RESUMEN
Organic anion transporters 1 and 3 (OAT1 and OAT3) play a crucial role in kidney function by regulating the secretion of multiple renally cleared small molecules and toxic metabolic by-products. Assessing the activity of these transporters is essential for drug development purposes as they can significantly impact drug disposition and safety. OAT1 and OAT3 are amongst the most abundant drug transporters expressed in human renal proximal tubules. However, their expression is lost when cells are isolated and cultured in vitro, which is a persistent issue across all human and animal renal proximal tubule cell models, including primary cells and cell lines. Although it is well known that the overall expression of drug transporters is affected in vitro, the underlying reasons for the loss of OAT1 and OAT3 are still not fully understood. Nonetheless, research into the regulatory mechanisms of these transporters has provided insights into the molecular pathways underlying their expression and activity. In this review, we explore the regulatory mechanisms that govern the expression and activity of OAT1 and OAT3 and investigate the physiological changes that proximal tubule cells undergo and that potentially result in the loss of these transporters. A better understanding of the regulation of these transporters could aid in the development of strategies, such as introducing microfluidic conditions or epigenetic modification inhibitors, to improve their expression and activity in vitro and to create more physiologically relevant models. Consequently, this will enable more accurate assessment for drug development and safety applications.
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Transportadores de Anión Orgánico Sodio-Independiente , Transportadores de Anión Orgánico , Animales , Humanos , Transportadores de Anión Orgánico Sodio-Independiente/genética , Proteína 1 de Transporte de Anión Orgánico/genética , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Riñón/metabolismo , Túbulos Renales Proximales/metabolismo , Células Epiteliales/metabolismo , Transportadores de Anión Orgánico/metabolismoRESUMEN
INTRODUCTION: Cisplatin is extensively used in the treatment of head and neck carcinomas. Cetuximab combination therapy is employed in recurrent and metastatic settings. Sunitinib showed positive results in the treatment of head and neck carcinomas, both as monotherapy or in combination with cetuximab. Nonetheless, the mechanism governing these pharmacological interactions is largely unresolved. This study investigates the impact of cetuximab on the cytotoxicity of cisplatin and sunitinib using cells representative of head and neck carcinoma and the oral epithelium. METHODS: The uptake and efflux activities of cells were determined using the prototypical fluorescent substrates 4-[4-[dimethylamino]styryl)-1-methyl pyridinium iodide, Hoechst 33342, and calcein-AM in the presence or absence of specific inhibitors in cells pretreated with cetuximab. The expression of key uptake and efflux drug transporters was analyzed using qPCR and immunofluorescence. Cisplatin and sunitinib cytotoxicities after cetuximab pretreatment were evaluated using the PrestoBlue viability assay. RESULTS: Both tumor and nontumor cells showed significant active drug transport activity. Cetuximab substantially deregulated the expression of key transporters involved in drug resistance in head and neck cancer cells. Transporter expression in the nontumor cell was unaffected. Upon cetuximab pretreatment, the half maximal effective toxic concentration of cisplatin was reduced by 0.75-fold and sunitinib by 0.82-fold in cancer cells. Nontumor cells were not sensitive to cisplatin or sunitinib under the conditions tested. CONCLUSION: Cetuximab regulates the expression and activity of key membrane drug transporters in head and neck cancer cells, involved in drug resistance. The deregulation of the transport mechanism behind cisplatin and sunitinib uptake reverses drug resistance and enhances the cytotoxicity of both drugs.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Humanos , Cetuximab/farmacología , Cetuximab/uso terapéutico , Cisplatino/farmacología , Sunitinib/farmacología , Sunitinib/uso terapéutico , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéuticoRESUMEN
Accurate prediction of pharmacokinetic parameters, such as renal clearance, is fundamental to the development of effective and safe new treatments for patients. However, conventional renal models have a limited ability to predict renal drug secretion, a process that is dependent on transporters in the proximal tubule. Improvements in microphysiological systems (MPS) have extended our in vitro capabilities to predict pharmacokinetic parameters. In this study a kidney-MPS model was developed that successfully recreated renal drug secretion. Human proximal tubule cells grown in the kidney-MPS, resembling an in vivo phenotype, actively secreted the organic cation drug metformin and organic anion drug cidofovir, in contrast to cells cultured in conventional culture formats. Metformin and cidofovir renal secretory clearance were predicted from kidney-MPS data within 3.3- and 1.3-fold, respectively, of clinically reported values by employing a semi-mechanistic drug distribution model using kidney-MPS drug transport parameters together with in vitro to in vivo extrapolation. This approach introduces an effective application of a kidney-MPS model coupled with pharmacokinetic modelling tools to evaluate and predict renal drug clearance in humans. Kidney-MPS renal clearance predictions can potentially complement pharma-cokinetic animal studies and contribute to the reduction of pre-clinical species use during drug development.
