Elevated glucose concentration in culture media decreases membrane trafficking of SGLT2 in LLC-PK1 cells via a cAMP/PKA-dependent pathway.

Na+-dependent glucose reabsorption in the renal proximal tubule is dynamically regulated by changes in blood glucose levels. There is, however, a disparity in reports studying the relationship between hyperglycemia and Na+-glucose-linked transporter (SGLT) function and expression. Similarly, manipulation of the glucose content in growth media of cultured renal cells has been shown to influence SGLT activity. In this investigation, SGLT activity was significantly lower in proximal tubule LLC-PK1 cells cultured in medium containing 17.5 than 5 mM glucose. α-Methyl d-glucopyranoside (AMG) transport kinetics showed reduced apparent Vmax and Km in cells grown in 17.5 mM glucose. SGLT2 was identified as the isoform responsible for glucose transport, and protein expression analyses showed decreased apical membrane localization of SGLT2 in cells grown in 17.5 mM glucose, explaining the reduced activity. Multiple signaling pathways have been implicated in regulation of SGLT activity and trafficking. Elevated media glucose decreased intracellular cAMP and PKA activation, leading to decreased SGLT2 trafficking into the plasma membrane, which was reversed after treatment with 1 µM forskolin. The effects of media glucose on SGLT activity were found to be dependent on p38 MAPK activation due to PKA-mediated signaling. Glucose-modulated AMG uptake is reversible and was associated with altered SGLT2 membrane trafficking and cAMP alterations. In summary, elevated glucose concentrations in culture medium decrease SGLT activity in LLC-PK1 cells by reducing membrane trafficking of SGLT2 via decreasing intracellular cAMP, resulting in a lowered PKA-dependent phosphorylation of p38 MAPK.

Blockade of the N-Methyl-D-Aspartate Glutamate Receptor Ameliorates Lipopolysaccharide-Induced Renal Insufficiency.

N-methyl-D-aspartate (NMDA) receptor activation in rat kidney reduces renal perfusion and ultrafiltration. Hypoperfusion-induced ischemia is the most frequent cause of functional insufficiency in the endotoxemic kidney. Here, we used non-hypotensive rat model of lipopolysaccharide-induced endotoxemia to examine whether NMDA receptor hyperfunction contributes to acute kidney injury. Lipopolysaccharide-induced renal damage via increased enzymuria and hemodynamic impairments were ameliorated by co-treatment with the NMDA receptor blocker, MK-801. The NMDA receptor NR1 subunit in the rat kidney mainly co-localized with serine racemase, an enzyme responsible for synthesizing the NMDA receptor co-agonist, D-serine. The NMDA receptor hyperfunction in lipopolysaccharide-treated kidneys was demonstrated by NR1 and serine racemase upregulation, particularly in renal tubules, and by increased D-serine levels. Lipopolysaccharide also induced cell damage in cultured tubular cell lines and primary rat proximal tubular cells. This damage was mitigated by MK-801 and by small interfering RNA targeting NR1. Lipopolysaccharide increased cytokine release in tubular cell lines via toll-like receptor 4. The release of interleukin-1ß from these cells are the most abundant. An interleukin-1 receptor antagonist not only attenuated cell death but also abolished lipopolysaccharide-induced NR1 and serine racemase upregulation and increases in D-serine secretion, suggesting that interleukin-1ß-mediated NMDA receptor hyperfunction participates in lipopolysaccharide-induced tubular damage. The results of this study indicate NMDA receptor hyperfunction via cytokine effect participates in lipopolysaccharide-induced renal insufficiency. Blockade of NMDA receptors may represent a promising therapeutic strategy for the treatment of sepsis-associated renal failure.

Design of Fexofenadine Prodrugs Based on Tissue-Specific Esterase Activity and Their Dissimilar Recognition by P-Glycoprotein.

