MDCK Cell Monolayers Research Articles

Disruption of gut barrier integrity and host–microbiome interactions underlie MASLD severity in patients with type-2 diabetes mellitus

ABSTRACT Aberration of the “gut-liver axis” contributes to the development and progression of metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we use multi-omics to analyze the gut microbiota composition and metabolic profile of patients with type-2 diabetes mellitus (T2DM). T2DM patients were screened for liver disease by blood tests, ultrasound, and liver stiffness measurements. Stool microbiota was analyzed by 16S rRNA gene sequencing; metabolomic profiling by Nuclear Magnetic Resonance spectroscopy and Ultra-High Performance-Mass Spectrometry. Microbiome and metabolic signatures were analyzed in the whole cohort and in matched subsets to identify signatures specific for steatosis (MASLD±) or fibrosis (Fibrosis±). Gut permeability was assessed in-vitro using monolayers of MDCK cells and trans-epithelial electric resistance (TEER). Cytokine profile was assessed in serum and stools. Overall, 285 patients were enrolled: 255 serum, 252 urine and 97 stool samples were analyzed. Anaeroplasma and Escherichia/Shigella ASVs were higher, while Butyricicoccus ASVs were lower in those with normal liver. In MASLD±, Butyricicoccus ASV was significantly higher in those with steatosis. In the Fibrosis±, Butyricicoccus ASV was significantly lower in those with fibrosis. Glycochenodeoxycholic acid-3-sulfate (G-UDCA-3S) appeared to be higher in MASLD with fibrosis. Fecal water from patients with MASLD and fibrosis caused the greatest drop in the TEER vs those with normal liver; this was reversed with protease inhibitors. Finally, fecal IL-13 was lower in MASLD with fibrosis. We identified microbiome signatures which were specific for steatosis and fibrosis and independent of other metabolic risk factors. Moreover, we conclude that protease-related gut permeability plays a role in those MASLD patients with fibrosis, and that disease progression is linked to a gut-liver axis which is at least partially independent of T2DM.

Pitfalls in evaluating permeability experiments with Caco-2/MDCK cell monolayers

When studying the transport of molecules across biological membranes, intrinsic membrane permeability (P0) is more informative than apparent permeability (Papp), because it eliminates external (setup-specific) factors, provides consistency across experiments and mechanistic insight. It is thus an important building block for modeling the total permeability in any given scenario. However, extracting P0 is often difficult, if not impossible, when the membrane is not the dominant transport resistance. In this work, we set out to analyze Papp values measured with Caco-2/MDCK cell monolayers of 69 literature references. We checked the Papp values for a total of 318 different compounds for the extractability of P0, considering possible limitations by aqueous boundary layers, paracellular transport, recovery issues, active transport, a possible proton flux limitation, and sink conditions. Overall, we were able to extract 77 reliable P0 values, which corresponds to about one quarter of the total compounds analyzed, while about half were limited by the diffusion through the aqueous layers. Compared to an existing data set of P0 values published by Avdeef, our approach resulted in a much higher exclusion of compounds. This is a consequence of stricter compound- and reference-specific exclusion criteria, but also because we considered possible concentration-shift effects due to different pH values in the aqueous layers, an effect only recently described in literature. We thus provide a consistent and reliable set of P0, e.g. as a basis for future modeling.

MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira, has an essential role during infection.

Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen dissemination and virulence mechanisms. Here we characterized the leptospiral Membrane Protein L36 (MPL36), a rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain, as an important virulence factor in pathogenic Leptospira. Our results confirmed that MPL36 is surface exposed and expressed during infection. Using recombinant MPL36 (rMPL36) we also confirmed previous findings of its high plasminogen (PLG)-binding ability determined by lysine residues of the C-terminal region of the protein, with ability to convert bound-PLG to active plasmin. Using Koch's molecular postulates, we determined that a mutant of mpl36 has a reduced PLG-binding ability, leading to a decreased capacity to adhere and translocate MDCK cell monolayers. Using recombinant protein and mutant strains, we determined that the MPL36-bound plasmin (PLA) can degrade fibrinogen. Finally, our mpl36 mutant had a significant attenuated phenotype in the hamster model for acute leptospirosis. Our data indicates that MPL36 is the major PLG binding protein in pathogenic Leptospira, and crucial to the pathogen's ability to attach and interact with host tissues during infection. The MPL36 characterization contributes to the expanding field of bacterial pathogens that explore PLG for their virulence, advancing the goal to close the knowledge gap regarding leptospiral pathogenesis while offering a novel potential candidate to improve diagnostic and prevention of this important zoonotic neglected disease.

Abstract 11898: Bovine Milk-Derived Extracellular Vesicles as a Novel Injury-Targeting Drug Delivery System Following Cardiac Injury

Introduction: A novel protocol provides large amounts of highly purified small extracellular vesicles (also called exosomes) from bovine milk (Marsh et al, PMID: 34367882). We sought to examine the targeting potential of these pure, highly concentrated bovine milk-derived extracellular vesicles (mEVs) to injured cells and tissues, with the intent of identifying an effective drug delivery platform capable of targeting injured cardiac tissue. Methods: Targeting of mEVs to injured cells and tissues was tested in vitro using a scratch assay on human dermal fibroblast (hDFs) and MDCK cell monolayers, and in vivo, using mouse models of skin wounding and cardiac injury - methods we have previously reported (PMID:34246197; PMID:29351451). mEVs were isolated using our published approach (PMID: 34367882), then fluorescently tagged with Cell Tracker Deep Red (CTDR). Labelled mEVs were then applied to cell cultures at 20 ug/mL for 15 minutes post wound; cells were then rinsed, fixed and stained for cell nuclei with Hoechst. Mice were provided 2 ug/kg loaded mEV’s by oral gavage before skin surgery and induction of cardiac ischemic reperfusion injury. Mice were sacrificed 4 hours post-surgery, fixed by perfusion with 4% paraformaldehyde followed by PBS rinsing, then organs were explanted. Organs were then embedded in O.C.T, sectioned on a cryostat, and stained for nuclei and actin with FITC-Phalloidin. Imaging was performed on a Leica SP8 laser scanning confocal microscope and quantified using mEV uptake normalized to cell nuclei on ImageJ. Results: mEV uptake was significantly increased in scratch wounded cultures of both hDFs and MDCK cells (p<0.001), over uninjured control cells. Similarly, injured heart and skin tissues exhibited significantly increased exosomal uptake (p<0.001), relative to sham injury controls and skin tissues remote from the injury. Conclusions: Our experiments indicate that injured cells and tissues show increased uptake of milk derived exosomes, with in vitro data suggesting that this enhanced uptake occurs, at least in part, in a cell autonomous manner. The data also supports that our isolation protocol provides a novel mEV-based drug delivery system that may preferentially target injured tissues, including ischemic cardiac tissue.

Polyubiquitination and SUMOylation Sites Regulate the Stability of ZO-2 Protein and the Sealing of Tight Junctions.

Tight junctions (TJs) regulate the transit of ions and molecules through the paracellular pathway in epithelial cells. Zonula occludens 2 (ZO-2) is a cytoplasmic TJ protein. Here, we studied the ubiquitination of hZO-2 employing mutants of SUMOylation site K730 present in the GuK domain and the putative ubiquitination residues K759 and K992 located at the GuK domain and proline-rich region, respectively. In immunoprecipitation experiments done with MDCK cells transfected with wild-type (WT) hZO-2 or the ubiquitination-site mutants hZO-2-K759R or -K992R, we observed diminished ubiquitination of the mutants, indicating that residues K759 and K992 in hZO-2 are acceptors for ubiquitination. Moreover, using TUBES, we found that residues K759 and K992 of hZO-2 are targets of K48 polyubiquitination, a signal for proteasomal degradation. Accordingly, compared to WT hZO-2, the half-life of hZO-2 mutants K759R and K992R augmented from 19.9 to 37.3 and 23.3 h, respectively. Instead, the ubiquitination of hZO-2 mutant K730R increased, and its half-life diminished to 6.7 h. The lack of these lysine residues in hZO-2 affects TJ sealing as the peak of TER decreased in monolayers of MDCK cells transfected with any of these mutants. These results highlight the importance of ZO-2 ubiquitination and SUMOylation to maintain a healthy and stable pool of ZO-2 molecules at the TJ.

