Heterogeneity of the Na(+)-H+ antiport systems in renal cells.

This study analyzes the differential characteristics of the Na(+)-H+ antiport systems observed in several epithelial and non-epithelial renal cell lines. Confluent monolayers of LLC-PK1A cells have a Na(+)-H+ antiport system located in the apical membrane of the cell. This system, however, is not expressed during cell proliferation or after incubation in the presence of different mitogenic agents. In contrast, confluent monolayers of MDCK4 express minimal Na(+)-H+ antiport activity in the confluent monolayer state but reach maximal antiport activity during cell proliferation or after activation of the cells by different mitogenic agents. Similar results were obtained with the renal fibroblastic cell line BHK. The system present in MDCK4 cells is localized in the basolateral membrane of the epithelial cell. In LLC-PK1A cells, an increase in the extracellular Na+ concentration produces a hyperbolic increase in the activity of the Na(+)-H+ antiporter. In MDCK4 and BHK cells, however, an increase in external Na+ produces a sigmoid activation of the system. Maximal activation of the system occur at a pHo 7.5 in LLC-PK1A cells and pHo 7.0 in MDCK4 cells. The Na(+)-H+ antiporter of LLC-PK1A cells is more sensitive to the inhibitory effect of amiloride (Ki 1.8 x 10(-7) M) than is the antiporter of MDCK4 cells (Ki 7.0 x 10(-6) M). Moreover, 5-(N-methyl-N-isobutyl)amiloride is the most effective inhibitor of Na(+)-H+ exchange in LLC-PK1A cells, but the least effective inhibitor in MDCK4 cells. Conversely, the analog, 5-(N,N-dimethyl)amiloride, is the most effective inhibitor of Na(+)-H+ exchange in MDCK4 cells, but is the least effective inhibitor in LLC-PK1A cells. These results support the hypothesis that Na(+)-H+ exchange observed in LLC-PK1A and other cell lines may represent the activity of different Na(+)-H+ antiporters.     

Increase in membrane fluidity and opening of tight junctions have similar effects on sodium-coupled uptakes in renal epithelial cells

Apical membranes of renal epithelial cells were shown to be more rigid than other plasma membranes, due in part to the abundance of sphingomyelin among their constituent phospholipids. Tight junctions play a key role in maintaining differences between the apical and the basolateral domains of the plasma membrane with respect to their lipid composition and fluidity. To evaluate the influence of alterations of membrane fluidity on the activity of two apically located transport systems, we compared the effect of opening of tight junctions, by a preincubation period in calcium-deprived medium and of increasing fluidity, with benzyl alcohol, on Na-dependent uptakes of Pi and alpha-methyl-D-glucopyranoside (MGP) in intact, confluent LLC-PK1 cells and MDCK cells. Benzyl alcohol, at 10 mM, increased the Vmax of Pi uptake by 55 and 42% in LLC-PK1 cells and MDCK cells, respectively, but decreased the Vmax of MGP uptake in LLC -PK1 cells by 23%. Similarly to 10 mM benzyl alcohol, opening of tight junctions also increased the Vmax of Pi uptake by 45 and 46% in LLC-PK1 cells and MDCK cells, respectively, and depressed MGP uptake in LLC-PK1 cells by inducing a 15% decrease of the Vmax. None of the two maneuvers (i.e. addition of benzyl alcohol or opening of tight junctions) affected the Km values of the transport systems. From these results it is concluded that (i) the increase in membrane fluidity, achieved either by benzyl alcohol or by opening of tight junctions, affects Na-Pi and Na-glucose cotransports differently, reflecting differences in the lipid environments of the two transport systems, and (ii) membrane fluidity might play a physiological role in the modulation of the activity of transport systems.     

