (mean⯱â¯SEM), underlining the placental accumulation found in vivo. During the 3â¯h ex vivo perfusion interval no placental transfer to the fetal circulation was observed. In addition, we found a non-homogeneous distribution of tacrolimus across the perfused cotyledons. In conclusion, we observed extensive accumulation of tacrolimus in placental tissue. This warrants further studies into potential effects on placental function and immune cells of the placenta.
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Inmunosupresores/farmacocinética , Trasplante de Riñón , Placenta/metabolismo , Tacrolimus/farmacocinética , Adulto , Femenino , Humanos , Perfusión , EmbarazoRESUMEN
Septic acute kidney injury (AKI) associates with poor survival rates and often requires renal replacement therapy. Glucocorticoids may pose renal protective effects in sepsis via stimulation of mitochondrial function. Therefore, we studied the mitochondrial effects of dexamethasone in an experimental inflammatory proximal tubule epithelial cell model. Treatment of human proximal tubule epithelial cells with lipopolysaccharide (LPS) closely resembles pathophysiological processes during endotoxaemia, and led to increased cytokine excretion rates and cellular reactive oxygen species levels, combined with a reduced mitochondrial membrane potential and respiratory capacity. These effects were attenuated by dexamethasone. Dexamethasone specifically increased the expression and activity of mitochondrial complex V (CV), which could not be explained by an increase in mitochondrial mass. Finally, we demonstrated that dexamethasone acidified the intracellular milieu and consequently reversed LPS-induced alkalisation, leading to restoration of the mitochondrial function. This acidification also provides an explanation for the increase in CV expression, which is expected to compensate for the inhibitory effect of the acidified environment on this complex. Besides the mechanistic insights into the beneficial effects of dexamethasone during renal cellular inflammation, our work also supports a key role for mitochondria in this process and, hence, provides novel therapeutic avenues for the treatment of AKI.
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Dexametasona/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Nefritis Intersticial/metabolismo , Biomarcadores , Línea Celular , Células Cultivadas , Citrato (si)-Sintasa/metabolismo , Endotoxemia/etiología , Endotoxemia/metabolismo , Endotoxemia/patología , Células Epiteliales/patología , Humanos , Concentración de Iones de Hidrógeno , Espacio Intracelular/metabolismo , Túbulos Renales Proximales/patología , Lipopolisacáridos/efectos adversos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Redes y Vías Metabólicas/efectos de los fármacos , Nefritis Intersticial/etiología , Nefritis Intersticial/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Organic anion-transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter, of which the polymorphic variant OATP1B1*15 (Asn130Asp and Val174Ala) has been associated with decreased transport activity. Rosuvastatin is an OATP1B1 substrate and often concomitantly prescribed with oral antidiabetics in the clinic. The aim of this study was to investigate possible drug-drug interactions between these drugs at the level of OATP1B1 and OATP1B1*15. We generated human embryonic kidney (HEK)293 cells stably overexpressing OATP1B1 or OATP1B1*15 that showed similar protein expression levels of OATP1B1 and OATP1B1*15 at the cell membrane as measured by liquid chromatography-tandem mass spectrometry. In HEK-OATP1B1*15 cells, the V(max) for OATP1B1-mediated transport of E(2)17ß-G (estradiol 17ß-d-glucuronide) was decreased >60%, whereas K(m) values (Michaelis constant) were comparable. Uptake of rosuvastatin in HEK-OATP1B1 cells (K(m) 13.1 ± 0.43 µM) was nearly absent in HEK-OATP1B1*15 cells. Interestingly, several oral antidiabetics (glyburide, glimepiride, troglitazone, pioglitazone, glipizide, gliclazide, and tolbutamide), but not metformin, were identified as significant inhibitors of the OATP1B1-mediated transport of rosuvastatin. The IC(50) values for inhibition of E(2)17ß-G uptake were similar between OATP1B1 and OATP1B1*15. In conclusion, these studies indicate that several oral antidiabetic drugs affect the OATP1B1-mediated uptake of rosuvastatin in vitro. The next step will be to translate these data to the clinical situation, as it remains to be established whether the studied oral antidiabetics indeed affect the clinical pharmacokinetic profile of rosuvastatin in patients.