The Entry Blocker Peptide Produced in Chlamydomonas reinhardtii Inhibits Influenza Viral Replication in vitro.

This year, a respiratory virus caused an emergency pandemic alert in health services around the world, showing the need for biotechnological approaches to fight these diseases. The influenza virus is one of the main viral agents that generate pandemic outbreaks. Currently, the majority of co-circulating influenza A virus (IAV) strains are adamantine‐ and oseltamivir-resistant strains, and the challenge is to find new antivirals for more efficient treatments. The antiviral entry blocker (EB) peptide is a promising candidate for blocking the virus entry into cells. The aim of this research was to express the EB peptide in the microalgae Chlamydomonas reinhardtii and test its antiviral activity against IAV in vitro. The EB peptide nucleotide sequence was introduced into the nuclear genome of microalgae using Agrobacterium tumefaciens transformation. The EB peptide amount produced in transformed microalgae was 4.99 ± 0.067% of the total soluble protein. In hemagglutination inhibition assays using influenza A/H1N1 pdm and influenza A H1N1/Virginia/ATCC/2009 strains, we reported that the EB peptide extract from the microalgae showed 100-fold higher efficiency than the EB synthetic peptide. In addition, both the EB peptide extract and synthetic peptide inhibited viral replication in MDCK cells (IC50 = 20.7 nM and IC50 = 754.4 nM, respectively); however, the EB peptide extract showed a 32-fold higher antiviral effectiveness than the synthetic peptide against influenza A/H1N1 pdm. Extracts from untransformed and transformed microalgae and synthetic peptide did not show cytotoxic effect on MDCK cell monolayers. Thus, C. reinhardtii may be a fast, safe, and effective expression platform for production of peptides with significant antiviral activity and can be used as a prophylactic treatment to reduce viral propagation.

Toxoplasma gondii excreted/secreted proteases disrupt intercellular junction proteins in epithelial cell monolayers to facilitate tachyzoites paracellular migration.

Toxoplasma gondii shows high dissemination and migration properties across biological barriers infecting immunologically privileged organs. Toxoplasma uses different routes for dissemination; however, the mechanisms are not fully understood. Herein, we studied the effects of proteases present in excretion/secretion products (ESPs) of Toxoplasma on MDCK cell monolayers. Ultrastructural analysis showed that ESPs of Toxoplasma disrupt the intercellular junctions (IJ) of adjacent cells. The tight junction (TJ) proteins ZO-1, occludin, and claudin-1 suffered a progressive decrease in protein levels upon ESPs treatment. In addition, ESPs induced mislocalization of such TJ proteins, along with the adherent junction protein E-cadherin, and this was prevented by pre-treating the ESPs with protease inhibitors. Reorganisation of cytoskeleton proteins was also observed. Endocytosis inhibitors, Dyngo®-4a and Dynasore, impeded the modifications, suggesting that TJ proteins internalisation is triggered by the ESPs proteases hence contributing to the loss of IJ. The observed disruption in TJ proteins went in line with a decrease in the transepithelial electrical resistance of the monolayers, which was significantly blocked by pre-treating ESPs with metalloprotease and serine protease inhibitors. Moreover, exposure of cell monolayers to ESPs facilitated paracellular migration of tachyzoites. Our results demonstrate that Toxoplasma ESPs contain proteases that can disrupt the IJ of epithelial monolayers and this could facilitate the paracellular route for Toxoplasma tissue dissemination and migration.