The aim of this study was to develop a suitable prodrug for fexofenadine (FXD), a model parent drug, that is resistant to intestinal esterase but converted to FXD by hepatic esterase. Carboxylesterases (CESs), human carboxylesterase 1 (hCE1) and human carboxylesterase 2 (hCE2), are the major esterases in human liver and intestine, respectively. These two CESs show quite different substrate specificities, and especially, hCE2 poorly hydrolyzes prodrugs with large acyl groups. FXD contains a carboxyl group and is poorly absorbed because of low membrane permeability and efflux by P-glycoprotein (P-gp). Therefore, two potential FXD prodrugs, ethyl-FXD and 2-hydroxyethyl-FXD, were synthesized by substitution of the carboxyl group in FXD. Both derivatives were resistant to intestinal hydrolysis, indicating their absorption as intact prodrugs. Ethyl-FXD was hydrolyzed by hepatic hCE1, but 2-hydroxyethyl-FXD was not. Both derivatives showed high membrane permeability in human P-gp-negative LLC-PK1 cells. In LLC-GA5-COL300 cells overexpressing human P-gp, ethyl-FXD was transported by P-gp, but its efflux was easily saturated. Whereas 2-hydroxyethyl-FXD showed more efficient P-gp-mediated transport than FXD. Although the structure of 2-hydroxyethyl-FXD only differs from ethyl-FXD by substitution of a hydroxyl group, 2-hydroxyethyl-FXD is unsuitable as a prodrug. However, ethyl-FXD is a good candidate prodrug because of good intestinal absorption and hepatic conversion by hCE1.

Disruption of renal tubular mitochondrial quality control by Myo-inositol oxygenase in diabetic kidney disease.

Diabetic kidney disease (DKD) is associated with oxidative stress and mitochondrial injury. Myo-inositol oxygenase (MIOX), a tubular-specific enzyme, modulates redox imbalance and apoptosis in tubular cells in diabetes, but these mechanisms remain unclear. We investigated the role of MIOX in perturbation of mitochondrial quality control, including mitochondrial dynamics and autophagy/mitophagy, under high-glucose (HG) ambience or a diabetic state. HK-2 or LLC-PK1 cells subjected to HG exhibited an upregulation of MIOX accompanied by mitochondrial fragmentation and depolarization, inhibition of autophagy/mitophagy, and altered expression of mitochondrial dynamic and mitophagic proteins. Furthermore, dysfunctional mitochondria accumulated in the cytoplasm, which coincided with increased reactive oxygen species generation, Bax activation, cytochrome C release, and apoptosis. Overexpression of MIOX in LLC-PK1 cells enhanced the effects of HG, whereas MIOX siRNA or d-glucarate, an inhibitor of MIOX, partially reversed these perturbations. Moreover, decreasing the expression of MIOX under HG ambience increased PTEN-induced putative kinase 1 expression and the dependent mitofusin-2-Parkin interaction. In tubules of diabetic mice, increased MIOX expression and mitochondrial fragmentation and defective autophagy were observed. Dietary supplementation of d-glucarate in diabetic mice decreased MIOX expression, attenuated tubular damage, and improved renal functions. Notably, d-glucarate administration also partially attenuated mitochondrial fragmentation, oxidative stress, and apoptosis and restored autophagy/mitophagy in the tubular cells of these mice. These results suggest a novel mechanism linking MIOX to impaired mitochondrial quality control during tubular injury in the pathogenesis of DKD and suggest d-glucarate as a potential therapeutic agent for the amelioration of DKD.

Effect of Danofloxacin on Reactive Oxygen Species Production, Lipid Peroxidation and Antioxidant Enzyme Activities in Kidney Tubular Epithelial Cell Line, LLC-PK1.

The purpose of this study was to investigate the possibility that oxidative stress was involved in danofloxacin-induced toxicity in renal tubular cells epithelial cell line (LLC-PK1). Confluent LLC-PK1 cells were incubated with various concentrations of danofloxacin. The extent of oxidative damage was assessed by measuring the reactive oxygen species (ROS) level, lipid peroxidation, cell apoptosis and antioxidative enzyme activities. Danofloxacin induced a concentration-dependent increase in the ROS production, not even cytotoxic conditions. Similarly, danofloxacin caused an about 4 times increase in the level of thiobarbituric acid reactive substances at the concentration of 400 µM for 24 hr, but it did not induce cytotoxicity and apoptosis. Antioxidant enzymes activities, such as superoxide dismutase (SOD) and catalase (CAT), were increased after treatment with 100, 200 and 400 µM of danofloxacin for 24 hr. The activity of glutathione peroxidase (GPX) was significantly decreased in a concentration-dependent manner. In addition, ROS production, lipid peroxidation and GPX decline were inhibited by additional glutathione and N-acetyl cysteine. These data suggested that danofloxacin could not induce oxidative stress in LLC-PK1 cells at the concentration (≤400 µM) for 24 hr. The increase levels of ROS and lipid peroxidation could be partly abated by the increase activities of SOD and CAT.