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Fluorobencenos/metabolismo , Hipoglucemiantes/metabolismo , Hipoglucemiantes/farmacología , Transportadores de Anión Orgánico/antagonistas & inhibidores , Pirimidinas/metabolismo , Sulfonamidas/metabolismo , Administración Oral , Cromatografía Liquida , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Estradiol/análogos & derivados , Estradiol/metabolismo , Células HEK293 , Humanos , Hipoglucemiantes/administración & dosificación , Cinética , Transportador 1 de Anión Orgánico Específico del Hígado , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Rosuvastatina Cálcica , Espectrometría de Masas en Tándem , TransfecciónRESUMEN
Fibroblast apoptosis plays a crucial role in normal and pathological scar formation and therefore we studied whether the putative apoptosis-inducing factor curcumin affects fibroblast apoptosis and may function as a novel therapeutic. We show that 25-microM curcumin causes fibroblast apoptosis and that this could be inhibited by co-administration of antioxidants N-acetyl-l-cysteine (NAC), biliverdin or bilirubin, suggesting that reactive oxygen species (ROS) are involved. This is supported by our observation that 25-microM curcumin caused the generation of ROS, which could be completely blocked by addition of NAC or bilirubin. Since biliverdin and bilirubin are downstream products of heme degradation by heme oxygenase (HO), it has been suggested that HO-activity protects against curcumin-induced apoptosis. Interestingly, exposure to curcumin maximally induced HO-1 protein and HO-activity at 10-15 microM, whereas, at a concentration of >20-microM curcumin HO-1-expression and HO-activity was negligible. NAC-mediated inhibition of 25-microM curcumin-induced apoptosis was demonstrated to act in part via restored HO-1-induction, since the rescuing effect of NAC could be reduced by inhibiting HO-activity. Moreover pre-induction of HO-1 using 5-microM curcumin protected fibroblasts against 25-microM curcumin-induced apoptosis. On a functional level, fibroblast-mediated collagen gel contraction, an in vitro wound contraction model, was completely prevented by 25-microM curcumin, while this could be reversed by co-incubation with NAC, an effect that was also partially HO-mediated. In conclusion, curcumin treatment in high doses (>25 microM) may provide a novel way to modulate pathological scar formation through the induction of fibroblast apoptosis, while antioxidants, HO-activity and its effector molecules act as a possible fine-tuning regulator.
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Apoptosis/efectos de los fármacos , Cicatriz/enzimología , Curcumina/farmacología , Fibroblastos/citología , Fibroblastos/enzimología , Hemo Oxigenasa (Desciclizante)/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Acetilcisteína/farmacología , Antioxidantes/farmacología , Bilirrubina/farmacología , Colágeno/metabolismo , Dermis/citología , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Geles , Glutatión/farmacología , Humanos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND AND PURPOSE: The xanthine oxidase inhibitors allopurinol and oxypurinol are used to treat hyperuricaemia, whereas loop and thiazide diuretics can cause iatrogenic hyperuricaemia. Some uricosuric drugs and salicylate have a bimodal action on urate renal excretion. The mechanisms of action of these hypo- and hyperuricaemic drugs on the handling of urate in renal tubules have not been fully elucidated. Recently, we identified the multidrug resistance protein (MRP) 4 as a luminal efflux transporter for urate in the proximal tubule. EXPERIMENTAL APPROACH: Here, we studied the effect of these drugs on [(14)C]urate transport using human embryonic kidney 293 cells overexpressing human MRP4 and in membrane vesicles isolated from these cells. KEY RESULTS: Allopurinol stimulated MRP4-mediated cellular urate efflux and allopurinol and oxypurinol both markedly stimulated urate transport by MRP4 in membrane vesicles. Bumetanide and torasemide had no effect, whereas furosemide, chlorothiazide, hydrochlorothiazide, salicylate, benzbromarone and sulfinpyrazone inhibited urate transport, at concentrations ranging from nanomolar up to millimolar. Probenecid stimulated urate transport at 0.1 microM and inhibited transport at higher concentrations. CONCLUSIONS AND IMPLICATIONS: These data suggest that inhibition of MRP4-mediated urate efflux by furosemide and thiazide diuretics could have an important function in their hyperuricaemic mechanisms. Furthermore, stimulation of MRP4-mediated renal urate efflux could be a new mechanism in the hypouricaemic action of allopurinol and oxypurinol. In conclusion, MRP4 may provide a potential target for drugs affecting urate homoeostasis, which needs to be further evaluated in vivo.