Vectorial transport of the arginine derivatives asymmetric dimethylarginine (ADMA) and l-homoarginine by OATP4C1 and P-glycoprotein studied in double-transfected MDCK cells

Elevated plasma concentrations of the uremic toxin asymmetric dimethylarginine (ADMA) and low plasma concentrations of l-homoarginine are independently associated with cardiovascular events and mortality. Key enzymes involved in the homeostasis of both arginine derivatives are expressed in proximal tubule cells of the kidney. To get access to these enzymes, transport proteins are important. One of the transporters mediating the transport of ADMA and l-homoarginine is the solute carrier superfamily (SLC) member OATP4C1, located in the basolateral membrane of proximal tubule cells. To gain insights into the role of export pumps in the transport of both substances, we established a double-transfected MDCK cell line expressing OATP4C1 and the export pump P-glycoprotein (P-gp). Using MDCK cell monolayers, we demonstrated in time-dependent and concentration-dependent vectorial transport experiments that ADMA and l-homoarginine are transported from the basolateral to the apical compartment of MDCK-OATP4C1-P-gp cells with significantly higher transport rates compared to single-transfected MDCK-OATP4C1, MDCK-P-gp and MDCK-VC (control) cells (e.g. transport ratio MDCK-OATP4C1-P-gp/MDCK-VC: for 50 µM ADMA = 2.0-fold, for 50 µM l-homoarginine = 3.4-fold). These results indicate that both OATP4C1 and P-gp transport the arginine derivatives ADMA and l-homoarginine and are, therefore, important for the homoeostasis of both substances.

Effect of ZO Protein Knockdown on Leak Pathway Pore Size in MDCK Renal Epithelial Cell Populations

The tight junction is the primary barrier to the paracellular movement of water and solutes across epithelial cells (paracellular permeability). Paracellular permeability is divided into two pathways: the Pore Pathway (small solute size, high capacity, charge‐selective) and the Leak Pathway (large solute size, low capacity, charge‐nonselective). Much has been learned about the molecular basis, properties, and regulation of the Pore Pathway. The Leak Pathway is much less well understood. Two basic questions about the Leak Pathway that remain controversial are: 1) does the Leak Pathway have a size limit; and 2) if so, how is the Leak Pathway size limit affected by manipulating specific tight junction proteins. We have initiated an investigation of these two questions by measuring the paracellular permeability of a size range of fluorescein dextrans (4 kDa to 70 kDa) and calculating pore size using the mathematical model, the Renkin sieving equation. In this mathematical model, calculation of pore size is independent of total flux rate, which is also affected by the pore volume fraction and pore length. We first used this approach to examine Leak Pathway permeability in the wild type MDCK renal epithelial cell line. The paracellular movement of all fluorescein dextrans across MDCK cell monolayers was linear with time. The apparent permeability (Papp) decreased with increasing solute Stokes radius. Using the Renkin sieving equation, the Leak Pathway pore size in wild type MDCK cell monolayers was calculated to be ~423 Angstroms. We then examined the effect of knockdown of either of the tight junction‐associated cytoplasmic proteins, ZO‐1 or ZO‐2, on pore size. The paracellular flux rates for all fluorescein dextrans were higher across ZO‐1 knockdown MDCK (ZO‐1 KD) cell populations than across either wild type MDCK cell populations or ZO‐2 knockdown MDCK (ZO‐2 KD) cell populations, which were similar. The paracellular flux of each fluorescein dextran was linear with time across populations of either ZO‐1 KD cells or ZO‐2 KD cells. Papp decreased with increasing Stokes radius for both knockdown cell lines. The calculated pore size for ZO‐2 KD cell populations was similar to that calculated for wild type MDCK cell populations. In contrast, the pore size for ZO‐1 KD cell populations was substantially smaller (~171 Angstroms) than was calculated for wild type MDCK cell populations despite the fact that the total flux rate for all fluorescein dextrans was significantly higher across ZO‐1 KD cell populations. These results indicate that knockdown of ZO‐1 in MDCK cells decreases the Leak Pathway pore size, whereas, knockdown of ZO‐2 does not alter Leak Pathway pore size. In addition, since the flux rate across ZO‐1 KD cell monolayers is higher than across monolayers of the other two cell lines, these results suggest ZO‐1 KD cell populations exhibit either a greater pore volume fraction and/or a shorter pore length than do either wild type MDCK cell populations or ZO‐2 KD cell populations.Support or Funding InformationNYITCOM internal funds

