Madin-Darby Canine Kidney Cell Monolayers Research Articles

The interactions of single-wall carbon nanohorns with polar epithelium.

Single-wall carbon nanohorns (SWCNHs), which have multitudes of horn interstices, an extensive surface area, and a spherical aggregate structure, offer many advantages over other carbon nanomaterials being used as a drug nanovector. The previous studies on the interaction between SWCNHs and cells have mostly emphasized on cellular uptake and intracellular trafficking, but seldom on epithelial cells. Polar epithelium as a typical biological barrier constitutes the prime obstacle for the transport of therapeutic agents to target site. This work tried to explore the permeability of SWCNHs through polar epithelium and their abilities to modulate transcellular transport, and evaluate the potential of SWCNHs in drug delivery. Madin-Darby canine kidney (MDCK) cell monolayer was used as a polar epithelial cell model, and as-grown SWCNHs, together with oxidized and fluorescein isothiocyanate-conjugated bovine serum albumin-labeled forms, were constructed and comprehensively investigated in vitro and in vivo. Various methods such as transmission electron microscopy and confocal imaging were used to visualize their intracellular uptake and localization, as well as to investigate the potential transcytotic process. The related mechanism was explored by specific inhibitors. Additionally, fast multispectral optoacoustic tomography imaging was used for monitoring the distribution and transport process of SWCNHs in vivo after oral administration in nude mice, as an evidence for their interaction with the intestinal epithelium. The results showed that SWCNHs had a strong bioadhesion property, and parts of them could be uptaken and transcytosed across the MDCK monolayer. Multiple mechanisms were involved in the uptake and transcytosis of SWCNHs with varying degrees. After oral administration, oxidized SWCNHs were distributed in the gastrointestinal tract and retained in the intestine for up to 36 h probably due to their surface adhesion and endocytosis into the intestinal epithelium. Overall, this comprehensive investigation demonstrated that SWCNHs can serve as a promising nanovector that can cross the barrier of polar epithelial cells and deliver drugs effectively.

Sectioning of cultured cell monolayer using photo-acid-generating substrate and micro-patterned light projection

A novel cell processing tool to cut and section a cultured cell monolayer was implemented by the micro-projection of grid pattern onto a substrate functionalized with photo-acid-generating polymer. In adequate conditions of irradiation dosage and line width of grid pattern, Madin-Darby canine kidney cell monolayer was cut into cell clumps with similar size corresponding to the projected grid pattern. The sectioned monolayers were collected as cell clumps with the similar size from the substrate maintaining their viability enough to outgrow after subculture to the other dish. Also the influence of cutout size on the growth rate of collected cell clumps after subculture was also investigated. Further, passage of human induced pluripotent stem cells with size uniformity of the colonies was achieved by using this sectioning method without critical influence on their viability and undifferentiated state.

Nasal Drug Absorption from Powder Formulations: Effect of Fluid Volume Changes on the Mucosal Surface.

The effect of changes in the mucosal fluid volume on the nasal drug absorption of powder formulations was evaluated using warfarin (WF), piroxicam (PXC), and norfloxacin (NFX) as model drugs. Lactose and sodium chloride (NaCl), which are water soluble and small-sized chemicals that increase osmotic pressure after dissolution, were used as excipients to change the mucosal fluid volume. The in vitro study using a Madin-Darby canine kidney (MDCK) cell monolayer indicated that lactose and NaCl, sprayed over the surface of air interface monolayers, increased the fluid volume on the monolayer surface and enhanced the transepithelial transport of the model drugs. The in vivo animal study indicated that the nasal absorption of PXC is enhanced by lactose and NaCl after nasal administration of the powder formulations. This is likely due to the enhanced dissolution of PXC on fluid-rich nasal mucosa and an increase in the effective surface area for drug permeation, which lead to better nasal absorption. However, both excipients failed to increase the nasal absorption of WF and NFX. To clarify the mechanism of the drug-dependent effect of lactose and NaCl, the nasal residence of the formulation was examined using FD70 as a non-absorbable marker. The nasal clearance of FD70 was enhanced by lactose and NaCl, leading to a decrease in the nasal drug absorption. Lactose and NaCl caused no damage to the nasal tissue. These results indicate that the addition of water-soluble excipients such as lactose to powder formulations can enhance the nasal absorption of highly permeable but poorly soluble drugs.

