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Influenza is an acute respiratory infection caused by the influenza virus, and vaccination against influenza is considered the best way to prevent the onset and spread. MDCK (Madin-Darby canine kidney) cells are typically used to isolate the influenza virus, however, their high tumorigenicity is the main controversy in the production of influenza vaccines. Here, MDCK-C09 and MDCK-C35 monoclonal cell lines were established, which were proven to be low in tumorigenicity. RNA-seq of MDCK-C09, MDCK-C35, and MDCK-W73 cells was performed to investigate the putative tumorigenicity mechanisms. Tumor-related molecular interaction analysis of the differentially expressed genes indicates that hub genes, such as CUL3 and EGFR, may play essential roles in tumorigenicity differences between MDCK-C (MDCK-C09 and MDCK-C35) and MDCK-W (MDCK-W73) cells. Moreover, the analysis of cell proliferation regulation-associated molecular interaction shows that downregulated JUN and MYC, for instance, mediate increased proliferation of these cells. The present study provides a new low-tumorigenic MDCK cell line and describes the potential molecular mechanism for the low tumorigenicity and high proliferation rate., (Copyright © 2020. Published by Elsevier Ltd.)

The coordination of cell proliferation and migration in growing tissues is crucial in development and regeneration but remains poorly understood. Here, we find that, while expanding with an edge speed independent of initial conditions, millimeter-scale epithelial monolayers exhibit internal patterns of proliferation and migration that depend not on the current but on the initial tissue size, indicating memory effects. Specifically, the core of large tissues becomes very dense, almost quiescent, and ceases cell-cycle progression. In contrast, initially-smaller tissues develop a local minimum of cell density and a tissue-spanning vortex. To explain vortex formation, we propose an active polar fluid model with a feedback between cell polarization and tissue flow. Taken together, our findings suggest that expanding epithelia decouple their internal and edge regions, which enables robust expansion dynamics despite the presence of size- and history-dependent patterns in the tissue interior., Competing Interests: MH, RA, JL, TZ, AK, DC No competing interests declared, (© 2020, Heinrich et al.)

A biopharmaceutics classification system class IV drug, hydrochlorothiazide (HCT), was combined with co-formers of L-and d-arginine (ARG) and sodium lauryl sulphate (SLS) by cryomilling in 1:1 molar ratio. Co-amorphization was observed with L- and D-ARG. These mixtures showed a single glass transition, evidence of possible salt formation and improved physical stability at elevated temperatures and/or humidity when compared with amorphous HCT. The co-amorphous formulations, along with the combinations of HCT and HCT:L-ARG with polyvinylpyrrolidone (PVP) in 1:1 mass ratio, were investigated with a simultaneous dissolution/permeation setup using parallel artificial membrane permeability assay (PAMPA) or Madine Darby kidney cells (MDCKII) as the permeation barrier. It was observed that co-amorphization with L-ARG and D-ARG was able to induce a supersaturated state for HCT, possibly through intermolecular interactions, but there was virtually no difference between the dissolution properties of the mixtures formed with the 2 optical isomers of ARG. The permeability of HCT was found to be dependent on the dissolution properties of the formulations in both PAMPA and cellular barrier experiments. Thus, co-amorphization of HCT with L- and D-ARG demonstrated the possibility to enhance the dissolution and thereby the permeation potential of a BCS class IV drug., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Purpose: MDCK cells are commonly used to assess drug permeability, but the existence of various strains merits a comparative functional study. Since metformin absorption is largely mediated by transporters and paracellular diffusion, we used it to functionally compare MDCK-wt and MDCK-II., Methods: Uptake, bidirectional transport and efflux experiments were performed using different buffers, pH, and a panel of transporter inhibitors. Relative contributions to total transport in both strains were estimated., Results: Metformin uptake into MDCK-wt was linear but saturable in MDCK-II. Uptake into MDCK-wt or -II was promoted at pH 5.4 or 8.4, respectively. Quinidine and cimetidine similarly inhibited uptake in both strains. Lopinavir (PMAT specific) at pH 5.4 or pyrimethamine (MATE specific) at pH 8.4 differentially inhibited MDCK-wt or -II, respectively. Transport at pH 7.4 was absorptive regardless of strains, but secretory (MDCK-II) or absorptive (MDCK-wt) at pH 5.4. Efflux was largely basolateral in both strains. While paracellular permeability was similar between strains, total transport was dominated by transporters in MDCK-II or paracellular diffusion in MDCK-wt., Conclusions: Metformin transport revealed functional differences between MDCK strains. Apical uptake was governed by MATE in MDCK-II or PMAT in MDCK-wt, such that metformin transport was either secretory or absorptive, respectively.

