The mechanism of the verapamil-digoxin interaction in renal tubular cells (LLC-PK1)

Verapamil, usually given as a racemic mixture, decreases in vivo and in vitro digoxin renal tubular secretion, which is suggested to be mediated by P-glycoprotein, an ATP-dependent multidrug efflux pump. Importantly, the two enantiomers of verapamil have been reported to similarly inhibit P-glycoprotein-mediated transport of chemotherapeutic agents. In this study, we examined effects of enantiomers of verapamil on digoxin transport across an LLC-PK1 cell monolayer, a model of proximal renal tubular cells. The results indicate that verapamil inhibition of digoxin transport is non-stereospecific. Furthermore, the verapamil-digoxin interaction is not competitive. The two drugs may not share a common initial step in the P-glycoprotein-mediated transport.     

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.     

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.     

Calcitonin stimulates plasminogen activator in porcine renal tubular cells: LLC-PK1

Plasminogen activators are highly selective proteases that activate the proenzyme plasminogen to the general protease, plasmin. We studied a porcine kidney cell line, originally isolated as a high producer of plasminogen activator, in which activities of cellular adenylate cyclase and cAMP-dependent protein kinase are increased in response to calcitonin. We found that salmon calcitonin, in the concentration range 0.03-300 nM, increased plasminogen activator production up to approximately 1,000-fold and concurrently inhibited cell multiplication; both of these effects were reversible. Human calcitonin was approximately 0.01 times as potent as salmon calcitonin, corresponding to potency differences observed in other biological systems. Plasminogen activator production was also stimulated by other agents that raise cellular cAMP levels such as cholera toxin, phosphodiesterase inhibitors, and vasopressin, but not to the same extent as by calcitonins. The rapidity and sensitivity of the plasminogen activator determination and other cellular responses may make it possible in the future to use this cell stain in a convenient bioassay for calcitonins and their analogues.     

Oxygen radicals alter the cell membrane potential in a renal cell line (LLC-PK1) with differentiated characteristics of proximal tubular cells

We employed a carbocyanine dye (1,1',3,3,3',3'-hexamethylindocarbocyanine iodide) to measure the plasma membrane potential of LLC-PK1 renal epithelial cells exposed to either xanthine oxidase-generated oxygen radicals or to hydrogen peroxide. Measurements were performed using a fluorescent-activated cell sorter to record fluorescence on a cell by cell basis. Initial exposure of cells to low concentrations of either H2O2 or xanthine oxidase resulted in a transient increase in membrane potential relative to control cells (P less than 0.001), followed by an exponential decline in potential (P less than 0.001). The addition of extracellular catalase diminished the H2O2-related decline in potential, consistent with a role for hydrogen peroxide in producing this effect. Pretreatment of cells with inhibitors of intracellular catalase and superoxide dismutase prior to exposure to xanthine oxidase caused an even larger decline in potential (P less than 0.001). Cells could be partially protected from the radical-mediated loss of potential by incubating them in a hypertonic (400 mosmolal) environment during radical exposure. Similarly, the loss of membrane potential was increased after incubation of cells in a hypotonic (200 mosmolal) environment during radical exposure. These observations are consistent with a reduction in membrane potential effected by exposure to oxygen radicals (including superoxide anion and hydrogen peroxide). This reduction may be prevented, in part, by radical scavenging enzymes and by reducing the degree of cellular swelling in response to oxygen radical exposure.     

Gene expression analysis in LLC-PK1 renal tubular cells by atrial natriuretic peptide (ANP): correlation of homologous human genes with renal response

We used human DNA microarray to explore the differential gene expression profiling of atrial natriuretic peptide (ANP)-stimulated renal tubular epithelial kidney cells (LLC-PK1) in order to understand the biological effect of ANP on renal kidney cell's response. Gene expression profiling revealed 807 differentially expressed genes, consisting of 483 up-regulated and 324 down-regulated genes. The bioinformatics tool was used to gain a better understanding of differentially expressed genes in porcine genome homologous with human genome and to search the gene ontology and category classification, such as cellular component, molecular function and biological process. Four up-regulated genes of ATP1B1, H3F3A, ITGB1 and RHO that were typically validated by real-time quantitative PCR (RT-qPCR) analysis serve important roles in the alleviation of renal hypertrophy as well as other related effects. Therefore, the human array can be used for gene expression analysis in pig kidney cells and we believe that our findings of differentially expressed genes served as genetic markers and biological functions can lead to a better understanding of ANP action on the renal protective system and may be used for further therapeutic application.     