Development of a Novel Formulation That Improves Preclinical Bioavailability of Tenofovir Disoproxil Fumarate

Tenofovir disoproxil fumarate (TDF), the bisphosphonate ester prodrug of tenofovir (TFV), has poor bioavailability due to intestinal degradation and efflux transport. Reformulation using U.S. Food and Drug Administration–approved esterase and efflux inhibitors to increase oral bioavailability could provide lower dose alternatives and reduce costs for patients with HIV in resource-limited settings. Inhibition of mucosal and intracellular esterases was studied in human and rat intestinal extracts (S9), where TDF was protected by the carboxylesterase inhibitor bis-para-nitrophenylphosphate, the ester mix EM1, and the generally recognized-as-safe (GRAS) excipient propylparaben. Permeability studies using Madin-Darby canine kidney and Caco-2 cell monolayers demonstrated that TDF was a substrate for the permeability glycoprotein with permeability glycoprotein inhibitors reducing basolateral to apical transport of TDF. These studies also showed that transport was increased by esterase inhibitors. TDF, TFV, and tenofovir monophosphonate ester transport across Caco-2 monolayers with esterase and efflux inhibitors revealed a maximum 38.7-fold increase in apical to basolateral TDF transport with the potent non-GRAS combination of EM1 and GF120918. Transport was increased 22.8-fold by the GRAS excipients, propylparaben, and d-a-tocopheryl polyethylene glycol 1000 succinate (a vitamin E derivative). TFV pharmacokinetics in rats following oral administration of TDF and GRAS esterase and efflux inhibitors confirmed enhanced bioavailability. Area under the curve increased 1.5- to 2.1-fold with various combinations of parabens and d-a-tocopheryl polyethylene glycol 1000 succinate. This significant inhibition of TDF hydrolysis and efflux in vivo exhibits the potential to safely increase TDF bioavailability in humans.

Enhanced replication of avian-origin H3N2 canine influenza virus in eggs, cell cultures and mice by a two-amino acid insertion in neuraminidase stalk.

Canine influenza virus (CIV) is a newly identified, highly contagious respiratory pathogen in dogs. Recent studies indicate that avian-origin H3N2 CIV are circulating in Chinese dogs. To investigate the effects of a two-amino acid (2-aa) insertion naturally occurring at the distal end of the neuraminidase (NA) stalk found in Chinese isolates since 2010 on virus replication and virulence, we rescued the CIV strain, A/canine/Jiangsu/06/2011(H3N2) and its NA mutant without the 2-aa insertion using reverse genetics. The NA stalk length affected virus growth in cell culture. Compared to the short stalk strain (without 2-aa insertion), the long stalk strain (with 2-aa insertion) exhibited higher peak titers and greater yields in Madin-Darby canine kidney (MDCK) cells, chicken embryo fibroblasts and canine bronchiolar epithelial cells, as well as much larger plaques in MDCK cell monolayers. Furthermore, mice inoculated with the long stalk strain showed more severe pathologic damage in lung and higher proportion of detectable viral RNA in tissues. The long stalk strain induced local IFN-γ production with faster kinetics and higher levels in mice. However, in chickens, the two viral strains showed no significant difference with nearly the same proportion of detectable viral RNA loads in tissues. These observations suggest that the 2-aa insertion in the NA stalk acquired by avian-origin H3N2 CIV helps to enhance viral replication and is likely a result of adaptive evolution in canine hosts.

Absorption mechanism of oxymatrine in cultured Madin–Darby canine kidney cell monolayers

Context Oxymatrine (OMT) is beneficial to human health by exerting various biological effects. Objective To investigate the absorption mechanism of OMT and discover absorption enhancers using Madin–Darby canine kidney (MDCK) cell monolayers.Materials and methods Concentration effects on the transport of OMT were measured in the range of 1.0 × 10−5–1.0 × 10−3 M in 2 h. Then, the effect of time, direction, temperature and pH on the transport of OMT at 10−4 M was studied. Moreover, Papp of OMT was determined in the absence/presence of cyclosporine and surfactants at 100 μM to further confirm the relative transport mechanism.Results The Papp AP→BL ranged from (3.040 ± 0.23) × 10−6 to (3.697 ± 0.19) × 10−6 cm/s as the concentration varied from 10−5 to 10−3 M. OMT showed similar Papp at 4 and 37 °C (p > 0.05). Increasing the apical pH 7.4 and 8.0 resulted in Papp versus pH 5.0 (p < 0.01). Furthermore, in the presence of cyclosporine and surfactants including sodium citrate, sodium dodecyl sulphate (SDS) and deoxysodium cholate, Papp was (0.318 ± 0.033) × 10−5, (0.464 ± 0.048) × 10−5, (0.897 ± 0.115) × 10−5 and (1.341 ± 0.122) × 10−5 cm/s, respectively. In the presence of surfactants, Papp significantly increased up to 1.5–4.3-fold (p < 0.05).Discussion and conclusion OMT transport across MDCK cell monolayers was by passive diffusion. Sodium citrate, SDS and deoxysodium cholate serve as excellent absorption enhancers which are useful for the related research improving the oral bioavailability of OMT.