Raman spectroscopy was applied with a high spectral resolution to a structural study of Influenza (type A) virus before and after its inoculation into Madin-Darby canine kidney cells. This study exploits the fact that the major virus and cell constituents, namely DNA/RNA, lipid, and protein molecules, exhibit peculiar fingerprints in the Raman spectrum, which clearly differed between cells and viruses, as well as before and after virus inoculation into cells. These vibrational features, which allowed us to discuss viral assembly, membrane lipid evolution, and nucleoprotein interactions of the virus with the host cells, reflected the ability of the virus to alter host cells' pathways to enhance its replication efficiency. Upon comparing Raman signals from the host cells before and after virus inoculation, we were also able to discuss in detail cell metabolic reactions against the presence of the virus in terms of compositional variations of lipid species, the formation of fatty acids, dephosphorylation of high-energy adenosine triphosphate molecules, and enzymatic hydrolysis of the hemagglutinin glycoprotein., (© 2019 Wiley Periodicals, Inc.)

Similar to growth factors aldosterone stimulates Na/H exchange in renal target cells leading to cytoplasmic alkalinization. An alkaline intracellular pH reduces the H bonds between repressor proteins and DNA leading to the destabilization of the nuclear chromatin. We observed that sustained alkaline stress 'per se' can lead to malignant transformation of Madin-Darby canine kidney (MDCK) cells. Cells grown for two weeks in alkaline culture medium (pH 7.8) developed multiple 'foci' composed of spindle-shaped pleomorphic cells lacking contact inhibition and exhibiting poor adhesion to the culture support, typical characteristics of dedifferentiated tumor cells. 'Focus' cells were cloned and grown in standard medium (pH 7.4). Cells maintained their abnormal growth pattern, indicating stable pH-induced genetic transformation. Cells were fused with polyethylene glycol to giant cells and impaled with microelectrodes. In contrast to non-transformed giant MDCK cells the plasma membrane potential showed spontaneous oscillations that could be virtually abolished by the omission of extracellular Ca or by the addition of the K channel blocker Ba. We conclude that sustained alkaline stress can induce malignant transformation in MDCK cells indicated by an abnormal growth pattern and by membrane potential oscillations most likely due to Ca activated K channels in the plasma membrane. [ABSTRACT FROM AUTHOR]

Stereo- and regioisomers of a series of N,N -bis(alkanol)amine aryl ester derivatives have been prepared and studied as multidrug resistance (MDR) modulators. The new compounds contain a 2-(methyl)propyl chain combined with a 3-, 5- or 7-methylenes long chain and carry different aromatic ester portions. Thus, these compounds have a methyl group on the 3-methylenes chain and represent branched homologues of previously studied derivatives. The introduction of the methyl group gives origin to a stereogenic center and consequently to (R) and (S) enantiomers. In the pirarubicin uptake assay on K562/DOX cell line these compounds showed good activity and efficacy and in many cases enantioselectivity was observed. Docking studies confirmed the influence of the stereocenter on the interaction in the P-gp pocket. The P-gp interaction mechanism and selectivity towards MRP1 and BCRP were also evaluated on MDCK transfected cells overexpressing the three transporters. Almost all these compounds inhibited both P-gp and BCRP, but only derivatives with specific structural characteristics showed MRP1 activity. Moreover, two compounds, (S)- 3 and (R)- 7 , showed the ability to induce collateral sensitivity (CS) against MDR cells. Therefore, these two CS-promoting agents could be considered interesting leads for the development of selective cytotoxic agents for drug-resistant cells. •Stereo- and regioisomers of amino aryl esters showed different MDR reversing activity profiles.•Compounds (S)- 3 (Ar = b, Ar1 = a, n = 3) and (R)- 7 (Ar = b, Ar1 = a, n = 5) induced collateral sensitivity (CS) against MDR cells. Image 1 • Stereo- and regioisomers of N,N -bis(alkanol)amine aryl esters were described. • The biological behavior of these compounds was studied by various tests. • They act as multidrug resistance (MDR) reversers. • Two compounds were identified as MDR1 or MRP1-overexpressing cells sensitizers. • Docking studies and stability tests in human plasma were performed. [ABSTRACT FROM AUTHOR]

