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Liver and Kidney on Chips: Microphysiological Models to Understand Transporter Function
Abstract
Due to complex cellular microenvironments of both the liver and kidney accurate modeling of transport function has remained a challenge, leaving a dire need for models that can faithfully recapitulate both the architecture and cell-cell interactions observed in vivo. The study of hepatic and renal transport function is a fundamental component of understanding the metabolic fate of drugs and xenobiotics however; there are few in vitro systems conducive for these types of studies. For both the hepatic and renal systems we provide an overview of the location and functions of the most significant phase I/II/III (transporter) enzymes then review current in vitro systems for transporter function study suitability and provide details on microphysiological systems that lead the field in these investigations. Microphysiological modeling of the liver and kidney using “organ-on-a-chip” technologies is rapidly advancing in transport function assessment and has emerged as a promising method to evaluate drug and xenobiotic metabolism. Future directions for the field are also discussed along with technical challenges encountered in complex multiple-organs-on-chips development. This article is protected by copyright. All rights reserved.
... These transporters mediate the uptake of various endogenous compounds (such as bile salts) and exogenous compounds from the blood into hepatocytes, thus playing an important role in the distribution of drugs and endogenous compounds [36]. In the liver, OATP1B1, OATP1B3, OATP2B1, OCT1, and OCT3 are specifically expressed on the basolateral membrane of hepatocytes which mediate the absorption of exogenous and endogenous substances (bile acids and cholesterol) in the liver [37,38]. ABC transporters including P-gp, BCRP, MRPs and BSEP excrete cholesterol, bile salts and other metabolites in the liver [39]. ...
... It has a wide range of substrate specificity, primarily transporting hydrophobic neutral and cationic compounds, and excreting drugs into bile for clearance [39]. MRP3 and MRP4 are expressed in the basolateral membrane of hepatocytes, which have a high affinity for endogenous substances, such as bile acids and steroid conjugates, indicating that they have a protective role in preventing cholestasis and hepatotoxicity [37]. BCRP is distributed on the apical membrane of human hepatocytes and mediates the clearance of drugs and metabolites in the liver. ...
As the use of herbs has become more popular worldwide, there are increasing reports of herb-drug interactions (HDIs) following the combination of herbs and drugs. The active components of herbs are complex and have a variety of pharmacological activities, which inevitably affect changes in the pharmacokinetics of chemical drugs in vivo. The absorption, distribution, metabolism, and excretion of drugs in vivo are closely related to the expression of drug transporters. When the active components of herbs inhibit or induce the expression of transporters, this can cause changes in substrate pharmacokinetics, resulting in changes in the efficacy and toxicity of drugs. In this article, the tissue distribution and physiological functions of drug transporters are summarized through literature retrieval, and the effects of herbs on drug transporters and the possible mechanism of HDIs are analyzed and discussed in order to provide ideas and a reference for further guiding of safe clinical drug use.
... 92,94 With increasing recognition of the clinical implications of transporters in drug disposition, toxicity, and efficacy, recapitulating or retaining the expression and functional activity of drug transporters in 3D-cell culture models are critically important. 95,96 Compared with spheroids and organoids, which are in microstructures, MPS provides a compartmentalized platform and is more suitable to study vectorial transport of substrates across apical and basolateral compartments of polarized cells mediated by multiple transporters in a dynamic, physiologically relevant microenvironments. Furthermore, MPS can be designed to incorporate multiple cell types to create more organ-like models and allow interconnectivity between different organ platforms to study transporter activity across different organ systems. ...
... Currently, the characterization and validation of these 3D models in ADME settings largely focus on the end points of morphologic features, gene expression profile, and metabolic activity, whereas the evaluation of transporters is still emerging, in particular at the functional level. 95,96 In Table 3, we summarized recent examples of liver, gut, kidney, and brain 3D/MPS platforms with the focus on the expression and functional characterization of drug transporters, as well as their applications and limitations for transporter research. For example, in a recently developed human duodenum intestine-Chip 97 established from the organoid-derived cells of three independent donors, mRNA expression of major intestinal efflux (P-gp, BCRP, MRP2, and MRP3) and uptake transporters (PepT1, OATP2B1, OCT1, and SLC40A1) on day 8 of chip culture are comparable to those in the freshly isolated human duodenum tissue. ...
Enabled by a plethora of new technologies, research in membrane transporters has exploded in the past decade. The goal of this state‐of‐the‐art manuscript is to describe recent advances in research on membrane transporters that are particularly relevant to drug discovery and development. This review covers advances in basic, translational and clinical research that has led to an increased understanding of membrane transporters at all levels. At the basic level, we describe the available crystal structures of membrane transporters in both the Solute Carrier (SLC) and ATP Binding Cassette (ABC) Superfamilies, which has been enabled by the development of cryogenic electron microscopy (cryo‐EM) methods. Next, we describe new research on lysosomal and mitochondrial transporters as well as recently deorphaned transporters in the SLC superfamily. The translational section includes a summary of proteomic research which has led to a quantitative understanding of transporter levels in various cell types and tissues and new methods to modulate transporter function such as allosteric modulators and targeted protein degraders of transporters. The section ends with a review of the effect of the gut microbiome on modulation of transporter function followed by a presentation of 3D cell cultures, which will enable in vivo predictions of transporter function. In the clinical section, we describe new genomic and pharmacogenomic research, highlighting important polymorphisms in transporters that are clinically relevant to many drugs. Finally, we describe new clinical tools, which are becoming increasingly available to enable precision medicine, with the application of tissue‐derived small extracellular vesicles and real‐world biomarkers.
... The proximal tubule is the primary functional site for transportmediated reabsorption and secretion of xenobiotics. Active transport of xenobiotics is achieved given the polarized configuration of the proximal tubule's epithelial cells and involves transporters found on the brush-border containing apical side and the basolateral side (Chang et al., 2016b). Therefore, several microphysiological devices have been developed to model the proximal tubule (Chang et al., 2016b;Sakolish et al., 2016). ...
... Active transport of xenobiotics is achieved given the polarized configuration of the proximal tubule's epithelial cells and involves transporters found on the brush-border containing apical side and the basolateral side (Chang et al., 2016b). Therefore, several microphysiological devices have been developed to model the proximal tubule (Chang et al., 2016b;Sakolish et al., 2016). An early kidney bioreactor design demonstrated the seeding and culture of canine (MacKay et al., 1998) and porcine (Humes et al., 1999) renal tubule cells into a hollow polysulferone filament suspended in an "extracapillary space" which aimed to mimic the vascular component of the tubule. ...
Kidney is a major route of xenobiotic excretion, but the accuracy of preclinical data for predicting in vivo clearance is limited by species differences and non-physiologic 2D culture conditions. Microphysiological systems can potentially increase predictive accuracy due to their more realistic 3D environment and incorporation of dynamic flow. We used a renal proximal tubule microphysiological device to predict renal reabsorption of five compounds: creatinine (negative control), perfluorooctanoic acid (positive control), cisplatin, gentamicin, and cadmium. We perfused compound-containing media to determine renal uptake/reabsorption, adjusted for non-specific binding. A physiologically-based parallel tube model was used to model reabsorption kinetics and make predictions of overall in vivo renal clearance. For all compounds tested, the kidney tubule chip combined with physiologically-based modeling reproduces qualitatively and quantitatively in vivo tubular reabsorption and clearance. However, because the in vitro device lacks filtration and tubular secretion components, additional information on protein binding and the importance of secretory transport is needed in order to make accurate predictions. These and other limitations, such as the presence of non-physiological compounds such as antibiotics and bovine serum albumin in media and the need to better characterize degree of expression of important transporters, highlight some of the challenges with using microphysiological devices to predict in vivo pharmacokinetics.
... Frontiers in Bioengineering and Biotechnology | www.frontiersin.org in microfluidic devices for the purposes of in vitro toxicity testing and drug screening (Theobald et al., 2017). Chang et al. developed an integrated liver-kidney microphysiological (MPS) system to identify nephrotoxic liver-metabolized chemicals using the connected liver-on-a-chip and kidney-on-a-chip (Chang et al., 2016). Their results show the important specific purpose of hepatic biotransformation in toxicity research and validation of in vitro and in vivo model comparisons (Chang et al., 2016). ...
... Chang et al. developed an integrated liver-kidney microphysiological (MPS) system to identify nephrotoxic liver-metabolized chemicals using the connected liver-on-a-chip and kidney-on-a-chip (Chang et al., 2016). Their results show the important specific purpose of hepatic biotransformation in toxicity research and validation of in vitro and in vivo model comparisons (Chang et al., 2016). ...
In vitro models are very important in medicine and biology, because they provide an insight into cells' and microorganisms' behavior. Since these cells and microorganisms are isolated from their natural environment, these models may not completely or precisely predict the effects on the entire organism. Improvement in this area is secured by organ-on-a-chip development. The organ-on-a-chip assumes cells cultured in a microfluidic chip. The chip simulates bioactivities, mechanics and physiological behavior of organs or organ systems, generating artificial organs in that way. There are several cell lines used so far for each tested artificial organ. For lungs, mostly used cell lines are 16HBE, A549, Calu-3, NHBE, while mostly used cell lines for liver are HepG2, Hep 3B, TPH1, etc. In this paper, state of the art for lung and liver organ-on-a-chip is presented, together with the established in vitro testing on lung and liver cell lines, with the emphasis on Calu-3 (for lung cell lines) and Hep-G2 (for liver cell lines). Primary focus in this review is to discuss different researches on the topics of lung and liver cell line models, approaches in determining fate and transport, cell partitioning, cell growth and division, as well as cell dynamics, meaning toxicity and effects. The review is finalized with current research gaps and problems, stating potential future developments in the field.
... To address this need, organotypic liver systems aim to incorporate the complexities of the native human liver in vitro, including three-dimensional (3D) architecture, multiple cell types, and the dynamic microenvironment stemming from blood flow through the sinusoids. The development of complex organotypic liver systems has been reviewed elsewhere, and this section of the review will focus on how these organotypic systems have been adapted for studying NAFL and NASH [15][16][17][18][19]. Though immortalized human cell lines, such as HUH7 and HepG2, as well as iPSC-derived hepatocytes, can be induced towards a steatotic phenotype in culture, multiple studies have shown that these cells poorly represent the native liver metabolic function, thus keeping primary human hepatocytes as the gold standard for in vitro studies [20][21][22]. ...
... Diverse approaches have been utilized to combine these different cell types into 3D tissues in order to mimic the architecture found in the intact liver, including layered co-cultures, spheroids, micro-patterned surfaces, human precision cut liver slices (hPCLS), and bioprinting [15][16][17][18][19]26]. Further, many of these tissues can be cultured within specialized devices (e.g. ...
Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the Western world, affecting about 1/3 of the US general population and remaining as a significant cause of morbidity and mortality. The hallmark of the disease is the excessive accumulation of fat within the liver cells (hepatocytes), which eventually paves the way to cellular stress, injury and apoptosis. NAFLD is strongly associated with components of the metabolic syndrome and is fast emerging as a leading cause of liver transplant in the USA. Based on clinico-pathologic classification, NAFLD may present as isolated lipid collection (steatosis) within the hepatocytes (referred to as non-alcoholic fatty liver; NAFL); or as the more aggressive phenotype (known as non-alcoholic steatohepatitis; NASH). There are currently no regulatory agency- approved medication for NAFLD, despite the enormous work and resources that have gone into the study of this condition. Therefore, there remains a huge unmet need in developing and utilizing pre-clinical models that will recapitulate the disease condition in humans. In line with progress being made in developing appropriate disease models, this review highlights the cutting-edge preclinical in vitro and animal models that try to recapitulate the human disease pathophysiology and/or clinical manifestations.
... The presence of different proteins with more or less ability to bind CO in each organ will also strongly influence the delivery and accumulation of CO in the organism. In addition, the high CO levels in the liver and kidney is in accordance with the putative role of these two organs in metabolism and excretion of drugs [57]. Although these measurements were conducted after acute administration of the compounds, our previous report showed that prolonged treatment with 401 during HFD in mice leads to reduced weight gain and improved insulin and glucose metabolism, including a decrease in liver steatosis [23]. ...