Modulation of Intercellular Junctions by Cyclic-ADT Peptides as a Method to Reversibly Increase Blood–Brain Barrier Permeability

It is challenging to deliver molecules to the brain for diagnosis and treatment of brain diseases. This is primarily because of the presence of the blood-brain barrier (BBB), which restricts the entry of many molecules into the brain. In this study, cyclic-ADT peptides (ADTC1, ADTC5, and ADTC6) have been shown to modify the BBB to enhance the delivery of marker molecules [e.g., (14) C-mannitol, gadolinium-diethylenetriaminepentacetate (Gd-DTPA)] to the brain via the paracellular pathways of the BBB. The hypothesis is that these peptides modulate cadherin interactions in the adherens junctions of the vascular endothelial cells forming the BBB to increase paracellular drug permeation. In vitro studies indicated that ADTC5 had the best profile to inhibit adherens junction resealing in Madin-Darby canine kidney cell monolayers in a concentration-dependent manner (IC50 = 0.3 mM) with a maximal response at 0.4 mM. Under the current experimental conditions, ADTC5 improved the delivery of (14) C-mannitol to the brain about twofold compared with the negative control in the in situ rat brain perfusion model. Furthermore, ADTC5 peptide increased in vivo delivery of Gd-DTPA to the brain of Balb/c mice when administered intravenously. In conclusion, ADTC5 has the potential to improve delivery of diagnostic and therapeutic agents to the brain.

Transcellular transport characteristics of huperzine alone or in combination with ginkgolide B across Caco-2 and Madin-Darby canine kidney cell monolayer.

To study the various processes involved in transcellular transport (TT) of huperzine A alone or in combination with ginkgolide B in Caco-2 and Madin-Darby canine renal (MDCK) cell monolayer. The transepithelial passage was assayed in the apical-to-basolateral (AP to BL) direction and opposite direction (BL to AP) in both cell lines. The determination of huperzine A and ginkgolide B were performed by high performance liquid chromatography (HPLC). The passage rates of huperzine A and ginkgolide B were calculated. Bi-directional TT (absorption and secretion) were taken in huperzine A and ginkgolide B in Caco-2 and MDCK cell monolayer. TT absorption and secretion kinetics of huperzine A and ginkgolide B across two cells existed at the same time. The passage rates of huperzine A were increased significantly with adding different concentrations of ginkgolide B. The compound preparations of HA in combination with GB for dementia caused by cerebral ischemic have synergistic effects on the pharmacodynamics, and improve the bioavailability through BBB.

Transport pathways of solid lipid nanoparticles across Madin-Darby canine kidney epithelial cell monolayer.

An understanding of drug delivery system transport across epithelial cell monolayer is very important for improving the absorption and bioavailability of the drug payload. The mechanisms of epithelial cell monolayer transport for various nanocarriers may differ significantly due to their variable components, surface properties, or diameter. Solid lipid nanoparticles (SLNs), conventionally formed by lipid materials, have gained increasing attention in recent years due to their excellent biocompatibility and high oral bioavailability. However, there have been few reports about the mechanisms of SLNs transport across epithelial cell monolayer. In this study, the molecular mechanisms utilized by SLNs of approximately 100 nm in diameter crossing intestinal epithelial monolayer were carefully studied using a simulative intestinal epithelial monolayer formed by Madin-Darby canine kidney (MDCK) epithelial cells. The results demonstrated that SLNs transportation did not induce a significant change on tight junction structure. We found that the endocytosis of SLNs into the epithelial cells was energy-dependent and was significantly greater than nanoparticle exocytosis. The endocytosis of SLNs was found to be rarely mediated via macropinocytosis, as confirmed by the addition of 5-(N-ethyl-N-isopropyl)amiloride (EIPA) as an inhibitory agent, and mainly depended on lipid raft/caveolae- and clathrin-mediated pathways. After SLNs was internalized into MDCK cells, lysosome was one of the main destinations for these nanoparticles. The exocytosis study indicated that the endoplasmic reticulum, Golgi complex, and microtubules played important roles in the transport of SLNs out of MDCK cells. The transcytosis study indicated that only approximately 2.5% of the total SLNs was transported from the apical side to the basolateral side. For SLNs transportation in MDCK cell monolayer, greater transport (approximately 4-fold) was observed to the apical side than to the basolateral side. Our findings may present a more comprehensive understanding on the transport of SLNs across epithelial cell monolayer.