Auto-fluorescent PAMAM-based dendritic molecules and their potential application in pharmaceutical sciences.

The epithelial permeation of water-soluble fluorescent PAMAM dendrons based on 7H-benz[de] benzimidazo [2,1-a] isoquinoline-7-one as a fluorescent core across epithelial cell models MDCK I and MDCK II has been quantified. Hydrodynamic radii have been derived from self-diffusion coefficients obtained via pulsed-gradient spin-echo Nuclear Magnetic Resonance (PGSE-NMR). Results indicate that these dendritic molecules are molecularly disperse, non-aggregating, and only slightly larger than their parent homologues. MDCK I permeability studies across epithelial barriers show that these dendritic molecules are biocompatible with the chosen epithelial in-vitro model and can permeate across MDCK cell monolayers. Permeability is demonstrated to be a property of dendritic size and cell barrier restrictiveness indicating that paracellular mechanisms play the predominant role in the transport of these molecules.

FITC labeling of human insulin and transport of FITC-insulin conjugates through MDCK cell monolayer

Fluorescein isothiocyanate-labeled insulin (FITC-insulin) has been widely used for bioanalytical applications. Due to the high cost of commercial FITC-insulin and tedious labeling procedures described in the literature, there is still a need to develop a cost effective, reliable and quick labeling method for insulin. The purpose of the present work was to develop a quick and affordable method for FITC labeling of human insulin and to determine the effect of different conjugations of FITC to human insulin on its permeability through the MDCK cell monolayer. FITC labeling of insulin gives mono-, di- or tri-conjugates depending on the reaction time and the molar ratio of FITC:insulin. Mono-conjugate with unlabeled insulin, mixture of di- and tri-conjugate, and tri-conjugate with very little amount of di-conjugate were synthesized in less than 4 h. Degree of conjugation had an effect on the permeability of insulin through the MDCK cell monolayer. Mono-conjugate had higher permeability than the unlabeled insulin due to increase in partition coefficient. However, tri-conjugate showed lower permeability than the unlabeled insulin due to the increase in molecular weight.

Lysosomal P-gp-MDR1 Confers Drug Resistance of Brentuximab Vedotin and Its Cytotoxic Payload Monomethyl Auristatin E in Tumor Cells.