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Diuréticos/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/efectos de los fármacos , Ácido Úrico/metabolismo , Uricosúricos/farmacología , Alopurinol/administración & dosificación , Alopurinol/farmacología , Transporte Biológico/efectos de los fármacos , Línea Celular , Diuréticos/administración & dosificación , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos , Homeostasis/efectos de los fármacos , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Oxipurinol/administración & dosificación , Oxipurinol/farmacología , Salicilatos/administración & dosificación , Salicilatos/farmacología , Uricosúricos/administración & dosificaciónRESUMEN
The Breast Cancer Resistance Protein (BCRP/ABCG2) is a transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. This transporter is highly expressed in many tissues; however, in human kidney, only the mRNA was found in contrast to the mouse kidney, where the transporter is abundant. In bcrp/abcg2((-/-)) mice, the expression of two sterol transporter genes, abcg5 and abcg8, was strongly increased in the kidney, perhaps as a compensatory mechanism to upregulate efflux. We found using immunohistochemical analysis clear localization of BCRP/ABCG2 to the proximal tubule brush border membrane of the human kidney comparable to that of other ABC transporters such as P-glycoprotein/ABCB1, MRP2/ABCC2, and MRP4/ABCC4. Hoechst 33342 dye efflux from primary human proximal tubule cells was significantly reduced by the BCRP/ABCG2 inhibitors fumitremorgin C and nelfinavir. Our study shows that in addition to other apical ABC transporters, BCRP/ABCG2 may be important in renal drug excretion.
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Transportadoras de Casetes de Unión a ATP/genética , Túbulos Renales Proximales/metabolismo , Proteínas de Neoplasias/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/análisis , Transportadoras de Casetes de Unión a ATP/fisiología , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Inmunohistoquímica , Túbulos Renales Proximales/química , Túbulos Renales Proximales/citología , Ratones , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/fisiología , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , RatasRESUMEN
The multidrug resistance gene 1 product, P-glycoprotein (P-gp), is expressed in several excretory organs, including the apical membrane of proximal tubules. After inducing acute renal failure, P-gp expression is upregulated and this might be a protective function by pumping out toxicants and harmful products of oxidative stress. We characterized renal function of P-gp knockout mice and studied its consequences in renal ischemic damage. Compared with wild-type mice, knockout mice have a lower glomerular filtration rate and renal plasma flow. An augmented urinary excretion of sodium, numerous amino acids, calcium, glucose, and low molecular weight proteins was observed along with an increased diuresis. A higher lithium plasma clearance in the knockout mice suggested proximal tubular dysfunction. Electron microscopy showed mitochondrial abnormalities in proximal tubular cells that could account for decreased adenosine triphosphate levels in the cortex. After inducing ischemia, wild-type mice showed a decrease in creatinine clearance and severe proximal tubular necrosis. In contrast, knockout mice had no signs of tubular damage. Our data indicate that P-gp knockout mice have impaired renal function but are protected against ischemic renal injury.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/deficiencia , Isquemia/prevención & control , Túbulos Renales Proximales/fisiopatología , Lesión Renal Aguda/fisiopatología , Aminoácidos/orina , Animales , Calcio/orina , Diuresis , Técnica del Anticuerpo Fluorescente , Tasa de Filtración Glomerular/genética , Glucosuria , Inmunohistoquímica , Isquemia/etiología , Túbulos Renales Proximales/ultraestructura , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Mitocondrias/patología , Mitocondrias/ultraestructura , Proteinuria , Circulación Renal/genética , Sodio/orinaRESUMEN
It has now been firmly established that, not only ischemia/reperfusion, but also cold itself causes damage during kidney transplantation. Iron chelators or anti-oxidants applied during the cold plus rewarming phase are able to prevent this damage. At present, it is unknown if these measures act only during the cold, or whether application during the rewarming phase also prevents damage. We aimed to study this after cold normoxic and hypoxic conditions. LLC-PK1 cells were incubated at 4 degrees C in Krebs-Henseleit buffer for 6 or 24h, followed by 18 or 6h rewarming, respectively. Cold preservation was performed under both normoxic (95% air/5% CO2) and hypoxic (95% N2/5% CO2) conditions. The iron chelator 2,2'-DPD (100 microM), anti-oxidants BHT (20 microM) or sibilinin (200 microM), and xanthine oxidase inhibitor allopurinol (100 microM) were added during either cold preservation plus rewarming, or rewarming alone. Cell damage was assessed by LDH release (n=3-9). Addition of 2,2'-DPD and BHT during cold hypoxia plus rewarming did, but during rewarming alone did not prevent cell damage. When added during rewarming after 6h cold normoxic incubation, BHT and 2,2'-DPD inhibited rewarming injury compared to control (p<0.05). Allopurinol did not prevent cell damage in any experimental set-up. Our data show that application of iron chelators or anti-oxidants during the rewarming phase protects cells after normoxic but not hypoxic incubation. Allopurinol had no effect. Since kidneys are hypoxic during transplantation, measures aimed at preventing cold-induced and rewarming injury should be taken during the cold.