Metal carbonyls have been developed as carbon monoxide-releasing molecules (CO-RMs) to deliver CO for therapeutic purposes. The manganese-based CORM-401 has been recently reported to exert beneficial effects in obese animals by reducing body weight gain, improving glucose metabolism and reprogramming adipose tissue towards a healthy phenotype. Here, we report on the synthesis and characterization of glyco-CORMs, obtained by grafting manganese carbonyls on dextrans (70 and 40kDa), based on the fact that polysaccharides facilitate the targeting of drugs to adipose tissue. We found that glyco-CORMs efficiently deliver CO to cells in vitro with higher CO accumulation in adipocytes compared to other cell types. Oral administration of two selected glyco-CORMs (5b and 6b) resulted in CO accumulation in various organs, including adipose tissue. In addition, glyco-CORM 6b administered for eight weeks elicited anti-obesity and positive metabolic effects in mice fed a high fat diet. Our study highlights the feasibility of creating carriers with multiple functionalized CO-RMs.
... The design of complex and controllable micro-devices relying on the recent advances in engineering technology mimics the environment close to in vivo physiological conditions (Chang et al., 2016;Maschmeyer et al., 2015;Li et al., 2016). Many studies have used living cells to model kidney tissue, such as renal tubules (Jang and Suh, 2010;Weber et al., 2016;Batchelder et al., 2015). ...
... The laboratory handling of 24-well plates is part of common training and ensures comfortable manipulation by the user. Similar developments have been initiated in recent years by leading manufacturers of laboratory products [115]. In our case, within the inserts, different microstructured scaffolds can be used. ...
Recent trends in 3D cell culturing has placed organotypic tissue models at another level. Now, not only is the microenvironment at the cynosure of this research, but rather, microscopic geometrical parameters are also decisive for mimicking a tissue model. Over the years, technologies such as micromachining, 3D printing, and hydrogels are making the foundation of this field. However, mimicking the topography of a particular tissue-relevant substrate can be achieved relatively simply with so-called template or morphology transfer techniques. Over the last 15 years, in one such research venture, we have been investigating a micro thermoforming technique as a facile tool for generating bioinspired topographies. We call them MatriGrid®s. In this research account, we summarize our learning outcome from this technique in terms of the influence of 3D micro morphologies on different cell cultures that we have tested in our laboratory. An integral part of this research is the evolution of unavoidable aspects such as possible label-free sensing and fluidic automatization. The development in the research field is also documented in this account.
... 14−17 Further new concepts in diagnostics and therapeutics such as "liver-on-a-chip" also rely on the use of human P450s. 18,19 In contrast to the mammalian P450s, bacterial P450s are soluble enzymes, show higher turnover, are readily expressed in E. coli, and use oxygen more efficiently (higher coupling efficiency). 10 These features make bacterial P450s attractive for applications in biocatalysis. ...
... In line, bladder cells need to maintain elevated mechanosensory competence together with sophisticated response to chemicals. Not surprisingly, in the development of novel organ-on-a-chip toxicological approaches, bladder and renal cells play a paramount role (Jang et al., 2013;Kim and Takayama, 2015;Xu et al., 2015;Chang et al., 2016;Wilmer et al., 2016;Musah et al., 2017;Vriend et al., 2019;Del Favero and Kraegeloh, 2020). Mechanistically, microfluidic devices can include shear stress or exclude it in order to create a solute gradient without a physical component (Lee et al., 2016;Oleaga et al., 2016). ...
Accumulation of xenobiotics and waste metabolites in the urinary bladder is constantly accompanied by shear stress originating from the movement of the luminal fluids. Hence, both chemical and physical cues constantly modulate the cellular response in health and disease. In line, bladder cells have to maintain elevated mechanosensory competence together with chemical stress response adaptation potential. However, much of the molecular mechanisms sustaining this plasticity is currently unknown. Taking this as a starting point, we investigated the response of T24 urinary bladder cancer cells to shear stress comparing morphology to functional performance. T24 cells responded to the shear stress protocol (flow speed of 0.03 ml/min, 3 h) by significantly their its surface area. When exposed to deoxynivalenol-3-sulfate (DON-3-Sulf), bladder cells increased this response in a concentration-dependent manner (0.1–1 µM). DON-3-Sulf is a urinary metabolite of a very common food contaminant mycotoxin (deoxynivalenol, DON) and was already described to enhance proliferation of cancer cells. Incubation with DON-3-Sulf also caused the enlargement of the endoplasmic reticulum (ER), decreased the lysosomal movement, and increased the formation of actin stress fibers. Similar remodeling of the endoplasmic reticulum and area spread after shear stress were observed upon incubation with the autophagy activator rapamycin (1–100 nM). Performance of experiments in the presence of chloroquine (chloroquine, 30 μM) further contributed to shed light on the mechanistic link between adaptation to the biomechanical stimulation and ER stress response. At the molecular level, we observed that ER reshaping was linked to actin organization, with the two components mutually regulating each other. Indeed, we identified in the ER stress–cytoskeletal rearrangement an important axis defining the physical/chemical response potential of bladder cells and created a workflow for further investigation of urinary metabolites, food constituents, and contaminants, as well as for pharmacological profiling.
... 29 Among them are "liver-on-a-chip", "kidney-on-achip", "muscle-on-a-chip", and "lymph node-on-a-chip". [30][31][32][33][34] A recent study demonstrated the feasibility of constructing a mimetic "skin-on-chip" (SoC) model in vitro, which was used for drug toxicity testing and disease studies. 35 However, no study has applied a SoC model for T cell migration studies. ...
A microfluidics-based three-dimensional skin-on-chip (SoC) model is developed in this study to enable quantitative studies of transendothelial and transepithelial migration of human T lymphocytes in mimicked skin inflammatory microenvironments and to test new drug candidates. The keys results include 1) CCL20-dependent T cell transmigration is significantly inhibited by an engineered CCL20 locked dimer (CCL20LD), supporting the potential immunotherapeutic use of CCL20LD for treating skin diseases such as psoriasis; 2) transepithelial migration of T cells in response to a CXCL12 gradient mimicking T cell egress from the skin is significantly reduced by a sphingosine-1-phosphate (S1P) background, suggesting the role of S1P for T cell retention in inflamed skin tissues; and 3) T cell transmigration is induced by inflammatory cytokine stimulated epithelial cells in the SoC model. Collectively, the developed SoC model recreates a dynamic multi-cellular micro-environment that enables quantitative studies of T cell transmigration at a single cell level in response to physiological cutaneous inflammatory mediators and potential drugs.
... The deposition of nanoparticles in these organs produces harmful effects. Therefore, liver and kidney cell lines, such as hepatocellular carcinoma cell line (Hep-G2) and renal proximal tubule cell line (LLC-PK1), are usually selected for organrelated toxicity studies [156]. Cell viability of these cell lines can be investigated by a variety of methods, including the measurement of loss of metabolic activity, membrane integrity, and monolayer adherence, and cell cycle analysis. ...
Introduction: The utilization of polymeric nanoparticles, as drug payloads, has been extensively
prevailed in cancer therapy. However, the precise distribution of these nanocarriers is restrained by
various physiological and cellular obstacles. Nanoparticles must avoid nonspecific interactions with
healthy cells and in vivo compartments to circumvent these barriers. Since in vivo interactions of
nanoparticles are mainly dependent on surface properties of nanoparticles, efficient control on surface constituents is necessary for the determination of nanoparticles’ fate in the body.
Areas covered: In this review, the surface-modified polymeric nanoparticles and their utilization in cancer treatment were elaborated. First, the interaction of nanoparticles with numerous in vivo barriers was highlighted. Second, different strategies to overcome these obstacles were described. Third, some inspiring examples of surface-modified nanoparticles were presented. Later, fabrication and characterization methods of surface-modified nanoparticles were discussed. Finally, the applications of these nanoparticles in different routes of treatments were explored.
Expert opinion: Surface modification of anticancer drug-loaded polymeric nanoparticles can enhance the efficacy, selective targeting, and biodistribution of the anticancer drug at the tumor site.
... Several working groups devoted their attention to the integration of organ-on-a-chip microfluidic models in the pharmacokinetics analysis. Numerous recent reviews summarize detailed information about the intestinal tract [176,177], liver [5,178] or multi-organ-systems [177,[179][180][181][182][183]. Indeed, cell cultivation in presence of biomechanical cues can help to reproduce in vitro processes that typically characterize the ADME in vivo. ...
Integration of biophysical stimulation in test systems is established in diverse branches of biomedical sciences including toxicology. This is largely motivated by the need to create novel experimental setups capable of reproducing more closely in vivo physiological conditions. Indeed, we face the need to increase predictive power and experimental output, albeit reducing the use of animals in toxicity testing. In vivo, mechanical stimulation is essential for cellular homeostasis. In vitro, diverse strategies can be used to model this crucial component. The compliance of the extracellular matrix can be tuned by modifying the stiffness or through the deformation of substrates hosting the cells via static or dynamic strain. Moreover, cells can be cultivated under shear stress deriving from the movement of the extracellular fluids. In turn, introduction of physical cues in the cell culture environment modulates differentiation, functional properties, and metabolic competence, thus influencing cellular capability to cope with toxic insults. This review summarizes the state of the art of integration of biophysical stimuli in model systems for toxicity testing, discusses future challenges, and provides perspectives for the further advancement of in vitro cytotoxicity studies.
... Efforts have been initiated in the right direction as highlighted by Chang et al. but demonstration of expression and activity of key enzymes and transporters in this system would be the next most logical step. 157 Weber et al. have demonstrated expression of metabolic enzymes and transporters including some data on function in a kidney MPS model. 158 Such advances can tremendously help in moving the field when translatability to the in vivo situation has been demonstrated. ...
Over the last decade, progress has been made on the development of microphysiological systems (MPS) for Absorption, Distribution, Metabolism, and Excretion (ADME) applications. Central to this progress has been proof of concept data generated by academic and industrial institutions followed by broader characterization studies, which provide evidence for scalability and applicability to drug discovery and development. In this review, we describe some of the advances made for specific tissue MPS and outline the desired functionality for such systems, which are likely to make them applicable for practical use in the pharmaceutical industry. Single organ MPS platforms will be valuable for modelling tissue-specific functions. However, dynamic organ crosstalk, especially in the context of disease or toxicity, can only be obtained with the use of inter-linked MPS models which will enable scientists to address questions at the intersection of pharmacokinetics (PK) and efficacy, or PK and toxicity. In the future, successful application of MPS platforms that closely mimic human physiology may ultimately reduce the need for animal models to predict ADME outcomes and decrease the overall risk and cost associated with drug development.
... The liver and the kidney are two very complex organs that together lead to the metabolism and excretion of drugs, respectively. To predict biotransformation (liver) and elimination (kidney) in humans is a rather difficult task to achieve using normal 2D cultures ( Chang et al. 2016); however, the combination of these two complex organs in an organ-on-a-chip platform made it possible to study the toxic effects of aristolochic acid I, a well-known nephrotoxicant that requires first hepatic bioactivation ( Chang et al. 2017). Table 3 summarizes the different in vitro models discussed to study drug-induced Fig. 4 Schematic figure of the different in vitro models developed for use in nephrotoxicity screenings ...
... The liver and the kidney are two very complex organs that together lead to the metabolism and excretion of drugs, respectively. To predict biotransformation (liver) and elimination (kidney) in humans is a rather difficult task to achieve using normal 2D cultures (Chang et al. 2016); however, the combination of these two complex organs in an organ-on-a-chip platform made it possible to study the toxic effects of aristolochic acid I, a well-known nephrotoxicant that requires first hepatic bioactivation (Chang et al. 2017). Table 3 summarizes the different in vitro models discussed to study drug-induced nephrotoxicity. ...