The present study was to investigate the protective effects of Zn supplementation in OTA-induced apoptosis of Madin-Darby canine kidney (MDCK) epithelial cells and explore the potential mechanisms. Aiming to provides a new insight into the treatment strategy of OTA-induced nephrotoxicity by nutritional regulation. Initially, through MTT and LDH assay revealed that Zn supplementation significantly suppressed OTA-induced cytotoxicity in MDCK cells. Then, the production of reactive oxygen species (ROS) was detected by using a DCFH-DA assay. Annexin V-FITC/PI, Hoechst 33258 staining and Flow cytometry were used to detect the apoptosis. The expressions of apoptosis-related molecules were determined by RT-PCR, Western blotting. Interestingly, OTA treatment slightly increased the levels of Metallothionein-1 (MT-1) and Metallothionein-2 (MT-2) by using RT-PCR, Western blotting assay; while Zn supplementation further improved the increase of MT-1 and MT-2 induced by OTA. However, the inhibitive effects of Zn supplementation were significantly blocked after double knockdown of MT-1 and MT-2 by using Small Interfering RNA (siRNA) Transfection method. Our study provides supportive data for the potential roles of Zn in reducing OTA-induced oxidative stress and apoptosis in MDCK cells. Zn is one of the key structural components of many proteins, which plays an important role in several physiological processes such as cell survival and apoptosis. This metal is expected to contribute to the conservative and adjuvant treatment of kidney disease and should therefore be investigated further. Unlabelled Image • Zn supplementation protected MDCK cells against OTA-induced cytotoxicity. • Zn supplementation alleviated OTA-induced ROS generation and apoptosis. • Zn supplementation further up-regulated MTs levels in MDCK cells. • The alleviating effect of Zn supplementation was abrogated after MTs knockdown. [ABSTRACT FROM AUTHOR]

Madin-Darby canine kidney(MDCK) cells can be used to prepare cell-based influenza vaccines; however, little is known regarding the effect of lncRNA regulatorson tumorigenicity. In the present study, two cell lines with low tumorigenicity were screened from highly tumorigenic MDCK cell lines using monoclonal cell technology. Accordingly, three groups of lncRNAs were extracted from three cell lines and investigated using strand-specific Ribo-Zero RNA sequencing, detecting 1092 known and 619 novel lncRNAs. Moreover, in pairwise comparisons between the libraries of the nominally tumorigenic clones and the highly tumorigenic parent cell line, a total of 344 transcripts were expressed differentially, which were validated by qPCR using six randomly selected lncRNA genes. Furthermore, 63 target genes were identified in the upstream and downstream 100 kb of lncRNAs and their relative functions were analyzed. It was found that ten GO terms and twelve KEGG terms related to tumor by target genes and functional items. Five lncRNA transcripts and the corresponding differentially expressed target genes were used for co-expression network analysis. In addition, certain classical tumor pathways were also activated by target genes, among which, lncRNA MSTRG.1056.2 directly regulates ERBB3 to activate the PI3K-Akt pathway, contributing to tumorigenesis. Consequently, direct evidence was obtained that lncRNA regulates tumorigenesis, and a variety of target genes regulated by lncRNA were elucidated, which may be significant for non-tumorigenic MDCK cells lines acquisition., Competing Interests: Declaration of Competing Interest None of the authors have any conflicts of interest to declare., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Missense variants are associated with various phenotypic traits and disorders in dogs. The canine P2RX7 gene, coding the ATP-gated P2X7 receptor ion channel, contains four known missense variants. The current study aimed to examine the presence of these variants in a random sample of pedigree and mixed-pedigree dogs. Exons 3, 8, 11 and 13 of the P2RX7 gene, encoding these four respective variants, in 65 dogs were assessed by Sanger sequencing and combined with existing sequencing data from another 69 dogs. The distribution of these variants was then evaluated in all 134 dogs combined and separately within individual breeds including 35 different pure breeds. The rs23314713 (p.Phe103Leu) and rs23315462 (p.Pro452Ser) variants were present in 47 and 40% of all dogs studied respectively, with the rs23314713 variant associated with brachycephalic breeds. Among pedigree dogs, the rs23314713 and rs23315462 variants were associated with brachycephalic and non-brachycephalic breeds respectively. The rs851148233 (p.Arg270Cys) and rs850760787 (p.Arg365Gln) variants were present only in dogs of Cocker Spaniel and Labrador Retriever pedigrees respectively. No other missense variants were found in exons 3, 8, 11 and 13 of the P2RX7 gene within the dogs. In conclusion, the rs23314713 and rs23315462 missense variants of the P2RX7 gene are present in a large proportion of dogs, with the rs23314713 variant associated with a number of brachycephalic breeds. However, the association of this variant with dogs of bulldog ancestry, not brachycephaly per se, cannot be excluded., (© 2019 Stichting International Foundation for Animal Genetics.)