Epithelial transport of Noscapine across cell monolayer and influence of absorption enhancers on in vitro permeation and bioavailability: implications for intestinal absorption

The purpose of this study was to investigate the permeation of Noscapine (Nos) across the Caco-2 and Madin–Darby canine kidney (MDCK) cell monolayers and to evaluate the influence of absorption enhancers on in vitro and in vivo absorption of Nos. The bidirectional transport of Nos was studied in Caco-2 and MDCK cell monolayers at pH 5.0–7.8. The effect of 0.5% w/v chitosan (CH) or Captisol (CP) on Nos permeability was investigated at pH 5.0 and 5.8. The effect of 1–5% w/v of CP on oral bioavailability of Nos (150 mg/kg) was evaluated in Sprague–Dawley rats. The effective permeability coefficients (Peff) of Nos across Caco-2 and MDCK cell monolayers was found to be in the order of pH 5.0 > 5.8 > 6.8 > 7.8. The efflux ratios of Peff < 2 demonstrated that active efflux does not limit the absorption of Nos. The use of CH or CP have shown significant (***, p < 0.001) enhancement in Peff of Nos across cell monolayer compared with the control group. The CP (1–5% w/v) based Nos formulations resulted in significant (***, p < 0.001) increase in the bioavailability of Nos compared with Nos solution. The use of CP represents viable approach for enhancing the oral bioavailability of Nos and reducing the required dose.

Permeability of exendin-4-loaded chitosan nanoparticles across MDCK cell monolayers and rat small intestine.

The purpose of this study was to investigate the permeability of exendin-4-loaded chitosan nanoparticles using the Madin Darby canine kidney (MDCK) cell monolayer as an in vitro model and the rat intestine as an ex vivo model of the human intestinal barrier. A series of formulations of sodium tripolyphosphate (TPP) and chitosan with different molecular weights and degrees of deacetylation was evaluated. The formulation consisting of 0.1% TPP and 0.2% chitosan (400 kDa, 95% degree of deacetylation), which gave optimized monodispersed particle size (303.1±10.36 nm), zeta potential (18.37±1.15 mV) and encapsulation efficiency (38.0±2.6%), was used for further analysis. After determining their biocompatibility, the transport potential of drug-loaded chitosan nanoparticles was evaluated and compared with free exendin-4 using both MDCK cell monolayers and different rat intestinal segments. Mechanisms underlying enhanced transport of exendin-4 in the cell model were also explored. Compared with free exendin-4, the absorption of optimized chitosan nanoparticles was enhanced by 4.7-fold in MDCK cell monolayers and by 2.0-2.78-fold in different rat intestinal segments, with no significant difference between the duodenum, jejunum and ileum. As supported by confocal laser scanning microscopic analysis, the lower enhancement of absorption in the intestine compared to the cell monolayer likely resulted from the chitosan nanoparticle-mediated opening of cellular tight junctions and not through intracellular transport. These findings suggest that the potential application of chitosan nanoparticles as delivery carriers of exendin-4 is limited and may need further modifications.