Antibody-drug conjugates (ADCs) are composed of an antibody linked to cytotoxic anticancer payloads. ADCs recognize tumor-specific cell surface antigens and are internalized into lysosomes where proteolytic enzymes release the cytotoxic payloads. Efflux transporters on plasma membrane that play a significant role on multi-drug resistance in chemotherapy can be internalized on lysosomal membrane and sequester the cytotoxic payloads. In the present study, ATP binding cassette (ABC) efflux transporters including breast cancer resistance protein (BCRP), P-glycoprotein (P-gp-MDR1), multidrug resistance protein (MRP) 2, MRP3 and MRP4 in lysosomal, and plasma membrane of tumor cells were quantified by targeted quantitative proteomics. The cytotoxicity of brentuximab vedotin and its cytotoxic payload monomethyl auristatin E (MMAE) to the tumor cell lines in the presence and absence of elacridar (P-gp-MDR1 inhibitor) or chloroquine (lysosomotropic agent) were evaluated. MMAE is a substrate for P-gp-MDR1, as the apparent efflux ratio in MDR1 transfected MDCK cell monolayers was 44.5, and elacridar abolished the MMAE efflux. Cell lines that highly express P-gp-MDR1 show higher EC50s toward the cell killing effects of MMAE. Co-incubation with chloroquine or elacridar resulted in left shift of MMAE EC50 by 2.9–16-fold and 4.2–22-fold, respectively. Similarly co-incubation with chloroquine or elacridar or in combination of chloroquine and elacridar increased cytotoxic effects of brentuximab vedotin by 2.8- to 21.4-fold on KM-H2 cells that express a specific tumor antigen CD30 and P-gp-MDR1. These findings demonstrate important roles of P-gp-MDR1 on cytotoxic effects of brentuximab vedotin and its payload MMAE. Collectively, ABC transporter-mediated drug extrusion and/or sequestration needs to be early assessed for selection of optimal payloads and linkers when developing ADCs.

Transient receptor potential V4 channel stimulation induces reversible epithelial cell permeability in MDCK cell monolayers.

The transient receptor potential V4 channel (TRPV4) is responsive to a variety of physical and chemical stimuli, including a synthetic agonist GSK1016790A (GSK). Here, we show that TRPV4 is functionally expressed in, and that GSK induces the reversible opening of tight junctions (TJs) in epithelial Madin-Darby canine kidney II monolayers. Stimulation of TRPV4 by GSK induces an increase in fluorescein isothiocyanate-conjugated dextran (4kDa) permeability and a reduction in transepithelial resistance, and these responses are blocked by pretreatment with the specific TRPV4 antagonist. Small conductance, but not large conductance Ca2+ -activated K+ channels, TRPA1 channel, and cofilin activation are involved in TRPV4-mediated reversible opening of TJs. These results suggest that a novel mechanism underlies TRPV4-mediated regulation of the tightness of epithelial barriers.

Erratum: Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips.

Erratum: Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips.

The function and mechanism of preactivated thiomers in triggering epithelial tight junctions opening

As a unique macromolecular permeation enhancer, thiolated polymers (thiomers), especially the preactivated thiomers, have demonstrated great merits in oral delivery of protein/peptide drugs by triggering epithelial tight junctions (TJs) opening. However, the underlying molecular mechanism remains unclear. To clarify this issue, preactivated thiomers were synthesized and their TJs opening function as well as signaling pathways on MDCK and Caco-2 cell monolayers was investigated. The results showed that preactivated thiomers could reduce TEER and increase the permeation of Na-Flu and FITC-Insulin over 2-fold and 4-fold on MDCK monolayers, respectively, indicating their huge potential as macromolecular permeation enhancers. The signaling pathway study showed that intracellular PTK Src but not FAK, involved in the TJs opening by claudin-4 disruption. Src activation was based on interaction between thiol group of thiomers and cysteine-riched Src upstream membrane receptors, EGFR and IGFR. The deep comprehension of the thiomers-mediated TJs opening mechanisms provides goodness in application of protein/peptide drugs for the oral delivery.

Integrating theoretical and experimental permeability estimations for provisional biopharmaceutical classification: Application to the WHO essential medicines.