Role of Dlg5/lp-dlg, a membrane-associated guanylate kinase family protein, in epithelial-mesenchymal transition in LLc-PK1 renal epithelial cells.

Discs large homolog 5 (Dlg5) is a member of the membrane-associated guanylate kinase adaptor family of proteins, some of which are involved in the regulation of epithelial-to-mesenchymal transition (EMT). Dlg5 has been described as a susceptibility gene for Crohn's disease; however, the physiological function of Dlg5 is unknown. We show here that transforming growth factor-ß (TGF-ß)-induced EMT suppresses Dlg5 expression in LLc-PK1 cells. Depletion of Dlg5 expression by knockdown promoted the expression of the mesenchymal marker proteins, fibronectin and α-smooth muscle actin, and suppressed the expression of E-cadherin. In addition, activation of JNK and p38, which are stimulated by TGF-ß, was enhanced by Dlg5 depletion. Furthermore, inhibition of the TGF-ß receptor suppressed the effects of Dlg5 depletion. These observations suggest that Dlg5 is involved in the regulation of TGF-ßreceptor-dependent signals and EMT.

[Establishment of BCRP expressed pig kidney cell line LLC-PK1/BCRP and its biological profile].

To establish a pig kidney cell line LLC-PK1/BCRP in which human breast cancer resistance protein was highly expressed, the expression vector pcDNA3.1(+)-BCRP which contained BCRP gene was constructed and transfected into LLC-PKI cells via liposomes. After selecting with G418, population doubling time, flow cytometry and Western blotting analysis were used to evaluate the cell line. MTT assays were employed to determine the drug resistance index of mitoxantrone and doxorubicin. Invert fluorescent microscope was used to observe the efflux of fluorescence dye Hoechst 33342 by BCRP, furthermore, the BCRP's inhibitor GF120918 was applied to reverse the efflux of Hoechst 33342. The experiment results showed that the expression of BCRP protein increased in LLC-PK1/BCRP cell. The population doubling time of LLC-PK1/BCRP cell was a little longer than that of the parental cell LLC-PK1. The resistance indexes to mitoxantrone and doxorubicin were 51.95 and 6.09 times, respectively, higher than LLC-PK1 cell. The efflux of Hoechst 33342 was significantly enhanced and could be reversed by GF120918. So a LLC-PK1/BCRP cell line was established, which highly expressed BCRP protein successfully. This cell line could be a valuable model to further investigate the biological profile of BCRP and select the substrate and inhibitor of BCRP.

In vitro transport profile of carbamazepine, oxcarbazepine, eslicarbazepine acetate, and their active metabolites by human P-glycoprotein.

PURPOSE: Antiepileptic drugs (AEDs) are widely used not only in the treatment of epilepsy but also as treatments for psychiatric disorders. Pharmacoresistance of AEDs in the treatment of epilepsy and psychiatric disorders is a serious problem. Transport of antiepileptic drugs by P-glycoprotein (Pgp, ABCB1, or MDR1), which is overexpressed in the blood-brain barrier, may be a mechanism for resistance of AEDs. For most AEDs, conflicting evidence precludes consensus on whether they are substrates of Pgp. The objective of this study was to evaluate whether analogs and metabolites of the AED carbamazepine are substrates of human Pgp. METHODS: Polarized cell lines MDCKII and LLC transfected with the human MDR1 gene were used in the bidirectional transport assay and concentration equilibrium transport assay. The expression of Pgp was detected by real-time polymerase chain reaction (PCR) and immunofluorescent staining. Rhodamine-123 uptake was also determined. KEY FINDINGS: Pgp did not transport carbamazepine, but it did transport its active metabolite carbamazepine-10,11-epoxide. Pgp also pumped eslicarbazepine acetate and oxcarbazepine, as well as their active metabolite (S)-licarbazepine. Transport of the drugs was in the order of ESL>OXC>S-LC>CBZ-E in concentration equilibrium conditions. The transport of these drugs was blocked by Pgp inhibitors tariquidar and verapamil. SIGNIFICANCE: All carbamazepine analogs or metabolites tested are Pgp substrates, except for carbamazepine. These data suggest that resistance to carbamazepine, oxcarbazepine, or eslicarbazepine acetate may be attributed to increased efflux function of Pgp because they or their active metabolites are Pgp substrates.