Comparative cytotoxicity of 5-aminosalicylic acid (mesalazine) and related compounds in different cell lines

Nonsteroidal anti-inflammatory drugs can cause serious side-effects such as tubulo-interstitial nephritis. Mesalazine (5-ASA, 5-aminosalicylic acid) is used for the treatment of colitis ulcerosa, Crohn disease, and other diseases; it has been found to induce necrosis of both proximal convoluted tubules and renal papillaries. The comparative cytotoxicity of 3-, 4-, and 5- aminosalicylic acid, acetylsalicylic acid (AcSA), and the parent compound salicylic acid (SA) was investigated for the free acids and for their sodium salts. The interaction with endogenous glutathione (GSH) was also investigated. Four established cell lines were used: MDCK, LLC-PK1, NRK as renal cells, and HepG2 as hepatic cells. The free acid compounds were less toxic than their corresponding salts. Acidic 5-ASA was the most toxic of the three isomers in MDCK and LLC-PK1 cells, while NRK and HepG2 were more susceptible to acidic 3-ASA. Addition of NaOH modified the relative toxicity of 3-ASA and 5-ASA. The LLC-PK1 and HepG2 cells were more sensitive to the test chemicals as their salts than were the NRK and MDCK cells. SA and 5-ASA decreased the GSH content in renal cells and increased it in HepG2. GSH depletion with l-buthionine-(S,R)-sulfoximine enhanced the toxicity only for SA in NRK and for 5-ASA and AcSA in HepG2. No correlation between endogenous GSH and the susceptibility of MDCK and LLC-PK1 to the test compounds was observed. The results suggest that no typical nephrotoxic effect occurred. No explanation could be found for the tubulo-interstitial nephritis caused by 5-ASA therapy.     

Biochemical properties of endothelin converting enzyme in renal epithelial cell lines.

This is the first report clearly demonstrating the presence of endothelin (ET) converting enzyme (ECE) in non-vascular cells (renal epithelial cell lines, MDCK and LLC-PK1). ECEs derived from these epithelial cells were very similar to the endothelial ECE in the following biochemical properties: 1) The optimum pH was 7.0; 2) the Km value for big ET-1 was approximately 30 microM; 3) the enzyme was potently inhibited by EDTA, o-phenanthroline and phosphoramidon; and 4) the enzyme did not convert big ET-2 or big ET-3. These data suggest that phosphoramidon-sensitive ECE is involved in the processing of big ET-1 to ET-1 in the renal tubule.     

Stress reaction of kidney epithelial cells to inorganic solid-core nanoparticles

A route of accumulation and elimination of therapeutic engineered nanoparticles (NPs) may be the kidney. Therefore, the interactions of different solid-core inorganic NPs (titanium-, silica-, and iron oxide-based NPs) were studied in vitro with the MDCK and LLC-PK epithelial cells as representative cells of the renal epithelia. Following cell exposure to the NPs, observations include cytotoxicity for oleic acid-coated iron oxide NPs, the production of reactive oxygen species for titanium dioxide NPs, and cell depletion of thiols for uncoated iron oxide NPs, whereas for silica NPs an apparent rapid and short-lived increase of thiol levels in both cell lines was observed. Following cell exposure to metallic NPs, the expression of the tranferrin receptor/CD71 was decreased in both cells by iron oxide NPs, but only in MDCK cells by titanium dioxide NPs. The tight association, then subsequent release of NPs by MDCK and LLC-PK kidney epithelial cells, showed that following exposure to the NPs, only MDCK cells could release iron oxide NPs, whereas both cells released titanium dioxide NPs. No transfer of any solid-core NPs across the cell layers was observed.     

Differential regulation of junctional complex assembly in renal epithelial cell lines

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and -catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner. calpeptin; tight junctions; adherens junctions; Rho; cadherin; p120ctn     

Cyclosporine A-induced toxicity in two renal cell culture models (LLC-PK1 and MDCK)