Apical and basolateral 4F2hc and the amino acid exchange of L-DOPA in renal LLC-PK1 cells

Summary. The present study aimed to examine the presence and define the role of 4F2hc, a glycoprotein associated with the LAT2 amino acid transporter, in L-DOPA handling by LLC-PK₁ cells. For this purpose we have measured the activity of the apical and basolateral inward and outward transport of [¹⁴C] L-DOPA in cell monolayers and examined the influence of 4F2hc antisense oligonucleotides on [¹⁴C] L-DOPA handling. The basal-to-apical transepithelial flux of [¹⁴C] L-DOPA progressively increased with incubation time and was similar to the apical-to-basal transepithelial flux. The spontaneous and the L-DOPA-stimulated apical fractional outflow of [¹⁴C] L-DOPA were identical to that through the basal cell side. The L-DOPA-induced fractional outflow of [¹⁴C] L-DOPA through the apical or basal cell side was accompanied by marked decreases in intracellular levels of [¹⁴C] L-DOPA. In cells treated with an antisense oligonucleotide complementary to 4F2hc mRNA for 72 h, [¹⁴C] L-DOPA inward transport and 4F2hc expression were markedly reduced. Treatment with the 4F2hc antisense oligonucleotide markedly decreased the spontaneous fractional outflow of [¹⁴C] L-DOPA through the apical or the basal cell side. It is likely that the Na⁺-independent and pH-sensitive uptake of L-DOPA include the hetero amino acid exchanger LAT2/4F2hc, which facilitates the trans-stimulation of L-DOPA and its outward transfer at both the apical and basal cell sides.     

Polarized distribution of the Na+/H+ exchange system in a renal cell line (LLC-PK1) with characteristics of proximal tubular cells

Studies of Na+ and H+ transport by confluent monolayers of the epithelial cell line LLC-PK1 were performed to verify the presence of a Na+/H+ exchange system. The presence of an outwardly directed H+ gradient produced a large stimulation of Na+ influx measured under net flux conditions. Amiloride (10(-3) M) completely inhibited Na+ influx stimulated by the H+ gradient and part of the Na+ influx measured in the absence of a pH gradient. Half-maximal inhibition of the Na+ influx stimulated by a pH gradient at 143 mM Na was observed at 5 microM amiloride. The presence of an inwardly oriented proton gradient also stimulated Na+ efflux from Na+-loaded cells. The stimulation was completely inhibited by the presence of 10(-3) M amiloride in the washout medium. These results indicate that this system could operate in the opposite direction depending on the orientation of the Na+ and H+ gradient. Incubation in Na+-free medium or in the presence of 10(-3) M ouabain resulted in a dramatic decrease of H+ release from LLC-PK1 cells. This H+ release was largely, although not completely, inhibited by 10(-4) M amiloride. Neither chloride substitution by the impermeable anion isethionate nor incubation in the presence of the ionophore valinomycin in high K+ medium affected Na+ influx by stimulated by a pH gradient. Inhibition of the Na+ influx by amiloride occurred only from the apical side of the monolayer. These results indicate that the Na+/H+ exchange system in LLC-PK1 monolayers is specifically localized in the apical membrane of the epithelial cells.     

Mechanisms of inflammatory injury of renal tubular cells in a cellular model of pyelonephritis

Previously, we have assembled a cellular model of pyelonephritis which contains a primary culture of renal tubular epithelial cells, mononuclear leukocytes, and bacterial lysate or lipopolysaccharide. After cocultivation of renal cells with leukocytes and bacterial lysate, proinflammatory changes were observed in the renal cells, followed by nitrosative and oxidative stress and cell death. The interaction of bacterial antigens not only with leukocytes, but also with epithelial cells of the renal tubules, was partially mediated by signaling pathways involving Toll-like receptors (TLR2 and TLR4). Activation of these receptors led to increased levels of oxidative stress and synthesis of proinflammatory cytokines (TNF, IL-6, IL-1α) in the renal epithelium, while TLR4 blockade decreased the severity of these processes. Apart from the fact that activation of inflammatory signaling in response to bacterial antigens is observed directly in the renal cells, the presence of leukocytes significantly amplifies the inflammatory response as measured by the level of cytokines generated in the ensemble. In the presence of activated leukocytes, higher expression of TLR2 on the surface of renal cells was observed in response to exposure to bacterial components, which might explain the increased inflammatory response in the presence of leukocytes. The synthesis of IL-1α in the epithelial cells of the renal tubules in this inflammatory model leads to its accumulation in the nuclei, which has been reduced by the TLR4 antagonist polymyxin. TLR2 agonists also led to increased levels of IL-1α. The elevation in the content of IL-1α in nuclei was accompanied by increased acetylation of nuclear proteins, which has been reduced to control values after exposure to protective agents (Trolox, mitochondria-targeted antioxidant SkQR1 or LiCl). The high level of acetylation of histones is probably regulated by proinflammatory cytokines, and to some extent it is a marker of inflammation, which can indirectly be reduced by protective agents.     

