Calcium transport systems in the LLC-PK1 renal epithelial established cell line

ATP-dependent calcium uptake was measured in membrane vesicles prepared from the renal epithelial LLC-PK1 established cell line. The relative contribution of the nonmitochondrial versus the mitochondrial calcium uptake is larger in LLC-PK1 cell homogenates than in homogenates from renal cortex. Two types of calcium pump, characterized by the formation of calcium-dependent phosphointermediates of 135 kDa and 115 kDa, were found in membrane fractions from LLC-PK1 cells. The 135 kDa calcium pump was also detected by 125I-labelled calmodulin overlay. Although the subcellular localization in LLC-PK1 cell membranes could not be unambiguously determined, it is conceivable that the 135 kDa and the 115 kDa molecules represent the plasma membrane calcium pump and the endoplasmic reticulum calcium pump respectively, in agreement with what was found for renal cortex preparations. Extravesicular sodium partially inhibits ATP-driven calcium uptake in a plasma-membrane-enriched fraction of the LLC-PK1 cells. The effect is potentiated by a vesicle inside-negative membrane potential. Although the effect is less pronounced than in renal cortex basal-lateral membranes, this observation suggests that an Na+-Ca2+ exchange mechanism is also present in LLC-PK1 cells. ATP-dependent calcium uptake in nonmitochondrial intracellular stores was investigated, using saponin-permeabilized cells. Permeabilized LLC-PK1 cells lowered the free calcium concentration in the medium to less than 0.4 microM. More than 60% of the accumulated calcium can be released by addition of inositol 1,4,5-trisphosphate. Our data indicate that the LLC-PK1 cell line can be successfully used as model system for the study of renal calcium handling.     

Polyamine transport systems in the LLC-PK1 renal epithelial established cell line

LLC-PK1 cells were brought to a quiescent state by treatment with DL-2-difluoromethylornithine (DFMO), a specific inhibitor of L-ornithine decarboxylase (ODC). The inhibition of ODC, which is the key enzyme for polyamine synthesis, strongly reduced the cellular content of putrescine and spermidine. The cells resumed DNA-synthesis followed by mitosis when exogenous putrescine was added. DFMO treatment strongly stimulated the putrescine uptake capability. A kinetic analysis of the initial uptake rates revealed a saturable Na+-dependent and a saturable Na+-independent pathway on top of non-saturable diffusion. The stimulation by DFMO was exclusively due to an effect on the Vmax values of the saturable pathways. The Na+-dependent transporter had a higher affinity for putrescine (apparent Km = 4.7 +/- 0.7 microM) than the Na+-independent transporter (apparent Km = 29.8 +/- 3.5 microM). As a consequence, although the latter transporter had a higher Vmax, the Na+-dependent transport was more important at a physiological putrescine concentration. Putrescine uptake by both transporters was inhibited with similar relative affinities by spermidine, spermine as well as by the antileukemic agent, methylglyoxal bis(guanylhydrazone), but not by amino acids. The activity of the Na+-dependent transporter was very much dependent on SH-group reagents, whereas the Na+-independent transporter was not affected. Both transporters were inhibited by metabolic inhibitors and by ionophores but the Na+-dependent transporter was affected to a greater extent. For both transporters there was a down-regulation in response to exogenous putrescine. This suggests that the polyamine transporters in LLC-PK1 are adaptively regulated and may contribute to the regulation of the cellular polyamine level and cellular proliferation.     

Calcitonin receptor-stimulating peptide-1 regulates ion transport and growth of renal epithelial cell line LLC-PK1

Calcitonin receptor-stimulating peptide-1 (CRSP-1) is a peptide recently identified from porcine brain by monitoring the cAMP production through an endogenous calcitonin (CT) receptor in the renal epithelial cell line LLC-PK(1). Here we investigated the effects of CRSP-1 on the ion transport and growth of LLC-PK(1) cells. CRSP-1 inhibited the growth of LLC-PK(1) cells with a higher potency than porcine CT. CRSP-1 enhanced the uptake of (22)Na(+) into LLC-PK(1) cells more strongly than did CT and slightly reduced the (45)Ca(2+) uptake. The enhancement of the (22)Na(+) uptake was abolished by 5-(N-ethyl-N-isopropyl) amiloride, a strong Na(+)/H(+) exchanger (NHE) inhibitor for NHE1, even at a concentration of 1x10(-8)M, although other ion transporter inhibitors did not affect the (22)Na(+) uptake. These results indicate that CRSP-1 enhances the (22)Na(+) uptake by the specific activation of NHE1. Taken together, CRSP-1 is considered to be a new regulator for the urinary ion excretion and renal epithelial cell growth.     

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.     