Renal damage caused by therapeutic treatment with cyclosporine A has been well documented. Clinical experiences have shown that cyclosporine A nephrotoxicity is determined by interstitial fibrosis with tubular atrophy. However, the exact mechanism by which this drug causes nephrotoxicity has not yet been clarified. This study used an in vitro model in an attempt to identify the cellular mechanisms underlying kidney cyclosporine A damage. We used two cell lines with the characteristics of proximal and distal tubule cells (pig kidney proximal tubular epithelial cell line [LLC-PK1] and Madin–Darby canine kidney cell line [MDCK]. The cell lines were treated with cyclosporine A for 24h. After the treatment, the cells were stained with Trypan Blue to estimate cell viability and processed by histochemical reactions to evaluate their cellular metabolism. Four enzymes (acid phosphatase, alkaline phosphatase, lactate dehydrogenase and succinate dehydrogenase) were considered. The cell viability assay showed that the LLC-PK1 cell line was more sensitive to cyclosporine A than MDCK. Remarkably, the LLC-PK1 cells disappeared with cyclosporine A treatment. As for the hydrolytic enzymes, only acid phosphatases showed an increased positivity in the treated LLC-PK1 cells. Similarly, lactate dehydrogenase showed a different activity histochemically. No statistically significant alterations were observed in the succinate dehydrogenase reaction.The cyclosporine A-treated MDCK cell lines did not show any difference in either their hydrolytic or succinate dehydrogenase enzyme positivity with respect to the control line. In contrast, there was a significant increase in lactate dehydrogenase activity. This study allowed the possible mechanism of cyclosporine A-induced damage in renal tubular cells to be evaluated. The enzymatic changes happened rapidly (during the 24h of treatment), suggesting that this alteration was one of the steps by which cyclosporine A induced toxicity. Moreover, since acid phosphatase is a marker of protein catabolism, the variation in the activity of this enzyme, in the LLC-PK1 line only, showed that cyclosporine can induce alterations leading to cellular toxicity. The modifications in lactate dehydrogenase activity, in both lines, suggested that this drug caused cell stress, inducing the production of lactic acid from glucose in the presence of oxygen. In conclusion, cyclosporine A treatment may force LLC-PK1 and MDCK cells to use anaerobic glycolysis preferentially. Further, these enzyme alterations may represent an epiphenomenon or a consequence of cyclosporine A toxicity.     

Intercellular ice propagation: experimental evidence for ice growth through membrane pores

Propagation of intracellular ice between cells significantly increases the prevalence of intracellular ice in confluent monolayers and tissues. It has been proposed that gap junctions facilitate ice propagation between cells. This study develops an equation for capillary freezing-point depression to determine the effect of temperature on the equilibrium radius of an ice crystal sufficiently small to grow through gap junctions. Convection cryomicroscopy and video image analysis were used to examine the incidence and pattern of intracellular ice formation (IIF) in the confluent monolayers of cell lines that do (MDCK) and do not (V-79W) form gap junctions. The effect of gap junctions on intracellular ice propagation was strongly temperature-dependent. For cells with gap junctions, IIF occurred in a directed wave-like pattern in 100% of the cells below -3 degrees C. At temperatures above -3 degrees C, there was a marked drop in the incidence of IIF, with isolated individual cells initially freezing randomly throughout the sample. This random pattern of IIF was also observed in the V-79W monolayers and in MDCK monolayers treated to prevent gap junction formation. The significant change in the low temperature behavior of confluent MDCK monolayers at -3 degrees C is likely the result of the inhibition of gap junction-facilitated ice propagation, and supports the theory that gap junctions facilitate ice nucleation between cells.     

Identification of basolateral membrane targeting signal of human sodium-dependent dicarboxylate transporter 3

Sodium-dependent dicarboxylate transporters (NaDC) include low-affinity NaDC1 and high-affinity NaDC3. Despite high similarities structurally and functionally, both are localized to opposite surfaces of renal tubular cells. The molecular mechanisms and localization signals leading to this polarized distribution remain unknown. In this study, distribution of NaDC3 in human kidney tissue was firstly observed by immunohistochemistry and immunofluorescence. Then, EGFP-fused wild-type, NH2- and COOH-terminal deletion and point mutants of NaDC3, and chimera between NaDC3 and NaDC1, were generated and transfected into polarized renal cells lines, LLC-PK1 and MDCK. Their subcellular localizations were analyzed by laser confocal microscopy. Immunolocalization results revealed that NaDC3 was expressed at basolateral membrane of human renal proximal tubular epithelia. Confocal examinations showed that wild-type NaDC3 was targeted to the basolateral membrane of MDCK and LLC-PK1. Deletion mutations indicated that the basolateral targeting signal of NaDC3 located within a short sequence AKKVWSARR of its amino-terminal cytoplasmic domain. Addition of this sequence could redirect apical NaDC1 to the basolateral membrane of LLC-PK1. Point mutagenesis revealed that mutation of either of two hydrophobic amino acids V and W in this short sequence largely redirected NaDC3 to both apical and basolateral surfaces of LLC-PK, indicating that the two hydrophobic amino acids are critical for the basolateral targeting of NaDC3. Our studies provide direct evidence of the localization of NaDC3 at the basolateral membrane of human renal proximal tubule cells and identify a di-hydrophobic amino acid motif VW as basolateral localization signal in the N-terminal cytoplasmic domain of NaDC3.     