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Metformina , Sistemas Microfisiológicos , Animales , Humanos , Cidofovir/farmacología , Riñón/metabolismo , Metformina/metabolismo , Metformina/farmacología , Vías de Eliminación de FármacosRESUMEN
The activity of drug transporters is central to the secretory function of the kidneys and a defining feature of renal proximal tubule epithelial cells (RPTECs). The expression, regulation, and function of these membrane-bound proteins is well understood under normal renal physiological conditions. However, the impact of drug transporters on the pathophysiology of kidney cancer is still elusive. In the present study, we employed different renal cell carcinoma (RCC) cell lines and a prototypical non-malignant RPTEC cell line to characterize the activity, expression, and potential regulatory mechanisms of relevant renal drug transporters in RCC in vitro. An analysis of the uptake and efflux activity, the expression of drug transporters, and the evaluation of cisplatin cytotoxicity under the effects of methylation or epidermal growth factor receptor (EGFR) inhibition showed that the RCC cells retained substantial drug transport activity. In RCC cells, P-glycoprotein was localized in the nucleus and its pharmacological inhibition enhanced cisplatin toxicity in non-malignant RPTECs. On the other hand, methylation inhibition enhanced cisplatin toxicity by upregulating the organic cation uptake activity in RCC cells. Differential effects of methylation and EGFR were observed in transporter expression, showing regulatory heterogeneity in these cells. Interestingly, the non-malignant RPTEC cell line that was used lacked the machinery responsible for organic cation transport, which reiterates the functional losses that renal cells undergo in vitro.
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Carcinoma de Células Renales , Neoplasias Renales , Carcinoma de Células Renales/metabolismo , Cationes/metabolismo , Cisplatino/metabolismo , Cisplatino/farmacología , Receptores ErbB/metabolismo , Humanos , Riñón/metabolismo , Neoplasias Renales/metabolismo , Túbulos Renales Proximales/metabolismo , Proteínas de Transporte de Membrana/metabolismoRESUMEN
MicroRNAs (miRNA) are ubiquitous non-coding RNAs that have a prominent role in cellular regulation. The expression of many miRNAs is often found deregulated in prostate cancer (PCa) and castration-resistant prostate cancer (CRPC). Although their expression can be associated with PCa and CRPC, their functions and regulatory activity in cancer development are poorly understood. In this study, we used different proteomics tools to analyze the activity of hsa-miR-3687-3p (miR-3687) and hsa-miR-4417-3p (miR-4417), two miRNAs upregulated in CRPC. PCa and CRPC cell lines were transfected with miR-3687 or miR-4417 to overexpress the miRNAs. Cell lysates were analyzed using 2D gel electrophoresis and proteins were subsequently identified using mass spectrometry (Maldi-MS/MS). A whole cell lysate, without 2D-gel separation, was analyzed by ESI-MS/MS. The expression of deregulated proteins found across both methods was further investigated using Western blotting. Gene ontology and cellular process network analysis determined that miR-3687 and miR-4417 are involved in diverse regulatory mechanisms that support the CRPC phenotype, including metabolism and inflammation. Moreover, both miRNAs are associated with extracellular vesicles, which point toward a secretory mechanism. The tumor protein D52 isoform 1 (TD52-IF1), which regulates neuroendocrine trans-differentiation, was found to be substantially deregulated in androgen-insensitive cells by both miR-3687 and miR-4417. These findings show that these miRNAs potentially support the CRPC by truncating the TD52-IF1 expression after the onset of androgen resistance.