Protective effect of sulforaphane against cisplatin-induced mitochondrial alterations and impairment in the activity of NAD(P)H: quinone oxidoreductase 1 and γ glutamyl cysteine ligase: studies in mitochondria isolated from rat kidney and in LLC-PK1 cells.

This work was designed to further study the mechanism by which sulforaphane (SFN) exerts a renoprotective effect against cisplatin (CIS)-induced damage. It was evaluated whether SFN attenuates the CIS-induced mitochondrial alterations and the impairment in the activity of the cytoprotective enzymes NAD(P)H: quinone oxidoreductase 1 (NQO1) and γ glutamyl cysteine ligase (γGCL). Studies were performed in renal epithelial LLC-PK1 cells and in isolated renal mitochondria from CIS, SFN or CIS+SFN treated rats. SFN effectively prevented the CIS-induced increase in reactive oxygen species (ROS) production and the decrease in NQO1 and γGCL activities and in glutathione (GSH) content. The protective effect of SFN on ROS production and cell viability was prevented by buthionine sulfoximine (BSO), an inhibitor of γGCL, and by dicoumarol, an inhibitor of NQO1. SFN was also able to prevent the CIS-induced mitochondrial alterations both in LLC-PK1 cells (loss of membrane potential) and in isolated mitochondria (inhibition of mitochondrial calcium uptake, release of cytochrome c, and decrease in GSH content, aconitase activity, adenosine triphosphate (ATP) content and oxygen consumption). It is concluded that the protection exerted by SFN on mitochondrial alterations and NQO1 and γGCL enzymes may be involved in the renoprotection of SFN against CIS.

The protection of selenium on ROS mediated-apoptosis by mitochondria dysfunction in cadmium-induced LLC-PK(1) cells.

Selenium, an essential trace element, showed the significant protective effects against liver and kidney damage induced by some heavy metals. However, the mechanism how selenium suppresses cadmium (Cd)-induced cytotoxicity remains unclear. In this study, we investigated the protective mechanism of selenium on Cd-induced apoptosis in LLC-PK(1) cells via reactive oxygen species (ROS) and mitochondria linked signal pathway. Studies of PI and Annexin V dual staining analysis demonstrated that 20 microM Cd-induced apoptosis as early as 18 h. A concomitant by the generation of ROS, the loss of mitochondrial membrane potential, cytochrome c (cyt c) release, activation of caspase-9, -3 and regulation of Bcl-2 and Bax were observed. N-acetylcysteine (NAC, 500 microM), a free radical scavenger, was used to determine the involvement of ROS in Cd-induced apoptosis. During the process, selenium played the same role as NAC. The anti-apoptosis exerted by selenium involved the blocking of Cd-induced ROS generation, the inhibition of Cd-induced mitochondrial membrane potential collapse, the prevention of cyt c release, subsequent inhibition of caspase activation and the changed level of Bcl-2 and Bax. Taken together, we concluded that Cd-induced apoptosis was mediated by oxidative stress and selenium produced a significant protection against Cd-induced apoptosis in LLC-PK(1) via ameliorating the mitochondrial dysfunction.

Lignans from the fruits of Forsythia suspensa.

