Madin-Darby Kidney Cells Research Articles

The high-throughput solid-phase extraction of cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) from Lactobacillus plantarum demonstrates efficacy against multidrug-resistant bacteria and influenza A (H3N2) virus.

Introduction: 2,5-diketopiperazines are the simplest forms of cyclic dipeptides (CDPs) and have diverse frameworks with chiral side chains that are useful for drug development. Previous research has investigated the antimicrobial properties of proline-linked CDPs and their combinations in the culture filtrate (CF) of Lactobacillus plantarum LBP-K10 using anion exchange chromatography (AEC). However, the quantity of CDPs showcasing notable anti-influenza virus activity derived from AECs was generally lower than those originating from Lactobacillus CF. Methods: To address this issue, the study aims to propose a more efficient method for isolating CDPs and to introduce the antiviral combinations of CDPs obtained using a new method. The study employed a novel technique entailing high-throughput C18-based solid-phase extraction with a methanol gradient (MeSPE). The MeSPE method involved increasing the methanol concentration from 5% to 50% in 5% increments. Results: The methanol SPE fractions (MeSPEfs) eluted with methanol concentrations between 35% and 45% evinced substantial efficacy in inhibiting the influenza A/H3N2 virus via plaque-forming assay. MeSPEf-45, the 45% MeSPEf, exhibited exceptional efficacy in preventing viral infections in Madin-Darby kidney cells, surpassing both individual CDPs and the entire set of MeSPEfs. To identify the specific antiviral components of MeSPEf-45, all MeSPEfs were further fractionated through preparative high-performance liquid chromatography (prep-HPLC). MeSPEf-45 fractions S8 and S11 presented the highest activity against multidrug-resistant bacteria and influenza A/H3N2 virus among all MeSPEfs, with 11 common fractions. Antiviral fractions S8 and S11 were identified as proline-based CDPs, specifically cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro), using gas chromatography-mass spectrometry. The combination of MeSPEf-45 fractions S8 and S11 displayed superior antibacterial and anti-influenza virus effects compared to the individual fractions S8 and S11. Discussion: High-throughput MeSPE-derived MeSPEfs and subsequent HPLC-fractionated fractions presents an innovative approach to selectively purify large amounts of potent antimicrobial CDPs from bacterial CF. The findings also show the effectiveness of physiologically bioactive combinations that utilize fractions not containing CDP. This study provides the initial evidence demonstrating the antimicrobial properties of CDPs acquired through high-throughput SPE techniques.

Lycopene improves methotrexate-induced functional alterations of the Madin-Darby kidney cells in a concentration-dependent manner.

Lycopene is one of the most potent antioxidants among carotenoids due to its ability to quench singlet oxygen and react with free radicals to reduce DNA damage. Methotrexate is widely used in the treatment of several types of cancers and autoimmune diseases. One of the most common side effects of a high-dose of methotrexate is kidney injury. In this study, we evaluated effects of lycopene on the Madin-Darby canine kidney cells (MDCK) treated with methotrexate through the estimation of their mitochondrial and lysosomal functions ((4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction assay and neutral red uptake assay) and changes in cell oxidative status (determination of advanced oxidized proteins concentrations and reduced glutathione levels) and lysosomal enzymes activity (β-N-acetyl glucosaminidase activity). Results of our study showed that lycopene applied in high concentration caused significant impairment of the MDCK function leading to cell death. Contrarily, in relatively low concentrations lycopene moderately ameliorated methotrexate-induced MDCK cell death estimated by both biochemical and microscopic analyses. It also prevented a significant decline in the MDCK cell lysosomal function estimated by neutral red accumulation ability and activity of the lysosomal enzyme β-N-acetyl glucosaminidase.

