Basement membrane and interstitial proteoglycans produced by MDCK cells correspond to those expressed in the kidney cortex

Multiple proteoglycans (PGs) are present in all basement membranes (BM) and may contribute to their structure and function, but their effects on cell behavior are not well understood. Their postulated functions include: a structural role in maintaining tissue histoarchitecture, or aid in selective filtration processes; sequestration of growth factors; and regulation of cellular differentiation. Furthermore, expression PGs has been found to vary in several disease states. In order to elucidate the role of PGs in the BM, a well-characterized model of polarized epithelium, Madin-Darby canine kidney (MDCK) cells has been utilized. Proteoglycans were prepared from conditioned medium by DEAE anion exchange chromatography. The eluted PGs were treated with heparitinase or chondroitinase ABC (cABC), separately or combined, followed by SDS-PAGE. Western blot analysis, using antibodies specific for various PG core proteins or CS stubs generated by cABC treatment, revealed that both basement membrane and interstitial PGs are secreted by MDCK cells. HSPGs expressed by MDCK cells are perlecan, agrin, and collagen XVIII. Various CSPG core proteins are made by MDCK cells and have been identified as biglycan, bamacan, and versican (PG-M). These PGs are also associated with mammalian kidney tubules in vivo.     

Subtypes of Madin-Darby canine kidney (MDCK) cells defined by immunocytochemistry: Further evidence for properties of renal collecting duct cells

The Madin-Darby canine kidney (MDCK) cell line has been proposed as a model for studying intercalated (IC) cells of the renal cortical collecting duct. The IC cells are characterized by peanut lectin (PNA) binding capacity, carbonic anhydrase (CA) activity and Cl^-–HCO ₃ ^- exchange mediated by a band 3-related protein. It has been suggested that these properties are also expressed in MDCK cells. So far however, the nature of the specific protein involved in Cl^-–HCO ₃ ^- exchange, the type of CA isozyme and the relationship between these two characteristics and PNA binding, have not been investigated in MDCK cells by immunocytochemical methods. Using two antibodies raised against human erythrocyte band 3 protein and two against human erythrocyte CA I and II isozymes, our study provides evidence that a protein related to band 3 is expressed in about 5% of cultured MDCK cells; these band 3-positive cells do not bind PNA and are not reactive for CAI or CAII. About 30% of the MDCK cells bind PNA, two-thirds of which are also CAII-positive. A majority (about 65%) of MDCK cells is not reactive for the three markers used; their density is increased after incubation with aldosterone. These data indicate (i) that the Cl^-–HCO ₃ ^- exchanger of the MDCK cells could be related to human erythrocyte band 3, (ii) that the CA activity of the MDCK cell line bears antigenic identity with the erythrocyte CA II isozyme and (iii) that the latter is always co-localized with PNA binding. These results provide immunocytochemical evidence for the heterogeneity of the MDCK cell line, which might reflect the cellular heterogeneity encountered in the renal cortical collecting duct. Our data also indicate that clonal selection will be required for future functional studies of the MDCK cells.     

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Sphingomyelin metabolism is involved in the differentiation of MDCK cells induced by environmental hypertonicity

Sphingolipids (SLs) are relevant lipid components of eukaryotic cells. Besides regulating various cellular processes, SLs provide the structural framework for plasma membrane organization. Particularly, SM is associated with detergent-resistant microdomains. We have previously shown that the adherens junction (AJ) complex, the relevant cell-cell adhesion structure involved in cell differentiation and tissue organization, is located in an SM-rich membrane lipid domain. We have also demonstrated that under hypertonic conditions, Madin-Darby canine kidney (MDCK) cells acquire a differentiated phenotype with changes in SL metabolism. For these reasons, we decided to evaluate whether SM metabolism is involved in the acquisition of the differentiated phenotype of MDCK cells. We found that SM synthesis mediated by SM synthase 1 is involved in hypertonicity-induced formation of mature AJs, necessary for correct epithelial cell differentiation. Inhibition of SM synthesis impaired the acquisition of mature AJs, evoking a disintegration-like process reflected by the dissipation of E-cadherin and β- and α-catenins from the AJ complex. As a consequence, MDCK cells did not develop the hypertonicity-induced differentiated epithelial cell phenotype.     