Literature review shows that various media have been used for in vitro transport study, but their impact on the monolayer integrity and permeability has been somehow neglected, and there is no systemic study on this subject. This study aims to investigate the impact of 6 commonly used media on Madin-Darby Canine Kidney cell viability and adherence, and the permeation of mannitol and propranolol. Dulbecco's modified Eagle medium (DMEM), cMedium (DMEM with serum), Hanks' balanced salt solution (HBSS), HBSS - (without Ca 2+ /Mg 2+ ), Dulbecco's phosphate-buffered saline (DPBS), and DPBS - (without Ca 2+ /Mg 2+ ) exhibited no cytotoxicity. Cell detachment was observed in 4 media in the order of DPBS - ≈ HBSS - > DPBS > HBSS. HBSS - /DPBS - caused 70% TEER reduction and 4-fold higher apparent permeability (P app ) of mannitol than the other media. The addition of D-glucose in DPBS - decreased the P app of mannitol. Cations and glucose negligibly influenced the P app of propranolol. The addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in HBSS increased the P app of both mannitol and propranolol for 2-3 folds without TEER reduction. In conclusion, the media compositions (Ca 2+ /Mg 2+ , glucose, vitamins, amino acids, and HEPES) can affect the monolayer integrity and permeability. DMEM and cMedium without HEPES are hence suggested as the media in transport study to avoid the potential impact on the monolayer integrity and permeability by the media., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

As an alternative to vitamin K antagonists (VKAs), direct oral anticoagulants (DOACs) are increasingly prescribed in combination with riociguat in the treatment of chronic thromboembolic pulmonary hypertension (CTEPH). Pharmacokinetics of riociguat and DOACs are influenced by efflux transporters, such as P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). This work aimed to assess P-gp and BCRP-mediated drug-drug interactions of riociguat with DOACs using in vitro models. Bidirectional permeabilities of apixaban and rivaroxaban were investigated across MDCK-MDR1 and MDCK-BCRP models, in the absence and in the presence of increasing concentrations of riociguat (0.5-100 μm). Calculated efflux ratios were subsequently used to determine riociguat inhibition percentages and half maximal inhibitory concentration (IC50). P-gp-mediated efflux of apixaban and rivaroxaban was inhibited by 8% and 21%, respectively, in the presence of 100 μm riociguat. BCRP-mediated transport of apixaban and rivaroxaban was inhibited by 36% and 77%, respectively. IC50s of riociguat on MDCK-MDR1 and MDCK-BCRP models were higher than 100 μm for apixaban and higher than 100 μm and 46.5 μm for rivaroxaban, respectively. This work showed an in vitro inhibition of BCRP-mediated DOACs transport by riociguat. In vivo studies may be required to determine the clinical relevance of these transporter-mediated interactions., (© 2019 Société Française de Pharmacologie et de Thérapeutique.)