Toxicological evaluation of aspartame against Madin–Darby canine kidney cells

Aspartame is most widely used as artificial sweeteners in more than 6000 food varieties. Aspartame digested into aspartic acid, phenylalanine, and methanol, and several peroxides, superoxide molecules also generated together. The kidney is the secondary site for the cellular metabolism. The present study examined whether the treatment of aspartame induces oxidative stress in the Madin–Darby kidney cells (MDCK). The effects of aspartame on MDCK cell viability were investigated by the sulphorhodamine-B assay and flow cytometry. Morphology of MDCK cells following aspartame exposure was observed. Mitochondria-derived reactive oxygen species (ROS) was also determined using 2′,7′-dichlorodihydrofluorescein diacetate. Lipid peroxidation (LPO), glutathione reduced (GSH) levels and activities of superoxide dismutase (SOD), and catalase enzymes were also determined. Cell viability was significantly altered following aspartame exposure. Morphology of MDCK cells did not change significantly. However, there was a marginal morphological change, including rounding, sporadic distribution and loss of adherence were observed at higher doses of aspartame exposure. Mitochondria-derived ROS was increased in a dose-dependent manner following aspartame exposure. A significant increase in LPO levels, whereas GSH level was reduced after 48 and 72 h of aspartame exposure. SOD and catalase enzyme activities were significantly reduced in a dose- and time-dependent manner. Taking all these data together, it is concluded that aspartame may induce oxidative stress in the MDCK cells.

Therapeutic efficacy of natural dipeptide carnosine against human cervical carcinoma cells.

Natural substances have been attracted several researchers in the recent years, because of its potential antioxidant, anti-inflammatory and anti-cancer properties. We have investigated the effect of carnosine on cell viability, apoptosis, DNA damage, reactive oxygen species (ROS) and caspase 3 enzyme expression in human cervical carcinoma and Madin-Darby Kidney Cells (MDCK) cells. Carnosine inhibited cancer cell growth up to 23%. ROS level was increased up to 30 and 31% in MDCK and HeLa cells respectively. Tunnel assay showed 42 and 14% of positive apoptotic cells in cancer and normal cells respectively. The alteration in mitochondrial and nuclear morphology was determined. The extended lace-like network of normal mitochondria found in control cells. Carnosine treatment significantly altered the mitochondrial morphology of normal cervical carcinoma cell. Mitochondria were condensed clump structures in carnosine treated cancer cells. Carnosine reduced the number of colonies of cervical carcinoma cells. Caspase 3 expression was corresponded to the appearance of immunofluorescence in the cytoplasm. Caspase 3 expression was gradually increased in cervical carcinoma cells. In Silico, docking study was performed to recognize the binding activity of carnosine against a subunit of the caspase 3, and carnosine was able to bind to the drug binding pocket of caspase 3. The glide energy is -5.2 kcal/mol, suggesting the high binding affinity of carnosine to caspase 3. Taking all these data together, the natural dipeptide L-carnosine could be a suitable antiproliferative agent in cervical carcinoma cells. Copyright © 2016 John Wiley & Sons, Ltd.

Differential Sensitivity of Madin-darby Canine Kidney (MDCK) Cells to Epinephrine.

Catecholamines regulate a variety of cellular functions in the mammalian kidney. The present study was aimed to investigate the differential sensitivity of Madin-Darby Kidney Cells (MDCK cells) to epinephrine in a dose-dependent manner. The loss of adhesion and altered cell shape were observed in MDCK cells. The presence of apoptosis and necrosis were studied by the fluorescence microscope and Confocal Laser Scanning Microscope (CLSM). Scanning Electron Microscope (SEM) analysis showed several surface microvilli, and cells were rounded having ruffled and crenated surface. Agarose gel electrophoresis study showed the presence of smearing, which further confirms the occurrence of necrosis. The fluorescence staining study showed the increased reactive oxygen species (ROS) level. Up-regulation of p53, bax, and caspase 3 mRNA expressions was evidenced by quantitative PCR (qPCR). Caspase 3 activity was also increased in epinephrine treated cells. Our experimental results do not imply that the epinephrine should not be used in the clinical treatments. However, our results add a research note of caution on the possible cytotoxic effect of maximal doses of epinephrine over a prolonged time.