The kidney is frequently involved in adverse effects caused by exposure to foreign compounds, including drugs. An early prediction of those effects is crucial for allowing novel, safe drugs entering the market. Yet, in current pharmacotherapy, drug-induced nephrotoxicity accounts for up to 25% of the reported serious adverse effects, of which one-third is attributed to antimicrobials use. Adverse drug effects can be due to direct toxicity, for instance as a result of kidney-specific determinants, or indirectly by, e.g., vascular effects or crystals deposition. Currently used in vitro assays do not adequately predict in vivo observed effects, predominantly due to an inadequate preservation of the organs’ microenvironment in the models applied. The kidney is highly complex, composed of a filter unit and a tubular segment, together containing over 20 different cell types. The tubular epithelium is highly polarized, and the maintenance of this polarity is critical for optimal functioning and response to environmental signals. Cell polarity is dependent on communication between cells, which includes paracrine and autocrine signals, as well as biomechanic and chemotactic processes. These processes all influence kidney cell proliferation, migration, and differentiation. For drug disposition studies, this microenvironment is essential for prediction of toxic responses. This review provides an overview of drug-induced injuries to the kidney, details on relevant and translational biomarkers, and advances in 3D cultures of human renal cells, including organoids and kidney-on-a-chip platforms.
... (continued) (Chang et al. 2016;Vriend et al. 2018) • Nephrotoxicity study (Kim et al. 2016;Li et al. 2017) 6. Pancreas-on-a-chip (a) Culture of pancreatic islet cells for insulin secretion ...
Over the past decades, stem cell technology has revolutionalized medical biotechnology due to the unlimited self-renewal ability and differentiation capacity of stem cells to generate cells and tissues of the entire human body. Many efforts have focused on providing cutting-edge stem cell therapies in order to repair or replace damaged cells or tissues, hoping to ultimately cure devastating diseases. Undoubtedly, this novel technology guarantees a serial entrepreneur’s confidence in the future prospects of stem cell-based products and services. Here, we describe the state of the art of several applications of adult stem cells, as well as of embryonic and induced pluripotent stem cells in biotechnology that represent entrepreneurial opportunities. Although the contribution of stem cells to medical research is enormous, several hurdles still have to be overcome, including ethical and regulatory issues, functional maturation of stem cell progenitors, stringent manufacturing guidelines, immune rejection, and tumorigenicity.
... Significantly more work is needed to advance these models, but such systems offer the potential to study drug metabolism and toxicity in a model that maintains considerably more three-dimensional architecture and mimics blood and urine flow through the kidney. 104 ...
The kidney plays a critical role in the elimination of many xenobiotics, and drug-induced kidney injury is a risk factor in drug discovery and development. In addition, accumulation of nephrotoxic compounds, a process often controlled by xenobiotic transporters, is often a prerequisite to kidney injury. Such adverse events are dependent on many transporters, particularly those in the solute carrier and adenosine triphosphate–binding cassette superfamilies. This review details the current understanding of how kidney transporters contribute to toxic outcomes and highlights critical knowledge gaps regarding species differences that account for some lack of predictivity between preclinical animal models and human beings. The basic classification, physiological roles, and species differences of solute carrier and adenosine triphosphate–binding cassette transporters is reviewed, along with mechanistic details for drug-induced kidney injury involving transporters. The use of preclinical data (in vitro and in vivo), clinical data, and conventional as well as emerging tools for studying kidney transporter function are summarized. Finally, we highlight some challenges and opportunities to improve experimental approaches to support preclinical and clinical studies of kidney transporters and their role in nephrotoxicity.
... MPS models take hepatocyte cultures a step further with the introduction of flow, and have begun to provide a platform to culture hepatocytes over extended periods of time and in doing so, these models demonstrate enhanced viability and activity under dynamic culture conditions. Liver MPS, alongside other hepatocyte in vitro models, have been the subject of recent reviews summarizing their advantages and giving an overview on current applications (Chang et al., 2016;Hughes et al., 2017;Beckwitt et al., 2018). Here we summarize advances made on the evaluation of hepatic drug transporters. ...
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 physiological conditions to in vitro cell culture models have enormous potential to better reproduce the morphology and transport activity across several organ models especially in tissues like the liver, kidney, intestine or the blood-brain-barrier where 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. Further, we analyse 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.
... Whether a drug is a substrate and/or an inhibitor of a particular transporter protein can be studied in vitro with transporter-overexpressing cells [5,6]. However, the in vivo consequences are not always easy to predict. ...
Physiologically based pharmacokinetic modelling (PBPK) is a powerful tool to predict in vivo pharmacokinetics based on physiological parameters and data from in vivo studies and in vitro assays. In vivo PBPK modelling in laboratory animals by noninvasive imaging could help to improve the in vivo-in vivo translation towards human pharmacokinetics modelling. We evaluated the feasibility of PBPK modelling with PET data from mice. We used data from two of our PET tracers under development, [ ¹¹ C]AM7 and [ ¹¹ C]MT107. PET images suggested hepatobiliary excretion which was reduced after cyclosporine administration. We fitted the time-activity curves of blood, liver, gallbladder/intestine, kidney, and peripheral tissue to a compartment model and compared the resulting pharmacokinetic parameters under control conditions ([ ¹¹ C]AM7 n=2 ; [ ¹¹ C]MT107, n=4 ) and after administration of cyclosporine ([ ¹¹ C]MT107, n=4 ). The modelling revealed a significant reduction in [ ¹¹ C]MT107 hepatobiliary clearance from 35.2±10.9 to 17.1±5.6 μ l/min after cyclosporine administration. The excretion profile of [ ¹¹ C]MT107 was shifted from predominantly hepatobiliary ( CLH / CLR = 3.8±3.0 ) to equal hepatobiliary and renal clearance ( CLH / CLR = 0.9±0.2 ). Our results show the potential of PBPK modelling for characterizing the in vivo effects of transporter inhibition on whole-body and organ-specific pharmacokinetics.
... These characteristics are mostly lacking in current in vitro screening models. Modular MPS models that mimic microphysiology of the hepatic lobule, 17 transport barriers to evaluate drug and xenobiotic metabolism, 18 and microvascular networks for realistic distribution systems 19 provide organ-specific structural and functional characteristics for translating local dosimetry into a quantitative response. ...
Microphysiological systems (MPS) and computer simulation models that recapitulate the underlying biology and toxicology of critical developmental transitions are emerging tools for developmental effects assessment of drugs/chemicals. Opportunities and challenges exist for their application to alternative, more public health relevant and efficient chemical toxicity testing methods. This is especially pertinent to children’s health research and the evaluation of complex embryological and reproductive impacts of drug/chemical exposure. Scaling these technologies to higher throughput is a key challenge and drives the need for in silico models for quantitative prediction of developmental toxicity to inform safety assessments. One example is cellular agent-based models, constructed from extant embryology, that produce data useful to simulate critical developmental transitions and thereby predict phenotypic consequences of disruption in silico. Biologically inspired MPS models built from human induced pluripotent stem (iPS)-derived cells and synthetic matrices that recapitulate organ-specific physiologies and native tissue architectures are providing exciting new research opportunities to advance the assessment of developmental toxicity and offer the possibility of deriving a full ‘human on a chip’ system, or a ‘Homunculus.’
Impact statement
This ‘commentary’ summarizes research needs and opportunities for engineered MPS models for developmental and reproductive toxicity testing. Emerging concepts can be taken forward to a virtual tissue modeling framework for assessing chemical (and non-chemical) stressors on human development. These models will advance children’s health research, both basic and translational and new ways to evaluate complex embryological and reproductive impacts of drug and chemical exposures to inform safety assessments.
... le celltypes may also prolong viability and functionality for long-term in vitro culture (You etal. ,2015;Berger etal. ,2015;Trask etal. ,2014;Bale etal. , 2014).We reviewed in vitro/ex vivo human hepatic cellsystems used in biotransformation and transporterstudies from traditional assays to the most recently advanced three-dimension (3D) cultures (Chang etal. ,2016). ...
The liver is the main site for drug and xenobiotics metabolism, including inactivation or bioactivation. In order to improve the predictability of drug safety and efficacy in clinical development, and to facilitate the evaluation of the potential human health effects from exposure to environmental contaminants, there is a critical need to accurately model human organ systems such as the liver in vitro. We are developing a microphysiological system (MPS) based on a new commercial microfluidic platform (Nortis, Inc.) that can utilize primary liver cells from multiple species (e.g., rat and human). Compared to conventional monolayer cell culture, which typically survives for 5–7 days or less, primary rat or human hepatocytes in an MPS exhibited higher viability and improved hepatic functions, such as albumin production, expression of hepatocyte marker HNF4α and canaliculi structure, for up to 14 days. Additionally, induction of Cytochrome P450 (CYP) 1A and 3A4 in cryopreserved human hepatocytes was observed in the MPS. The acute cytotoxicity of the potent hepatotoxic and hepatocarcinogen, aflatoxin B1, was evaluated in human hepatocytes cultured in an MPS, demonstrating the utility of this model for acute hepatotoxicity assessment. These results indicate that MPS-cultured hepatocytes provide a promising approach for evaluating chemical toxicity in vitro.
... Yet, due to complex cellular microenvironments of human organs (including the liver and kidneys), accurate modeling of transport function in vitro has not been satisfactorily achieved. In this issue, Chang et al. 11 review details of novel microphysiological systems that lead the field in these investigations. Microphysiological modeling of the liver and kidneys using "organ-on-a-chip" technologies is rapidly advancing in human transport function assessment, and has emerged as a promising method to evaluate drug and xenobiotic metabolism. ...
Drug transporter research conducted over the last several decades has led to a greatly advanced understanding of the mechanisms underlying the principles of drug absorption and disposition. Although many transporters remain poorly characterized, there is ample evidence that the drug transporter field will ultimately provide vital support to routine patient management, and will play a key role in the discovery, development, and evaluation of innovative, cutting-edge therapies. © 2016 American Society for Clinical Pharmacology and Therapeutics
Predicting transporter-based drug clearance (CL) and tissue concentrations (TC) in humans is important to reduce the risk of failure during drug development. In addition, when transporters are present at the tissue:blood interface (e.g., in the liver, blood-brain barrier), predicting TC is important to predict the drug's efficacy and safety. With the advent of quantitative targeted proteomics, in vitro to in vivo extrapolation (IVIVE) of transporter-based drug CL and TC is now possible using transporter-expressing models (cells lines, membrane vesicles) and the in vivo to in vitro relative expression of transporters (REF) as a scaling factor. Unlike other approaches based on physiological scaling, the REF approach is not dependent on the availability of primary cells. Here, we review the REF approach and compare it with other IVIVE approaches such as the relative activity factor approach and physiological scaling. For each of these scaling approaches, we review their underlying principles, assumptions, methodology, predictive performance, as well as advantages and limitations. Finally, we discuss current gaps in IVIVE of transporter-based CL and TC and propose possible reasons for these gaps as well as areas to investigate to bridge these gaps.
Acute liver failure (ALF) is a rapidly progressive disease with high morbidity and mortality rates. Liver transplantation and artificial liver support systems, such as artificial livers (ALs) and bioartificial livers (BALs), are the two major therapies for ALF. Compared to ALs, BALs are composed of functional hepatocytes that provide essential liver functions, including detoxification, metabolite synthesis, and biotransformation. Furthermore, BALs can potentially provide effective support as a form of bridging therapy to liver transplantation or spontaneous recovery for patients with ALF. In this review, we systematically discussed the currently available state-of-the-art designs and manufacturing processes for BAL support systems. Specifically, we classified the cell sources and bioreactors that are applied in BALs, highlighted the advanced technologies of hepatocyte culturing and bioreactor fabrication, and discussed the current challenges and future trends in developing next generation BALs for large scale clinical applications.
The bark extract of Rhizophora mucronata (BERM) was recently reported for its prominent in vitro protective effects against liver cell line toxicity caused by various toxicants, including ethanol. Here, we aimed to verify the in vivo hepatoprotective effects of BERM against ethanol intoxication. An oral administration of different concentrations (100, 200, and 400 mg/kg) of BERM prior to high-dose ethanol via intraperitoneal injection was performed in mice. On the 7th day, liver and kidney sections were dissected out for histopathological examination. The ethanol intoxication caused large areas of liver necrosis while the kidneys were not affected. Pre-BERM administration decreased ethanol-induced liver injury, as compared to the mice treated with ethanol alone. In addition, the pre-BERM administration resulted in a decrement in the level of ethanol-induced oxidative stress, revealed by a concomitant increase of GSH and a decrease of MDA hepatic levels. The BERM extract also reversed the ethanol-induced liver injury and hepatotoxicity, characterized by the low detection of TNF-α gene expression level and fragmented DNA, respectively. Altogether, BERM extract exerts antioxidative activities and present promising hepatoprotective effects against ethanol intoxication. The identification of the related bioactive compounds will be of interest for future use at physiological concentrations in ethanol-intoxicated individuals.