Influence of prior pandemic A(H1N1)2009 virus infection on invasion of MDCK cells by community-associated methicillin-resistant Staphylococcus aureus

Secondary bacterial pneumonia due to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a highly publicized cause of death associated with influenza. In this study, we performed the gentamicin-killing assay using Madin-Darby canine kidney (MDCK) cells and MRSA strains to investigate whether prior infection from pandemic A(H1N1)2009 virus (A[H1N1]pdm09) lead to increased invasion of MDCK cells by MRSA. We found that the invasion rate of two MRSA strains (ATCC BAA-1680 [USA 300] and ATCC BAA-1699 [USA 100]) into intact MDCK cell monolayers was 0.29±0.15% and 0.007±0.002%, respectively (p<0.01, n≥3). In addition, the relative invasion rate of both ATCC BAA-1680 and ATCC BAA-1699 was significantly increased by prior A(H1N1)pdm09 infection of MDCK monolayers from 1±0.28 to 1.38±0.02 and from 1±0.24 to 1.73±0.29, respectively (p<0.01). These results indicate that ATCC BAA-1680 displays much stronger invasiveness of MDCK cells than ATCC BAA-1699, although invasion of both strains was increased by prior A(H1N1)pdm09 infection. In conclusion, this study provided the first evidence that prior A(H1N1)pdm09 infection facilitates the invasion of MDCK cells by MRSA, presumably due to cellular injury caused by the virus.

Meprin A impairs epithelial barrier function, enhances monocyte migration, and cleaves the tight junction protein occludin

Meprin metalloproteases are highly expressed at the luminal interface of the intestine and kidney and in certain leukocytes. Meprins cleave a variety of substrates in vitro, including extracellular matrix proteins, adherens junction proteins, and cytokines, and have been implicated in a number of inflammatory diseases. The linkage between results in vitro and pathogenesis, however, has not been elucidated. The present study aimed to determine whether meprins are determinative factors in disrupting the barrier function of the epithelium. Active meprin A or meprin B applied to Madin-Darby canine kidney (MDCK) cell monolayers increased permeability to fluorescein isothiocyanate-dextran and disrupted immunostaining of the tight junction protein occludin but not claudin-4. Meprin A, but not meprin B, cleaved occludin in MDCK monolayers. Experiments with recombinant occludin demonstrated that meprin A cleaves the protein between Gly(100) and Ser(101) on the first extracellular loop. In vivo experiments demonstrated that meprin A infused into the mouse bladder increased the epithelium permeability to sodium fluorescein. Furthermore, monocytes from meprin knockout mice on a C57BL/6 background were less able to migrate through an MDCK monolayer than monocytes from their wild-type counterparts. These results demonstrate the capability of meprin A to disrupt epithelial barriers and implicate occludin as one of the important targets of meprin A that may modulate inflammation.

Activation of liver X receptors reduces CFTR-mediated Cl−transport in kidney collecting duct cells

Liver X receptors (LXRs) are transcription factors belonging to the nuclear receptor super family, which act as regulators of lipid and glucose metabolism. However, LXRs have been shown to regulate the function of transporters in the kidney, including the Na-Pi cotransporter, organic anion transporter, and epithelial Na(+) channel. In this report, we demonstrated the ability of LXR ligands, both endogenous [22 (R)-hydroxycholesterol] and synthetic (T0901317 and GW3965), to reduce CFTR-mediated Cl(-) secretion in a type I Madin-Darby canine kidney (MDCK) cell line and in murine primary inner medullary collecting duct (IMCD) cells, based on measurements of [Arg(8)]-vasopressin-induced Cl(-) current. However, treatment of MDCK cell monolayers with 5 μM T0901317 for 24 h did not alter ouabain-senstive current or Na(+)-K(+)-ATPase-α protein content. Furthermore, basolateral membranes permeabilization of MDCK cell monolayers still resulted in a decrease in apical Cl(-) current stimulated by both [Arg(8)]-vasopressin and 8-cholorophenyl-thio-cAMP, indicating that the factor(s) encoded by the target gene(s) of agonist-activated LXRs might be located at the apical membrane. Western blot analysis revealed that inhibition of Cl(-) secretion occurred via a decrease in CFTR protein, which was not due to downregulation of its mRNA expression. In addition, both synthetic LXR agonists significantly retarded the growth of forskolin-induced cysts formed in MDCK cells cultured in collagen gel. This is the first evidence showing that ligand-activated LXRs are capable of downregulating CFTR-mediated Cl(-) secretion of kidney cells and of retarding cyst growth in a MDCK cell model.

