Madin-Darby Canine Kidney Cell Line Research Articles

Diagnostic tools in the detection of Canine parvovirus (CPV) infection in dogs

Canine parvovirus-2 (CPV-2) is one of the most common viral infections among all the enteric pathogens of dogs affecting all age groups causing high morbidity (100 percent) and frequent mortality up to 10 percent in adult dogs and 91 percent in pups. Early detection of the infection is of significant importance for the confirmation of the Canine parvovirus. In the present study, it was aimed to detect the parvovirus using various diagnostic tools like haemato-biochemical studies, pathological changes, isolation in cell cultures, transmission electron microscopy and molecular characterization by polymerase chain reaction (PCR). A total of 59 samples were screened out of which 39 tested positive for CPV-2 by the molecular assay polymerase chain reaction and an amplicon size of 630 bp was obtainedwith VP2 (Viral protein) specific primers. The common haematological andbiochemical findings were anaemia,thrombocytopenia, pancytopenia andhypoproteinaemia. The virus produced amarked cytopathic effect in Madindarby canine kidney (MDCK)cell lines and transmission electronic microscopy (TEM) revealed spherical virus like particles of approximately 20 nm in diameter. Thus, the above tools used in the diagnosis of Canine parvovirus (CPV) were very useful for providing a better therapy in protecting the dogs.

Characterization of MDCK cells and evaluation of their ability to respond to infectious and non-infectious stressors.

The Madin-Darby Canine Kidney (MDCK) cell line is widely used as epithelial cell model in studies ranging from viral infection to environmental pollutants, and vaccines production. However, little is known about basal expression of genes involved in innate immunity, and the ability to respond to infectious and non-infectious stressors. Therefore, the aims of our study were to evaluate the basal level of expression of pivotal genes in the innate immune response and cell cycle regulation, as well as to evaluate the ability of this cell line to respond to infectious or non-infectious stressors. As surmised in our working hypothesis, we demonstrated the constitutive expression of genes involved in the innate immune response and cell defense alike, including TLRs, Interleukins, Myd88, p65/NF-kB and p53. Moreover, we described the ability of this cell line to respond to LPS and cadmium (Cd2+) in terms of gene expression and cytokine release. These data confirm the possibility of using this cell line as a model in studies of host/pathogen interaction and response to non-infectious stressors.

Fluorescent Terpolymers via In Situ Allocation of Aliphatic Fluorophore Monomers: Fe(III) Sensor, High‐Performance Removals, and Bioimaging

Herein, purely aliphatic intrinsically fluorescent terpolymers, i.e., 1 and 2, are synthesized through one-pot solution polymerization via N-H functionalized and multi C-C/C-N coupled in situ protrusion of fluorescent monomers using two nonemissive monomers. These scalable terpolymers are suitable for highly selective Fe(III) sensing, high-performance exclusion of Fe(III), logic function and the imaging of normal mammalian Madin-Darby canine kidney and human osteosarcoma cancer cell lines. The structures of terpolymers, in situ attachment of fluorescent monomers, clusteroluminescence, adsorption-mechanism, and cell-imaging abilities are understood via unadsorbed and/or adsorbed microstructural analyses using 1 H/13 C NMR, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, UV-vis spectroscopy, atomic absorption spectroscopy, thermogravimetric analysis, high-resolution transmission electron microscopy, dynamic light scattering, fluorescence imaging, and fluorescence lifetime. The geometries, electronic structures, location of fluorophores, and singlet-singlet absorption and emission of terpolymers are examined using density functional theory (DFT) and time-dependent DFT. For the precise identification of fluorophores, transition from occupied natural transition orbitals (NTOs) to unoccupied NTOs is computed. For 1/2, limit of detection (LOD) values and adsorption capacities are 6.0 × 10-7 /8.0 × 10-7 m and 147.82/120.56 mg g-1 at pHi = 7.0 and 303 K, respectively. The overall properties of 1 are more advantageous compared to 2 in sensing, cell imaging, and adsorptive exclusion of Fe(III).

High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses.