Introduction
The utilization of polymeric nanoparticles, as drug payloads, has been extensively prevailed in cancer therapy. However, the precise distribution of these nanocarriers is restrained by various physiological and cellular obstacles. Nanoparticles must avoid non-specific interactions with healthy cells and in vivo compartments to circumvent these barriers. Since in vivo interactions of nanoparticles are mainly dependent on surface properties of nanoparticles, efficient control on surface constituents is necessary for the determination of nanoparticles’ fate in the body.
Areas covered
In this review, the surface modified polymeric nanoparticles and their utilization in cancer treatment were elaborated. First, the interaction of nanoparticles with numerous in vivo barriers was highlighted. Second, different strategies to overcome these obstacles were described. Third, some inspiring examples of surface modified nanoparticles were presented. Later, fabrication and characterization methods of surface modified nanoparticles were discussed. Finally, the applications of these nanoparticles in different routes of treatments were explored.
Expert opinion
Surface modification of anticancer drug loaded polymeric nanoparticles can enhance the efficacy, selective targeting, and biodistribution of the anticancer drug at the tumor site.
Background
Dug-metabolizing enzymes and transporters play key roles in drug disposition and drug interactions. The alterations of their expression will influence drug pharmacokinetics and pharmacodynamics. However, the changes of the expression of enzymes and transporters in the disease state are still unclear.
Objective
Our study was to investigate the changes of the expression of main enzymes and drug transporters distributed in Adriamycin nephropathy rat liver, kidney and intestine.
Method
An intravenous injection with a single dose of Adriamycin (6mg/kg) was made to establish Adriamycin nephropathy (AN) model and normal groups were injected with normal saline. Serum was collected for lipid metabolism, renal and hepatic function measurement. The real-time PCR and western blot were applied to determine the mRNA and protein expression of drug enzymes and transporters.
Results
In kidney, a greater expression of Mdr1, Mrp2, Mrp4 Oat2 and Oct2 mRNA was found in AN rats as compared with control rats. In liver, the expression of Bcrp mRNA was more doubled or trebled than control groups and a downregulation of Mdr1, Mrp2, Mrp4 and Bsep gene expression was found in AN rats. Besides, we observed a downward trend of Cyp1a2, Cyp3a4 and Cyp2c9 mRNA levels in AN groups. In duodenum, the expression of Mdr1 and Mrp3 mRNA level was decreased, while Bcrp and Mrp2 mRNA was increased.
Conclusion
The changes of drug-metabolizing enzymes and transporters expression in AN rats were clarified, which may be beneficial for understanding the altered pharmacokinetics and pharmacodynamics of clinical drugs and reduce unexpected clinical findings for nephropathy patients.
Introduction: Transporters and enzymes play an important role in absorption, distribution, clearance and elimination of drugs. Involvement of transporter-enzyme interplay in the hepatic disposition and pharmacokinetics of drugs is increasingly appreciated.
Areas covered: This review provides an overview of the extended clearance concept and usefulness of extended clearance classification system (ECCS) in early identification of predominant clearance mechanisms. Clinical studies demonstrating transporter-enzyme interplay are highlighted, and the in vitro tools and challenges in scaling clearance are discussed. The utility of animal models and modelling approaches for evaluating hepatic clearance and drug-drug interactions are reviewed.
Expert opinion: Clinical evidence exists supporting organic anion transporting peptide (OATP)1B and drug metabolizing enzymes involvement in clearance of ECCS class 1B drugs. Emerging evidence point towards contribution of organic cation transporter (OCT)1 to hepatic uptake of cationic drugs. Although, limited clinical evidence is presented, preclinical studies and modeling suggests organic anion transporter (OAT)2-enzyme interplay in clearance of class 1A drugs. Data from in vitro assays and preclinical models coupled with physiologically-based modelling approaches are key for understanding transporter-enzyme interplay, enabling prediction of pharmacokinetics, tissue exposure and drug interactions. Current methodologies incur certain limitations, and emphasis should be placed on the development of physiologically relevant in vitro models (eg. micro-physiological systems, albumin in incubations) and characterize in vivo animal models to inform mechanistic modeling and improve confidence in prospective predictions.
In this study, canine adipose-derived mesenchymal stem cells (cADSCs) therapeutic potential was investigated in artificially induced acute liver injury model by CCl4 in canines. The primary cADSCs cells were cultured and then intravenously administered into the canine animal model. Six cross-breed dogs were divided into three groups including blank control group, CCl4 model group, CCl4 induced cADSCs transplantation group.
The results showed that after intraperitoneal injection of CCl4 solution, the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Albumin (ALB) in peripheral blood of experimental canines confirmed the correct induction of acute liver injury. Moreover, the liver structure showed clear macroscopic damage. The cADSCs were homed in the liver of the administered animals. The AST, ALT and ALB in the peripheral blood rapidly decreased. H&E and PAS histological evaluation showed that both the structure of canine liver tissue and the ability to synthesize hepatic glycogen could be restored to the control level after cADSCs transplantation. Therefore, cADSCs can play a therapeutic role in the recovery of liver injury. Overall, this study demonstrates that the primary cADSCs transplantation into the acute liver injury model induced by intravenous injection can play a certain therapeutic role in the recovery of liver in canines. These results may provide a new treatment idea for acute liver disease in pets clinically.
Liver plays a major role in drug metabolism and is one of the main sites of drug adverse effects. Microphysiological systems (MPS), also known as organs‐on‐a‐chip, are a class of microfluidic platforms that recreate properties of tissue microenvironments. Among different properties, the liver microenvironment is three‐dimensional, fluid flows around its cells, and different cell types regulate its function. Liver MPS aim to recreate these properties and enable drug testing and measurement of functional endpoints. Tests with these systems have demonstrated their potential for predicting clinical drug effects. Properties of liver MPS that improve the physiology of cell culture are reviewed, specifically focusing on the importance of recreating a physiological microenvironment to evaluate and model drug effects. Advances in modeling hepatic function by leveraging MPS are addressed, noting the need for standardization in the use, quality control, and interpretation of data from these systems. This article is protected by copyright. All rights reserved.
The products resulting from the techniques and processes of biotechnology continue to grow at an exponential rate, and the expectations are that an even greater percentage of drug development and clinically-utilized pharmaceuticals worldwide will be classified as biologics. A recent Pharmaceutical Research and Manufacturers of America report (PhRMA, 2017 industry profile: medicines are transforming the trajectory of disease. Available at http://phrma-docs.phrma.org/industryprofile/pdfs/2017IndustryProfile_MedicinesareTransforming.pdf, 2017) notes that there are currently about 7000 medicines in clinical development globally and 80% in the pipeline have the potential to be first-in-class treatments. Most pertinent to this textbook, the majority of these medicines in development were impacted directly or indirectly by biotechnologies at one or more points during their lifetime via: target identification, and/or lead identification, and/or lead optimization, and/or clinical development and evaluation and/or product production. Pharmaceutical biotechnology techniques are at the core of most methodologies used today for drug discovery and development of both biologics and small molecules. While recombinant DNA technology and hybridoma techniques were the major methods utilized in pharmaceutical biotechnology through most of its historical timeline, our ever-widening understanding of human cellular function and disease processes and a wealth of additional and innovative biotechnologies have been, and will continue to be, developed in order to harvest the information found in the human genome. These technological advances will provide a better understanding of the relationship between genetics and biological function, unravel the underlying causes of disease, explore the association of genomic variation and drug response, enable personalized and precision medicine, enhance pharmaceutical research, and fuel the discovery and development of new and novel biopharmaceuticals. These revolutionary technologies and additional biotechnology-related techniques are improving the very competitive and costly process of drug development of new medicinal agents, diagnostics, and medical devices. Some of the technologies and techniques described in this chapter are both well established and commonly used applications of biotechnology producing clinically-utilized medicines as well as potential therapeutic products now in the developmental pipeline. New techniques are emerging at a rapid and unprecedented pace and their full impact on the future of molecular medicine will turn dreams into realities.
Renal disease is a global problem with unsustainable health-care costs. There currently exists a lack of accurate human renal disease models that take into account the complex microenvironment of these tissues. Here, we present a reusable microfluidic model of the human proximal tubule and glomerulus, which allows for the growth of renal epithelial cells in a variety of conditions that are representative of renal disease states including altered glomerular filtration rate, hyperglycemia, nephrolithiasis, and drug-induced nephrotoxicity (cisplatin and cyclosporine). Cells were exposed to these conditions under fluid flow or in traditional static cultures to determine the effects of a dynamic microenvironment on the pathogenesis of these renal disease states. The results indicate varying stress-related responses (α-smooth muscle actin (α-SMA) expression, alkaline phosphatase activity, fibronectin, and neutrophil gelatinase-associated lipocalin secretion) to each of these conditions when comparing cells that had been grown in static and dynamic conditions, potentially indicating more realistic and sensitive predictions of human responses and a requirement for a more complex "fit for purpose" model.
Recently, microfluidic organomimetic technology with precise spatiotemporal fluid control has offered unprecedented benefits to create physiologically-relevant in vitro organ models by recapitulating subtle organ-specific variations. The fundamental design principle of the microfluidic organ-on-a-chip (OoC) platform is founded on ‘reverse engineering’ living organs, which are deconstructed to recapitulate their essential function. In addition, OoC has leveraged recapitulation of multiorgan-level function with inter-connection and has modeled human pathophysiology. This review aims to highlight recent advances of the microphysiological dynamic OoC platform, exploring its biomedical and personalized medicine applications. We will discuss the critical aspects of OoC development and provide guidance to researchers to build physiologically-relevant OoCs in terms of cell source, perfusion flow, micro-sized biomimetic organ architecture, and mechanobiological motion. Finally, future directions for multi-OoCs are discussed along with the technical challenges encountered in drug development pipelines of the pharmaceutical industry. © 2018 The Korean Society of Industrial and Engineering Chemistry
Protocols have been established to direct the differentiation of human induced pluripotent stem (iPS) cells into nephron progenitor cells and organoids containing many types of kidney cells, but it has been difficult to direct the differentiation of iPS cells to form specific types of mature human kidney cells with high yield. Here, we describe a detailed protocol for the directed differentiation of human iPS cells into mature, post-mitotic kidney glomerular podocytes with high (>90%) efficiency within 26 d and under chemically defined conditions, without genetic manipulations or subpopulation selection. We also describe how these iPS cell-derived podocytes may be induced to form within a microfluidic organ-on-a-chip (Organ Chip) culture device to build a human kidney Glomerulus Chip that mimics the structure and function of the kidney glomerular capillary wall in vitro within 35 d (starting with undifferentiated iPS cells). The podocyte differentiation protocol requires skills for culturing iPS cells, and the development of a Glomerulus Chip requires some experience with building and operating microfluidic cell culture systems. This method could be useful for applications in nephrotoxicity screening, therapeutic development, and regenerative medicine, as well as mechanistic study of kidney development and disease.
The liver and the kidney are key toxicity target organs during drug development campaigns, as they typically carry the burden of drug transport and metabolism. Primary hepatocytes and proximal tubule epithelial cells grown in traditional in vitro 2-D culture systems do not maintain transporter and metabolic functions, thus limiting their utility for nonclinical toxicology investigations. We have developed a renal and hepatic microphysiological system (MPS) platform that uses a commercially available MPS device as the core cell culture platform for our methodologies. We describe protocols for isolating and propagating human proximal epithelial cells and how to seed and culture a renal MPS to recapitulate the human proximal tubule. We present two methods to culture hepatocytes within an MPS and the steps required to connect a renal MPS to a liver MPS. © 2017 by John Wiley & Sons, Inc.