Antiapoptotic activity of bovine herpesvirus type-1 (BHV-1) UL14 protein

Viruses have evolved different strategies to interfere with apoptotic pathways in order to halt cellular responses to infection. One previous study showed that transient transfection of bovine herpesvirus type-1 (BHV-1) UL14 protein is efficient in protecting Madin Darby kidney (MDBK) and human chronic myelogenous leukemia (K562) cells from sorbitol-induced apoptosis. This protein corresponds to a putative protein of BHV-1, which shares aminoacid sequence with a part of the peptide-binding domain conserved in human heat shock protein (HSP70) family. The pBK-CMV-UL14 plasmid transfected MDBK cells treated with sorbitol did not show caspase-3 and caspase-9 activation with respect to non-transfected MDBK cells (UL14 negative). Furthermore, we report that the expression of the full length sequence of BHV-1 UL14 is evident after 7 h of infection of BHV-1 on MDBK cells which were then treated with sorbitol. These results indicate that UL14 gene product has important implications to enhance cell survival in response to apoptotic stimuli.

Apparent active transport of MDMA is not mediated by P‐glycoprotein: a comparison with MDCK and Caco‐2 monolayers

Amphetamines and their methylenedioxy derivatives generically display similar behavioral, physiologic and toxic effects. Inconsistent pharmacokinetic and toxicity data for methylenedioxymethamphetamine (MDMA) may suggest that active drug transporters are interacting with these compounds, and thus altering drug absorption and tissue distribution. In vitro models of CNS accumulation and intestinal drug transport were used to assess efflux transport of MDMA. Madin-Darby kidney cell epithelial (MDCK) monolayers displayed a 4-fold increase in accumulation in the basolateral to apical orientation relative to the apical to basolateral orientation, although no differential accumulation was noted between MDCK-WT and MDCK-MDR1 monolayers. Caco-2 monolayers demonstrated an approximate 2-fold increase in accumulation of MDMA. Exposure of various inhibitors of active drug transporters demonstrated mixed results; ritonavir, progesterone and indomethacin produced an approximately 50% reduction of MDMA transport, while verapamil, MK-571 and probenecid had no effect. Based on these data, it is concluded that MDMA efflux is mediated via the activity of a transporter distinct from P-glycoprotein. The possible inhibitory effects of amphetamines on rhodamine-123 transport were also assessed. MDMA, methylenedioxyamphetamine, amphetamine and methamphetamine, at physiologically relevant concentrations, did not significantly alter the transport of rhodamine-123 in Caco-2 monolayers or the LS180 cell line, suggesting that these compounds do not alter the function of P-glycoprotein.

Polycystin as Mechanosensor

Polycystin 1 (PC1) is a transmembrane protein that is the product of the PKD1 gene, which is one of two genes that causes autosomal-dominant polycystic kidney disease (ADPKD). Low et al. report that the cytoplasmic domain of PC1 was cleaved, producing an unstable C-terminal fragment that localized to the nucleus. The abundance of this fragment was increased in renal specimens from ADPKD patients. In cell lines expressing either a labeled PC1 protein or a chimeric molecule, the abundance of the C-terminal fragment was increased if the proteasome was inhibited. Coimmunoprecipitation experiments indicated that the C terminus of PC1 interacted with the transcriptional regulator P100 and also with STAT6 (signal transducer and activator of transcription 6). Furthermore, cells cotransfected with the soluble C-terminal PC1 fragment and a STAT6 reporter showed increased STAT6 reporter gene activity compared with that in cells not expressing the PC1 fragment. In confluent Madin-Darby kidney cells subjected to flow, endogenous STAT6 was redistributed from the nucleus to the primary cilia, which is also where a small fraction of P100 is localized and the site of the PC1 full-length protein. However, MDCK cells cultured in the absence of flow exhibited nuclear-localized STAT6. STAT6 abundance was increased in ADPKD renal tissue. Rapidly fixed mouse kidney tissue that had been perfused showed the ciliary localization of STAT6, whereas tissue samples isolated under normal conditions that lack flow (similar to those used for human kidney samples) showed STAT6 in the nucleus. Finally, injection of the soluble PC1 C-terminal tail into zebrafish embryos caused pronephric cyst formation. The authors propose that under normal conditions of flow, PC1 in the cilia sequesters P100 and STAT6, preventing transcription of STAT6-dependent genes. In the absence of flow, the PC1 C-terminal tail is cleaved, releasing a fragment that interacts with P100 and STAT6, translocates to the nucleus, and regulates gene expression. Details of this model and its relevance for ADPKD remain to be fully elucidated. S. H. Low, S. Vasanth, C. H. Larson, S. Mukherjee, N. Sharma, M. T. Kinter, M. E. Kane, T. Obara, T. Weimbs, Polycystin-1, STAT6, and P100 function in a pathway that transduces ciliary mechanosensation and is activated in polycystic kidney disease. Dev. Cell 10 , 57-69 (2006). [PubMed]