Victoria and Yamagata lineages of influenza B viruses are globally circulating in seasonal epidemics. Madin–Darby canine kidney (MDCK) cells are permissive for viral isolation and vaccine manufacture. Nevertheless, the interplay between influenza B viruses and host microRNAs has not been investigated in this cell line. Therefore, the present study aims at high-throughput analysis of canine microRNA profile upon infection of influenza B viruses. Briefly, MDCK cells were infected with Victoria or Yamagata lineage at MOI of 0.01. After being harvested at 6, 12 and 24 h post infection, microRNAs were subjected to high-throughput sequencing based on MiSeq platform (Illumina). The results demonstrated that five microRNAs including cfa-miR-197, cfa-miR-215, cfa-miR361, cfa-miR-1841, and cfa-miR-1842 were overexpressed in both Victoria and Yamagata lineage infections. Interestingly, computational prediction showed that karyopherin alpha 6 (KPNA6) was targeted by cfa-miR-197 and cfa-miR-215. Moreover, the binding sites of both microRNAs were assessed by 3′-UTR reporter assay. The results showed that only cfa-miR-197 could bind to the target sites of KPNA6, leading to suppressing luciferase activity. Additionally, silencing of KPNA6 was confirmed by overexpression of cfa-miR-197. This study provides canine microRNA responses to seasonal influenza B viruses, suggesting that virus-mediated microRNAs might play crucial roles in host gene regulation.

Characterization of Adult Canine Kidney Epithelial Stem Cells That Give Rise to Dome-Forming Tubular Cells.

Dome formation can occur in cultured tubular epithelial cells originating from various tissues, including the mammary gland and the kidney. The isolation and characterization of normal kidney epithelial stem cells that give rise to dome-forming tubular cells have never been reported. We attempted to isolate and characterize canine kidney epithelial stem cells using a simple cell culture method that we have previously used to isolate other adult human stem cells. Dome-forming kidney epithelial cells were derived from dissociated adult canine kidney tissues that were cultured in a modified keratinocyte serum-free medium supplemented with N-acetyl-l-cysteine, l-ascorbic acid 2-phosphate, nicotinamide, and fetal bovine serum. These cells exhibited high self-renewal capacity in long-term culture (growth for >13 months and 30 cumulative population doublings) and exhibited characteristics of stem cells, including (1) deficiency in gap junctional intercellular communication, (2) anchorage-independent growth, (3) expression of stem cell markers octamer-binding transcription factor 4 and SRY (sex determining region Y)-box 2, (4) expression of cell surface markers CD24 and CD133, and (5) multipotent differentiation into osteoblasts, adipocytes, chondrocytes, and dome-forming tubular cells. Most of these characteristics are shared by the well-known canine renal tubule-derived immortalized Madin-Darby Canine Kidney cell line. Furthermore, the putative canine kidney stem cells developed in this study formed budding tubule-like organoids on Matrigel and required high cell density (>4,000 cells/cm2) for sustained growth and confluency for dome formation. The signal transducer and activator of transcription-3 (STAT3) phosphorylation inhibitor, AG490, inhibited colony-forming efficiency and dome formation, whereas lipopolysaccharide, an activator of STAT3, increased colony-forming efficiency in a dose-dependent manner. These results are consistent with the hypothesis that high cell density induces STAT3 expression, which promotes both stem cell self-renewal and differentiation into tubular cells. Our novel cell culture method should be useful for the future development of normal human kidney stem cells for clinical applications and for studying mechanisms of nephrotoxicity.

Genome-wide identification and characterization of long non-coding RNAs in MDCK cell lines with high and low tumorigenicities

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.

A humanized MDCK cell line for the efficient isolation and propagation of human influenza viruses.

Here, we developed hCK, a Madin-Darby canine kidney (MDCK) cell line that expresses high levels of human influenza virus receptors and low levels of avian virus receptors. hCK cells supported human A/H3N2 influenza virus isolation and growth much more effectively than conventional MDCK or human virus receptor-overexpressing (AX4) cells. A/H3N2 viruses propagated in hCK cells also maintained higher genetic stability than those propagated in MDCK and AX4 cells.