To date, tissue engineering and organ-on-a-chip devices become more powerful as replacements for animal testings in high throughput drug screenings. However, the majority of the devices are either based on static 2D or 3D cell cultures or on microfluidic channel systems that are usually rectangular and do not fit to the geometry of natural blood vessels. Therefore, a semicircular microfluidic blood vessel scaffold (vasQchip) with a surrounding microfluidic compartment for vascularized 3D cell culture was developed, which resembles the curvature of natural blood vessels. vasQchip is composed of a porous microchannel which is produced by micro-thermoforming of polycarbonate membranes. The pores generated by ion track technology allow for the support of nutrients and gases as well as for the exchange of growth factors or immune cells with the surrounding compartment. Eventually the surrounding compartment can be used for the establishment of 3D cell cultures in order to reconstruct vascularized tissues. Here, the vasQchip for its biocompatibility is characterized to somatic primary cells, the diffusion of molecules through the artificial blood vessels and its suitability for 3D cell culture. In addition, shear stress and flow regimes are simulated in order to mimic the natural environment of vascularized tissues.
Introduction:
The progressive disease spectrum of non-alcoholic fatty liver disease (NAFLD), which includes non-alcoholic steatohepatitis (NASH), is a rapidly emerging public health crisis with no approved therapy. The diversity of various therapies under development highlights the lack of consensus around the most effective target, underscoring the need for better translatable preclinical models to study the complex progressive disease and effective therapies. Areas covered: This article reviews published literature of various mouse models of NASH used in preclinical studies, as well as complex organotypic in vitro and ex vivo liver models being developed. It discusses translational challenges associated with both kinds of models, and describes some of the studies that validate their application in NAFLD. Expert opinion: Animal models offer advantages of understanding drug distribution and effects in a whole body context, but are limited by important species differences. Human organotypic in vitro and ex vivo models with physiological relevance and translatability need to be used in a tiered manner with simpler screens. Leveraging newer technologies, like metabolomics, proteomics, and transcriptomics, and the future development of validated disease biomarkers will allow us to fully utilize the value of these models to understand disease and evaluate novel drugs in isolation or combination.
Microphysiological systems (MPS) provide relevant physiological environments in vitro for studies of pharmacokinetics, pharmacodynamics and biological mechanisms for translational research. Designing multi-MPS platforms is essential to study multi-organ systems. Typical design approaches, including direct and allometric scaling, scale each MPS individually and are based on relative sizes not function.
This study’s aim was to develop a new multi-functional scaling approach for integrated multi-MPS platform design for specific applications. We developed an optimization approach using mechanistic modeling and specification of an objective that considered multiple MPS functions, e.g., drug absorption and metabolism, simultaneously to identify system design parameters.
This approach informed the design of two hypothetical multi-MPS platforms consisting of gut and liver (multi-MPS platform I) and gut, liver and kidney (multi-MPS platform II) to recapitulate in vivo drug exposures in vitro. This allows establishment of clinically relevant drug exposure-response relationships, a prerequisite for efficacy and toxicology assessment.
Design parameters resulting from multi-functional scaling were compared to designs based on direct and allometric scaling. Human plasma time-concentration profiles of eight drugs were used to inform the designs, and profiles of an additional five drugs were calculated to test the designed platforms on an independent set. Multi-functional scaling yielded exposure times in good agreement with in vivo data, while direct and allometric scaling approaches resulted in short exposure durations.
Multi-functional scaling allows appropriate scaling from in vivo to in vitro of multi-MPS platforms, and in the cases studied provides designs that better mimic in vivo exposures than standard MPS scaling methods.
Man-made xenobiotics, whose potential toxicological effects are not fully understood, are oversaturating the already-contaminated environment. Due to the rate of toxicant accumulation, unmanaged disposal, and unknown adverse effects to the environment and the human population, there is a crucial need to screen for environmental toxicants. Animal models and in vitro models are ineffective models in predicting in vivo responses due to inter-species difference and/or lack of physiologically-relevant 3D tissue environment. Such conventional screening assays possess limitations that prevent dynamic understanding of toxicants and their metabolites produced in the human body. Organ-on-a-chip systems can recapitulate in vivo like environment and subsequently in vivo like responses generating a realistic mock-up of human organs of interest, which can potentially provide human physiology-relevant models for studying environmental toxicology. Feasibility, tunability, and low-maintenance features of organ-on-chips can also make possible to construct an interconnected network of multiple-organs-on-chip toward a realistic human-on-a-chip system. Such interconnected organ-on-a-chip network can be efficiently utilized for toxicological studies by enabling the study of metabolism, collective response, and fate of toxicants through its journey in the human body. Further advancements can address the challenges of this technology, which potentiates high predictive power for environmental toxicology studies.
Nephrotoxicity is often underestimated because renal clearance in animals is higher compared to in humans. This paper aims to illustrate the potential to fill in such pharmacokinetic gaps between animals and humans using a microfluidic kidney model. As an initial demonstration, we compare nephrotoxicity of a drug, administered at the same total dosage, but using different pharmacokinetic regimens. Kidney epithelial cell, cultured under physiological shear stress conditions, are exposed to gentamicin using regimens that mimic the pharmacokinetics of bolus injection or continuous infusion in humans. The perfusion culture utilized is important both for controlling drug exposure and for providing cells with physiological shear stress (1.0 dyn cm(-2)). Compared to static cultures, perfusion culture improves epithelial barrier function. We tested two drug treatment regimens that give the same gentamycin dose over a 24 h period. In one regimen, we mimicked drug clearance profiles for human bolus injection by starting cell exposure at 19.2 mM of gentamicin and reducing the dosage level by half every 2 h over a 24 h period. In the other regimen, we continuously infused gentamicin (3 mM for 24 h). Although junctional protein immunoreactivity was decreased with both regimens, ZO-1 and occludin fluorescence decreased less with the bolus injection mimicking regimen. The bolus injection mimicking regimen also led to less cytotoxicity and allowed the epithelium to maintain low permeability, while continuous infusion led to an increase in cytotoxicity and permeability. These data show that gentamicin disrupts cell-cell junctions, increases membrane permeability, and decreases cell viability particularly with prolonged low-level exposure. Importantly a bolus injection mimicking regimen alleviates much of the nephrotoxicity compared to the continuous infused regimen. In addition to potential relevance to clinical gentamicin administration regimens, the results are important in demonstrating the general potential of using microfluidic cell culture models for pharmacokinetics and toxicity studies.
The bioartificial kidney (BAK) aims at improving dialysis by developing 'living membranes' for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) as a fluorescent substrate. Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.
Nephrotoxicity due to drugs and environmental chemicals accounts for significant patient mortality and morbidity, but there is no high throughput in vitro method for predictive nephrotoxicity assessment. We show that primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells rendering them desirable to use in such in vitro systems. To identify a reliable biomarker of nephrotoxicity, we conducted multiplexed gene expression profiling of HPTECs after exposure to six different concentrations of nine human nephrotoxicants. Only overexpression of the gene encoding heme oxygenase-1 (HO-1) significantly correlated with increasing dose for six of the compounds, and significant HO-1 protein deregulation was confirmed with each of the nine nephrotoxicants. Translatability of HO-1 increase across species and platforms was demonstrated by computationally mining two large rat toxicogenomic databases for kidney tubular toxicity and by observing a significant increase in HO-1 after toxicity using an ex vivo three-dimensional microphysiologic system (kidney-on-a-chip). The predictive potential of HO-1 was tested using an additional panel of 39 mechanistically distinct nephrotoxic compounds. Although HO-1 performed better (area under the curve receiver-operator characteristic curve [AUC-ROC]=0.89) than traditional endpoints of cell viability (AUC-ROC for ATP=0.78; AUC-ROC for cell count=0.88), the combination of HO-1 and cell count further improved the predictive ability (AUC-ROC=0.92). We also developed and optimized a homogenous time-resolved fluorescence assay to allow high throughput quantitative screening of nephrotoxic compounds using HO-1 as a sensitive biomarker. This cell-based approach may facilitate rapid assessment of potential nephrotoxic therapeutics and environmental chemicals.
Copyright © 2015 by the American Society of Nephrology.
Human hepatocytes, with complete hepatic metabolizing enzymes, transporters and cofactors, represent the gold standard for in vitro evaluation of drug metabolism, drug-drug interactions, and hepatotoxicity. Successful cryopreservation of human hepatocytes enables this experimental system to be used routinely. The use of human hepatocytes to evaluate two major adverse drug properties: drug-drug interactions and hepatotoxicity, are summarized in this review. The application of human hepatocytes in metabolism-based drug-drug interaction includes metabolite profiling, pathway identification, P450 inhibition, P450 induction, and uptake and efflux transporter inhibition. The application of human hepatocytes in toxicity evaluation includes in vitro hepatotoxicity and metabolism-based drug toxicity determination. A novel system, the Integrated Discrete Multiple Organ Co-culture (IdMOC) which allows the evaluation of nonhepatic toxicity in the presence of hepatic metabolism, is described.
Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a three-dimensional human microphysiological hepatocyte-Kupffer cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and was assessed by the release of proinflammatory cytokines, interleukin 6 and tumor necrosis factor α. A sensitive and specific reversed-phase-ultra high-performance liquid chromatography-quadrupole time of flight-mass spectrometry method was used to evaluate hydrocortisone disappearance and metabolism at near physiologic levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for 2 days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10% of the loss, and 45-52% consisted of phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life, rate of elimination, clearance, and area under the curve, were 23.03 hours, 0.03 hour(-1), 6.6 × 10(-5) l⋅hour(-1), and 1.03 (mg/l)*h, respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized, and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically relevant tool for investigating hepatic function in an inflamed liver.
1. The quantitative prediction of the pharmacokinetic parameters of a drug from data obtained using human in vitro systems remains a significant challenge i.e. prediction of metabolic clearance in humans and estimation of the relative contribution of enzymes involved in the clearance. This has become particularly problematic for low turnover compounds. 2. Having human hepatocytes with stable cellular function over several days that adequately mimic the complexity of the physiological environment would be a major advance. Thus, we evaluated human hepatocytes, maintained in culture during 7 days in the microfluidic LiverChip™ system, in terms of morphological appearance, relative mRNA expression of phase I and II enzymes and transporters as a function of time, and metabolic capacity using probe substrates. 3. The results showed that mRNA levels of the major genes for enzymes involved in drug metabolism were well-maintained over a 7-day period of culture. Furthermore, after 4 days of culture, in the Liverchip™ device, human hepatocytes exhibited higher or similar CYPs activities compared to 1 day of culture in 2D-static conditions. 4. The functional data were supported by light/electron microscopies and immunohistochemistry showing viable tissue structure and well-differentiated human hepatocytes: presence of cell junctions, glycogen storage, and bile canaliculi.
In the last few years, scientists have made important progress in developing systems using human cells to test the effects of drugs and other substances. These systems have the potential to improve toxicity testing beyond currently available tools. The innovative new tools, which are known as microsystems, microphysiological systems, or organs on a chip, can aid in the development of medical products so that toxicity may be identified earlier in product development. This may lower costs and speed new treatments to patients. Experts believe that these systems may eventually enable scientists to test more environmental compounds more efficiently.
The National Institutes of Health has partnered with the US Food and Drug Administration and the Defense Advanced Research Projects Agency to accelerate the development of human microphysiological systems (MPS) that address challenges faced in predictive toxicity assessment and efficacy analysis of new molecular entities during the preclinical phase of drug development. Use of human MPS could provide better models for predicting the efficacy of new molecular entities in clinical trials. It is also anticipated that improvements in predicting drug toxicities early in the drug development process through the use of MPS or human organs-on-a-chip will decrease the need to withdraw new therapies from the market and minimize or eliminate deaths due to unidentified drug toxicities.
Kidney disease is a public health problem that affects more than 20 million people in the US adult population, yet little is understood about the impact of kidney disease on drug disposition. Consequently there is a critical need to be able to model the human kidney and other organ systems, to improve our understanding of drug efficacy, safety, and toxicity, especially during drug development. The kidneys in general, and the proximal tubule specifically, play a central role in the elimination of xenobiotics. With recent advances in molecular investigation, considerable information has been gathered regarding the substrate profiles of the individual transporters expressed in the proximal tubule. However, we have little knowledge of how these transporters coupled with intracellular enzymes and influenced by metabolic pathways form an efficient secretory and reabsorptive mechanism in the renal tubule. Proximal tubular secretion and reabsorption of xenobiotics is critically dependent on interactions with peritubular capillaries and the interstitium. We plan to robustly model the human kidney tubule interstitium, utilizing an ex vivo three-dimensional modular microphysiological system with human kidney-derived cells. The microphysiological system should accurately reflect human physiology, be usable to predict renal handling of xenobiotics, and should assess mechanisms of kidney injury, and the biological response to injury, from endogenous and exogenous intoxicants.