Preparation, characterization, and in vitro and in vivo investigation of chitosan-coated poly (d,l-lactide-co-glycolide) nanoparticles for intestinal delivery of exendin-4

BackgroundExendin-4 is an incretin mimetic agent approved for type 2 diabetes treatment. However, the required frequent injections restrict its clinical application. Here, the potential use of chitosan-coated poly (d,l-lactide-co-glycolide) (CS-PLGA) nanoparticles was investigated for intestinal delivery of exendin-4.Methods and resultsNanoparticles were prepared using a modified water–oil–water (w/o/w) emulsion solvent-evaporation method, followed by coating with chitosan. The physical properties, particle size, and cell toxicity of the nanoparticles were examined. The cellular uptake mechanism and transmembrane permeability were performed in Madin-Darby canine kidney-cell monolayers. Furthermore, in vivo intraduodenal administration of exendin-4-loaded nanoparticles was carried out in rats. The PLGA nanoparticle coating with chitosan led to a significant change in zeta potential, from negative to positive, accompanied by an increase in particle size of ~30 nm. Increases in both the molecular weight and degree of deacetylation of chitosan resulted in an observable increase in zeta potential but no apparent change in the particle size of ~300 nm. Both unmodified PLGA and chitosan-coated nanoparticles showed only slight cytotoxicity. Use of different temperatures and energy depletion suggested that the cellular uptake of both types of nanoparticles was energy-dependent. Further investigation revealed that the uptake of PLGA nanoparticles occurred via caveolin-mediated endocytosis and that of CS-PLGA nanoparticles involved both macropinocytosis and clathrin-mediated endocytosis, as evidenced by using endocytic inhibitors. However, under all conditions, CS-PLGA nanoparticles showed a greater potential to be transported into cells, as shown by flow cytometry and confocal microscopy. Transmembrane permeability analysis showed that unmodified and modified PLGA nanoparticles could improve the transport of exendin-4 by up to 8.9- and 16.5-fold, respectively, consistent with the evaluation in rats.ConclusionThe chitosan-coated nanoparticles have a higher transport potential over both free drug and unmodified particles, providing support for their potential development as a candidate oral delivery agent for exendin-4.

Highly Stabilized Curcumin Nanoparticles Tested in an In Vitro Blood–Brain Barrier Model and in Alzheimer’s Disease Tg2576 Mice

The therapeutic effects of curcumin in treating Alzheimer's disease (AD) depend on the ability to penetrate the blood-brain barrier. The latest nanoparticle technology can help to improve the bioavailability of curcumin, which is affected by the final particle size and stability. We developed a stable curcumin nanoparticle formulation to test in vitro and in AD model Tg2576 mice. Flash nanoprecipitation of curcumin, polyethylene glycol-polylactic acid co-block polymer, and polyvinylpyrrolidone in a multi-inlet vortex mixer, followed by freeze drying with β-cyclodextrin, produced dry nanocurcumin with mean particle size <80 nm. Nanocurcumin powder, unformulated curcumin, or placebo was orally administered to Tg2576 mice for 3 months. Before and after treatment, memory was measured by radial arm maze and contextual fear conditioning tests. Nanocurcumin produced significantly (p=0.04) better cue memory in the contextual fear conditioning test than placebo and tendencies toward better working memory in the radial arm maze test than ordinary curcumin (p=0.14) or placebo (p=0.12). Amyloid plaque density, pharmacokinetics, and Madin-Darby canine kidney cell monolayer penetration were measured to further understand in vivo and in vitro mechanisms. Nanocurcumin produced significantly higher curcumin concentration in plasma and six times higher area under the curve and mean residence time in brain than ordinary curcumin. The P(app) of curcumin and tetrahydrocurcumin were 1.8×10(-6) and 1.6×10(-5)cm/s, respectively, for nanocurcumin. Our novel nanocurcumin formulation produced highly stabilized nanoparticles with positive treatment effects in Tg2576 mice.

Characterization of Two Mammalian Cortical Collecting Duct Cell Lines with Hopping Probe Ion Conductance Microscopy

We morphologically and physiologically characterized Madin-Darby canine kidney (MDCK) cell and mouse principal cell of kidney in cortical collecting duct (mpkCCD) via hopping probe ion conductance microscopy, transepithelial electrical resistance (TEER) measurements, and single-channel recordings. The specific membrane structures of microvilli and tight junctions were clearly observed in MDCK and mpkCCD cell monolayers. The electrophysiological functions of epithelial Na(+) channel in MDCK and mpkCCD cells were further characterized by measuring amiloride-sensitive TEER values for the whole-cell monolayer and detecting the ion channel activities with patch clamping. Our results provide more morphological and functional information to help better utilize these two mammalian CCD cell lines for mechanism studies of sodium absorption and reabsorption in the distal nephron.