Validation of Human MDR1-MDCK and BCRP-MDCK Cell Lines to Improve the Prediction of Brain Penetration

It is of great challenge to predict human brain penetration for substrates of multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP), 2 major efflux transporters at blood-brain barrier. Thus, a physiologically based pharmacokinetic (PBPK) model with the incorporation of in vitro MDR1 and BCRP transporter function data and transporter protein expression levels has been developed. As such, it is crucial to generate MDR1 and BCRP substrate data with a high fidelity. In this study, 2 widely used human MDR1 cell lines from Borst and National Institutes of Health laboratories were evaluated using rodent brain penetration data, and the study suggested that the MDR1 expressed in Madin-Darby canine kidney (MDCK) cell line from National Institutes of Health laboratory predicted brain penetration better, particularly for compounds with a high passive permeability. In addition, human BCRP-MDCK cell line with 1 μM PSC833, a specific MDR1 inhibitor, demonstrated the ability to identify BCRP substrates without the confounding of endogenous canine Mdr1. Comparison of human BCRP and mouse Bcrp transporter functions revealed that the functional differences of BCRP between the 2 species is minimal. The incorporation of both the validated MDR1 and BCRP assays into our brain PBPK model has significantly improved the prediction for the brain penetration of MDR1 and BCRP substrates across species.

Unlabeled imaging of primary cilia by scanning ion conductance microscopy

Primary cilia are non-motile cilia consisting of a centriole-derived basal body and a microtubule-based axoneme. In recent years, the structure and function of primary cilia have been attracting attention due to the relation with the onset of ciliary disease. Scanning ion conductance microscopy (SICM) is a probe microscopy used to measure the topography and functions of living cells at nanoscale. Furthermore, the labelling procedure is not necessary for SICM measurement compare to fluorescence imaging. We compared the structures of primary cilia of human retinal pigment epithelial cell line (RPE-1 cells) and Madin-Darby canine kidney cell line (MDCK cells) at nanoscale by using SICM. In addition, high resolution SICM images have also succeeded in visualizing ciliary pockets that difficult to be fluorescently labeled.

Increased osmolality enhances the tight junction-mediated barrier function in a cultured renal epithelial cell line.

Osmotic alterations are associated with several human diseases, including diabetic nephropathy. We have previously shown that high glucose, which is a well-known osmotic agent, induces significant disruption of the tight junction (TJ)-mediated tubular barrier of the Madin-Darby canine kidney (MDCK) cell line. In this study, we investigated the effect of acute (24 h) and chronic (72 h) exposure to increased osmolality (with a 14.5 mM mannitol solution) on TJ-mediated barrier function in MDCK cells. The treatment with mannitol significantly increased the transepithelial electrical resistance (TEER) and accelerated the TEER recovery after Ca2+ switch assay in comparison with control monolayers. Immunofluorescence and Western blot analyses showed that mannitol treatment induced a significant increase in the tight junctional and cellular content of claudin-1 (a barrier-forming claudin) as well as a significant decrease in claudin-2 (a pore-forming claudin) junctional and cellular contents. These data suggest that an increased osmolality induces enhancement of the TJ-mediated barrier of MDCK cells, and that, therefore, the negative effect of high glucose on the epithelial paracellular barrier cannot be attributed to its osmotic actions. In addition, a subtle increase in osmolality may have an impact on kidney function and renal-related diseases.

Inhibitory effects of the phytohormone inhibitors fluridone and inabenfide against Babesia gibsoni in vitro

Pharmacological options to treat canine babesiosis caused by Babesia gibsoni, are limited. To address this challenge, screening for novel drug candidates and drug targets against B. gibsoni is urgently needed. In this study, we explored the inhibitory effects of two phytohormone inhibitors, fluridone (FLU) and inabenfide (INA), against B. gibsoni in vitro. The half-maximal inhibitory concentration (IC50) values of FLU and INA against B. gibsoni were 60.6 ± 3.4 and 4.3 ± 0.3 μM, respectively. Parasitemia and viability at 24, 48, and 72 h after FLU and INA treatments were significantly lower than those in the control group. The cytotoxicity of FLU and INA was evaluated using the dog-derived Madin-Darby canine kidney (MDCK) cell line; both FLU and INA were less toxic to the MDCK cells than to the control cells. The selectivity index of FLU and INA were higher than 16.5 and 232.6, respectively. In summary, the present study demonstrated that FLU and INA were effective against B. gibsoni infection in vitro and that these compounds might have potential as candidate drugs for the treatment of B. gibsoni.