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Antioxidantes/farmacología , Quelantes del Hierro/farmacología , Soluciones Preservantes de Órganos/farmacología , Preservación de Órganos/métodos , 2,2'-Dipiridil/farmacología , Alopurinol/farmacología , Animales , Hidroxitolueno Butilado/farmacología , Etanol/farmacología , Hipoxia/patología , Hipoxia/fisiopatología , L-Lactato Deshidrogenasa/metabolismo , Células LLC-PK1/efectos de los fármacos , Daño por Reperfusión/prevención & control , Recalentamiento/métodos , Silibina , Silimarina/farmacología , Hidróxido de Sodio/farmacología , PorcinosRESUMEN
In this study, the release of rhBMP-2 loaded porous Ca-P cement was studied in vitro and in vivo. We hypothesized that adsorption sites of Ca-P ceramic with high affinity for rhBMP-2 can be blocked by pretreatment of the ceramic with albumin prior to rhBMP-2 loading, which would result in weaker rhBMP-2 binding and enhanced release of rhBMP-2. Preset porous Ca-P cement discs with a diameter of 6.35 mm (volume: 75 mm3) were pretreated by incubation in a solution of 10% rat serum albumin for 24 h or in ddH2O (control group) prior to administration of 5 mug radiolabeled 131I-rhBMP-2. Release was assessed in vitro in phosphate buffered saline (PBS) and fetal calf serum and in vivo by longitudinal scintigraphic imaging of radiolabeled 131I-rhBMP-2 and gamma counting of dissected implants. In vitro release from pretreated discs was higher during the first day. For both formulations, release in PBS was limited compared to release in serum. In vivo release considerably exceeded in vitro release. In vivo release kinetics showed no significant difference of half-lives between pretreated and control discs. Both formulations showed sustained release during at least 4 weeks. Ex vivo gamma counting of retrieved samples confirmed scintigraphic results and showed that the capsule and surrounding tissues only contained a minor fraction rhBMP-2. We conclude that 1. scintigraphy of 131I-labeled rhBMP-2 provides a reliable method for longitudinal measurement of rhBMP-2 release kinetics in vivo. 2. albumin pretreatment of porous Ca-P cement does not results in relevant increase of initial release of rhBMP-2 in vivo, and 3. preset rhBMP-2 loaded porous Ca-P cement discs exhibit one phase exponential release kinetics in the rat ectopic model, characterized by a retention of 20-30% after 4 weeks.
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Albúminas , Materiales Biocompatibles , Proteínas Morfogenéticas Óseas/administración & dosificación , Fosfatos de Calcio , Sistemas de Liberación de Medicamentos , Proteínas Recombinantes/administración & dosificación , Factor de Crecimiento Transformador beta/administración & dosificación , Animales , Proteína Morfogenética Ósea 2 , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Renal ATP binding cassette (ABC) transporters have an important role in the elimination of metabolic waste products and compounds foreign to the body. The kidney has the ability to tightly control the expression of these efflux transporters to maintain homeostasis, and as a major mechanism of adaptation to environmental stress. In the present study, we investigated the expression of 45 ABC transporter genes in the mouse kidney under basal conditions, after induction of ischemia and after regeneration. Two days after clamping, mice showed a 76% decrease in renal creatinine clearance, which improved clearly within 7 days. This was confirmed by histological examinations. Seven days after ischemia, real-time quantitative Polymerase chain reaction data showed that transcript abundance of abcb1, abcb11, and abcc4 was increased, and that of abca3, abcc2, and abcg2 decreased. Expression of all transporters returned to baseline after 14 days, except for abcb11, which was reduced. Abcb11 is the major liver canalicular bile salt export pump. Here we show for the first time expression in the kidney and localization of the transporter to the apical membrane of proximal tubules. The presence of another novel renal transporter, abca3, was confirmed by Western blotting. Immunohistochemistry showed that abca3 is localized to the peritubular capillaries and apical membrane of proximal tubules. In conclusion, after inducing ischemic reperfusion injury in the kidney, ABC transporters appear to be differentially regulated, which might be associated with the renal regeneration process. Furthermore, we showed for the first time expression and subcellular localization of abcb11 and abca3 in mouse kidney.