Involvement of DMT1 in uptake of Cd in MDCK cells: role of protein kinase C.

The involvement of iron (Fe) transporters in the uptake of cadmium (Cd) was examined in Madin-Darby kidney cells (MDCK). The uptake of Cd displayed properties that are associated with the Fe transporter divalent metal transporter 1 (DMT1). For example, the uptake of Cd and Fe was reduced by altering the cell membrane potential. The uptake of Cd was blocked by Fe, and the uptake of Fe was blocked by Cd. Also, the uptake of Cd and Fe was higher in MDCK cells bathed in a buffer at low pH. Increased uptake of Fe and Cd was observed in the HEK-293 cell line overexpressing DMT1. Overnight treatment of MDCK cells with the protein kinase C activator phorbol 12,13-dibutyrate (PDBu) resulted in increased uptake of Cd and Fe and an increase in DMT1 mRNA. An increase in newly transcribed DMT1 mRNA was not observed, suggesting that PDBu does not increase DMT1 mRNA by activating transcription. Rather, the increase was most likely due to greater stability of DMT1 mRNA, because the rate of degradation of DMT1 mRNA was slower in MDCK cells treated with PDBu. Our results suggest that Fe and Cd are transported in MDCK cells by a transporter with biochemical properties similar to those of DMT1.

Enhanced resolution of membranes in cultured cells by cryoimmobilization and freeze-substitution.

Investigations of cellular processes demand immediate arresting of the process at any given time and excellent retention of cellular material and excellent visibility of membranes. To achieve this goal we used cryofixation to arrest cellular processes instantly and tested diverse freeze-substitution protocols. Madin-Darby kidney cells and Vero cells were grown on carbon-coated sapphire disks. For cryofixation the sapphire disks covered with a cell monolayer were injected with the aid of a guillotine into liquid propane or ethane or a mixture of both cooled by liquid nitrogen. Freezing of the cryogen was prevented by using a partially insulated cylinder and by vigorous stirring that results in a substantial decrement of the freezing point of the cryogen. Cell monolayers can be cryofixed successfully using the guillotine in a safety hood at ambient temperature and humidity or at 37 degrees C and 45% humidity. The freezing unit can also be placed in a laminar flow for working under biohazard conditions. For visualizing cell membranes at high contrast and high resolution, cells were substituted in the presence of various concentrations of glutaraldehyde and osmium tetroxide and the temperature was raised to diverse final temperatures. Substitution for 4 hours at -90 degrees C in anhydrous acetone containing 0.25% anhydrous glutaraldehyde and 0.5% osmium tetroxide followed by a temperature rise of 5 degrees C/hour to 0 degrees C and final incubation for 1 hour at 0 degrees C resulted in high contrast and excellent visibility of subcellular components at the level of the membrane bilayer. The high spatial and temporal resolution makes this methodology an excellent tool for studying cell membrane-bound processes, such as virus-cell interactions.