Hyperosmolality is found under physiological conditions in the kidneys, whereas hyperosmolality in other tissues may be associated with pathological conditions. In such tissues an association between inflammation and hyperosmolality has been suggested. During hyperosmotic stress, an important phenomenon is upregulation of solute carriers (SLCs). We hypothesize that hyperosmolality affects the expression of many SLCs as well as ABC transporters. Through RNA-sequencing and topological pathway analysis, the cell cycle, the cytokine-cytokine receptor interaction pathway, and the chemokine-signaling pathway were significantly activated in MDCK I cells after hyperosmotic treatment (Δ200 mOsm) with raffinose or NaCl. 9065, 8052 and 5018 genes were significantly regulated by raffinose, NaCl or urea supplementation (500 mOsm), respectively, compared to control (300 mOsm). Cytokines, that have not previously been associated with hyperosmolality, were identified. We further provide an overview of transport proteins that could be of relevance in tissues exposed to hyperosmolality. Especially Slc5a8 was found highly up-regulated., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Kidney disease prevalence increases with age, with a common feature of the disease being defects in the epithelial tight junctions. Emerging evidence suggests that the desmosomal adhesion protein Desmoglein-3 (Dsg3) functions beyond the desmosomal adhesion and plays a role in regulating the fundamental pathways that govern cell fate decisions in response to environmental chemical and mechanical stresses. In this study, we explored the role of Dsg3 on dome formation, reactive oxygen species (ROS) production and transepithelial electrical resistance (TER) in MDCK cells, a kidney epithelial cell model widely used to study cell differentiation and tight junction formation and integrity. We show that overexpression of Dsg3 constrained nuclear ROS production and cellular doming in confluent cell cultures and these features coincided with augmented TER and enhanced tight junction integrity. Conversely, cells expressing dominant-negative Dsg3ΔC mutants exhibited heightened ROS production and accelerated doming, accompanied by increased apoptosis, as well as cell proliferation, with massive disruption in F-actin organization and accumulation, and alterations in tight junctions. Inhibition of actin polymerization and protein synthesis was able to sufficiently block dome formation in mutant populations. Taken together, these findings underscore that Dsg3 has a role in controlling cellular viability and differentiation as well as the functional integrity of tight junctions in MDCK cells., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The influenza A virus (IAV) binds to α-2,3- and α-2,6-linked sialic acid (SA) receptors expressed by Madin-Darby canine kidney (MDCK) cells. The receptor distribution may therefore be important in regulating IAV propagation. Serum-free medium (SFM) avoids variability in conventional culture medium containing fetal bovine serum (FBS), which can have variable composition and may contain endotoxins. However, little is known about the distribution of SA receptors on cells maintained in SFM., Objectives: We assessed the influence of culture media on MDCK cell SA receptor distribution along with the effect of SA receptor distribution on IAV recovery. We hypothesized that SFM would increase the proportion of α-2,6-linked SA receptors present and alter isolate recovery., Methods: Madin-Darby canine kidney cells were cultured in medium containing FBS and two SFMs. Cell surface distribution of α-2,6- and α-2,3-linked receptors was determined using flow cytometry. Recovery of swine- and avian-lineage IAVs from MDCK cells maintained in each medium was quantified as TCID 50 ., Results: Madin-Darby canine kidney cells cultured in UltraMDCK SFM expressed both SA receptors and supported the growth of both swine- and avian-lineage IAVs. Cells maintained in other medium inconsistently expressed each receptor and the avian IAV grew to lower titers in cells cultured with FBS., Conclusions: Medium conditions altered the distribution of SA receptors present on MDCK cells and affected IAV recovery. Culture in UltraMDCK SFM resulted in cells expressing both receptors and IAVs grew to higher titers than in the other culture condition, indicating that this medium may be useful for culturing IAV from multiple species., (© 2019 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Antithrombin is a serine protease inhibitor that exerts a crucial role in hemostasis as the main inhibitor of the coagulation cascade. It plays also critical roles in other processes, such as inflammation and cancer. Here we show that exosomes released by Madin-Darby canine kidney (MDCK) cells cultured in the presence of heparin incorporate antithrombin from the serum. Exosomal antithrombin is found complexed with the serine protease high temperature requirement A1 (HTRA1), whose cellular levels are increased after serum deprival, the condition used to collect exosomes. Although the biological relevance of the presence of antithrombin in exosomes remains to be investigated, our results suggest a functional interplay between antithrombin and HTRA1., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Introduction: Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na + /H + exchange and H 2 O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously., Methods: We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport., Results: piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport., Conclusions: Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy., (© This is a U.S. government work and its text is not subject to copyright protection in the United States; however, its text may be subject to foreign copyright protection 2019.)

Tight junction (TJ) modulation is a promising approach for improving drug bioavailability by enhancing the absorption of active pharmaceutical ingredients. However, the application of many different test methods to determine the efficacy of new TJ modulators (TJMs) or to assess different compounds is accompanied by a lack of comparable results, reducing the rational evaluation and commercial marketing of these pharmaceutical excipients. The establishment of unified testing methods can fill this gap and offers the opportunity to compare results from different laboratories. Furthermore, the calculation of a TJ modulation score allows the objective comparison of TJ modulators and facilitates the selection of appropriate candidates. In this study, eight well-known TJ modulators were tested with a focus on four different in vitro bioassays carried out with MDCK cells. The extent of TJ modulation was determined by transepithelial electric resistance (TEER) measurements and permeability studies with mannitol. To evaluate tolerability, cell viability (MTT) and cytotoxicity (CellTox ™ Green) assays were performed, and TEER regeneration was monitored for 24 h after exposure. With the exception of labradimil, seven TJ modulators caused significant TEER reduction of up to 100 %. For five compounds, an enhancement of mannitol permeation was observed. As expected, first-generation enhancers exhibited lower cell compatibility than mechanism-based modulators. Based on the experimental results of this study, for the first time, an evaluation system (tight junction modulator scoring system, TJMSS) is presented that provides a ranking of the tested modulators depending on weighted parameters. Such a system offers the possibility of rational formulation development for drugs requiring improved absorption., (Copyright © 2019 Elsevier B.V. All rights reserved.)