Sodium-dependent phosphate transport by apical membrane vesicles from a cultured renal epithelial cell line (LLC-PK1)

Apical membrane vesicles were prepared from confluent monolayers of LLC-PK1 cells grown upon microcarrier beads. The final membrane preparation, obtained by a modified divalent cation precipitation technique, was enriched in alkaline phosphatase, leucine aminopeptidase and trehalase (8-fold compared to the initial homogenate). Analysis of phosphate uptake into the vesicles identified a specific sodium-dependent pathway. Lithium and other cations were unable to replace sodium. At 100 mmol/l sodium and pH 7.4, an apparent Km for phosphate of 99 +/- 19 mumol/l and an apparent Ki for arsenate of 1.9 mmol/l were found. Analysis of the sodium activation of phosphate uptake gave an apparent Km for sodium of 32 +/- 12 mmol/l and suggested the involvement of two sodium ions in the transport mechanism. Sodium modified the apparent Km of the transport system for phosphate. The rate of sodium-dependent phosphate uptake was higher at pH 6.4 than at pH 7.4. At both pH values, an inside negative membrane potential (potassium gradient plus valinomycin) had no stimulatory effect on the rate of the sodium-dependent component of phosphate uptake. It is concluded that the apical membrane of LLC-PK1 cells contains a sodium-phosphate cotransport system with a stoichiometry of 2 sodium ions: 1 phosphate anion.     

Sugar transport in the LLC-PK1 renal epithelial cell line: Similarity to mammalian kidney and the influence of cell density

Cells of confluent cultures of the established pig renal epithelial line, LLC-PK₁, accumulate α-methyl-D-glucoside against a concentration gradient. This transport system is strongly inhibited by phlorizin and 6-deoxy-D-glucose, moderately inhibited by phloretin, and only weakly inhibited by 3-0-methyl-D-glucose, paralleling the situation in mammalian kidney. The time course for the uptake of α-methyl-D-glucoside and for the carrier-mediated but passive uptake of 3-0-methyl-D-glucose are identical to those seen in mammalian kidney. Subconfluent cultures of LLC-PK₁ cells are unable to accumulate α-methyl-D-glucoside, and their transport of this glucose analog is less sensitive to phlorizin inhibition than is the transport system in confluent cultures. Transmission electron micrographs show that cells from subconfluent cultures lack the microvillous surface seen in cells from confluent cultures. Cell density is thus a factor in the occurrence of structural and functional differentiated properties related to transport in these cells.     

Induction of microvillar hydrolase activities by cell density and exogenous differentiation inducers in an established kidney epithelial cell line (LLC-PK1)

Several hydrolase activities characteristic of the apical brush border membrane of renal proximal tubule, leucine aminopeptidase, gamma-glutamyl transpeptidase, alkaline phosphatase, maltase, and trehalase, were identified in cultures of the LLC-PK1 kidney epithelial cell line. A coordinate increase in activities of these enzymes was observed upon development of a confluent cell density and functional membrane polarization. Further large progressive increases in individual hydrolase activities were induced after the addition of compounds known as differentiation inducers. Hexamethylene bisacetamide preferentially induced increased trehalase and maltase activities. Induced trehalase activity exhibited an increased Vmax but a similar Km compared with activity in control extracts. Induction required protein synthesis and was dependent on inducer concentration and exposure time. Treatment of confluent cultures with N,N'-dimethylformamide triggered an induction of maltase, trehalase, alkaline phosphatase, and gamma-glutamyl transpeptidase activities, whereas dimethylsulfoxide induced trehalase and gamma-glutamyl transpeptidase activities. Increased leucine aminopeptidase and maltase activities were observed after addition of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. Induction of trehalase activity by N,N'-dimethylformamide was reversible over a 4-day period after removal of inducer, but effects of hexamethylene bisacetamide were irreversible. These results suggest that the LLC-PK1 cell line reproducibly develops differentiation-specific characteristics under defined conditions in cell culture, which can be individually modulated by chemicals known as inducers of cell differentiation.     

Inhibition of oxidant-induced lipid peroxidation in cultured renal tubular epithelial cells (LLC-PK1) by quercetin

The protective effect of quercetin against oxidant-induced cell injury (hypoxanthine/xanthine oxidase system) was studied in the renal tubular epithelial cell line LLC-PK₁. Pretreatment with quercetin provided protection from structural and functional cell damage in a concentration-dependent manner (10-100 μM). Comparison with structural variants revealed that the protective property of quercetin depends on the number of hydroxyl substituents in the B-ring, the presence of an extended C-ring chromophore, 3-D-planarity and lipophilicity, indicating that membrane affinity is essential for protection. The hypothesis that quercetin exerts its protective effects via inhibition of lipid peroxidation was further examined. Protection by quercetin was found when lipid peroxidation, assessed by the release of malondialdehyde, was initiated by H₂O₂ or by the combination of 1-chloro-2,4-dinitrobenzene and aminotriazole. In contrast, the bioflavonoid was not protective when oxidative cell damage was induced by menadione and occurred in the absence of lipid peroxidation. These data suggest that cytoprotective effects of quercetin are related to membrane affinity and may be explained by interruption of membrane lipid peroxidation rather than by intracellular scavenging of oxygen free radicals.     