An increased appreciation of the importance of transporter and enzyme interplay in drug clearance and a desire to delineate these mechanisms necessitates the utilization of models which contain a full complement of enzymes and transporters at physiologically relevant activities. Additionally, the development of drugs with longer half-lives requires in vitro systems with extended incubation times that allow characterization of metabolic pathways for low clearance drugs. A recently developed co-culture hepatocyte model, HepatoPac, has been applied to meet these challenges. Faldaprevir is a drug in late stage development for the treatment of hepatitis C. Faldaprevir is a low clearance drug with the somewhat unique characteristic of being slowly metabolized, producing two abundant hydroxylated metabolites (M2a and M2b) in feces (approximately 40% of the dose) without exhibiting significant levels of circulating metabolites in humans. The human HepatoPac model was investigated to characterize the metabolism and transport of faldaprevir. In human HepatoPac cultures, M2a and M2b were the predominant metabolites formed, with extents of formation comparable to in vivo. Direct glucuronidation of faldaprevir was shown to be a minor metabolic pathway. HepatoPac studies also demonstrated that faldaprevir is concentrated in liver with active uptake by multiple transporters (including OATP1B1 and Na(+)-dependant transporters). Overall, human HepatoPac cultures provided valuable insights into the metabolism and disposition of faldaprevir in humans and demonstrated the importance of enzyme and transporter interplay on the clearance of the drug.
Human in vitro-manufactured tissue and organ models can serve as powerful enabling tools for the exploration of fundamental questions regarding cell, matrix and developmental biology as well as the study of drug delivery dynamics and kinetics. To date, the development of a human model of the renal proximal tubule (PT) has been hindered by the lack of an appropriate cell source and scaffolds that allow epithelial monolayer formation and maintenance. Using extracellular matrices or matrix proteins, an in vivo-mimicking environment can be created that allows epithelial cells to exhibit their typical phenotype and functionality. Here, we describe an in vitro-engineered PT model. We isolated highly proliferative cells from cadaveric human kidneys (hKDCs), which express markers that are associated with renal progenitor cells. Seeded on small intestinal submucosa (SIS), hKDCs formed a confluent monolayer and displayed the typical phenotype of PT epithelial cells. PT markers, including N-cadherin, were detected throughout the hKDC culture on the SIS, whereas markers of later tubule segments were weakly (E-cadherin) or not (aquaporin-2) expressed. Basement membrane and microvilli formation demonstrated a strong polarization. We conclude that the combination of hKDCs and SIS is a suitable cell-scaffold composite to mimic the human PT in vitro.
Since drug induced liver injury (DILI) remains a major reason for late stage drug attrition, predictive assays are needed that can be deployed throughout the drug discovery process Clinical DILI can be predicted with a sensitivity of ~50% and a false positive rate of ~5% using 24 hour cultures of primary sandwich cultured human hepatocytes and imaging of four cell injury endpoints (Xu et al., 2008). We hypothesized that long-term drug dosing in a stable model of primary hepatocytes (micropatterned co-cultures or MPCCs) could provide for increased predictivity over short-term dosing paradigms. We used MPCCs with either primary human or rat hepatocytes to understand possible species differences along with standard end-points (glutathione levels, ATP levels, albumin and urea secretion) to test 45 drugs either known or not known to cause clinical DILI. Human MPCCs correctly detected 23 of 35 compounds known to cause DILI (65.7% sensitivity) with a false positive rate of 10% for the 10 negative compounds tested. Rat MPCCs correctly detected 17 of 35 DILI compounds (48.6% sensitivity) and had a higher false positive rate than human MPCCs (20% versus 10%). For drugs with the most DILI concern, human MPCCs displayed a sensitivity of 100%. Furthermore, MPCCs were able to detect relative clinical toxicities of structural drug analogs. In conclusion, MPCCs showed superiority over conventional short-term cultures for predictions of clinical DILI and human MPCCs were more predictive for human liabilities than their rat counterparts.
Current 2-dimensional hepatic model systems often fail to predict chemically induced hepatotoxicity due to the loss of a hepatocyte-specific phenotype in culture. For more predictive in vitro models, hepatocytes have to be maintained in a 3-dimensional environment that allows for polarization and cell–cell contacts. Preferably, the model will reflect an in vivo-like multi-cell type environment necessary for liver-like responses. Here, we report the characterization of a multi-cell type microtissue model, generated from primary human hepatocytes and liver-derived non-parenchymal cells. Liver microtissues were stable and functional for 5 weeks in culture enabling, for example, long-term toxicity testing of acetaminophen and diclofenac. In addition, Kupffer cells were responsive to inflammatory stimuli such as LPS demonstrating the possibility to detect inflammation-mediated toxicity as exemplified by the drug trovafloxacin. Herewith, we present a novel 3D liver model for routine testing in 96-well format capable of reducing the risk of unwanted toxic effects in the clinic.
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Prediction of chemical-induced hepatotoxicity in humans from in vitro data continues to be a significant challenge for the pharmaceutical and chemical industries. Generally, conventional in vitro hepatic model systems (i.e. 2-D static monocultures of primary or immortalized hepatocytes) are limited by their inability to maintain histotypic and phenotypic characteristics over time in culture, including stable expression of clearance and bioactivation pathways, as well as complex adaptive responses to chemical exposure. These systems are less than ideal for longer-term toxicity evaluations and elucidation of key cellular and molecular events involved in primary and secondary adaptation to chemical exposure, or for identification of important mediators of inflammation, proliferation and apoptosis. Progress in implementing a more effective strategy for in vitro-in vivo extrapolation and human risk assessment depends on significant advances in tissue culture technology and increasing their level of biological complexity. This article describes the current and ongoing need for more relevant, organotypic in vitro surrogate systems of human liver and recent efforts to recreate the multicellular architecture and hemodynamic properties of the liver using novel culture platforms. As these systems become more widely used for chemical and drug toxicity testing, there will be a corresponding need to establish standardized testing conditions, endpoint analyses and acceptance criteria. In the future, a balanced approach between sample throughput and biological relevance should provide better in vitro tools that are complementary with animal testing and assist in conducting more predictive human risk assessment.
The levels of metabolizing enzymes and transporters expressed in hepatocytes are decisive factors for hepatobiliary disposition of most drugs. Induction via nuclear receptor activation can significantly alter those levels, with the coregulation of multiple enzymes and transporters occurring to different extents. Here, we report the use of a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for concurrent quantification of multiple cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and transporter proteins in cultured primary human hepatocytes. The effects of culture format (i.e., sandwich culture versus conventional culture) and of dexamethasone (DEX) media concentrations on mRNA, protein, and activity levels were determined for three donors, and protein expression was compared with that in liver. In general, P450 and UGT expression was lower in hepatocyte cultures than that in liver, and CYP2C9 was found to be the most abundant P450 isoform expressed in cultured hepatocytes. The sandwich culture format and 0.1 μM DEX in media retained the protein expression in the hepatocytes closest to the levels found in liver. However, higher in vitro expression was observed for drug transporters, especially for multidrug resistance protein 1 and breast cancer resistance protein. Direct protein quantification was applied successfully to study in vitro induction in sandwich cultured primary hepatocytes in a 24-well format using the prototypical inducers rifampicin, omeprazole, and phenobarbital. We conclude that targeted absolute LC-MS/MS quantification of drug-metabolizing enzymes and transporters can broaden the scope and significantly increase the impact of in vitro drug metabolism studies, such as induction, as an important supplement or future alternative to mRNA and activity data.
HepaRG cells, derived from a female hepatocarcinoma patient, are capable of differentiating into biliary epithelial cells and hepatocytes. More importantly, differentiated HepaRG cells are able to maintain activities of many xenobiotic-metabolizing enzymes, and expression of the metabolizing enzyme genes can be induced by xenobiotics. The ability of these cells to express and induce xenobiotic-metabolizing enzymes is in stark contrast to the frequently used HepG2 cells. The previous studies have mainly focused on a set of selected genes; therefore, it is of significant interest to know the extent of similarity of gene expression at whole genome levels in HepaRG cells and HepG2 cells compared with primary human hepatocytes and human liver tissues. To accomplish this objective, we used Affymetrix (Santa Clara, CA) U133 Plus 2.0 arrays to characterize the whole genome gene expression profiles in triplicate biological samples from HepG2 cells, HepaRG cells (undifferentiated and differentiated cells), freshly isolated primary human hepatocytes, and frozen liver tissues. After using similarity matrix, principal components, and hierarchical clustering methods, we found that HepaRG cells globally transcribe genes at levels more similar to human primary hepatocytes and human liver tissues than HepG2 cells. In particular, many genes encoding drug-processing proteins are transcribed at a more similar level in HepaRG cells than in HepG2 cells compared with primary human hepatocytes and liver samples. The transcriptomic similarity of HepaRG with primary human hepatocytes is encouraging for use of HepaRG cells in the study of xenobiotic metabolism, hepatotoxicology, and hepatocyte differentiation.
In vitro models that capture the complexity of in vivo tissue and organ behaviors in a scalable and easy-to-use format are desirable for drug discovery. To address this, we have developed a bioreactor that fosters maintenance of 3D tissue cultures under constant perfusion and we have integrated multiple bioreactors into an array in a multiwell plate format. All bioreactors are fluidically isolated from each other. Each bioreactor in the array contains a scaffold that supports formation of hundreds of 3D microscale tissue units. The tissue units are perfused with cell culture medium circulated within the bioreactor by integrated pneumatic diaphragm micropumps. Electronic controls for the pumps are kept outside the incubator and connected to the perfused multiwell by pneumatic lines. The docking design and open-well bioreactor layout make handling perfused multiwell plates similar to using standard multiwell tissue culture plates. A model of oxygen consumption and transport in the circulating culture medium was used to predict appropriate operating parameters for primary liver cultures. Oxygen concentrations at key locations in the system were then measured as a function of flow rate and time after initiation of culture to determine oxygen consumption rates. After seven days of culture, tissue formed from cells seeded in the perfused multiwell reactor remained functionally viable as assessed by immunostaining for hepatocyte and liver sinusoidal endothelial cell (LSEC) phenotypic markers.
The organic solute and steroid transporter, Ost alpha-Ost beta, is an unusual heteromeric carrier that appears to play a central role in the transport of bile acids, conjugated steroids, and structurally-related molecules across the basolateral membrane of many epithelial cells. The transporter's substrate specificity, transport mechanism, tissue distribution, subcellular localization, transcriptional regulation, as well as the phenotype of the recently characterized Ost alpha-deficient mice all strongly support this model. In particular, the Ost alpha-deficient mice display a marked defect in intestinal bile acid and conjugated steroid absorption; a decrease in bile acid pool size and serum bile acid levels; altered intestinal, hepatic and renal disposition of known substrates of the transporter; and altered serum triglyceride, cholesterol, and glucose levels. Collectively, the data indicate that Ost alpha-Ost beta is essential for bile acid and sterol disposition, and suggest that the carrier may be involved in human conditions related to imbalances in bile acid or lipid homeostasis.
Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.