Phosphate uptake by kidney epithelial (LLC-PK1) cells

Phosphate uptake by the cultured kidney epithelial cell (LLC-PK1) was studied. The uptake was Na+ dependent, saturable with respect to phosphate and Na+, and energy dependent. The characteristics of the cell uptake system resembled the properties of phosphate transport in the kidney. Parathyroid hormone, dibutyryl cyclic AMP, and forskolin decreased Na+-dependent phosphate uptake. These agonists did not affect Na+-dependent alpha-methylglucoside uptake. Vasopressin and isoproterenol, which do not affect renal phosphate transport, did not inhibit phosphate uptake by the cell. These findings suggest that the cultured cell system may be a useful experimental model for studies of renal phosphate transport and its regulation.     

Localisation of Alanine uptake by cultured renal epithelial cells (LLC-PK1) to the basolateral membrane

Alanine uptake by LLC-PK¹ cells has previously been demonstrated to be almost exclusively sodium dependent. We here confirm that when the cells are grown on an impermeable substratum there is a marked fall in uptake as confluence is reached. By applying an autoradiographic technique to visualize transported alanine, it is clear, however, that even in subconfluent cultures there is marked cellular inhomogeneity with regard to uptake, which takes place predominantly in those cells at the periphery of growing islands and not those at the interior. In contrast, when cells are grown on permeable substrata, a uniform distribution of silver grains is found. In two other types of experiment, we found that when confluent cell monolayers on an impermeable support were treated briefly with a chelating agent or suspended by mechanical treatment, there was a marked increase per cell in sodium-dependent alanine uptake and in ouabain-sensitive potassium uptake. We conclude that the apparent fall in alanine uptake as cells reach confluence on an impermeable support is due to masking of transport sites, which are predominantly, if not exclusively, located at the basolateral membrane.     

LLC-PK1 epithelia as a model for in vitro assessment of proximal tubular nephrotoxicity

Summary LLC-PK₁ cells, an established epithelial cell line derived from pig kidney, were used as a model system for assessment of nephrotoxic side effects of three cephalosporin antibiotics: cephaloridine, ceftazidime, and cefotaxime. Toxic effects of these xenobiotics were monitored on confluent monolayers by light and electron microscopy and by the release of cellular marker enzyme activities into the culture medium. In addition, LLC-PK₁ cells were grown on microporous supports, and cephalosporin-induced alteration of epithelial functional integrity was monitored by a novel electrophysiologic approach. For this purpose, an Ussing chamberlike experimental setup was used. The dose-dependent effects on transepithelial ionic permselectivity were monitored under conditions in which defined fractions of the apical culture medium NaCl contents were replaced iso-osmotically by mannitol. This method of determining the functional intactness of the epithelial barrier by measuring dilution potentials was found to be far more sensitive than monitoring cell injury by means of morphology or measurement of enzyme release. As expected from animal experimental data, a dose-dependent disruption of monolayer integrity was detected with all three methodologies applied. Cephaloridine was found the most toxic compound followed by ceftazidime, where a 3-fold, and cefotaxime, where a 10-fold dose of that of cephaloridine was needed to produce cell injury. Measurement of transepithelial dilution potentials was more sensitive as compared to the release of the apical plasma membrane marker enzyme activities alkaline phosphatase andγ-glutamyltranspeptidase, the cytosolic lactate dehydrogenase, or the mitochondrial glutamate dehydrogenase. The data were compared to the effects of the aminoglycoside antibiotic gentamicin, which at least with respect to its effects on LLC-PK₁ morphology and enzyme release, but not transepithelial electrical properties, was already investigated.     