Bioactivity-guided fractionation of the methanol extract of the fruits of Forsythia suspensa Vahl has led to the isolation of two new monoepoxylignans, forsythialan A (1) and B (2), together with a known tetrahydrofurofuran lignan, phillyrin (3). The structures of compounds 1 and 2 were elucidated by spectroscopic methods. The antioxidant activities of these lignans have been assessed by evaluating their protective effects against peroxynitrite-induced oxidative stress. The compounds 1 and 2 showed protective effects against renal epithelial cell injury by 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator. The relatively stronger antioxidant activities of compounds 1 and 2 may be associated with the presence of aromatic hydroxy function.

Ezrin promotes functional expression and parathyroid hormone-mediated regulation of the sodium-phosphate cotransporter 2a in LLC-PK1 cells.

The sodium-phosphate cotransporter 2a (NPT2a) is the principal phosphate transporter expressed in the brush border of renal proximal tubules and is downregulated by parathyroid hormone (PTH) through an endocytic mechanism. Apical membrane expression of NPT2a is dependent on interactions with the sodium-hydrogen exchanger regulatory factor 1 (NHERF-1). An LLC-PK1 renal cell line stably expressing the PTH receptor (PTH1R) and NHERF-1, termed B28-N1, fails to functionally express NPT2a. In B28-N1 cells, NHERF-1 and NPT2a are inappropriately localized to the cytoplasm. Ezrin, in the activated state, is capable at linking NHERF-1-assembled complexes to the actin cytoskeleton. Early-passage LLC-PK1 cells stably transfected with either empty vector or wild-type ezrin express a comparable level of the active, T567 phosphorylated form of ezrin and are capable of functionally expressing NPT2a. Colocalization of the PTH1R, NPT2a, and ezrin exists and is prominently associated with actin-containing microvilli in apical domains of these cells. Upon PTH treatment, the PTH1R, NPT2a, NHERF-1, and ezrin colocalize to endocytic vesicles and NPT2a-dependent phosphate uptake is markedly inhibited. LLC-PK1 cells expressing the constitutively active ezrin (T567D) display enhanced NPT2a functional expression and PTH-mediated regulation of phosphate. Expression of a dominant-negative ezrin, consisting of the NH(2)-terminal half of the protein, markedly disrupts NPT2a-dependent phosphate uptake. PTH does not appear to alter ezrin phosphorylation at T567. Instead, PTH perhaps initiates NPT2a endocytosis by inducing reorganization of the actin-containing microvilli in a process that is blocked by the actin-stabilizing compound jasplakinolide.

Inhibition of P-glycoprotein-mediated transport by terpenoids contained in herbal medicines and natural products.

Terpenoids form a large and structurally diverse family of natural products and are ingredients of various herbal medicines. We have investigated possible interactions between herbal medicines and conventional medicines, and recently reported that monoterpenoids contained in Zanthoxyli Fructus can be potent inhibitors of P-glycoprotein (P-gp). In the present study, the influence of 70 kinds of terpenoids present in natural products on P-gp-mediated efflux transport was investigated. LLC-GA5-COL150 cells transfected with human MDR1 cDNA encoding P-gp were used to screen the terpenoids. Large increases in the intracellular accumulation of [(3)H]digoxin were observed in the presence of (R)-(+)-citronellal, (S)-(-)-beta-citronellol, alpha-terpinene, terpinolene, (-)-beta-pinene, abietic acid, ophiobolin A, cucurbitacin I, and glycyrrhetic acid. A study of the concentration-dependency revealed that the IC(50) of ophiobolin A, glycyrrhetic acid, (R)-(+)-citronellal, abietic acid, and cucurbitacin I was smaller than that of verapamil. The transcellular transport of [(3)H]digoxin across Caco-2 cell monolayers was then examined in the presence of (R)-(+)-citronellal, abietic acid, and glycyrrhetic acid. Significant increases in the apical-to-basolateral transport and decreases in the basolateral-to-apical transport and efflux ratio were demonstrated. These findings suggest that some natural products containing these terpenoids may inhibit P-gp-mediated transport and interact with P-gp substrates in the intestinal absorption process.

Cyclosporin A stimulates apical Na+/H+ exchange in LLC-PK1/PKE20 proximal tubular cells.

