Established Renal Epithelial Cell Line Research Articles

Ciglitazone induces caspase-independent apoptosis via p38-dependent AIF nuclear translocation in renal epithelial cells

Peroxisome proliferator-activated receptor γ (PPARγ) agonists have been reported to induce apoptosis in a variety of cell types including renal proximal epithelial cells. However, the underlying mechanism of cell death induced by PPARγ agonists has not been clearly defined in renal proximal tubular cells. This study was therefore undertaken to determine the mechanism by which ciglitazone, a synthetic PPARγ agonist, induces apoptosis in opossum kidney (OK) cells, an established renal epithelial cell line. Ciglitazone treatment induced apoptotic cell death in a dose- and time-dependent manner. Ciglitazone caused a transient activation of ERK and sustained activation of p38 MAP kinase. Ciglitazone-mediated cell death was attenuated by the p38 inhibitor SB203580 and transfection of dominant-negative form of p38, but not by the MEK inhibitor U0126, indicating that p38 MAP kinase activation is involved in the ciglitazone-induced cell death. Although ciglitazone-induced caspase-3 activation, the ciglitazone-mediated cell death was not affected by the caspase-3 inhibitor DEVD-CHO. Ciglitazone-induced mitochondrial membrane depolarization and apoptosis-inducing factor (AIF) nuclear translocation and these effects were prevented by the p38 inhibitor. These results suggest that ciglitazone induces caspase-independent apoptosis through p38 MAP kinase-dependent AIF nuclear translocation in OK renal epithelial cells.

PROTECTIVE EFFECT OF A PROTEIN KINASE INHIBITOR ON CELLULAR INJURY INDUCED BY CEPHALORIDINE IN THE PORCINE KIDNEY CELL LINE LLC-PK1

We investigated the effects of a protein kinase C inhibitor and a tyrosine kinase inhibitor on the cellular injury induced by cephaloridine in an established renal epithelial cell line, LLC-PK(1). Cephaloridine increased the leakage of lactate dehydrogenase (LDH) from LLC-PK(1) cells into the medium and also caused an increase in the level of lipid peroxide (index of oxidative stress) in the cells. Treatment of the cells with a hydroxyl radical scavenger, dimethylthiourea (DMTU), inhibited the increases in LDH leakage and lipid peroxidation in LLC-PK(1) cells exposed to cephaloridine. A protein kinase C inhibitor, H-7, and tyrosine kinase inhibitors, genistein and lavendustinA, inhibited the increases in LDH leakage and lipid peroxidation in LLC-PK(1) cells exposed to cephaloridine. These results suggest that a signaling pathway which involves protein kinase C and tyrosine kinase plays a role in the generation of reactive oxygen species in LLC-PK(1) cells damaged by cephaloridine.

Na +-dependent and -independent uridine uptake in an established renal epithelial cell line, OK, from the opossum kidney

The characteristics of Na +-dependent and Na +-independent uridine uptake at 22°C were determined for monolayers of OK renal epithelial cells. The majority of uridine influx in subconfluent to early confluent (day 1 postconfluency) OK monolayers was mediated via a facilitated-diffusion pathway (apparent K m 160 ± 41 μM, V max 610 ± 100 pmol/mg protein per min). This system was inhibited with high affinity by nitrobenzylthioinosine (NBMPR) (IC 50 value 1.5 nM) and by purine and pyrimidine nucleosides. Specific [ 3H]NBMPR binding sites were detected in OK monolayers (apparent K d 0.67 ± 0.25 nM, B max 90 ± 19 fmol/mg protein) yielding a turnover number for the carrier of 112 uridine molecules/site per s at 22°C. Na +-dependent uridine uptake was minor in subconfluent OK monolayers, but increased 8-fold with time after confluency reaching a stable plateau at 5 days postconfluency. Inhibition of Na +-dependent 1 μM uridine uptake by inosine, guanosine, adenosine and uridine was biphasic with approx. 40% of the total uptake inhibited with high affinity (IC 50 value 2 to 14 μM). Concentrations of thymidine and cytidine up to 1mM had no effect on Na +-dependent uridine uptake and no Na +-dependent thymidine influx by confluent OK monolayers was detected. Using cell monolayers grown on a permeable filter support, Na +-dependent uridine uptake occurred preferentially from the apical surface. This high affinity component of Na +-dependent uridine uptake is suggested to represent the Na +-dependent purine preferring N1 nucleoside transporter. The Na +/uridine stoichiometry for this system was consistent with 1:1. The remaining component of Na +-dependent uridine uptake was inhibited by some nucleosides, such as guanosine and inosine, with low affinity (IC 50 values of 0.6 to 5 mM). Other nucleosides showed little specific inhibition. We propose that this component of uridine uptake represents a mutated carrier that binds nucleosides but is defective in the translocation of permeant.