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MicroARNs , Neoplasias de la Próstata Resistentes a la Castración , Andrógenos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , MicroARNs/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Proteómica , Espectrometría de Masas en TándemRESUMEN
The main physiological functions of renal proximal tubule cells in vivo are reabsorption of essential nutrients from the glomerular filtrate and secretion of waste products and xenobiotics into urine. Currently, there are several established cell lines of human origin available as in vitro models of proximal tubule. However, these cells appeared to be limited in their biological relevance, because essential characteristics of the original tissue are lost once the cells are cultured. As a consequence of these limitations, primary human proximal tubule cells constitute a suitable and a biologically more relevant in vitro model to study this specific segment of the nephron and therefore, these cells can play an important role in renal regenerative medicine applications. Here, we describe a protocol to isolate proximal tubule cells from human nephrectomies. We explain the steps performed for an in-depth characterization of the cells, including the study of markers from others segments of the nephron, with the goal to determine the purity of the culture and the stability of proteins, enzymes, and transporters along time. The human proximal tubule cells isolated and used throughout this study showed many proximal tubule characteristics, including monolayer organization, cell polarization with the expression of tight junctions and primary cilia, expression of proximal tubule-specific proteins, such as megalin and sodium/glucose cotransporter 2, among others. The cells also expressed enzymatic activity for dipeptidyl peptidase IV, as well as for gamma glutamyl transferase 1, and expressed transporter activity for organic anion transporter 1, P-glycoprotein, multidrug resistance proteins, and breast cancer resistance protein. In conclusion, characterization of our cells confirmed presence of putative proximal tubule markers and the functional expression of multiple endogenous organic ion transporters mimicking renal reabsorption and excretion. These findings can constitute a valuable tool in the development of bioartificial kidney devices.
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Técnicas de Cultivo de Célula/métodos , Túbulos Renales Proximales/citología , Terapia de Reemplazo Renal , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Nefrectomía , Transportadores de Anión Orgánico/metabolismo , gamma-Glutamiltransferasa/metabolismoRESUMEN
Antisense oligonucleotide therapy has been reported to be associated with renal injury. Here, the mechanism of reversible proteinuria was investigated by combining clinical, pre-clinical, and in vitro data. Urine samples were obtained from Duchenne muscular dystrophy (DMD) patients treated with drisapersen, a modified 2'O-methyl phosphorothioate antisense oligonucleotide (6 mg/kg). Urine and kidney tissue samples were collected from cynomolgus monkeys (Macaca fascicularis) dosed with drisapersen (39 weeks). Cell viability and protein uptake were evaluated in vitro using human conditionally immortalized proximal tubule epithelial cells (ciPTECs). Oligonucleotide treatment in DMD patients was associated with an increase in urinary alpha-1-microglobulin (A1M), which returned to baseline following treatment interruptions. In monkeys, increased urinary A1M correlated with dose-dependent accumulation of oligonucleotide in kidney tissue without evidence of tubular damage. Furthermore, oligonucleotides accumulated in the lysosomes of ciPTECs and reduced the absorption of A1M, albumin, and receptor-associated protein, but did not affect cell viability when incubated for up to 7 days. In conclusion, phosphorothioate oligonucleotides appear to directly compete for receptor-mediated endocytosis in proximal tubules. We postulate that oligonucleotide-induced low molecular weight proteinuria in patients is therefore a transient functional change and not indicative of tubular damage.
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Organ-Chips are micro-engineered systems that aim to recapitulate the organ microenvironment. Implementation of Organ-Chips within the pharmaceutical industry aims to improve the probability of success of drugs reaching late stage clinical trial by generating models for drug discovery that are of human origin and have disease relevance. We are adopting the use of Organ-Chips for enhancing pre-clinical efficacy and toxicity evaluation and prediction. Whilst capturing cellular phenotype via imaging in response to drug exposure is a useful readout in these models, application has been limited due to difficulties in imaging the chips at scale. Here we created an end-to-end, automated workflow to capture and analyse confocal images of multicellular Organ-Chips to assess detailed cellular phenotype across large batches of chips. By automating this process, we not only reduced acquisition time, but we also minimised process variability and user bias. This enabled us to establish, for the first time, a framework of statistical best practice for Organ-Chip imaging, creating the capability of using Organ-Chips and imaging for routine testing in drug discovery applications that rely on quantitative image data for decision making. We tested our approach using benzbromarone, whose mechanism of toxicity has been linked to mitochondrial damage with subsequent induction of apoptosis and necrosis, and staurosporine, a tool inducer of apoptosis. We also applied this workflow to assess the hepatotoxic effect of an active AstraZeneca drug candidate illustrating its applicability in drug safety assessment beyond testing tool compounds. Finally, we have demonstrated that this approach could be adapted to Organ-Chips of different shapes and sizes through application to a Kidney-Chip.