Cyclosporin A (CyA) causes renal Na(+) retention which may lead to arterial hypertension. The apical Na(+)/H(+) exchanger (NHE3) is responsible for bulk proximal tubular Na(+) reabsorption. The aim of this study was to investigate the effects of CyA on the NHE3 of polarized proximal tubular cells to evaluate cellular mechanisms of CyA-associated arterial hypertension. The change of the intracellular pH (Delta-[pH](i)/min) was determined as a measure of the activity of the NHE in LLC-PK(1)/PKE(20) cells using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). The NHE activity was identified as the apical NHE3 since it could be inhibited by the inhibitor S3226, but not by inhibitors of the basolateral isoform (NHE1) amiloride or HOE 694. CyA stimulated the NHE3 activity dose dependently. The mean increase stimulated by relevant CyA concentrations was 61+/-11%. A 24-h application of CyA also stimulated an increase of NHE3 activity which did not seem to be mediated by an increase of NHE3 RNA expression. The less immunosuppressive derivatives cyclosporin H and cyclosporin G caused NHE3 activation as well. Carbachol and ATP, which both induce a Ca(2+) release from internal Ca(2+) stores, also increased the NHE3 activity. The Ca(2+) chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,-N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) abolished the CyA-associated NHE3 stimulation, whereas low extracellular Ca(2+) had no effect. CyA-associated effects did not seem to be mediated via inhibition of protein kinase C (PKC). CyA had no additive effects on the angiotensin II-associated NHE3 stimulation. Concurrent application of losartan did not impair the CyA-induced NHE3 stimulation. In conclusion CyA stimulates the apical NHE3 in proximal tubular cells. This is mediated by Ca(2+) release from intracellular stores but is independent of the action of angiotensin II or PKC.

Aristolochic acid I-induced DNA damage and cell cycle arrest in renal tubular epithelial cells in vitro.

DNA damage is a critical event preceding cellular apoptosis or necrosis. This study was carried out to investigate the effect of aristolochic acid I (AAI) on DNA damage and cell cycle in porcine proximal tubular epithelial cell lines (LLC-PK1 cells). LLC-PK1 cells were stimulated with AAI at the concentrations of 80, 320, and 1,280 ng/ml for 24 h. DNA damage was examined by comet assay and the cell cycle was assayed by flow cytometry (FCM), cellular apoptosis and lysis were examined simultaneously. Cellular nuclear changes were observed by electron microscopy and the expression of wild-type p53 protein and mRNA were measured by FCM and RT-PCR. We found that AAI-induced DNA damage prior to apoptosis and lysis in LLC-PK1 cells in a dose-dependent manner (P<0.01). The percentage of cells in the G2/M phase that were treated with AAI (320 and 1,280 ng/ml) for 24 h increased significantly (P<0.01). Electron micrographs showed the nuclear abnormalities in AAI-treated cells. The expression of p53 protein and mRNA did not change in the AAI-treated cells. AAI may cause DNA damage and cell cycle arrest in LLC-PK1 cells through a wild-type p53-independent pathway, prior to apoptosis or necrosis. This study on the molecular mechanism of AAI-induced toxicity may explain why tubular epithelial cells present limited proliferation and regeneration abilities in the clinical presentation of AAI-associated nephrotoxicity.

Development and characterization of LLC-PK1 cells containing Sprague-Dawley rat Abcb1a (Mdr1a): comparison of rat P-glycoprotein transport to human and mouse.