The accuracy of the provisional estimation of the Biopharmaceutics Classification System (BCS) is heavily influenced by the permeability measurement. In this study, several theoretical and experimental models currently employed for BCS permeability classification have been analysed. The experimental models included the in situ rat intestinal perfusion, the ex vivo rat intestinal tissue in an Ussing chamber, the MDCK and Caco-2 cell monolayers, and the parallel artificial membrane (PAMPA). The theoretical models included the octanol-water partition coefficient and the QSPeR (Quantitative Structure-Permeability Relationship) model recently developed. For model validation, a dataset of 43 compounds has been recompiled and analysed for the suitability for BCS permeability classification in comparison with the use of human intestinal absorption and oral bioavailability values. The application of the final model, based on a majority voting system showed a 95.3% accuracy for predicting human permeability. Finally, the present approach was applied to the 186 orally administered drugs in immediate-release dosage forms of the WHO Model List of Essential Medicines. The percentages of the drugs that were provisionally classified as BCS Class I and Class III was 62.4%, suggesting that in vivo bioequivalence (BE) may potentially be assured with a less expensive and more easily implemented in vitro dissolution test, ensuring the efficiency and quality of pharmaceutical products. The results of the current study improve the accuracy of provisional BCS classification by combining different permeability models.

Contribution of multidrug and toxin extrusion protein 1 (MATE1) to renal secretion of trimethylamine-N-oxide (TMAO)

Trimethylamine-N-oxide (TMAO) gained considerable attention because of its role as a cardiovascular risk biomarker. Organic cation transporter 2 (OCT2) mediates TMAO uptake into renal proximal tubular cells. Here we investigated the potential role of multidrug and toxin extrusion protein 1 (MATE1) for translocation of TMAO across the luminal membrane of proximal tubular cells. HEK293 cells stably expressing OCT2 (HEK-OCT2) or MATE1 (HEK-MATE1) were used for uptake studies. Transcellular transport of TMAO was investigated using monolayers of MDCK control cells (MDCK-Co) as well as single- (MDCK-OCT2, MDCK-MATE1) and double-transfected cells (MDCK-OCT2-MATE1). In line with previous studies, HEK-OCT2 cells revealed a 2.4-fold uptake of TMAO compared to control cells (p < 0.001), whereas no significant uptake was observed in HEK-MATE1. In monolayers of MDCK cells, polarised TMAO transcellular transport was not significantly different between MDCK-Co and MDCK-OCT2 cells, but significantly increased in MDCK-MATE1 (p < 0.05) and MDCK-OCT2-MATE1 cells (p < 0.001). The OCT/MATE inhibitor trimethoprim abolished TMAO translocation in MDCK-OCT2-MATE1 cells (p < 0.05). The present data suggest that MATE1 contributes to renal elimination of TMAO. For selected MATE substrates, such as TMAO, uptake studies using non-polarised MATE-expressing cells can reveal false negative results compared to studies using polarised monolayers.

Hepatobiliary Disposition of Atovaquone: A Case of Mechanistically Unusual Biliary Clearance.

Atovaquone, an antiprotozoal and antipneumocystic agent, is predominantly cleared by biliary excretion of unchanged parent drug. Atovaquone is ≥10,000-fold concentrated in human bile relative to unbound plasma. Even after correcting for apparent nonspecific binding and incomplete solubility in bile, atovaquone is still concentrated ≥100-fold in bile, consistent with active biliary excretion. Mechanisms of atovaquone hepatobiliary disposition were studied using a multiexperimental in vitro and in vivo approach. Atovaquone uptake was not elevated in HEK293 cells singly overexpressing OATP1B1, OATP1B3, OATP2B1, OCT1, NTCP, or OAT2. Hepatocyte uptake of atovaquone was not impaired by OATP and OCT inhibitor cocktail (rifamycin and imipramine). Atovaquone liver-to-blood ratio at distributional equilibrium was not reduced in Oatp1a/1b and Oct1/2 knockout mice. Atovaquone exhibited efflux ratios of approximately unity in P-gp and BCRP overexpressing MDCK cell monolayers and did not display enhanced uptake in MRP2 vesicles. Biliary and canalicular clearance were not decreased in P-gp, Bcrp, Mrp2, and Bsep knockout rats. In the present study, we rule out the involvement of major known basolateral uptake and bile canalicular efflux transporters in the hepatic uptake and biliary excretion of atovaquone. This is the first known example of a drug cleared by biliary excretion in humans, with extensive biliary concentration, which is not transported by the mechanisms investigated herein.