Ferroportin 1 is expressed basolaterally in rat kidney proximal tubule cells and iron excess increases its membrane trafficking

Ferroportin 1 (FPN1) is an iron export protein expressed in liver and duodenum, as well as in reticuloendothelial macrophages. Previously, we have shown that divalent metal transporter 1 (DMT1) is expressed in late endosomes and lysosomes of the kidney proximal tubule (PT), the nephron segment responsible for the majority of solute reabsorption. We suggested that following receptor mediated endocytosis of transferrin filtered by the glomerulus, DMT1 exports iron liberated from transferrin into the cytosol. FPN1 is also expressed in the kidney yet its role remains obscure. As a first step towards determining the role of renal FPN1, we localized FPN1 in the PT. FPN1 was found to be located in association with the basolateral PT membrane and within the cytosolic compartment. FPN1 was not expressed on the apical brush‐border membrane of PT cells. These data support a role for FPN1 in vectorial export of iron out of PT cells. Furthermore, under conditions of iron loading of cultured PT cells, FPN1 was trafficked to the plasma membrane suggesting a coordinated cellular response to export excess iron and limit cellular iron concentrations.     

The MAL proteolipid is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin in Madin-Darby canine kidney cells

The MAL (MAL/VIP17) proteolipid is a nonglycosylated integral membrane protein expressed in a restricted pattern of cell types, including T lymphocytes, myelin-forming cells, and polarized epithelial cells. Transport of the influenza virus hemagglutinin (HA) to the apical surface of epithelial Madin-Darby canine kidney (MDCK) cells appears to be mediated by a pathway involving glycolipid- and cholesterol- enriched membranes (GEMs). In MDCK cells, MAL has been proposed previously as being an element of the protein machinery for the GEM-dependent apical transport pathway. Using an antisense oligonucleotide-based strategy and a newly generated monoclonal antibody to canine MAL, herein we have approached the effect of MAL depletion on HA transport in MDCK cells. We have found that MAL depletion diminishes the presence of HA in GEMs, reduces the rate of HA transport to the cell surface, inhibits the delivery of HA to the apical surface, and produces partial missorting of HA to the basolateral membrane. These effects were corrected by ectopic expression of MAL in MDCK cells whose endogenous MAL protein was depleted. Our results indicate that MAL is necessary for both normal apical transport and accurate sorting of HA.     

Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells

Claudin-5 is a transmembrane protein reported to be primarily present in tight junctions of endothelia. Unexpectedly, we found expression of claudin-5 in HT-29/B6 cells, an epithelial cell line derived from human colon. Confocal microscopy showed colocalization of claudin-5 with occludin, indicating its presence in the tight junctions. By contrast, claudin-5 was absent in the human colonic cell line Caco-2 and in Madin-Darby canine kidney cells (MDCK sub-clones C7 and C11), an epithelial cell line derived from the collecting duct. To determine the contribution of claudin-5 to tight junctional permeability in cells of human origin, stable transfection of Caco-2 with FLAG-claudin-5 cDNA was performed. In addition, clone MDCK-C7 was transfected. Synthesis of the exogenous FLAG-claudin-5 was verified by Western blot analysis and confocal fluorescent imaging by employing FLAG-specific antibody. FLAG-claudin-5 was detected in transfected cells in colocalization with occludin, whereas cells transfected with the vector alone did not exhibit specific signals. Resistance measurements and mannitol fluxes after stable transfection with claudin-5 cDNA revealed a marked increase of barrier function in cells of low genuine transepithelial resistance (Caco-2). By contrast, no changes of barrier properties were detected in cells with a high transepithelial resistance (MDCK-C7) after stable transfection with claudin-5 cDNA. We conclude that claudin-5 is present in epithelial cells of colonic origin and that it contributes to some extent to the paracellular seal. Claudin-5 may thus be classified as a tight-junctional protein capable of contributing to the "sealing" of the tight junction.     