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Dispositivos Laboratorio en un Chip , Imagen Óptica/instrumentación , Animales , Automatización , Evaluación Preclínica de Medicamentos , Humanos , Riñón/diagnóstico por imagen , Riñón/efectos de los fármacos , Hígado/diagnóstico por imagen , Hígado/efectos de los fármacos , RatasRESUMEN
Transmembrane flux of a drug within a tissue or organ frequently involves a complex system of transporters from multiple families that have redundant and overlapping specificities. Current in vitro systems poorly represent physiology, with reduced expression and activity of drug transporter proteins; therefore, novel models that recapitulate the complexity and interplay among various transporters are needed. The development of microphysiological systems that bring simulated physiologic conditions to in vitro cell culture models has enormous potential to better reproduce the morphology and transport activity across several organ models, especially in tissues such as the liver, kidney, intestine, or the blood-brain barrier, in which drug transporters play a key role. The prospect of improving the in vitro function of organ models highly prolific in drug transporters holds the promise of implementing novel tools to study these mechanisms with far more representative biology than before. In this short review, we exemplify recent developments in the characterization of perfused microphysiological systems involving the activity of drug transporters. Furthermore, we analyze the challenges and opportunities for the implementation of such systems in the study of transporter-mediated drug disposition and the generation of clinically relevant physiology-based in silico models incorporating relevant drug transport activity.
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Transporte Biológico/fisiología , Preparaciones Farmacéuticas/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Técnicas de Cultivo de Célula , Humanos , Riñón/metabolismo , Hígado/metabolismo , Proteínas de Transporte de Membrana/metabolismoRESUMEN
The combination of methotrexate with epidermal growth factor receptor (EGFR) recombinant antibody, cetuximab, is currently being investigated in treatment of head and neck carcinoma. As methotrexate is cleared by renal excretion, we studied the effect of cetuximab on renal methotrexate handling. We used human conditionally immortalized proximal tubule epithelial cells overexpressing either organic anion transporter 1 or 3 (ciPTEC-OAT1/ciPTEC-OAT3) to examine OAT1 and OAT3, and the efflux pumps breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp) in methotrexate handling upon EGF or cetuximab treatment. Protein kinase microarrays and knowledge-based pathway analysis were used to predict EGFR-mediated transporter regulation. Cytotoxic effects of methotrexate were evaluated using the dimethylthiazol bromide (MTT) viability assay. Methotrexate inhibited OAT-mediated fluorescein uptake and decreased efflux of Hoechst33342 and glutathione-methylfluorescein (GS-MF), which suggested involvement of OAT1/3, BCRP, and MRP4 in transepithelial transport, respectively. Cetuximab reversed the EGF-increased expression of OAT1 and BCRP as well as their membrane expressions and transport activities, while MRP4 and P-gp were increased. Pathway analysis predicted cetuximab-induced modulation of PKC and PI3K pathways downstream EGFR/ERBB2/PLCg. Pharmacological inhibition of ERK decreased expression of OAT1 and BCRP, while P-gp and MRP4 were increased. AKT inhibition reduced all transporters. Exposure to methotrexate for 24 h led to a decreased viability, an effect that was reversed by cetuximab. In conclusion, cetuximab downregulates OAT1 and BCRP while upregulating P-gp and MRP4 through an EGFR-mediated regulation of PI3K-AKT and MAPKK-ERK pathways. Consequently, cetuximab attenuates methotrexate-induced cytotoxicity, which opens possibilities for further research into nephroprotective comedication therapies.