Atrial natriuretic factor and cGMP inhibit amiloride-sensitive Na+ transport in the cultured renal epithelial cell line, LLC-PK1

The renal cell culture model, LLC-PK1, which contains an amiloride-sensitive conductive Na+ transport pathway and a Na+/H+ exchanger, was utilized to examine the direct effects of atriopeptin II and cGMP on Na+ transport in epithelial cells. Exposure of cells to atriopeptin II (10(-7) M) increased cGMP production within 2 min of addition to cells in monolayer. Atriopeptin II (10(-7) M) or exogenous 8-bromo-cGMP (10(-3) M) maximally inhibited the uptake of 22Na+ through the conductive pathway which accounted for up to 60% of total 22Na+ uptake. The apparent Ki for this inhibition by atriopeptin II was 2 X 10(-11) M. Amiloride inhibited 22Na+ uptake to a similar extent as atriopeptin II, and the effects of the presence of both agents was not additive. In contrast, neither atriopeptin II nor cGMP blunted the increment in 22Na+ uptake induced by a pH gradient. Thus atriopeptin II can directly inhibit Na+ transport in renal epithelial cells, probably through its stimulation of cGMP.     

Spontaneous reversal of polarity of the voltage across LLC-PK1 renal epithelial cell sheets

While sterilely monitoring transepithelial voltage (potential difference) across LLC-PK cell sheets over a 24-hr period, we noted that the apical-negative, transepithelial voltage, a key property of the LLC-PK1 renal epithelial cell line, reverses polarity to become apical-positive. This spontaneous change of polarity of electrical potential difference (PD) across LLC-PK1 cell sheets cultured on permeable filters was observed to occur approximately 12 hr after refeeding. Unlike the apical (luminal)-negative PD, the apical-positive PD was insensitive to phlorizin and ouabain. Both were insensitive to the diuretics amiloride, furosemide, and 4-acetamido-4-isothiocynato-stilbene-2,2-disulfonic acid (SITS). A pH gradient existed across apical-positive cell sheets (apical medium more acidic by 0.3 units) but an osmotic gradient did not. Unlike the temperature-sensitive apical-negative PD, the apical positive PD was unaffected by brief exposure to 4 degrees C temperature. Junctional disruptive agents such as the tumor promotor, TPA, dissipated both types of PD with similar time courses. The formation of the apical-positive PD correlated in time with apical glucose levels falling below the reported Km of the Na+-sugar contransporter. A high glycolytic rate per se may not be essential for this PD polarity reversal since the reversal could occur in glucose-free medium with a normal time course and magnitude. The lysis with time of floating cells with consequent release of KCl into the apical compartment was also considered as a possible cause of the polarity reversal, but the turnover of even 2 X 10(6) cells in 12 hr was found not to raise apical KCl sufficiently to produce the polarity shift. Although a significant K+ gradient did not exist across cell sheets with apical-positive PD values, a sizable gradient of Cl- did exist, directed apical to basolateral. This gradient, coupled with anion-selective tight junctions, should contribute to the observed apical positive voltage. The voltage polarity shift seen in these cell cultures with time is not unlike the polarity shift occurring in the renal proximal convoluted tubule, with distance from the glomerulus.     

Effects of the pig renal epithelial cell line LLC-PK1 and its conditioned medium on the phenotype of porcine microglia in vitro

Microglia are dispersed throughout the central nervous system. Under physiological circumstances they display a 'ramified' resting phenotype. In different neuropathologies microglia reversibly transform into the activated form, an amoeboid phagocyte with a broad spectrum of immune effector functions. In this study, a coculture of porcine microglia and the pig renal epithelial cell line LLC-PK1 was used to investigate microglial cell biology. The morphology of the cocultures was elucidated as well as the functionality of the microglia cells by proliferation, superoxide and phagocytosis assays. Our results demonstrate that direct intercellular contact between the two cell types was necessary for microglia to acquire a ramified morphology. Moreover, the conditioned medium of the renal cells promoted proliferation of microglia, inhibited giant cell formation and stimulated microglia to retain their capability to generate superoxide and to perform phagocytosis. In conclusion, we have constructed a cell culture system showing differentiation of microglia in vitro and keeping them in optimal conditions.     