Augmented sensitivity to methotrexate by curcumin induced overexpression of folate receptor in KG-1 cells

Folate receptors are targets of various strategies aimed at efficient delivery of anti-cancer drugs. Folate receptors also play a role in the uptake of antifolate drugs which are used for therapeutic intervention in leukemia. Therefore, it is important to identify compounds which regulate expression of folate receptors in leukemic cells. The present study examined if curcumin could modulate the uptake and cytotoxicity of the antifolate drug methotrexate, in KG-1 leukemic cells. This is the first report to show that curcumin (10–50 μM) causes a significant, dose-dependent, 2–3 fold increase in uptake of radiolabelled folic acid and methotrexate into KG-1 cells both at 24 h and 48 h of treatment. Interestingly, pre-treatment of KG-1 leukemic cells with curcumin (10 μM and 25 μM) also caused a statistically significant enhancement in the cytotoxicity of methotrexate. We performed Real Time Quantitative RT-PCR to confirm the upregulation of FRβ mRNA in curcumin treated cells. Immunocytochemistry and Western blotting showed that curcumin caused increased expression of folate receptor βin KG-1 cells. Our data show that the mechanism of curcumin action involves up-regulation of folate receptor β mRNA and protein in KG-1 cells. Therefore, combination of non-toxic concentrations of curcumin and methotrexate, may be a viable strategy for therapeutic intervention for leukemias using a folate receptor-targeted drug delivery system.     

The role of PAK1 in the sensitivity of kidney epithelial cells to ischemia-like conditions

Kidney ischemia, characterized by insufficient supply of oxygen and nutrients to renal epithelial cells, is the main cause of acute kidney injury and an important contributor to mortality world-wide. Earlier research implicated a G-protein coupled receptor (NK1R) in the death of kidney epithelial cells in ischemia-like conditions. P21-associated kinase 1 (PAK1) is involved in signalling by several G-proteins. We explored the consequences of PAK1 inhibition for cell survival under the conditions of reduced glucose and oxygen. Inhibition of PAK1 by RNA interference, expression of a dominant-negative mutant or treatment with small molecule inhibitors greatly reduced the death of cultured kidney epithelial cells. Similar protection was achieved by treating the cells with inhibitors of MEK1, in agreement with the prior reports on PAK1-MEK1 connection. Concomitant inhibition of NK1R and PAK1 offered no better protection than inhibition of NK1R alone, consistent with the two proteins being members of the same pathway. Furthermore, NK1R, PAK and MEK inhibitors reduced the induction of TRAIL in ischemia-like conditions. Considering the emerging role of TRAIL in ischemia-mediated cell death, this phenomenon may contribute to the protective effects of these small molecules. Our findings support further exploration of PAK and MEK inhibitors as possible agents to avert ischemic kidney injury.     

Irreversible inhibitors of methotrexate transport in L1210 cells: Characteristics of inhibition by an N-hydroxysuccinimide ester of methotrexate

Methotrexate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide react to form an activated ester of methotrexate which is a potent irreversible inhibitor of methotrexate transport in L1210 cells. In cells treated with the reagent at 37°C, inhibition was rapid ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{< 1}\text{min}$), optimal at pH 6.8, half-maximal at an inhibitor concentration of 20 nM, and complete at high levels of the reagent. Specificity was indicated by the fact that excess methotrexate added during the pretreatment step protected the transport system against inactivation. Irreversible inhibition was also observed in cells exposed to the reagent at 4°C. Inactivation in this case was qualitatively similar to the corresponding process at 37°C; it appeared rapidly, was half-maximal at 20 nM, and could be prevented by the addition of high concentrations of the substrate. The extent of the inhibition, however, reached a maximum of only 75%, even in samples containing excess or multiple additions of reagent. The latter findings suggest that at 4°C the transport protein exists in two forms, one (75% of the total) containing binding sites which are accessible to the active ester, and the other (25% of the total) with inaccessible sites. The identity of these sites is suggested to be transport proteins which have outward and inward orientations, respectively.     

Conversion of proteins from a non-polarized to an apical secretory pattern in MDCK cells

Previously it was shown that fusion proteins containing the amino terminus of an apical targeted member of the serpin family fused to the corresponding carboxyl terminus of the non-polarized secreted serpin, antithrombin, are secreted mainly to the apical side of MDCK cells. The present study shows that this is neither due to the transfer of an apical sorting signal from the apically expressed proteins, since a sequence of random amino acids acts the same, nor is it due to the deletion of a conserved signal for correct targeting from the non-polarized secreted protein. Our results suggest that the polarity of secretion is determined by conformational sensitive sorting signals.     

Calcium transport in epithelial cells of the intestine and kidney

The central role of 1α,25-dihydroxyvitamin D₃ in the regulation of calcium balance is well established. By increasing the absorption of calcium in the intestine and the reabsorption of filtered calcium in the kidney tubule, the hormone maintains an appropriate calcium balance. The cellular mechanisms that underlie the increase in calcium transport in epithelial cells in response to 1α,25-dihydroxyvitamin D₃ are beginning to be defined. These events include an increase in the movement of calcium across the apical membrane of the cell, an increase in the movement of calcium across the cell, and an increase in the extrusion of calcium at the basolateral portion of the cell. In this Prospects article, I will discuss the nature of the various processes and proteins involved in transcellular calcium movement, and I will attempt to highlight various future areas of research.     