Sequential and synchronized hypertonicity-induced activation of Rel-family transcription factors is required for osmoprotection in renal cells

NF-κB and TonEBP belong to the Rel-superfamily of transcription factors. Several specific stimuli, including hypertonicity which is a key factor for renal physiology, are able to activate them. It has been reported that, after hypertonic challenge, NF-κB activity can be modulated by TonEBP, considered as the master regulator of transcriptional activity in the presence of changes in environmental tonicity. In the present work we evaluated whether hypertonicity-induced gene transcription mediated by p65/RelA and TonEBP occurs by an independent action of each transcription factor or by acting together. To do this, we evaluated the expression of their specific target genes and cyclooxygenase-2 (COX-2), a common target of both transcription factors, in the renal epithelial cell line Madin-Darby canine kidney (MDCK) subjected to hypertonic environment. The results herein indicate that hypertonicity activates the Rel-family transcription factors p65/RelA and TonEBP in MDCK cells, and that both are required for hypertonic induction of COX-2 and of their specific target genes. In addition, present data show that p65/RelA modulates TonEBP expression and both colocalize in nuclei of hypertonic cultures of MDCK cells. Thus, a sequential and synchronized action p65/RelA → TonEBP would be necessary for the expression of hypertonicity-induced protective genes.

Influenza virus A (H1 and H3) and B co-circulation among patient presenting with acute respiratory tract infection in Ibadan, Nigeria.

Influenza is an acute respiratory disease that continues to cause global epidemics and pandemics in human with significant mortality and morbidity. This study was designed to identify the circulating influenza virus in Ibadan, Nigeria during the 2006/2007 season. Throat swab samples were collected from patients presenting with acute respiratory tract infection at the Out-Patient Departments of major hospitals in Ibadan over a period of seven months from November 2006 to May 2007. Isolation of influenza virus was performed using Madin-Darby Canine Kidney cell line and 10 days old chicken embryonated egg. Isolates was identified by haemagglutination and haemagglutination-inhibition assays using selected CDC Influenza reference antisera (A, B, subtype H1 and H3). Out of 128 patients tested, 21(16.4%) yielded positive for virus isolation. Identification of the isolates showed that 19(14.8%) were positive for influenza virus out of which 11(8.6%) and 8(6.2%) were influenza A and B viruses respectively. Influenza A virus 6(4.7%) were subtype H1; 4(3.1%) were co-subtype H1 and H3; and 1(0.8%) was not inhibited by subtype H1 and H3. The circulation of influenza virus A and B in this study is important to contributing knowledge and data to influenza epidemiology and surveillance in Nigeria.

Neurological and psychological status of multiple sclerosis patients after a year in program of personalized psychological interventions

Madin-Darby canine kidney (MDCK) II cells stably transfected with transport proteins are commonly used models for drug transport studies. However, endogenous expression of especially canine MDR1 (cMDR1) confounds the interpretation of such studies. Here we have established an MDCK cell line stably overexpressing the human MDR1 transporter (hMDR1; P-glycoprotein), and used CRISPR-Cas9 gene editing to knockout the endogenous cMDR1. Genomic screening revealed the generation of a clonal cell line homozygous for a 4-nucleotide deletion in the canine ABCB1 gene leading to a frameshift and a premature stop codon. Knockout of cMDR1 expression was verified by quantitative protein analysis and functional studies showing retained activity of the human MDR1 transporter. Application of this cell line allowed unbiased reclassification of drugs previously defined as both substrates and non-substrates in different studies using commonly used MDCK-MDR1 clones. Our new MDCK-hMDR1 cell line, together with a previously developed control cell line, both with identical deletions in the canine ABCB1 gene and lack of cMDR1 expression represent excellent in vitro tools for use in drug discovery.

Correction to: Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization, by Uzarski JS, Bijonowski BM, Wang B, Ward HH, Wandinger-Ness A, Miller WM, and Wertheim JA. Tissue Eng Part C Methods 2015;21(10):1032–1043. DOI: 10.1089/ten.tec.2014.0665

[This corrects the article DOI: 10.1089/ten.tec.2014.0665.].