Models to Predict Unbound Intracellular Drug Concentrations in the Presence of Transporters

Knowledge of free drug intracellular concentration is necessary to predict the impacts of drugs on intracellular targets. The goal of this study was to develop models to predict free intracellular drug concentrations in the presence of apical efflux transporters. The apical efflux transporter P-glycoprotein (P-gp), encoded by human gene multidrug resistance 1 (MDR1), was studied. Apparent permeabilities for 10 compounds in Madin-Darby canine kidney (MDCK) and MDR1-MDCK cell monolayers were obtained experimentally. Six of these compounds were evaluated additionally in the presence of the P-gp inhibitor cyclosporine A. A three-compartment model was developed, and passive and apical efflux clearances (CL(d) and CL(ae), respectively) were estimated. Endogenous canine transporters also were delineated. The three-compartment model was unable to simulate experimentally observed lag times and exhibited systematic bias across the simulations. Next, a five-compartment model with explicit membrane compartments was developed. This model resulted in lower systematic errors and simulated the lag time observed experimentally. Apical efflux was modeled out of the cell or out of the membrane. The five-compartment model with apical efflux out of the membrane predicted marked differences in unbound intracellular concentrations between the apical-to-basolateral and the basolateral-to-apical directions. Upon apical drug addition, large decreases in intracellular concentrations were observed with the efflux transporter. No such difference was predicted upon basolateral drug addition. This is consistent with experimental differences in the impact of P-gp on hepatic and brain distribution and supports the hypothesis that apical efflux occurs out of the apical membrane.

Simvastatin represses translocation of &lt;I&gt;Pseudomonas aeruginosa&lt;/I&gt; across Madin-Darby canine kidney cell monolayers

Pseudomonas aeruginosa causes both invasive (bacteremic) and chronic noninvasive infections. An increase in intestinal epithelial permeability is a characteristic of severe sepsis. Alterations in the normal barrier function of the gut mucosa may result in the translocation of microbial cells and products. On the otherhand, it has been demonstrated that statin use is associated with a lower risk of mortality from bloodstream infections. Therefore, we investigated the ability of P. aeruginosa PAO1 to translocate across the Madin-Darby canine kidney (MDCK) cell monolayers in the presence and absence of simvastatin. The bacteria readily translocated across MDCK cell monolayers after 3 h of infection irrespective of the presence or absence of the drug in the medium. However, the bacteria were less able to penetrate the MDCK monolayers in the presence of simvastatin than in its absence. A gentamicin survival assay demonstrated that simvastatin did not affect the bacteria's invasive behavior in the MDCK cells.

Investigation of Transport Mechanism of Exendin-4 across Madin Darby Canine Kidney Cell Monolayers

The purpose of this study was to investigate the transport mechanism of exendin-4 using Madin Darby canine kidney (MDCK) cell monolayer as an in vitro model of the human intestinal barrier. The roles of active and passive mechanisms of exendin-4 in the cell models were well studied and the corresponding contributions of the transcelluar and paracellular pathway to exendin-4 transport were also evaluated. Moreover, the apparent permeability coefficient (P(app)) values of exendin-4 were determined in the presence of chitosan, sodium decanoate and ethylenediaminetetraacetic acid (EDTA) to further confirm the relative transport mechanism and to evaluate their potential utility in future formulation design. The results revealed the low transport capacity of exendin-4 (P(app), 0.10±0.06×10(-6) cm/s). And exendin-4 transport across the cell models was time and concentration-dependence, direction and energy-independence, and similar to the passive transport marker. Drug efflux and active transport were not observed. In the presence of absorption enhancers, the P(app) value significantly increased up to 2.2-11.9 folds without apparent cytotoxicity, which is comparable to that of the paracellular transport marker. And the order of enhancement was to the effect of chitosan>EDTA>sodium decanoate, and the order of safety was sodium decanoate≈chitosan>EDTA. These findings demonstrated that exendin-4 transport across MDCK cell monolayer mainly by passive paracellular pathway, which agrees with the result of confocal laser scanning microscopy. And these absorption enhancers can be used as potential safe ingredients to improve oral efficacy of exendin-4.