Salt-inducible kinase 1 is present in lung alveolar epithelial cells and regulates active sodium transport

Salt-inducible kinase 1 (SIK1) in epithelial cells mediates the increases in active sodium transport (Na⁺, K⁺-ATPase-mediated) in response to elevations in the intracellular concentration of sodium. In lung alveolar epithelial cells increases in active sodium transport in response to β-adrenergic stimulation increases pulmonary edema clearance. Therefore, we sought to determine whether SIK1 is present in lung epithelial cells and to examine whether isoproterenol-dependent stimulation of Na⁺, K⁺-ATPase is mediated via SIK1 activity. All three SIK isoforms were present in airway epithelial cells, and in alveolar epithelial cells type 1 and type 2 from rat and mouse lungs, as well as from human and mouse cell lines representative of lung alveolar epithelium. In mouse lung epithelial cells, SIK1 associated with the Na⁺, K⁺-ATPase α-subunit, and isoproterenol increased SIK1 activity. Isoproterenol increased Na⁺, K⁺-ATPase activity and the incorporation of Na⁺, K⁺-ATPase molecules at the plasma membrane. Furthermore, those effects were abolished in cells depleted of SIK1 using shRNA, or in cells overexpressing a SIK1 kinase-deficient mutant. These results provide evidence that SIK1 is present in lung epithelial cells and that its function is relevant for the action of isoproterenol during regulation of active sodium transport. As such, SIK1 may constitute an important target for drug discovery aimed at improving the clearance of pulmonary edema.     

Modulation of the epithelial barrier by dexamethasone and prolactin in cultured Madin-Darby canine kidney (MDCK) cells

Glucocorticoids and prolactin (PRL) have a direct effect on the formation and maintenance of tight junctions (TJs) in cultured endothelial and mammary gland epithelial cells. In this work, we investigated the effect of a synthetic glucocorticoid dexamethasone (DEX) and PRL on the paracellular barrier function in MDCK renal epithelial cells. DEX (4 microM)+PRL (2 microg/ml) and DEX alone increased significantly the transepithelial electrical resistance after chronic treatment (4 days) of confluent MDCK monolayers or after 24 h treatment of subconfluent monolayers. Immunoblotting and immunocytochemistry revealed no changes in the expression and distribution of TJ-associated proteins occludin, ZO-1 and claudin-1 in confluent monolayers after hormone addition. However, a marked increase in junctional content for occludin and ZO-1 with no changes in their total expression was observed in subconfluent MDCK monolayers 24 h exposed to DEX or DEX+PRL. No change in cell proliferation/growth was detected at subconfluent conditions following hormone treatment. An increase in the total number of viable cells was observed only in confluent MDCK monolayers after exposure to DEX+PRL suggesting that the main effect of these hormones on already established barrier may be associated with the inhibition of cell death. In conclusion, our data suggest that these hormones (specially dexamethasone) have an effect on TJ structure and function only during the formation of MDCK epithelial barrier by probably modulating the localization, stability or assembly of TJ proteins to membrane sites of intercellular contact.     

Dominant and recessive distal renal tubular acidosis mutations of kidney anion exchanger 1 induce distinct trafficking defects in MDCK cells

Distal renal tubular acidosis (dRTA), a kidney disease resulting in defective urinary acidification, can be caused by dominant or recessive mutations in the kidney Cl-/HCO3- anion exchanger (kAE1), a glycoprotein expressed in the basolateral membrane of alpha-intercalated cells. We compared the effect of two dominant (R589H and S613F) and two recessive (S773P and G701D) dRTA point mutations on kAE1 trafficking in Madin-Darby canine kidney (MDCK) epithelial cells. In contrast to wild-type (WT) kAE1 that was localized to the basolateral membrane, the dominant mutants (kAE1 R589H and S613F) were retained in the endoplasmic reticulum (ER) in MDCK cells, with a few cells showing in addition some apical localization. The recessive mutant kAE1 S773P, while misfolded and largely retained in the ER in non-polarized MDCK cells, was targeted to the basolateral membrane after polarization. The other recessive mutants, kAE1 G701D and designed G701E, G701R but not G701A or G701L mutants, were localized to the Golgi in both non-polarized and polarized cells. The results suggest that introduction of a polar mutation into a transmembrane segment resulted in Golgi retention of the recessive G701D mutant. When coexpressed, the dominant mutants retained kAE1 WT intracellularly, while the recessive mutants did not. Coexpression of recessive G701D and S773P mutants in polarized cells showed that these proteins could interact, yet no G701D mutant was detected at the basolateral membrane. Therefore, compound heterozygous patients expressing both recessive mutants (G701D/S773P) likely developed dRTA due to the lack of a functional kAE1 at the basolateral surface of alpha-intercalated cells.     