Epsilon toxin from Clostridium perfringens induces cytotoxicity in FRT thyroid epithelial cells

Epsilon toxin (Etx) is produced by Clostridium perfringens and induces enterotoxemia in ruminants. Etx crosses the blood-brain barrier, binds to myelin structures, and kills oligodendrocytes, inducing central nervous system demyelination. In addition, Etx has a cytotoxic effect on distal and collecting kidney tubules. There are few cell lines sensitive to Etx. At present, the most sensitive in vitro model for Etx is the Madin-Darby canine kidney (MDCK) cell line, where Etx oligomerizes and forms a pore with consequent ion efflux and cell death. Although the Etx receptor has not yet been fully clarified, it is known that caveolin 1 and 2 potentiate Etx cytotoxicity and oligomerization, and more recently, the myelin and lymphocyte (MAL) protein has been implicated in Etx binding and activity. Here, we studied the effect of Etx on Fischer rat thyroid cells (FRT) and observed similar effects as those seen in MDCK cells. Etx incubated with FRT cells showed binding to the plasma membrane, and western blotting assays revealed oligomeric complex formation. Moreover, cytotoxic assays on FRT cells after Etx incubation indicated cell death at a similar level as in MDCK cells. In addition, a luminescent ATP detection assay revealed ATP depletion in FRT cells after Etx exposure. Previous studies have reported that FRT cells do not express caveolins and do not form caveolae but express MAL protein in glycolipid-enriched membrane microdomains. Our results indicate that caveolins are not directly implicated in Etx cytotoxicity, supporting the notion that the MAL protein is involved in Etx action. In addition, a cell line of thyroid origin is described for the first time as a good model to study Etx action.

In vitro evaluation of a simulated pneumoperitoneum environment using carbon dioxide on canine transitional cell carcinoma.

To assess the impact of a simulated CO2 pneumoperitoneum environment on the viability and proliferation of canine transitional cell carcinoma (TCC) cells in vitro. In vitro study. A control Madin-Darby canine kidney (MDCK) cell line and 3 canine TCC cell lines were exposed to 100% CO2 at pressure of 0, 5, 10, or 15 mmHg for 2 hours by using an airtight chamber and a mechanical insufflator at 37°C. Culture media pH was measured. Viability and proliferation were assessed by using a resazurin assay and trypan blue dye, respectively. The pH in the media significantly decreased immediately after CO2 exposure but returned to normal within 1 hour. The viability of the cell lines was variably affected at the evaluated pressures. Insufflation pressure of 10 mmHg resulted in significantly decreased cell viability compared with control. The impact of 15 mmHg CO2 was comparable to 0 mmHg and control. CO2 insufflation pressure had no significant effects on proliferation up to 7 days postexposure. Conclusion/Clinical significance: A positive pressure CO2 environment significantly decreased the viability of TCC and MDCK cells under specific conditions without influencing their proliferation up to 7 days postexposure. Investigating these effects in clinical patients undergoing CO2 laparoscopy is essential to assess for port site metastasis or peritoneal carcinomatosis in order to translate these in vitro results to clinical recommendations.

Dual function of active constituents from bark of Ficus racemosa L in wound healing

BackgroundDifferent parts including the latex of Ficus racemosa L. has been used as a medicine for wound healing in the Ayurveda and in the indigenous system of medicine in Sri Lanka. This plant has been evaluated for its wound healing potential using animal models. The aim of this study was to obtain an insight into the wound healing process and identify the potential wound healing active substance/s present in F. racemosa L. bark using scratch wound assay (SWA) as the in-vitro assay method.MethodStem bark extracts of F. racemosa were evaluated using scratch wound assay (SWA) on Baby Hamster Kidney (BHK 21) and Madin-Darby Canine Kidney (MDCK) cell lines and Kirby Bauer disc diffusion assay on common bacteria and fungi for cell migration enhancing ability and antimicrobial activity respectively. Dichloromethane and hexanes extracts which showed cell migration enhancement activity on SWA were subjected to bioactivity directed fractionation using column chromatography followed by preparative thin layer chromatography to identify the compounds responsible for the cell migration enhancement activity.ResultsDichloromethane and hexanes extracts showed cell migration enhancement activity on both cell lines, while EtOAc and MeOH extracts showed antibacterial activity against Staphylococcus and Bacillus species and antifungal activity against Saccharomyces spp. and Candida albicans. Lupeol (1) and β-sitosterol (2) were isolated as the potential wound healing active compounds which exhibited significant cell migration enhancement activity on BHK 21 and MDCK cell lines (> 80%) in par with the positive control, asiaticoside at a concentration of 25 μM. The optimum concentration of each compound required for the maximum wound healing has been determined as 30 μM and 35 μM for 1 and 2 respectively on both cell lines. It is also established that lupeol acetate (3) isolated from the hexanes extract act as a pro-drug by undergoing hydrolysis into lupeol in the vicinity of cells.ConclusionDifferent chemical constituents present in stem bark of Ficus racemosa L show enhancement of cell migration (which corresponds to the cell proliferation) as well as antimicrobial activity. This dual action of F. racemosa stem bark provides scientific support for its traditional use in wound healing.