Differential modulation of cadherin-mediated cell-cell adhesion by platelet endothelial cell adhesion molecule-1 isoforms through activation of extracellular regulated kinases

The role of platelet endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cell-cell interactions and its contribution to cadherin-mediated cell adhesion are poorly understood. Such studies have been difficult because all known endothelial cells express PECAM-1. We have used Madin-Darby canine kidney (MDCK) cells as a model system in which to evaluate the role of PECAM-1 isoforms that differ in their cytoplasmic domains in cell-cell interactions. MDCK cells lack endogenous PECAM-1 but form cell-cell junctions similar to those of endothelial cells, in which PECAM-1 is concentrated. MDCK cells were transfected with two isoforms of murine PECAM-1, Delta15 and Delta14&15, the predominant isoforms expressed in vivo. Expression of the Delta15 isoform resulted in apparent dedifferentiation of MDCK cells concomitant with the loss of adherens junctions, down-regulation of E-cadherin, alpha- and beta-catenin expression, and sustained activation of extracellular regulated kinases. The Delta15 isoform was not concentrated at cell-cell contacts. In contrast, the Delta14&15 isoform localized to sites of cell-cell contact and had no effect on MDCK cell morphology, cadherin/catenin expression, or extracellular regulated kinase activity. Thus, the presence of exon 14 in the cytoplasmic domain of PECAM-1 has dramatic effects on the ability of cells to maintain adherens junctions and an epithelial phenotype. Therefore, changes in the expression of exon 14 containing PECAM-1 isoforms, which we have observed during development, may have profound functional consequences.     

Translocation of MARCKS and reorganization of the cytoskeleton by PMA correlates with the ion selectivity,the confluence,and transformation state of kidney epithelial cell lines

The role of protein kinase C (PKC) in the regulation of the cytoskeleton of epithelial cells with tightly sealed contacts, poor contacts, and without contacts were investigated by incubating them with a protein kinase C activator phorbol myristoyl acetate (PMA). The morphology and organization of the membrane skeleton and stress fibers as well as the localization of an actin-bundling PKC substrate MARCKS in confluent MDCK cells originating from the distal tubulus of dog kidney, LLC-PK1 cells originating from the proximal tubulus of pig kidney, src-transformed MDCK cells, epidermoid carcinoma A431 cells, and MDCK cells grown in low calcium medium (LC medium) in low density were visualized with phase contrast and immunofluorescence microscopy. Four different responses to the PMA-treatment in actin-based structures of cultured epithelial cells were observed: 1) disintegration of the membrane skeleton in confluent MDCK cells; 2) depolymerization of the stress fibers in confluent MDCK and LLC-PK1 cells; 3) formation of the membrane skeleton in A431 cells, and 4) formation of the stress fibers and membrane skeleton in LC-MDCK cells. Thus, it seems that in fully confluent tightly sealed epithelium, activation of PKC has a deleterious effect on actin-based structures, whereas in cells without contacts or loose contacts, activation of PKC by PMA results in improvement of actin-based cytoskeletal structures. The main difference between the two kidney cell lines used is their selectivity to ion transport: the monolayer of LLC-PK1 cells is anion selective and MDCK cells cation selective. We propose a model where alterations in the ionic milieu within the MDCK cells by means of cation channels affect the disintegration of the membrane skeleton. The distribution of MARCKS followed the distribution of fodrin in both cell lines upon PMA-treatment, suggesting that phosphorylation of MARCKS by PKC may contribute in the regulation of the integrity of the membrane skeleton. J. Cell. Physiol. 181:83–95, 1999. © 1999 Wiley-Liss, Inc.     