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Transportadoras de Casetes de Unión a ATP/genética , Perfilación de la Expresión Génica , Riñón/patología , Daño por Reperfusión/genética , Daño por Reperfusión/patología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/análisis , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Western Blotting , Regulación de la Expresión Génica , Inmunohistoquímica , Riñón/química , Túbulos Renales Proximales/química , Túbulos Renales Proximales/patología , Masculino , Proteínas de Transporte de Membrana/análisis , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Endogámicos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/análisis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The release kinetics of recombinant human bone morphogenetic protein-2 (rhBMP-2) loaded poly(dl-lactic-co-glycolic acid)/calcium phosphate cement (PLGA/Ca-P cement) composites were studied in vivo. RhBMP-2 was radiolabeled with (131)I and entrapped within PLGA microparticles or adsorbed onto the microparticle surface. PLGA microparticles were prepared of high molecular weight (HMW) PLGA (weight average molecular weight [M(w)] 49,100+/-1700) or low molecular weight (LMW) PLGA (M(w) 5,900+/-300) and used for preparation of 30:70 wt.% PLGA/Ca-P cement composite discs. Release of 131I-rhBMP-2 loaded composites was assessed by scintigraphic imaging according to a 2(2) two-level full factorial design in the rat ectopic model during four weeks. In vivo release kinetics varied among formulations. All formulations showed slow release without initial burst, and displayed a linear release from 3 to 28 days. Release of LMW entrapped rhBMP-2 composites (1.7+/-0.3%/day) was significantly faster than release from other formulations (p<0.01). After 28 days, retention within the composites was 65+/-5%, 75+/-4%, 50+/-4% and 70+/-6% of the initial rhBMP-2 for HMW entrapped, HMW adsorbed, LMW entrapped and LMW adsorbed rhBMP-2 composites, respectively. Release from the composite was probably slowed down by an interaction of rhBMP-2 and Ca-P cement after rhBMP-2 release from PLGA microparticles. We conclude that PLGA/Ca-P cement composites can be considered as sustained slow release vehicles and that the release and retention of rhBMP-2 can be modified according to the desired profile to a limited extent.
Asunto(s)
Cementos para Huesos , Proteínas Morfogenéticas Óseas/administración & dosificación , Fosfatos de Calcio/química , Glicolatos/química , Proteínas Recombinantes/administración & dosificación , Factor de Crecimiento Transformador beta/administración & dosificación , Adsorción , Animales , Materiales Biocompatibles , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/farmacocinética , Preparaciones de Acción Retardada , Portadores de Fármacos , Ácido Láctico , Masculino , Microesferas , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/farmacocinéticaRESUMEN
AIM: In diabetes, perinatal morbidity is significantly increased. This may partly be related to functional changes in the foetoplacental vascular bed. In diabetes models, a defect in the cyclo-oxygenase pathway is a common observation. Therefore, we hypothesized that the human foetoplacental circulation of diabetic patients is characterized by dysfunction of the cyclo-oxygenase pathway, as well. METHODS: We performed ex-vivo perfusions of isolated placental cotyledons from healthy women (n = 14) and from patients with Type 1 diabetes (n = 9). The contribution of cyclo-oxygenase products to foetoplacental vascular tone was quantified by measuring the response to the cyclo-oxygenase inhibitor indomethacin. RESULTS: Baseline foetoplacental arterial pressure was comparable between controls and diabetic women (mean +/- sem, 21.7 +/- 1.2 vs. 24.4 +/- 2.0 mmHg). Maximum foetoplacental arterial pressure at highest dose of indomethacin was 32.8 +/- 3.0 mmHg in controls vs. 27.3 +/- 2.3 mmHg in diabetic women. The indomethacin-induced increase in pressure was reduced in diabetes (2.9 +/- 0.7 vs. 11.2 +/- 2.4 mmHg in controls, P = 0.01). CONCLUSIONS: Under baseline conditions, the net effect of all cyclo-oxygenase products in the foetoplacental vascular bed is vasodilation. In diabetes, this vasodilator effect seems significantly impaired.