Sol-gel TiO2 colloidal suspensions and nanostructured thin films: structural and biological assessments

The role of substrate topography in phenotype expression of in vitro cultured cells has been widely assessed. However, the production of the nanostructured interface via the deposition of sol-gel synthesized nanoparticles (NPs) has not yet been fully exploited. This is also evidenced by the limited number of studies correlating the morphological, structural and chemical properties of the grown thin films with those of the sol-gel ‘brick’ within the framework of the bottom-up approach. Our work intends to go beyond this drawback presenting an accurate investigation of sol-gel TiO2 NPs shaped as spheres and rods. They have been fully characterized by complementary analytical techniques both suspended in apolar solvents, by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) and after deposition on substrates (solid state configuration) by transmission electron microscopy (TEM) and powder x-ray diffraction (PXRD). In the case of suspended anisotropic rods, the experimental DLS data, analyzed by the Tirado-Garcia de la Torre model, present the following ranges of dimensions: 4–5 nm diameter (∅) and 11–15 nm length (L). These results are in good agreement with that obtained by the two solid state techniques, namely 3.8(9) nm ∅ and 13.8(2.5) nm L from TEM and 5.6(1) ∅ and 13.3(1) nm L from PXRD data. To prove the suitability of the supported sol-gel NPs for biological issues, spheres and rods have been separately deposited on coverslips. The cell response has been ascertained by evaluating the adhesion of the epithelial cell line Madin–Darby canine kidney. The cellular analysis showed that titania films promote cell adhesion as well clustering organization, which is a distinguishing feature of this type of cell line. Thus, the use of nanostructured substrates via sol-gel could be considered a good candidate for cell culture with the further advantages of likely scalability and interfaceability with many different materials usable as supports.

Regulation of Kv1.4 potassium channels by PKC and AMPK kinases

ABSTRACTOver the last years extensive kinase-mediated regulation of a number of voltage-gated potassium (Kv) channels important in cardiac electrophysiology has been reported. This includes regulation of Kv1.5, Kv7.1 and Kv11.1 cell surface expression, where the kinase-mediated regulation appears to center around the ubiquitin ligase Nedd4-2. In the present study we examined whether Kv1.4, constituting the cardiac Ito,s current, is subject to similar regulation. In the epithelial Madin-Darby Canine Kidney (MDCK) cell line, which constitutes a highly reproducible model system for addressing membrane targeting, we find, by confocal microscopy, that Kv1.4 cell surface expression is downregulated by activation of protein kinase C (PKC) and AMP-activated protein kinase (AMPK). In contrast, manipulating the activities of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and serum and glucocorticoid-regulated kinase 1 (SGK1) were without effect on channel localization. The PKC and AMPK-mediated downregulation of Kv1.4 membrane surface localization was confirmed by two-electrode voltage clamp in Xenopus laevis oocytes, where pharmacological activation of PKC and AMPK reduced Kv1.4 current levels. We further demonstrate that unlike related Kv channels, Kv1.4 current levels in Xenopus laevis oocytes are not reduced by co-expression of Nedd4-2, or the related Nedd4-1 ubiquitin ligase. In conclusion, we demonstrate that the surface expression of Kv1.4 is downregulated by the two kinases AMPK and PKC, but is unaffected by PI3K-SGK1 signaling, as well as Nedd4-1/Nedd4-2 activity. In the light of previous reports, our results demonstrate an impressive heterogeneity in the molecular pathways controlling the surface expression of highly related potassium channel subunits.