The Entamoeba histolytica TBP and TRF1 transcription factors are GAAC-box binding proteins, which display differential gene expression under different stress stimuli and during the interaction with mammalian cells

BackgroundEntamoeba histolytica is the protozoan parasite responsible for human amebiasis. It causes up to 100,000 deaths worldwide each year. This parasite has two closely related basal transcription factors, the TATA-box binding protein (EhTBP) and the TBP-related factor 1 (EhTRF1). TBP binds to the canonical TATTTAAA-box, as well as to different TATA variants. TRF1 also binds to the TATTTAAA-box. However, their binding capacity to diverse core promoter elements, including the GAAC-element, and their role in gene regulation in this parasite remains unknown.MethodsEMSA experiments were performed to determine the binding capacity of recombinant TBP and TRF1 to TATA variants, GAAC and GAAC-like boxes. For the functional analysis under different stress stimuli (e.g. growth curve, serum depletion, heat-shock, and UV-irradiation) and during the interaction with mammalian cells (erythrocytes, MDCK cell monolayers, and hepatocytes of hamsters), RT-qPCR, and gene knockdown were performed.ResultsBoth transcription factors bound to the different TATA variants tested, as well as to the GAAC-boxes, suggesting that they are GAAC-box-binding proteins. The KD values determined for TBP and TRF1 for the different TATA variants and GAAC-box were in the range of 10-12 M to 10-11 M. During the death phase of growth or in serum depletion, Ehtbp mRNA levels significantly increased, whereas the mRNA level of Ehtrf1 did not change under these conditions. Ehtrf1 gene expression was negatively regulated by UV-irradiation and heat-shock stress, with no changes in Ehtbp gene expression. Moreover, Ehtrf1 gene also showed a negative regulation during erythrophagocytosis, liver abscess formation, and a transient expression level increase at the initial phase of MDCK cell destruction. Finally, the Ehtbp gene knockdown displayed a drastic decrease in the efficiency of erythrophagocytosis in G3 trophozoites.ConclusionsTo our knowledge, this study reveals that these basal transcription factors are able to bind multiple core promoter elements. However, their immediate change in gene expression level in response to different stimuli, as well as during the interaction with mammalian cells, and the diminishing of erythrophagocytosis by silencing the Ehtbp gene indicate the different physiological roles of these transcription factors in E. histolytica.

Predicting apparent passive permeability of Caco-2 and MDCK cell-monolayers: A mechanistic model.

Experimentally derived apparent permeabilities, Papp, through cell monolayers such as Caco-2 and MDCK are considered to be an in-vitro gold standard for assessing the uptake efficiency of drugs. Here, we present a mechanistic model that describes ‘passive’ Papp values (i.e., neglecting active transport) by accounting for the different resistances solutes encounter when permeating a cell monolayer. We described three parallel permeation pathways, namely a cytosolic-, paracellular-, and lateral route, each of which consists of a number of serial resistances. These resistances were accounted for via a mechanistic depiction of the underlying processes that are largely based on literature work. For the present Papp dataset, about as much chemicals are dominated by the cytosolic route as were dominated by the paracellular route, while the lateral route was negligible. For the cytosolic route by far the most chemicals found their main resistance in the various water layers and not in the membrane. Although correlations within the subclasses of chemicals dominated by a specific permeation route were rather poor, we could overall satisfyingly predict Papp for 151 chemicals at a pH of 7.4 (R2 = 0.77, RMSE = 0.48). For a specific evaluation of the intrinsic membrane permeability, Pm, a second experimental dataset based on experiments with black lipid membranes, BLM, was evaluated. Pm could be predicted for 37 chemicals with R2 = 0.91 and RMSE = 0.64 log units.