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Diabetes Mellitus Tipo 1/enzimología , Circulación Placentaria , Embarazo en Diabéticas/enzimología , Prostaglandina-Endoperóxido Sintasas/fisiología , Adulto , Presión Sanguínea/efectos de los fármacos , Inhibidores de la Ciclooxigenasa , Diabetes Mellitus Tipo 1/fisiopatología , Femenino , Humanos , Técnicas In Vitro , Indometacina , Circulación Placentaria/efectos de los fármacos , Embarazo , Embarazo en Diabéticas/fisiopatología , Transducción de Señal/efectos de los fármacos , Vasoconstricción/efectos de los fármacosRESUMEN
Maternal folate deficiency is associated with fetal growth restriction, however, transfer of folate across placentae of pregnancies complicated by fetal growth restriction has never been investigated. We studied whether maternal to fetal 5-methyltetrahydrofolate (5MTF) transport in the ex vivo dually perfused isolated cotyledon, binding of [(3)H] folate (PteGlu) to the syncytial microvillous membrane, and protein expression of folate receptor alpha (FR-alpha) and reduced folate carrier (RFC) in these placentae are disturbed. Placental clearance of 5MTF from the maternal perfusate appeared to be non-saturable over a range of 50 to 500 nm, independent of albumin and flow-independent. No statistically significant differences between placentae complicated with fetal growth restriction and uncomplicated pregnancies were observed. Binding characteristics of [(3)H-]PteGlu to microvillous membranes of fetal growth restriction versus control placentae were similar: B(max)of 3.9+/-2.0 (mean+/-s.d.) versus 4.0+/-1.6 pmol/mg protein and a K(d)of 0.037+/-0.010 versus 0.040+/-0.018 nm. Expression of FR-alpha and RFC were not different in placentae of both groups studied. In conclusion, fetal growth restriction appears not to be associated with impaired maternal to fetal placental folate transport, placental receptor binding, or expression of FR-alpha and RFC.
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Retardo del Crecimiento Fetal/metabolismo , Ácido Fólico/metabolismo , Placenta/metabolismo , Adulto , Transporte Biológico , Proteínas Portadoras/análisis , Proteínas Portadoras/metabolismo , Femenino , Receptores de Folato Anclados a GPI , Humanos , Immunoblotting , Cinética , Proteínas de Transporte de Membrana/análisis , Proteínas de Transporte de Membrana/metabolismo , Microvellosidades/química , Microvellosidades/metabolismo , Embarazo , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/metabolismo , Proteína Portadora de Folato Reducido , Tetrahidrofolatos/metabolismo , Tritio , Trofoblastos/ultraestructuraRESUMEN
OBJECTIVE: To study the pathophysiological changes of renal tubular drug transport mechanisms in congenital renal obstruction, by developing a model for perfusing the isolated kidney (IPK) after neonatal surgical induction of partial ureteric obstruction in Hanover Wistar rats. MATERIAL AND METHODS: Moderately severe obstruction of the right kidney of male rats was created by burying a segment of the right ureter under the psoas fascia at 5-7 days after birth. Different fluorescent substrates for renal organic anion and cation drug transport systems were added to the IPK, and the concentration of these substances with time analysed in perfusate and urine. RESULTS: The reproducibility in all groups of the glomerular filtration rate (GFR) and drug excretion was remarkably good. GFR was significantly lower in obstructed kidneys than in unobstructed kidneys. 123Rhodamine, a marker for organic cation and P-glycoprotein transport, had a significantly lower maximum excretion rate in the obstructed than in unobstructed kidneys. Renal fractional clearance (123rhodamine clearance corrected for diminished GFR) was also significantly lower in obstructed kidneys. There was no significant difference in maximum excretion (absolute and corrected GFR) for Lucifer Yellow, a marker for sodium-dependent organic anion transport. The maximum excretion rate of calcein, a marker for sodium-independent organic anion transport, was significantly lower in the obstructed than in the unobstructed kidneys, but significantly higher after correcting for reduced GFR. CONCLUSION: The IPK is a good model for studying the effect of neonatal renal obstruction on tubular drug transport. These results show that organic anion and cation transport mechanisms are affected differently by obstruction.