Regulation of mOAT-mediated organic anion transport by okadaic acid and protein kinase C in LLC-PK(1) cells

Organic anion transporters in the kidney proximal tubule play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites, thereby preventing their potentially toxic effects within the body. We have previously cloned a cDNA encoding an organic anion transporter from mouse kidney (mOAT) (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478; Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519-1524). In the present study, we assessed the potential for regulation of this transporter by heterologous expression of mOAT in the pig proximal tubule-like cell line, LLC-PK(1). We report here that both protein phosphatase (PP1/PP2A) inhibitor, okadaic acid, and protein kinase C (PKC) activators down-regulate mOAT-mediated transport of para-aminohippuric acid (PAH), a prototypic organic anion, in a time- and concentrationdependent manner. However their mechanisms of action for this down-regulation are distinct. Okadaic acid modulated PAH transport, at least in part, through phosphorylation/dephosphorylation of mOAT; phosphoamino acid analysis indicated this phosphorylation occurs on serine. In contrast, PKC activation induced a decrease in the maximum transport velocity (V(max)) of PAH transport without direct phosphorylation of the transporter protein. Together these results provide the first demonstration that regulation of organic anion transport by mOAT is likely to be tightly controlled directly and indirectly by phosphatase PP1/PP2A and PKC. Our results also suggest that kinases other than PKC are involved in this process.     

Parallel changes in amino acid transport and protein kinase C localization in LLC-PK1 cells treated with TPA or diradylglycerols

Protein kinase C is considered to be a major target for tumor promoting phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA). We have analyzed the correlation between A-system amino acid transport and the distribution of protein kinase C (PKC) between a membrane-rich fraction (100,000 g pellet) and cytosol (supernatant) from homogenized LLC-PK1 cells, a pig kidney epithelial cell line grown in culture. During log growth 1 day after seeding the cells onto culture plates, PKC activity is high in the membrane fraction and low in the cytosol. As the cells become confluent the PKC distribution shifts to a cytosolic pool. Concomitantly, A-system amino acid transport, as measured by methylaminoisobutyric acid [14C]MeAIB uptake, decreases. TPA (0.01-1.0 microM) induces a shift of PKC activity from the cytosol back to the membrane-rich fraction in post-confluent cells with a concomitant 2-3 fold stimulation of MeAIB uptake. The same responses can be achieved by treating cells with certain diradylglycerols, either diacylglycerols such as 1-oleyl-2-acetyl-sn-glycerol (OAG) or alkylacylglycerols such as 1-hexadecenyl-2-oleyl-sn-glycerol. Both responses to TPA are blocked by cytochalasin B, but cycloheximide inhibits the transport response without affecting PKC redistribution. It is suggested that the redistribution may be a necessary but not sufficient concomitant to the transport activation.     

Apolipoprotein E3 (apoE3) safeguards pig proximal tubular LLC-PK1 cells against reduction in SGLT1 activity induced by gentamicin C

Megalin, a family of endocytic receptors related to the low-density lipoprotein (LDL) receptor, is a major pathway for proximal tubular aminoglycoside accumulation. We previously reported that aminoglycoside antibiotics reduce SGLT1-dependent glucose transport in pig proximal tubular epithelial LLC-PK1 cells in parallel with the order of their nephrotoxicity. In this study, using a model of gentamicin C (GMC)-induced reduction in SGLT1 activity, we examined whether ligands for megalin protect LLC-PK1 cells from the GMC-induced reduction in SGLT1 activity. We employed apolipoprotein E3 (apoE3) and lactoferrin as ligands for megalin. Then the cells were treated with various concentrations of apoE3, lactoferrin and bovine serum albumin with or without 100 microg/ml of GMC, and the SGLT1-dependent methyl alpha-D-glucopyranoside (AMG) uptake and levels of SGLT1 expression were determined. As a result, we demonstrated that the apoE3 significantly protects these cells from GMC-induced reduction in AMG uptake, but neither lactoferrin nor albumin does. In accord with a rise in AMG uptake activity, the mRNA and protein levels of SGLT1 were apparently up-regulated in the presence of apoE3. Furthermore, we found that the uptake of [3H] gentamicin is decreased by apoE3, and that apoE3 showed obvious protection against the GMC-dependent N-acetyl-beta-D-glucosamidase (NAG) release from LLC-PK1 cells. Thus, these results indicate that apoE3 could be a valuable tool for the prevention of aminoglycoside nephrotoxicity.     