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Cetuximab/farmacología , Factor de Crecimiento Epidérmico/metabolismo , Metotrexato/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Bencimidazoles/metabolismo , Supervivencia Celular/efectos de los fármacos , Glutatión/análogos & derivados , Glutatión/metabolismo , Células HEK293 , Humanos , Compuestos de Metilmercurio/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismoRESUMEN
The relevance of membrane transporters gained momentum in recent years and it is now widely recognized that transporters are key players in drug disposition and chemoresistance. As such, the kidneys harbor a variety of drug transporters and are one of the main routes for xenobiotic excretion. The breast cancer resistance protein (BCRP/ABCG2) is widely accepted as a key mediator of anticancer drug resistance and is a prominent renal drug transporter. Here, we review the role of BCRP in both processes and present a multitude of variables that can influence its activity. An increasing number of renally cleared chemotherapeutics, including tyrosine kinase inhibitors, described as BCRP substrates can modulate its activity via transcription factors and cellular signaling pathways, such as the phosphoinositide 3-kinase (PI3K) pathway. In addition to pharmacological actions, genetic variations, as well as differences between species and gender can affect BCRP function, which are also discussed. Furthermore, the role of BCRP in light of cancer treatments and the implications for novel therapeutic interventions that take into account renal function are discussed.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/fisiología , Riñón/metabolismo , Proteínas de Neoplasias/metabolismo , Eliminación Renal/fisiología , Antineoplásicos/farmacocinética , Humanos , Proteínas de Transporte de Membrana/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Factores Sexuales , Transducción de Señal/fisiología , Factores de Transcripción/metabolismoRESUMEN
Apical transport is key in renal function, and the activity of efflux transporters and receptor-mediated endocytosis is pivotal in this process. The conditionally immortalized proximal tubule epithelial cell line (ciPTEC) endogenously expresses these systems. Here, we used ciPTEC to investigate the activity of three major efflux transporters, viz., breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp), as well as protein uptake through receptor-mediated endocytosis, using a fluorescence-based setup for transport assays. To this end, cells were exposed to Hoechst33342, chloromethylfluorescein-diacetate (CMFDA), and calcein-AM in the presence or absence of model inhibitors for BCRP (KO143), P-gp (PSC833), or MRPs (MK571). Overexpression cell lines MDCKII-BCRP and MDCKII-P-gp were used as positive controls, and membrane vesicles overexpressing one transporter were used to determine substrate and inhibitor specificities. Receptor-mediated endocytosis was investigated by determining the intracellular accumulation of fluorescently labeled receptor-associated protein (RAP-GST). In ciPTEC, BCRP and P-gp showed similar expressions and activities, whereas MRP4 was more abundantly expressed. Hoechst33342, GS-MF, and calcein are retained in the presence of KO143, MK571, and PSC833, showing clearly redundancy between the transporters. Noteworthy is the fact that both KO143 and MK571 can block BCRP, P-gp, and MRPs, whereas PSC833 appears to be a potent inhibitor for BCRP and P-gp but not the MRPs. Furthermore, ciPTEC accumulates RAP-GST in intracellular vesicles in a dose- and time-dependent manner, which was reduced in megalin-deficient cells. In conclusion, fluorescent-probe-based assays are fast and reproducible in determining apical transport mechanisms, in vitro. We demonstrate that typical substrates and inhibitors are not specific for the designated transporters, reflecting the complex interactions that can take place in vivo. The set of tools we describe are also compatible with innovative kidney culture models and allows studying transport mechanisms that are central to drug absorption, disposition, and detoxification.
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Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Túbulos Renales Proximales/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Células Cultivadas , Perros , Endocitosis/fisiología , Fluorescencia , Humanos , Túbulos Renales Proximales/citología , Células de Riñón Canino Madin DarbyRESUMEN
The uremic solutes p-cresyl sulfate (pCS) and p-cresyl glucuronide (pCG) accumulate in patients with chronic kidney disease (CKD), and might contribute to disease progression. Moreover, retention of these solutes may directly be related to renal tubular function. Here, we investigated the role of the efflux transporters Multidrug Resistance Protein 4 (MRP4) and Breast Cancer Resistance Protein (BCRP) in pCS and pCG excretion, and studied the impact of both solutes on the phenotype of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC). Our results show that p-cresol metabolites accumulate during CKD, with a shift from sulfation to glucuronidation upon progression. Moreover, pCS inhibited the activity of MRP4 by 40% and BCRP by 25%, whereas pCG only reduced MRP4 activity by 75%. Moreover, BCRP-mediated transport of both solutes was demonstrated. Exposure of ciPTEC to pCG caused epithelial-to-mesenchymal transition, indicated by increased expression of vimentin and Bcl-2, and diminished E-cadherin. This was associated with altered expression of key tubular transporters. In conclusion, BCRP is likely involved in the renal excretion of both solutes, and pCG promotes phenotypical changes in ciPTEC, supporting the notion that uremic toxins may be involved in CKD progression by negatively affecting renal tubule cell phenotype and functionality.