Maitotoxin stimulates Cd influx in Madin-Darby kidney cells by activating Ca-permeable cation channels

This study examined the role of calcium channels for the uptake of cadmium (Cd) into Madin-Darby canine kidney (MDCK) cells. Maitotoxin, an activator of different types of calcium channels, increased accumulation of 109Cd and 45Ca in MDCK cells. We found that maitotoxin increased accumulation by stimulating 109Cd influx because it did not affect efflux. An inhibitor of store-operated Ca channels, SKF96365, partially blocked 45Ca influx but did not affect 109Cd influx. Ni and Mn, and loperamide and proadifen (SKF 525a), inhibited 45Ca and 109Cd influx in cells stimulated with maitotoxin, but La and nifedipine did not. Overnight treatment with phorbol 12, 13-ibutyrate (PDBu) to activate protein kinase C resulted in a decrease in the concentration of maitotoxin needed to stimulate 45Ca and 109Cd influx. The effect of PDBu was blocked by treating cells with the protein kinase C inhibitor GF109203X. Additionally, the effect of PDBu was lost in cells treated with an inhibitor of RNA synthesis actinomycin D. These results suggest that a Ca permeable cation channel different from voltage-dependent and store-operated Ca channels mediates the uptake of Cd in MDCK cells. The expression of this channel is regulated by protein kinase C.

Relationship of Surface Characteristics to Cellular Attachment in PU and PHEMA

Adhesion characteristics of fibroblastic baby hamster kidney (BHK) cells and epithelial Madine Darby kidney (MDBK) cells on polyurethane (PU) and polyhydroxyethylmethacrylate (PHEMA) based surfaces have been studied. PU surfaces were prepared by a classical solvent-casting procedure of Pellethane solution that contains different types of solvents, i.e., THF, dioxane and their compositions. PHEMA based surfaces were obtained by bulk polymerization of respective comonomers (HEMA, acrylic acid, AA, and dimethylaminoethylmethacrylate, DMAEMA) in the presence of the crosslinker. Thus, a number of polymeric surfaces were obtained with different surface charges (COO- and NH+4) and with different surface free energies in a range between 60-82 ergs/cm2. Surface properties of these membranes were characterized by equilibrium water contents, air and octane contact angles, surface free energies, SEM photographs and ATR-FTIR spectra. Interactions of BHK and MDBK cells with the surfaces were examined in stationary culture conditions which were carried out in MEM supplemented with fetal calf serum. The observations strongly suggested that the chemical and/or physical properties of membrane surface and morphology of the cell control the degree of cell adhesion to the PU and PHEMA based membranes.

Cytotoxicity of Mycoplasma mycoides Subspecies mycoides for Cultured Endothelial Cells

Toxic effect of 5 SC- and 4 LC-type strains of Mycoplasma mycoides subspecies mycoides has been demonstrated in cultured endothelial cells. The SC strains are agents of contagious bovine pleuropneumonia and presumably specific for cattle although occasionally isolated from goats. The LC strains produce an acute septicemic disease in goats and a few strains have been reported in cattle. The tissue culture cytotoxic dose causing 75% cell death (TCCD75) was calculated for each strain. Cytotoxicity ranged from 0.9 x 109 to 12.0 x 10 9 when strains were tested in bovine endothelial cells with the SC strains being twice as cytotoxic as the LC strains on average. Strains G175/78 and D44 representing the most cytotoxic SC and LC strains respectively, were selected for comparative experiments using bovine, caprine and porcine endothelial cells, bovine embryonic lung fibroblast cells (BELF) and the bovine cell line Madin-Darby kidney cells (MDBK). Strain G175/78 was significantly more cytotoxic for bovine endothelial cells than caprine and porcine, suggesting that the cytotoxicity reflects specificity for the bovine species. This strain was also cytotoxic for BELF but not for MDBK cells indicating that not all bovine cells are susceptible. Conversely, cytotoxicity of strain D44 was not significantly different in any of the endothelial cells tested, although cytotoxicity for BELF was significantly different, the cytotoxicity of the LC strains seems to be of less specificity.