This paper describes the development and characterization of a microphysiology platform for drug safety and efficacy in liver models of disease that includes a human, 3D, microfluidic, four-cell, sequentially layered, self-assembly liver model (SQL-SAL); fluorescent protein biosensors for mechanistic readouts; as well as a microphysiology system database (MPS-Db) to manage, analyze, and model data. The goal of our approach is to create the simplest design in terms of cells, matrix materials, and microfluidic device parameters that will support a physiologically relevant liver model that is robust and reproducible for at least 28 days for stand-alone liver studies and microfluidic integration with other organs-on-chips. The current SQL-SAL uses primary human hepatocytes along with human endothelial (EA.hy926), immune (U937) and stellate (LX-2) cells in physiological ratios and is viable for at least 28 days under continuous flow. Approximately, 20% of primary hepatocytes and/or stellate cells contain fluorescent protein biosensors (called sentinel cells) to measure apoptosis, reactive oxygen species (ROS) and/or cell location by high content analysis (HCA). In addition, drugs, drug metabolites, albumin, urea and lactate dehydrogenase (LDH) are monitored in the efflux media. Exposure to 180 μM troglitazone or 210 μM nimesulide produced acute toxicity within 2-4 days, whereas 28 μM troglitazone produced a gradual and much delayed toxic response over 21 days, concordant with known mechanisms of toxicity, while 600 µM caffeine had no effect. Immune-mediated toxicity was demonstrated with trovafloxacin with lipopolysaccharide (LPS), but not levofloxacin with LPS. The SQL-SAL exhibited early fibrotic activation in response to 30 nM methotrexate, indicated by increased stellate cell migration, expression of alpha-smooth muscle actin and collagen, type 1, alpha 2. Data collected from the in vitro model can be integrated into a database with access to related chemical, bioactivity, preclinical and clinical information uploaded from external databases for constructing predictive models.
© 2015 by the Society for Experimental Biology and Medicine.
Improving the effectiveness of preclinical predictions of human drug responses is critical to reducing costly failures in clinical trials. Recent advances in cell biology, microfabrication and microfluidics have enabled the development of microengineered models of the functional units of human organs - known as organs-on-chips - that could provide the basis for preclinical assays with greater predictive power. Here, we examine the new opportunities for the application of organ-on-chip technologies in a range of areas in preclinical drug discovery, such as target identification and validation, target-based screening, and phenotypic screening. We also discuss emerging drug discovery opportunities enabled by organs-on-chips, as well as important challenges in realizing the full potential of this technology.
Severe cholestasis may result in end-stage liver disease with the need of liver transplantation (LTX). In children, about 10 % of LTX are necessary because of cholestatic liver diseases. Apart from bile duct atresia, three types of progressive familial intrahepatic cholestasis (PFIC) are common causes of severe cholestasis in children. The three subtypes of PFIC are defined by the involved genes: PFIC-1, PFIC-2, and PFIC-3 are due to mutations of P-type ATPase ATP8B1 (familial intrahepatic cholestasis 1, FIC1), the ATP binding cassette transporter ABCB11 (bile salt export pump, BSEP), or ABCB4 (multidrug resistance protein 3, MDR3), respectively. All transporters are localized in the canalicular membrane of hepatocytes and together mediate bile salt and phospholipid transport. In some patients with PFIC-2 disease, recurrence has been observed after LTX, which mimics a PFIC phenotype. It could be shown by several groups that inhibitory anti-BSEP antibodies emerge, which most likely cause disease recurrence. The prevalence of severe BSEP mutations (e.g., splice site and premature stop codon mutations) is very high in this group of patients. These mutations often result in the complete absence of BSEP, which likely accounts for an insufficient auto-tolerance against BSEP. Although many aspects of this "new" disease are not fully elucidated, the possibility of anti-BSEP antibody formation has implications for the pre- and posttransplant management of PFIC-2 patients. This review will summarize the current knowledge including diagnosis, pathomechanisms, and management of "autoimmune BSEP disease."
Microphysiological systems (MPS), consisting of interacting organs-on-chips or tissue-engineered, 3D organ constructs that use human cells, present an opportunity to bring new tools to biology, medicine, pharmacology, physiology, and toxicology. This issue of Experimental Biology and Medicine describes the ongoing development of MPS that can serve as in-vitro models for bone and cartilage, brain, gastrointestinal tract, lung, liver, microvasculature, reproductive tract, skeletal muscle, and skin. Related topics addressed here are the interconnection of organs-on-chips to support physiologically based pharmacokinetics and drug discovery and screening, and the microscale technologies that regulate stem cell differentiation. The initial motivation for creating MPS was to increase the speed, efficiency, and safety of pharmaceutical development and testing, paying particular regard to the fact that neither monolayer monocultures of immortal or primary cell lines nor animal studies can adequately recapitulate the dynamics of drug-organ, drug-drug, and drug-organ-organ interactions in humans. Other applications include studies of the effect of environmental toxins on humans, identification, characterization, and neutralization of chemical and biological weapons, controlled studies of the microbiome and infectious disease that cannot be conducted in humans, controlled differentiation of induced pluripotent stem cells into specific adult cellular phenotypes, and studies of the dynamics of metabolism and signaling within and between human organs. The technical challenges are being addressed by many investigators, and in the process, it seems highly likely that significant progress will be made toward providing more physiologically realistic alternatives to monolayer monocultures or whole animal studies. The effectiveness of this effort will be determined in part by how easy the constructs are to use, how well they function, how accurately they recapitulate and report human pharmacology and toxicology, whether they can be generated in large numbers to enable parallel studies, and if their use can be standardized consistent with the practices of regulatory science.
Adenosine triphosphate (ATP)-binding cassette, sub-family B, member 4 (ABCB4), also called multidrug resistance 3 (MDR3), is a member of the ATP-binding cassette transporter superfamily, which is localized at the canalicular membrane of hepatocytes, and mediates the translocation of phosphatidylcholine into bile. Phosphatidylcholine secretion is crucial to ensure solubilization of cholesterol into mixed micelles and to prevent bile acid toxicity towards hepatobiliary epithelia. Genetic defects of ABCB4 may cause progressive familial intrahepatic cholestasis type 3 (PFIC3), a rare autosomic recessive disease occurring early in childhood that may be lethal in the absence of liver transplantation, and other cholestatic or cholelithiasic diseases in heterozygous adults. Development of therapies for these conditions requires understanding of the biology of this transporter and how gene variations may cause disease. This review focuses on our current knowledge on the regulation of ABCB4 expression, trafficking and function, and presents recent advances in fundamental research with promising therapeutic perspectives.
The liver is a heterogeneous organ with many vital functions, including metabolism of pharmaceutical drugs and is highly susceptible to injury from these substances. The etiology of drug-induced liver disease is still debated although generally regarded as a continuum between an activated immune response and hepatocyte metabolic dysfunction, most often resulting from an intermediate reactive metabolite. This debate stems from the fact that current animal and in vitro models provide limited physiologically relevant information, and their shortcomings have resulted in "silent" hepatotoxic drugs being introduced into clinical trials, garnering huge financial losses for drug companies through withdrawals and late stage clinical failures. As we advance our understanding into the molecular processes leading to liver injury, it is increasingly clear that (a) the pathologic lesion is not only due to liver parenchyma but is also due to the interactions between the hepatocytes and the resident liver immune cells, stellate cells, and endothelial cells; and (b) animal models do not reflect the human cell interactions. Therefore, a predictive human, in vitro model must address the interactions between the major human liver cell types and measure key determinants of injury such as the dosage and metabolism of the drug, the stress response, cholestatic effect, and the immune and fibrotic response. In this mini-review, we first discuss the current state of macro-scale in vitro liver culture systems with examples that have been commercialized. We then introduce the paradigm of microfluidic culture systems that aim to mimic the liver with physiologically relevant dimensions, cellular structure, perfusion, and mass transport by taking advantage of micro and nanofabrication technologies. We review the most prominent liver-on-a-chip platforms in terms of their physiological relevance and drug response. We conclude with a commentary on other critical advances such as the deployment of fluorescence-based biosensors to identify relevant toxicity pathways, as well as computational models to create a predictive tool.
Madin-Darby canine kidney (MDCK) cells transfected with human MDR1 gene (MDCK-MDR1) encoding for P-glycoprotein (hPgp, ABCB1) are widely used for transport studies to identify drug candidates as substrates of this efflux protein. Therefore, it is necessary to rely on constant and comparable expression levels of Pgp to avoid false negative or positive results. We generated a cell line with homogenously high and stable expression of hPgp through sorting single clones from a MDCK-MDR1 cell pool using fluorescence-activated cell sorting (FACS). To obtain control cell lines for evaluation of cross-interactions with endogenous canine Pgp (cPgp) wild-type cells were sorted with a low expression pattern of cPgp in comparison with the MDCK-MDR1. Expression of other transporters was also characterized in both cell lines by quantitative real-time PCR and Western blot. Pgp function was investigated applying the Calcein-AM assay as well as bidirectional transport assays using (3) H-Digoxin, (3) H-Vinblastine, and (3) H-Quinidine as substrates. Generated MDCK-MDR1 cell lines showed high expression of hPgp. Control MDCK-WT cells were optimized in showing a comparable expression level of cPgp in comparison with MDCK-MDR1 cell lines. Generated cell lines showed higher and more selective Pgp transport compared with parental cells. Therefore, they provide a significant improvement in the performance of efflux studies yielding more reliable results. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
Many cell lines, despite the fact that they are easy to culture, tend to lose some of their in vivo characteristics in vitro, we therefore decided to investigate whether culturing HK-2 cells on Kidney derived micro scaffolds (KMS) could improve proximal tubule functionality to these cells. Kidney Derived Micro Scaffolds (KMS) have been prepared that, due to the fact that they are only 300µm in depth, allow for transfer of gasses and nutrients via diffusion whilst maintaining the kidney's intricate microstructure. Culturing HK-2 on KMS shows significant increase in expression of AQP-1, ATP1B1, SLC23A1 and SLC5A2 after 1,2 and 3 weeks compared with HK-2 grown under standard tissue culture conditions. Additionally, very high levels of expression of CCL-2 (15-30 fold increase) and LRP-2 (25-200 fold increase) were observed when the HK-2 were grown on KMS compared with HK-2 grown under standard tissue culture conditions. Furthermore, HK-2 cells grown under standard conditions released higher levels of Il-6 and Il-8 compared with primary tubule cells (Asterand AS-9-2) and secreted no MCP-1 or RANTES as opposed to primary cells that released MCP-1 and RANTES following stimulation. However, HK-2 grown on KMS showed both a marked decrease in Il-6/Il-8 secretion in line with the primary cells and secreted MCP-1 as well. These results show that the micro environment of the KMS assists in restoring in vivo like properties to the HK-2 cells.
The kidney plays a fundamental role in maintaining body salt and fluid balance and blood pressure homeostasis through the actions of its proximal and distal tubular segments of nephrons. However, proximal tubules are well recognized to exert a more prominent role than distal counterparts. Proximal tubules are responsible for reabsorbing approximately 65% of filtered load and most, if not all, of filtered amino acids, glucose, solutes, and low molecular weight proteins. Proximal tubules also play a key role in regulating acid-base balance by reabsorbing approximately 80% of filtered bicarbonate. The purpose of this review article is to provide a comprehensive overview of new insights and perspectives into current understanding of proximal tubules of nephrons, with an emphasis on the ultrastructure, molecular biology, cellular and integrative physiology, and the underlying signaling transduction mechanisms. The review is divided into three closely related sections. The first section focuses on the classification of nephrons and recent perspectives on the potential role of nephron numbers in human health and diseases. The second section reviews recent research on the structural and biochemical basis of proximal tubular function. The final section provides a comprehensive overview of new insights and perspectives in the physiological regulation of proximal tubular transport by vasoactive hormones. In the latter section, attention is particularly paid to new insights and perspectives learnt from recent cloning of transporters, development of transgenic animals with knockout or knockin of a particular gene of interest, and mapping of signaling pathways using microarrays and/or physiological proteomic approaches. © 2013 American Physiological Society. Compr Physiol 3:1079-1123, 2013.
Cholangiocytes are epithelial cells that line the intra- and extrahepatic ducts of the biliary tree. The main physiologic function of cholangiocytes is modification of hepatocyte-derived bile, an intricate process regulated by hormones, peptides, nucleotides, neurotransmitters, and other molecules through intracellular signaling pathways and cascades. The mechanisms and regulation of bile modification are reviewed herein. © 2013 American Physiological Society. Compr Physiol 3:541-565, 2013.