INTRODUCTION: P-glycoprotein is localized in numerous tissues throughout the body and plays an important role in the disposition of many xenobiotics. The contribution of P-glycoprotein-mediated drug transport is being evaluated in early drug discovery stages, particularly for compounds targeted to the central nervous system, using in vitro tools including cell lines expressing P-glycoprotein. Previous work in our laboratory suggests there are species differences in P-glycoprotein transport activity between humans and animals. The rat Abcb1a form of P-glycoprotein (formerly known as Mdr1a), the predominate isoform in the brain, has not been described in a functional cell system. Here, we describe the development and characterization of LLC-PK1 cells expressing rat Abcb1. METHODS: We cloned rat Abcb1a and generated a stable LLC-PK1 cell line. Expression and function of the cells were evaluated by immunoblot analysis, cytotoxicity analysis, cellular accumulation assays, and transcellular transport of probe substrates. The transport ratios of structurally diverse compounds obtained from parental cells or cells stably transfected with human ABCB1, mouse Abcb1a or rat Abcb1a were compared. RESULTS: Two forms of rat Abcb1a were cloned from Sprague-Dawley cDNA that differ by six amino acids and a base pair deletion. The intact form was stably transfected in LLC-PK1 cells. Immunoblot analysis demonstrated expression of the protein. The cells demonstrated P-glycoprotein-mediated function by directional transport of dexamethasone, ritonavir, and vinblastine in a transwell assay that was inhibited in the presence of cyclosporin A, verapamil, or quinidine. Likewise, the cells showed reduced cellular accumulation of Rh123 by FACS analysis that was reversed in the presence of cyclosporin A. These cells showed >or=350-fold resistance to colchicine, doxorubicin, vinblastine, and taxol and were sensitized in the presence of verapamil or cyclosporin A. Of 179 chemically diverse compounds evaluated, approximately 20% of the compounds evaluated were predicted to be substrates in one species but not in other species. DISCUSSION: Taken together, these data suggest these cells will be useful for evaluation of rat Abcb1a-mediated transport and for evaluation of species-specific P-glycoprotein-mediated transport.

Vectorial transport of unconjugated and conjugated bile salts by monolayers of LLC-PK1 cells doubly transfected with human NTCP and BSEP or with rat Ntcp and Bsep.

Na(+)-taurocholate-cotransporting peptide (NTCP)/SLC10A1 and bile salt export pump (BSEP)/ABCB11 synergistically play an important role in the transport of bile salts by the hepatocyte. In this study, we transfected human NTCP and BSEP or rat Ntcp and Bsep into LLC-PK1 cells, a cell line devoid of bile salts transporters. Transport by these cells was characterized with a focus on substrate specificity between rats and humans. The basal to apical flux of taurocholate across NTCP- and BSEP-expressing LLC-PK1 monolayers was 10 times higher than that in the opposite direction, whereas the flux across the monolayer of control and NTCP or BSEP single-expressing cells did not show any vectorial transport. The basal to apical flux of taurocholate was saturated with a K(m) value of 20 microM. Vectorial transcellular transport was also observed for cholate, chenodeoxycholate, ursodeoxycholate, their taurine and glycine conjugates, and taurodeoxycholate and glycodeoxycholate, whereas no transport of lithocholate was detected. To evaluate the respective functions of NTCP and BSEP and to compare them with those of rat Ntcp and Bsep, we calculated the clearance by each transporter in this system. A good correlation in the clearance of the examined bile salts (cholate, chenodeoxycholate, ursodeoxycholate, and their taurine or glycine conjugates) was observed between transport by human and that of rat transporters in terms of their rank order: for NTCP, taurine conjugates > glycine conjugates > unconjugated bile salts, and for BSEP, unconjugated bile salts and glycine conjugates > taurine conjugates. In conclusion, the substrate specificity of human and rat NTCP and BSEP appear to be very similar at least for monovalent bile salts under physiological conditions.

Interaction of N1,N12-diacetylspermine with polyamine transport systems of polarized porcine renal cell line LLC-PK1.

LLC-PK(1) cells grown on porous membrane filters were employed as a model system to explore the renal transport of polyamines. The polarity of LLC-PK(1) monolayers was confirmed by the exclusive appearance of a Na(+)-dependent alpha-methylglucoside transport system on the apical surface. The uptake of free polyamines from the basolateral side of monolayers was consistent with the existence of a single class of transport system, while the existence of two kinetically distinct polyamine transport systems with higher and lower affinities on apical membranes was suggested. The results of competition studies indicated that each of these transporters was able to interact with putrescine, spermidine and spermine. LLC-PK(1) cells incorporated monoacetylspermine from the apical surface of monolayers at about half the rate of spermine uptake. Monoacetylspermine inhibited spermidine uptake, indicating that free polyamine transport systems also recognized the monoacetylated derivative. In contrast, N(1),N(12)-diacetylspermine did not inhibit spermidine uptake, nor was it incorporated into the cells, indicating the absence of transport systems that recognize N(1),N(12)-diacetylspermine on the apical membranes of LLC-PK(1) cells. These results may be relevant as to our previous observation that the content of diacetylpolyamines in urine is relatively constant, and may explain the excellence of N(1),N(12)-diacetylspermine as a tumor marker.