Organization of the endoplasmic reticulum in renal cell lines MDCK and LLC-PK1

The spatial organization of the endoplasmic reticulum has been studied in two renal cell lines, MDCK and LLC-PK1, which originate from the distal and proximal portions of the mammalian nephron, respectively, and which form a polarized epithelium when they reach confluence in tissue culture. The two renal cell lines, grown to confluence on either solid or permeable supports, were investigated by fluorescence microscopy, confocal microscopy, and transmission electron microscopy. Fluorescence labeling of the endoplasmic reticulum was achieved using the cationic fluorescent dye DIOC₆ (3). In order to differentiate fluorescent labeling of the endoplasmic reticulum from that of the mitochondria, cells were also labeled with rhodamine 123. For electron microscopy, the spatial organization of the endoplasmic reticulum was examined in thick sections using the long-duration osmium impregnation technique or the ferrocyanide/osmium technique. In both cell lines, the endoplasmic reticulum formed an abundant tubular network of canaliculi that frequently abutted the basolateral domain of the plasma membrane and occasionally the apical membrane. Elements of the endoplasmic reticulum were also found in close proximity to mitochondria that, as in the nephron, formed branched structures. Canaliculi appeared circular or flattened and had an inner diameter of 10–70 nm for MDCK cells and 20–90 nm for LLC-PK1 cells. Such a three-dimensional organization might facilitate the translocation of defined lipid species between the endoplasmic reticulum and the plasma membrane, and between the endoplasmic reticulum and mitochondria.     

Adaptive changes in sulfoglycolipids of kidney cell lines by culture in anisosmotic media

(1) The effects of osmolarity environments on renal glycolipid composition were examined using established renal cell lines. The profile of glycosphingolipids of Madin-Darby canine kidney cells (MDCK) in culture with anisosmotic media showed that a hyposomotic medium reduced the concentration of GalCer I3-sulfate and LacCer II3-sulfate. (2) The concentrations of sulfoglycolipids were increased by maintaining the culture in a hyperosmotic media prepared by the addition of various sodium salts to the control isosmotic medium, while the contents of most of the neutral glycolipids were reduced. The hyperosomotic medium supplemented with nonelectrolytes, mannitol, sucrose or urea, also increased the concentration of sulfoglycolipids. (3) Both sulfoglycolipids were increased linearly with gradual increases of sodium chloride in the medium. Hyperosmolarity produced by the addition of a nonelectrolyte, mannitol, also increased the levels of sulfoglycolipids. In both series of media, the most prominent accumulation was observed in LacCer II3-sulfate. (4) The incorporation of radioactive sulfate into sulfoglycolipids was elevated in cells adapted to high NaCl or mannitol. The increase of the label was observed not only in MDCK but also in three other established cell lines of renal tubular origin, JTC-12, LLC-PK1 and MDBK. (5) It was established, using the culture system of homogeneous cell lines, that the mechanism of increasing the amount of sulfoglycolipids is independent of the integral regulatory mechanism of animals and resides in the renal epithelial cell itself. These results suggest that by culture in hyperosmotic media, the elevated level of intracellular cations stimulated the activity of GalCer and LacCer sulfotransferase, inducing the increased expression of sulfoglycolipids.     

Transepithelial transport of vinblastine by kidney-derived cell lines. Application of a new kinetic model to estimate in situ Km of the pump