Effect of aristolochic acid on intracellular calcium concentration and its links with apoptosis in renal tubular cells

Aristolochic acid (AA) has been demonstrated to play a causal role in Chinese herbs nephropathy. However, the detailed mechanism for AA to induce apoptosis of renal tubular cells remains obscure. In this study, we show that AA evokes a rapid rise in the intracellular Ca²⁺ concentration of renal tubular cells through release of intracellular endoplasmic reticulum Ca²⁺ stores and influx of extracellular Ca²⁺, which in turn causes endoplasmic reticulum stress and mitochondria stress, resulting in activation of caspases and finally apoptosis. Ca²⁺ antagonists, including calbindin-D_28k (an intracellular Ca²⁺ buffering protein) and BAPTA-AM (a cell-permeable Ca²⁺ chelator), are capable of ameliorating endoplasmic reticulum stress and mitochondria stress, and thereby enhance the resistance of the cells to AA. Moreover, we show that overexpression of the anti-apoptotic protein Bcl-2 in combination with BAPTA-AM treatment can provide renal tubular cells with almost full protection against AA-induced cytotoxicity. In conclusion, our results demonstrate an impact of AA to intracellular Ca²⁺ concentration and its link with AA-induced cytotoxicity. Yi-Hong Hsin and Chi-Hung Cheng are equally contributed to this work.     

Regulation of the Na(+)-dependent and the Na(+)-independent polyamine transporters in renal epithelial cells (LLC-PK1)

We have studied the regulation of the Na(+)-dependent and Na(+)-independent polyamine transport pathways in the renal LLC-PK1 cell line. Most of the experiments were performed in the presence of 5 mM DL-2-difluoromethylornithine (DFMO) in order to inhibit the cellular synthesis of polyamines. The activity of both transporters as measured by putrescine uptake was increased by growth-promoting stimuli and decreased by exogenous polyamines. The time course of the increase in uptake activity induced by fetal calf serum could be fitted by a single exponential, and the process was three times faster for the Na(+)-dependent than for the Na(+)-independent transporter. Maximum activity was reached after more than 24 h. This increase could be inhibited by actinomycin D and by cycloheximide. Other growth-promoting stimuli, such as subconfluent cell density, as well as growth factors also induced an increase in the transport activity. Particularly, there was a marked stimulation of the Na(+)-dependent pathway by epidermal growth factor in combination with insulin. On the other hand, the transport activity decayed very rapidly upon addition of exogenous polyamines (t1/2 less than 60 min). The diamine putrescine was much less effective in this respect than the polyamines spermidine and spermine. The non-metabolizable substrate methylglyoxal bis(guanylhydrazone) did not induce a decay of the transport activity, but it protected the Na(+)-dependent pathway against the polyamine-induced decay. Inhibition of the protein synthesis by cycloheximide did not induce a rapid decrease of the transport activity; neither did it affect the polyamine-induced decay. These observations suggest that this polyamine-induced decay is not owing to an inhibitory effect on the rate of synthesis of the transporters, but rather to a degradation or an inactivation of the transporters. The polyamine-induced decay slowed down at lower cell density. This effect was particularly pronounced for the Na(+)-dependent transporter. Since the uptake of polyamines was increased at low cell density, the decreased rate of decay in this condition pleads against a simple mechanism of transinhibition by the substrate. In conclusion, both transport pathways were similarly affected by the regulatory parameters, but the Na(+)-dependent transporter was more rapidly and more effectively regulated. The numerous interacting regulatory steps furthermore suggest a physiological role for these transporters, such as an involvement in urinary polyamine disposal.     

Mitochondrial cytochrome c oxidase as a target site for cephalosporin antibiotics in renal epithelial cells (LLC-PK(1)) and renal cortex

We reported previously that treatment of the pig kidney proximal tubular epithelial cell line LLC-PK(1) with cephaloridine (CLD) decreased the activity of cytochrome c oxidase in the mitochondria of the cells followed by increases in lipid peroxidation and cell necrosis. In this study, we investigated the effects of CLD on the activity of cytochrome c oxidase in mitochondria isolated from LLC-PK(1) cells and purified the enzyme from mitochondria of the rat renal cortex. The activity of cytochrome c oxidase in the isolated mitochondria from LLC-PK(1) cells was significantly decreased from 1 h after addition of 1 mM CLD. Other cephalosporin antibiotics, cefazolin and cefalotin, also decreased the activity of cytochrome c oxidase in the isolated mitochondria. The activity of cytochrome c oxidase purified from the mitochondria of the rat renal cortex was also decreased from 2 h after addition of 1 mM CLD in a non-competitive manner. These results suggest that the direct inhibition of cytochrome c oxidase activity in the mitochondrial electron transport chain by cephlosporins may result from the observed nephrotoxicity.