Cationic amino acid transport by two renal epithelial cell lines: LLC-PK1 and MDCK cells

LLC-PK1 and MDCK cells take up cationic amino acids (lysine and arginine) by a specific sodium independent transport system. Uptake is inhibited by ornithine in LLC-PK1 and MDCK cells either in the presence or absence of sodium and by glutamine or homoserine in MDCK cells in the presence of sodium. Trans-stimulation of uptake occurs in the presence of intracellular cationic amino acids. Experiments with valinomycin or with different extracellular potassium concentrations suggest that uptake is dependent on the membrane potential of these cells. These transport features are similar to those previously ascribed to a transport system denominated y+ in other cells. Further experiments suggested that this carrier system is localised to the basolateral membrane in each cell type.     

Ecto-lysophospholipase C controls lysophospholipid uptake and metabolism in porcine kidney epithelial cell line LLC-PK1

Lysophosphatidylcholine (LPC) has diverse biological activities through different mechanisms including its conversion into other types of lipid mediators such as lysophosphatidic acid and 2-arachidonoylglycerol. Previously, we found that a large portion of the fluorescent analog of alkyl type LPC (Bodipy-lysoPAF) on porcine kidney epithelial cells (LLC-PK1) was degraded to monoalkylglycerol by lysophospholipase C-like activity and then quickly internalized into the cells. In this study, we investigated whether exogenous fluorescently labeled LPC (NBD-LPC) itself was also metabolized and internalized by a similar mechanism. LLC-PK1 cells converted NBD-LPC to either NBD-MG, possibly due to lysophospholipase C-like activity of ecto-nucleotide pyrophosphatase/phosphodiesterase-6, or to free fatty acid (FA), due to lysophospholipase activity in the culture medium at both sites. The resultant NBD-MG was further degraded to NBD-FA by lipase activity before or after its uptake into the cells, and a portion of NBD-FA was finally released into the culture medium on the opposite side.     

Comparison of reactive oxygen scavenging systems between a cetacean (DKN1) and a porcine renal epithelial cell line (LLC-PK1)

A transformed renal epithelial cell line, (DKN(1)), from an Atlantic Bottlenose Dolphin, Tursiops truncatus was established in this laboratory and has been used for in vitro genomic analysis and initial toxicological evaluations of dolphin cells. Studies were initiated to compare maintenance of normal antioxidant mechanisms in DKN(1) with similar mechanisms in cells of a pig kidney line, LLC-PK(1). Levels of catalase, glutathione peroxidase, and of reduced glutathione in these dolphin cells were significantly lower than in the porcine cells. Both cell lines were then challenged with hydrogen peroxide at 0.01, 0.1, and 1.0 mM concentrations. The dolphin cells exhibited increased cytotoxicity with a concurrent increase in apoptosis at lower concentrations (0.1 mM) than those required to initiate cytotoxicity in the porcine cells (1.0 mM). Taken together, these results would indicate that the dolphin cells are more susceptible to the damaging effects of certain reactive oxygen species than their terrestrial counterparts.     

Regulation of amino acid transport in the renal epithelial cell line NBL-1

Summary The activities of the transport systems A, B° and X_AG- are induced by various forms of stress in renal epithelial cells. Amino acid deprivation induces System A and X_AG- in a protein-synthesis dependent process. In the case of System X_AG- evidence is presented that induction of transport does not involve an increase in the amount of mRNA for the transporter or of the amount of transport protein. Preliminary evidence for the existence of a novel glycoprotein which is induced in parallel to the induction of these transport systems is presented. It is suggested that the induction of amino acid transport proteins and of some of the so-called stress proteins may be triggered by a common molecular mechanism.     

Specific binding activities and cyclic GMP responses by atrial natriuretic polypeptide in kidney epithelial cell line (LLC-PK1)

Receptor binding activities and cyclic GMP responses by alpha-human atrial natriuretic polypeptide (alpha-hANP) and its fragments were studied in a kidney epithelial cell line (LLC-PK1). Binding of 125I-alpha-hANP to the cells at 0 degrees C was saturable, time-dependent and reversible, indicating the presence of a single class of binding sites. alpha-hANP (7-23)NH2 fragment inhibited most effectively the specific binding of 125I-alpha-hANP to the LLC-PK1 cells, followed by alpha-hANP (17-28) and alpha-hANP (8-22), while alpha-hANP (1-6) and alpha-hANP (24-28) did not. alpha-hANP stimulated the formation of cyclic GMP in the LLC-PK1 cells dose-dependently. Although no fragments of alpha-hANP used were effective for cyclic GMP formation in the LLC-PK1 cells, alpha-hANP (7-23) NH2 antagonized the action of alpha-hANP on cyclic GMP formation. These data suggest that the LLC-PK1 cells retain specific receptors for atrial natriuretic polypeptide (ANP) and respond to ANP by stimulating cyclic GMP formation, and therefore this cell line may be useful for studying the mechanism of action for ANP in renal tubular cells.