We present a new transport model that may be useful for many kinds of transepithelial transport experiments. The model permits estimation of a pump Km and pump activity solely on the basis of transepithelial tracer fluxes. We apply the model to studies of a multidrug efflux pump, P-glycoprotein, which is normally located in the apical plasma membrane of certain transporting epithelia such as kidney proximal tubule cells. To determine the functional properties of this multidrug transporter in an epithelium, we studied the transepithelial transport of the chemotherapeutic drug, vinblastine, in epithelia formed by the kidney cell lines MDCK, LLC-PK1, and OK. We have previously shown that basal to apical flux of 100 nM vinblastine was about five times higher than apical to basal flux in MDCK epithelia, indicating that there is a net transepithelial transport of vinblastine across MDCK epithelia. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer in a concentration-dependent manner, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. The model permits estimation of a pump Km and pump activity solely on the basis of transepithelial tracer fluxes. According to the transport model the apical membrane pump has Michaelis-Menten kinetics with an apparent Km = 1.1 microM. Net basal to apical transport of vinblastine was also observed in LLC-PK1 cells and OK cells which are other kidney-derived cell lines. The order of potency of the transport is LLC-PK1 greater than MDCK greater than OK cells. The organic cation transporter is not involved in this vinblastine transport because vinblastine transport in MDCK cells was not affected by 3 mM tetramethyl- or tetraethylammonium. Inhibitors of vinblastine transport in MDCK cells was not affected by potency, were verapamil greater than vincristine greater than actinomycin D greater than daunomycin. The transport pattern we observed is that predicted to result from the function of the multidrug transporter in the apical plasma membrane.     

The MDCK cell line is made up of populations of cells with diverse resistive and transport properties

The MDCK cell line has been used as a model for the renal distal tubule. When grown on permeable supports, the cells form tight junctions and transport sodium and calcium. Widely different values have, however, been obtained for resistance and transports by different investigators. Since the MDCK cell line is genetically heterogeneous, one potential reason for observed differences is the use of different populations of cells. Dilutional techniques have been used to obtain eleven MDCK cell clones. The clones exhibit a range of values for resistance (300-4000 ohms cm2), net Ca2+ transport (3.2-81 pmol/cm2/min) and net Na+ transport (0.4-6.0 nmol/cm2/min). To determine if the characteristics of clonal cell lines changed with increasing passage number, resistance was measured at relatively early and late passage number. Resistance increased with increasing passage number in two of six lines and decreased in one. These studies demonstrate that differences between previous studies may be due to differences in cell population, length of time since passage, and passage number. Clonal cell lines should provide a useful tool to investigate ion transport and mechanisms of regulation of transport in this model of renal distal tubule.     

Development of intercellular communication during the epithelial reorganization of a renal cell line (LLC-PK1)

Junctional permeability determinations after microinjection of the fluorescent tracer, Lucifer Yellow CH, show that the cells in confluent monolayers of the renal epithelial cell lines LLC-PK1 and A6 are interconnected by intercellular junctions. This cell-to-cell communication network permits the fluorescent dye to diffuse from the microinjected cell into multiple adjacent neighboring cells. Cell-to-cell diffusion of the fluorescent dye was not observed at pH 6.0. Full recovery occurred, however, when the pH of the extracellular medium was adjusted to 7.4. To provide a sensitive index of the averaged efficacy of junctional communication, we measured the number of cells that survived ouabain treatment in a 50% mixture of wild and ouabain-resistant mutant LLC-PK1 cells. Electron probe microanalysis in uncoupled cells showed that ouabain treatment produced two populations of cells, with totally different intracellular Na+ and K+ content. Under this condition, only 50% of the population survived after 48 h of treatment. When ouabain treatment was initiated 24 h after plating, however, 100% survival was observed, and the cells contained uniform intracellular Na+ and K+ concentration. This finding is consistent with the theory that this protective effect is mediated through the presence of the functional communicating intercellular junctions. When ouabain was applied at different times after plating, full protection is reached by 2 h. The early development of cell-to-cell communication, which precedes the development of the occluding junctions and several transport systems by several hours, is consistent with the involvement of the intercellular junctions in the synchronization of the polarization process.     

Renal epithelial cell lines (BSC-1, MDCK, LLC-PK1) express 11 beta-hydroxysteroid dehydrogenase activity

Renal tissue of several species has been shown to express considerable 11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) activity. However, it is uncertain as to which renal cell types exhibit 11-HSD activity. In the present study, we investigated corticosterone metabolism in BSC-1 cells, a continuous renal epithelial cell line derived from the African green monkey (Cercopithecus aethiops). In incubation experiments using 3H-labelled corticosterone and HPLC, we have demonstrated oxidative 11-HSD activity in intact monolayers of BSC-1 cells as well as in BSC-1 cell homogenates. 11-HSD activity in cell homogenates could be stimulated 7-9-fold by the addition of exogenous NADP+ (1 mM). In contrast, no reductive 11-HSD could be detected either in intact cells or in cell homogenates under various experimental conditions which were designed to favor reductive 11-HSD activity. Pilot experiments were performed in cell homogenates from two other renal epithelial cell lines derived from canine (MDCK) and porcine (LLC-PK1) kidney. They also revealed oxidative but no reductive 11-HSD activity. The data provide evidence for an epithelial localization of renal oxidative 11-HSD activity.     