Evaluation of proliferation and functional differentiation of LLC-PK1 cells on porous polymer membranes for the development of a bioartificial renal tubule device.

To develop a bioartificial renal tubule system using renal tubular cells and porous polymer membrane hollow fibers, long-term maintenance of a confluent monolayer and the functionally differentiated condition of cells is essential. We examined the proliferation and functional differentiation of LLC-PK1 (Lewis-lung cancer porcine kidney 1) cells on two types of membranes: polysulfone and cellulose acetate. Cell proliferation was significantly higher on the polysulfone membrane than on the cellulose acetate membrane, and was enhanced by coating the membranes with various extracellular matrices. Confluent monolayer formation of cells was observed on matrix-coated polysulfone membrane but not on matrix-coated cellulose acetate membrane within 1 week. Cell proliferation continued for 3 weeks after confluent monolayer formation. Messenger RNA (mRNA) expression of glucose transporters, indicators of the functional differentiation of the LLC-PK1 cells, was observed in the polysulfone and cellulose acetate membrane groups, but was not observed in the nonporous polystyrene plate group under subconfluent conditions. Expression of glucose transporters mRNA was maintained for 3 weeks after confluent monolayer formation. Polysulfone membrane is more suitable than cellulose acetate membrane for a bioartificial renal tubule system with regard to LLC-PK1 cell proliferation. Extracellular matrix coating of the membrane further improves cell proliferation.

O papel da heme oxigenase-1 na lesão celular desencadeada pelo sulfato de polimixina B em células LLC-PK1 / The role of heme oxygenase-1 in the cellular injury caused by polymyxin B sulphate on LLC-PK1 cells

A heme-oxigenase-1 (HO-1) é uma enzima induzível envolvida na degradação do grupo prostético heme, produzindo compostos com funções anti-oxidante, anti-inflamatória, anti-apoptótica e modulatória do sistema imune no rim. A importância de sua indução está associada à resposta adaptativa ao estresse oxidativo e à inflamação envolvidos na gênese da insuficiência renal aguda. O sulfato de polimixina B é um antibiótico usado no tratamento de infecções Gram-negativas e que apresenta um efeito nefrotóxico ainda não completamente elucidado. O objetivo deste estudo foi verificar a viabilidade e apoptose de células LLC-PK1 submetidas ao tratamento com polimixina B, com tempos de exposição diferentes, e pré-tratadas com hemin (indutor de heme oxigenase-1) ou protoporfirina de zinco (inibidor de heme oxigenase-1). Células renais de porco, LLC-PK1, foram cultivadas com polimixina B durante 24, 48 e 72 horas. A apoptose e viabilidade celular foram avaliadas usando diferentes doses do antibiótico: Controle (CTL, 0 µM); G1 (12,5µM); G2 (37,5µM); G3 (75µM); G4 (125µM) e G5 (375µM). O hemin (25µM) e a protoporfirina de zinco (10µM) foram administrados uma hora antes da polimixina B. Foram utilizados os métodos Acridine orange/ brometo de etídio (viabilidade) e Hoescht 33342 (apoptose). Os resultados demonstraram redução linear de viabilidade induzida pela polimixina B quando a dose e o tempo de exposição foram aumentados, isto foi confirmado pela variação inversa de apoptose. O hemin aumentou a viabilidade e reduziu apoptose na presença de polimixina B, sugerindo um efeito protetor da HO-1 neste modelo. O efeito observado para a protoporfirina de zinco foi semelhante ao descrito para o hemin. O estudo confirmou a citotoxicidade da polimixina B em células renais e constatou que esse efeito pode ser mediado pela HO-1 considerando o efeito obtido no tratamento com o indutor daquela enzima