N-Glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in Madin-Darby canine kidney cells.

Glycosyl-phosphatidylinositol (GPI)- anchored proteins are preferentially transported to the apical cell surface of polarized Madin-Darby canine kidney (MDCK) cells. It has been assumed that the GPI anchor itself acts as an apical determinant by its interaction with sphingolipid-cholesterol rafts. We modified the rat growth hormone (rGH), an unglycosylated, unpolarized secreted protein, into a GPI-anchored protein and analyzed its surface delivery in polarized MDCK cells. The addition of a GPI anchor to rGH did not lead to an increase in apical delivery of the protein. However, addition of N-glycans to GPI-anchored rGH resulted in predominant apical delivery, suggesting that N-glycans act as apical sorting signals on GPI-anchored proteins as they do on transmembrane and secretory proteins. In contrast to the GPI-anchored rGH, a transmembrane form of rGH which was not raft-associated accumulated intracellularly. Addition of N-glycans to this chimeric protein prevented intracellular accumulation and led to apical delivery.     

Effect of anisotonic media on volume,ion and amino-acid content and membrane potential of kidney cells (MDCK) in culture

Summary Effects of anisotonic media on a monolayer of confluent kidney cells in culture (MDCK) were studied by measuring: cell thickness and cross-section changes, ion and amino-acid content and membrane potential. The volume was also determined with cells in suspension. When cells in a monolayer were incubated in hypotonic media, the lateral and the apical membranes were rapidly stretched. Afterwards the lateral membranes returned to their initial state while the apical membranes remained stretched. This partial regulatory volume decrease (RVD) was verified with cells in suspension. RVD was accompanied by a loss of K⁺, Cl^– and amino acids, but there was no loss of inorganic phosphate. Also a transient hyperpolarization of the membrane potential was observed, suggesting an increase of the K⁺ conductance during RVD. Upon restoring the isotonic medium, a regulatory volume increase (RVI) was observed accompanied by a rapid Na⁺ and Cl^– increase and followed by a slow recovery of the initial K⁺ and Na⁺ content while amino acids remained at their reduced content. A transient depolarization of the membrane potential was measured during this RVI, suggesting that Na⁺ and Cl^– conductance could have increased. In hypertonic media, only a small and slow RVI was observed accompanied by an increase in K⁺ and Cl^– content but without any change of membrane potential. Quinine partly inhibited RVD in hypotonic media with cells in a monolayer while inhibiting RVD completely with cells in suspension. Incubation during four hours in a Ca²⁺ free medium had no effect on RVD. Furosemide and amiloride had no effect on RVD and RVI. Volume regulation, RVD or RVI, was not affected by replacing Cl^– by nitrate. When cells in a monolayer were incubated in a hypotonic K₂SO₄ medium, no RVD was observed. From these results, it seems that MDCK cells in a confluent monolayer regulate their volume by activating specific ion and amino-acid transport pathways. Selective K⁺ and Na⁺ conductances are activated during RVD and RVI, while the activated anion conductance has a low selectivity. The controlling mechanism might not be the free intracellular Ca²⁺ concentration.     

转谷氨酰胺酶2抑制H1亚型流感病毒在MDCK细胞中的增殖

组织转谷酰胺酶(transglutaminase 2,TGM2)是一种普遍存在的多功能蛋白,与不同细胞的粘附和肿瘤形成有关.有证据表明,TGM2参与了宿主细胞与病毒间的相互作用,但是对于流感病毒在细胞内增殖的影响还未有报道.为了探究MDCK细胞中TGM2对H1N1亚型流感病毒增殖的影响,本研究构建了TGM2过表达和敲除...