Kidney toxicity is one of the most frequent adverse events reported during drug development. The lack of accurate predictive cell culture models and the unreliability of animal studies have created a need for better approaches to recapitulate kidney function in vitro. Here, we describe a microfluidic device lined by living human kidney epithelial cells exposed to fluidic flow that mimics key functions of the human kidney proximal tubule. Primary kidney epithelial cells isolated from human proximal tubule are cultured on the upper surface of an extracellular matrix-coated, porous, polyester membrane that splits the main channel of the device into two adjacent channels, thereby creating an apical 'luminal' channel and a basal 'interstitial' space. Exposure of the epithelial monolayer to an apical fluid shear stress (0.2 dyne cm(-2)) that mimics that found in living kidney tubules results in enhanced epithelial cell polarization and primary cilia formation compared to traditional Transwell culture systems. The cells also exhibited significantly greater albumin transport, glucose reabsorption, and brush border alkaline phosphatase activity. Importantly, cisplatin toxicity and Pgp efflux transporter activity measured on-chip more closely mimic the in vivo responses than results obtained with cells maintained under conventional culture conditions. While past studies have analyzed kidney tubular cells cultured under flow conditions in vitro, this is the first report of a toxicity study using primary human kidney proximal tubular epithelial cells in a microfluidic 'organ-on-a-chip' microdevice. The in vivo-like pathophysiology observed in this system suggests that it might serve as a useful tool for evaluating human-relevant renal toxicity in preclinical safety studies.
Acquiring a mechanistic understanding of the processes underlying the renal clearance of drug molecules in man has been hampered by a lack of robust in vitro models of human proximal tubules. Several human renal epithelial cell lines derived from the renal cortex are available, but few have been characterised in detail in terms of transporter expression. This includes the HK-2 proximal tubule cell line, which has been used extensively as a model of nephrotoxicity. The aim of this study was to investigate the expression and function of drug transporters in HK-2 cells and their suitability as an in vitro model of the human proximal tubule. qPCR showed no mRNA expression of the SLC22 transporter family (OAT1, OAT3, OCT2) in HK-2 cells compared to renal cortex samples. In contrast, SLC16A1 (MCT1), which is important in the uptake of monocarboxylates, and SLCO4C1 (OATP4C1) were expressed in HK-2 cells. The functional expression of these transporters was confirmed by uptake studies using radiolabelled prototypic substrates DL-lactate and digoxin, respectively. The mRNA expression of apical membrane efflux transporters ABCB1 (MDR1) and several members of the ABCC family (multidrug resistance proteins, MRPs) was shown by qPCR. ABCG1 (BCRP) was not detected. The efflux of Hoechst 33342, a substrate for MDR1, was blocked by MDR1 inhibitor cyclosporin A, suggesting the functional expression of this transporter. Similarly, the efflux of the MRP-specific fluorescent dye glutathione methylfluorescein was inhibited by the MRP inhibitor MK571. Taken together, the results of this study suggest that HK-2 cells are of limited value as an in vitro model of drug transporter expression in the human proximal tubule.
Microscale engineering technologies provide unprecedented opportunities to create cell culture microenvironments that go beyond current three-dimensional in vitro models by recapitulating the critical tissue-tissue interfaces, spatiotemporal chemical gradients, and dynamic mechanical microenvironments of living organs. Here we review recent advances in this field made over the past two years that are focused on the development of 'Organs-on-Chips' in which living cells are cultured within microfluidic devices that have been microengineered to reconstitute tissue arrangements observed in living organs in order to study physiology in an organ-specific context and to develop specialized in vitro disease models. We discuss the potential of organs-on-chips as alternatives to conventional cell culture models and animal testing for pharmaceutical and toxicology applications. We also explore challenges that lie ahead if this field is to fulfil its promise to transform the future of drug development and chemical safety testing.
Acute kidney injury (AKI), accompanied by the development of systemic inflammatory response syndrome and multiorgan dysfunction syndrome, is associated with a high risk of death. Bioartificial renal tubule device (BTD) is a cell therapy that improves the conditions common to artificial kidney recipients treated for kidney diseases. In this paper, we describe the establishment of BTD with lifespan-extended human renal proximal tubular epithelial cells.
AKI goats were established by performing bilateral nephrectomy followed by lipopolysaccharide administration. The AKI goats were treated with BTD or sham-BTD, and the two groups of animals were compared by measuring the respective life spans and the levels of blood urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and serum electrolytes. The expression levels of inflammatory cytokines were detected by reverse transcription-polymerase chain reaction, and plasma interleukin (IL)-6 levels were measured by enzyme-linked immunosorbent assay.
The life span of AKI goats was extended: the lifetime with the BTD treatment compared with sham-BTD. BTD and sham-BTD showed a similar degree of small solute clearance. The expression levels of inflammatory cytokines and plasma IL-6 levels were decreased by the BTD treatment.
BTD treatment results in less damage from endotoxin shock and increased life span in AKI goats. These results suggest that BTD may be a useful component of bioartificial kidneys and should be considered in the next generation of renal replacement therapies.
OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, l-glutamate, l-proline, l-alanine, and α-methyl-glucopyranoside (α-methyl d-glucoside) were accumulated through Na+-dependent and Na+-independent transport pathways. d-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na+. Influx of inorganic phosphate was only observed in the presence of Na+. Na+/α-methyl d-glucoside uptake was preferentially inhibited by phlorizin and d-glucose uptake by cytochalasin B. An amiloride-sensitive Na+-transport was also identified. In isolated apical vesicles (enriched 8-fold in γ-glutamyltransferase), l-glutamate, l-proline, l-alanine, α-methyl d-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na+-gradient as compared to an inwardly directed K+-gradient. l-Glutamate transport required additionally intravesicular K+. d-Glucose transport was similar in the presence of a Na+- and a K+-gradient. Na+/α-methyl d-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na+/d-glucose transport. An amiloride-sensitive Na+/H+ exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na+-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. d-Glucose appears a poor substrate for the Na+/hexose transport system.
In the process of drug development it is of high importance to test the safety of new drugs with predictive value for human toxicity. A promising approach of toxicity testing is based on shifts in gene expression profiling of the liver. Toxicity screening based on animal liver cells cannot be directly extrapolated to humans due to species differences. The aim of this study was to evaluate precision-cut human liver slices as in vitro method for the prediction of human specific toxicity by toxicogenomics. The liver slices contain all cell types of the liver in their natural architecture. This is important since drug-induced toxicity often is a multi-cellular process. Previously we showed that toxicogenomic analysis of rat liver slices is highly predictive for rat in vivo toxicity. In this study we investigated the levels of gene expression during incubation up to 24 h with Affymetrix microarray technology. The analysis was focused on a broad spectrum of genes related to stress and toxicity, and on genes encoding for phase-I, -II and -III metabolizing enzymes and transporters. Observed changes in gene expression were associated with cytoskeleton remodeling, extracellular matrix and cell adhesion, but for the ADME-Tox related genes only minor changes were observed. PCA analysis showed that changes in gene expression were not associated with age, sex or source of the human livers. Slices treated with acetaminophen showed patterns of gene expression related to its toxicity. These results indicate that precision-cut human liver slices are relatively stable during 24h of incubation and represent a valuable model for human in vitro hepatotoxicity testing despite the human inter-individual variability.
The importance of membrane transporters for drug pharmacokinetics has been increasingly recognized during the last decade. Organic anion transporting polypeptide 1B1 (OATP1B1) is a genetically polymorphic influx transporter expressed on the sinusoidal membrane of human hepatocytes, and it mediates the hepatic uptake of many endogenous compounds and xenobiotics. Recent studies have demonstrated that OATP1B1 plays a major, clinically important role in the hepatic uptake of many drugs. A common single-nucleotide variation (coding DNA c.521T>C, protein p.V174A, rs4149056) in the SLCO1B1 gene encoding OATP1B1 decreases the transporting activity of OATP1B1, resulting in markedly increased plasma concentrations of, for example, many statins, particularly of active simvastatin acid. The variant thereby enhances the risk of statin-induced myopathy and decreases the therapeutic indexes of statins. However, the effect of the SLCO1B1 c.521T>C variant is different on different statins. The same variant also markedly affects the pharmacokinetics of several other drugs. Furthermore, certain SLCO1B1 variants associated with an enhanced clearance of methotrexate increase the risk of gastrointestinal toxicity by methotrexate in the treatment of children with acute lymphoblastic leukemia. Certain drugs (e.g., cyclosporine) potently inhibit OATP1B1, causing clinically significant drug interactions. Thus, OATP1B1 plays a major role in the hepatic uptake of drugs, and genetic variants and drug interactions affecting OATP1B1 activity are important determinants of individual drug responses. In this article, we review the current knowledge about the expression, function, substrate characteristics, and pharmacogenetics of OATP1B1 as well as its role in drug interactions, in parts comparing with those of other hepatocyte-expressed organic anion transporting polypeptides, OATP1B3 and OATP2B1.
Within the scope of developing an in vitro culture model for pharmacological research on human liver functions, a three-dimensional multicompartment hollow fiber bioreactor proven to function as a clinical extracorporeal liver support system was scaled down in two steps from 800 mL to 8 mL and 2 mL bioreactors. Primary human liver cells cultured over 14 days in 800, 8, or 2 mL bioreactors exhibited comparable time-course profiles for most of the metabolic parameters in the different bioreactor size variants. Major drug-metabolizing cytochrome P450 activities analyzed in the 2 mL bioreactor were preserved over up to 23 days. Immunohistochemical studies revealed tissue-like structures of parenchymal and nonparenchymal cells in the miniaturized bioreactor, indicating physiological reorganization of the cells. Moreover, the canalicular transporters multidrug-resistance-associated protein 2, multidrug-resistance protein 1 (P-glycoprotein), and breast cancer resistance protein showed a similar distribution pattern to that found in human liver tissue. In conclusion, the down-scaled multicompartment hollow fiber technology allows stable maintenance of primary human liver cells and provides an innovative tool for pharmacological and kinetic studies of hepatic functions with small cell numbers.
The human breast cancer resistance protein (BCRP/ABCG2) is the second member of the G subfamily of the large ATP-binding cassette (ABC) transporter superfamily. BCRP was initially discovered in multidrug resistant breast cancer cell lines where it confers resistance to chemotherapeutic agents such as mitoxantrone, topotecan and methotrexate by extruding these compounds out of the cell. BCRP is capable of transporting non-chemotherapy drugs and xenobiotiocs as well, including nitrofurantoin, prazosin, glyburide, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. BCRP is frequently detected at high levels in stem cells, likely providing xenobiotic protection. BCRP is also highly expressed in normal human tissues including the small intestine, liver, brain endothelium, and placenta. Therefore, BCRP has been increasingly recognized for its important role in the absorption, elimination, and tissue distribution of drugs and xenobiotics. At present, little is known about the transport mechanism of BCRP, particularly how it recognizes and transports a large number of structurally and chemically unrelated drugs and xenobiotics. Here, we review current knowledge of structure and function of this medically important ABC efflux drug transporter.
P-glycoprotein (Pgp) efflux assays are widely used to identify Pgp substrates. The kidney cell lines Madin-Darby canine kidney (MDCK)-II and LLC-PK1, transfected with human MDR1 (ABCB1) are used to provide recombinant models of drug transport. Endogenous transporters in these cells may contribute to the activities of recombinant transporters, so that drug transport in MDR1-transfected cells is often corrected for the transport obtained in parental (wildtype) cells. However, expression of endogenous transporters may vary between transfected and wildtype cells, so that this correction may cause erroneous data. Here, we have measured the expression of endogenous efflux transporters in transfected and wildtype MDCK-II or LLC cells and the consequences for Pgp-mediated drug transport.
Using quantitative real-time RT-PCR, we determined the expression of endogenous Mdr1 mRNA and other efflux transporters in wildtype and MDR1-transfected MDCK-II and LLC cells. Transcellular transport was measured with the test substrate vinblastine.
In MDR1-transfected MDCK cells, expression of endogenous (canine) Mdr1 and Mrp2 (Abcc2) mRNA was markedly lower than in wildtype cells, whereas MDR1-transfected LLC cells exhibited comparable Mdr1 but strikingly higher Mrp2 mRNA levels than wildtype cells. As a consequence, transport of vinblastine by human Pgp in efflux experiments was markedly underestimated when transport in MDR1-transfected MDCK cells was corrected for transport obtained in wildtype cells. This problem did not occur in LLC cells.
Differences in the expression of endogenous efflux transporters between transfected and wildtype MDCK cells provide a potential bias for in vitro studies on Pgp-mediated drug transport.