Immortalized mouse brain endothelial cells are ultrastructurally similar to endothelial cells and respond to astrocyte-conditioned medium

Summary Studies of brain microvessel endothelial cell physiology and blood-brain barrier properties are often hampered by the requirement of repeatedly producing and characterizing primary endothelial cell cultures. The use of viral oncogenes to produce several immortalized brain microvessel cell lines has been reported. The resulting cell lines express many properties of the blood-brain barrier phenotype but do not completely mimic primary endothelial cells in culture. As immortalized brain microvessel endothelial cell lines have not yet been produced from mice, we transformed mouse brain endothelial cells with the adenovirus E1A gene using a retroviral vector (DOL). Eight of 11 clones produced exhibited an endothelial-like cobblestone morphology and were characterized as endothelial with a panel of antibodies, lectins, and ultrastructural criteria. These cells are endothelial in origin and share ultrastructural features with primary cultures of endothelial cells. Examination of freeze fracture and transmission electron micrographs show adherens junctions exist between the transformed cells, and culture in astrocyte-conditioned medium induces the formation of gap junctions. This is one indication that responses to astrocyte-derived factors are retained by the transformed cell lines.     

Cadmium (Cd2+) disrupts Ca(2+)-dependent cell-cell junctions and alters the pattern of E-cadherin immunofluorescence in LLC-PK1 cells.

Recent findings from our laboratories have shown that Cd2+ has relatively specific damaging effects on the adhering and occluding junctions in the established porcine renal epithelial cell line, LLC-PK1. Results of the present studies show that the junction-perturbing effects of Cd2+ in LLC-PK1 cells are more pronounced when Cd2+ is applied to the basolateral cell surface than when it is applied to the apical surface, and that the severity of the effects is inversely related to the concentration of Ca2+ in the medium. Additional results show that exposure to sublethal concentrations of Cd2+ decreases the amount of E-cadherin that is associated with cell-cell contacts. These results suggest that Cd2+ damages Ca(2+)-dependent cell-cell junctions in LLC-PK1 cells by interacting with E-cadherin or a similar Ca(2+)-sensitive site that is oriented toward the basolateral cell surface.     

The role of c-Fes in vascular endothelial growth factor-A-mediated signaling by endothelial cells

c-Fes plays pivotal roles in angiogenic cellular responses of endothelial cells. Here we examined the role of c-Fes in vascular endothelial growth factor-A (VEGF-A)-mediated signaling pathways in endothelial cells. We introduced either wild-type or kinase-inactive c-Fes in porcine aortic endothelial (PAE) cell lines, which endogenously express VEGF receptor (VEGFR)-1, and PAE cells ectopically expressing VEGFR-2 (denoted KDR/PAE cells) and generated stable cell lines. VEGF-A induced autophosphorylation of c-Fes only in KDR/PAE cells, suggesting that VEGFR-2 was required for its activation. Expression of kinase-inactive c-Fes failed to demonstrate dominant negative effect on VEGF-A-induced chemotaxis and capillary morphogenesis. Phosphoinositide 3-kinase (PI3-kinase) was activated in KDR/PAE cells and c-Fes contributed to this process in a kinase activity-dependent manner. However, VEGFR-2, insulin receptor substrate-1, and c-Src were also involved in VEGF-A-induced activation of PI3-kinase, resulting in the compensation in cells expressing kinase-inactive c-Fes. Interestingly, overexpression of wild-type c-Fes in PAE cells induced VEGF-A-independent capillary morphogenesis. Considered collectively, VEGF-A activated PI3-kinase partly through c-Fes and increase in c-Fes kinase activity enhanced capillary morphogenesis by yet unknown signaling pathways.