Na+-dependent nucleoside uptake in an established renal epithelial cell line, OK

Na+-dependent nucleoside uptake in an established renal epithelial cell line, OK

Parathyroid hormone regulation of Na+/H+ exchange in opossum kidney cells: polarity and mechanisms

In previous work we have shown that parathyroid hormone (PTH) inhibits Na+/H+ exchange in cellular suspensions of OK (opossum kidney) cells (an established renal epithelial cell line) in a dose-dependent manner. PTH effects could be mimicked by pharmacological activation of both protein kinase A and protein kinase C (Helmle-Kolb et al. 1990). In the present paper we extend these observations and analyze the PTH-dependent control of Na+/H+ exchange in OK cells kept in epithelial configuration (monolayer). Na+/H+ exchange activity is examined by microfluorometry using the intracellularly trapped pH-sensitive dye 2'7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein. Cells recovered from an acid load (NH4Cl prepulse) after addition of apical Na+. Ethylisopropylamiloride inhibits Na(+)-dependent pHi recovery at micromolar concentrations. PTH leads to an inhibition of apical Na+/H+ exchange activity; inhibition is observed even at a concentration of 5 pM PTH. PTH given at maximally effective concentrations (24 nM) reduces the total Na+/H+ exchange capacity by 60%-70%. Apical as well as basolateral hormone additions elicit an inhibitory response at low (5 pM) or high (24 nM) concentrations. Forskolin (activation of protein kinase A) and phorbol esters (activation of protein kinase C) lead to an inhibition of Na+/H+ exchange activity (60%-70% inhibition). These observations suggest that Na+/H+ exchange activity is preferentially located in the apical membranes of OK cells kept in monolayer configuration.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of attachment substrates on the growth and differentiation of LLC-PK1 cells

The growth and differentiation of an established renal epithelial cell line, LLC-PK1, on membrane bound mussel adhesive protein (MAP), collagen, and extracellular matrix (ECM) in serum-containing medium was studied. Cell attachment and growth on uncoated- vs. protein-coated cellulose nitrate and acetate membranes did not differ significantly, and confluence was achieved on all membranes. However, cells remained in a single monolayer only when plated on collagen or ECM. LLC-PK1 monolayers grown on ECM-coated membranes displayed the highest transepithelial D-glucose transport (333 +/- 22 ng.cm-2.min-1) whereas cells plated on collagen-coated membranes displayed the lowest (94 +/- 23 ng.cm-2.min-1). Glucose flux values increased with age of the culture, reaching a plateau at 28 d postseeding. These results indicate that the underlying substratum and cell age can affect differentiation of renal epithelial cells in vitro.

The Na+/Pi-cotransporter of OK cells: reaction and tentative identification with N-acetylimidazole

Using an established renal epithelial cell line (OK cells) the effect of the amino-acid side-chain modifying reagent N-acetylimidazole (NAI) upon the sodium-dependent transport of phosphate (Pi) was investigated. After an incubation with 10 mM NAI for 20 min, cellular Na+/Pi uptake was inhibited by 70%. The presence of 5 mM Pi protected this transport function from being affected by NAI by 80 to 100%. Since the presence of sulfate was unable to protect the Na+/Pi transport inactivation by NAI and since the presence of Pi did not affect NAI inhibition of other transport systems, it is suggested that NAI interacts with the Pi transporter directly. The protective effect of Pi was used as a criterion to identify Pi-protectable [3H]NAI labelling of OK cell plasma membrane proteins. Pi protection was observed in four molecular mass regions: 31, 53, 104 and 176 kDa. Since the incorporation of [3H]NAI into these proteins was also affected by parathyroid hormone at 10−10 M, it is concluded that the identified proteins represent possible candidates for the renal Na+/Pi cotransported.

Characteristics of Na +-dependent hexose transport in OK, an established renal epithelial cell line

The characteristics of Na +-dependent hexose uptake were determined for monolayers of OK, an established renal epithelial cell line derived from an opossum kidney. A comparison is made with other cultured cells, particularly LLC-PK 1. The capacity to accumulate α-methyl d-glucoside (AMG) in OK cells develops with time, reaching a maximum level of 18 nmol/mg protein per h, 3 days after confluency. In contrast to LLC-PK 1, this level is not influenced by the medium d-glucose concentration. AMG uptake in OK cells was characterized by an apparent K m of 2.9 mM and a V max of 17.1 nmol/mg protein per min. For Na +-dependent phlorisin binding, a K D of 0.025 μM and a B max of 1.5 pmol/mg protein were found. A turnover frequency of 158/s was derived from our data. The hexose carrier of OK shares with the carrier of LLC-PK 1 a high level of expression, its substrate specificity and turnover frequency. It differs however with respect to the substrate binding site. The affinity for AMG and d-glucose is 3- and 10-fold lower, whereas the affinity for phlorizin is 3-times higher in OK than in LLC-PK 1. The Na + dependence of AMG uptake was also different for both cell lines and suggested for OK cells a 1:1, Na +:substrate stoichiometry. In OK cells, the phlorizin-sensitive uptake rate of d-glucose is much lower than the one for AMG. Nevertheless, d-glucose interacts with the AMG binding site in a competitive way and with an affinity similar to AMG. This could indicate a malfunction of the carrier with d-glucose as a substrate at the level of the translocation step.

Cellular mechanisms of inorganic phosphate transport in kidney.

articleCellular mechanisms of inorganic phosphate transport in kidneyP. Gmaj, and H. MurerP. GmajSearch for more papers by this author, and H. MurerSearch for more papers by this authorPublished Online:01 Jan 1986https://doi.org/10.1152/physrev.1986.66.1.36MoreSectionsPDF (6 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByDisorders of the Endocrine SystemEthyl Chloride18 June 2004Phosphate absorption in Aplysiacalifornica gut: glucocorticoid inhibitionCanadian Journal of Physiology and Pharmacology, Vol. 81, No. 4Regulation of Na/Pi Transporter in the Proximal TubuleAnnual Review of Physiology, Vol. 65, No. 1pH dependence of Na+/myo-inositol cotransporters in rat thick limb cellsKidney International, Vol. 62, No. 6Thyroid-hormone-induced phosphate absorption in Aplysia californica gut is mediated through protein synthesisCanadian Journal of Physiology and Pharmacology, Vol. 80, No. 12Basolateral Phosphate Transport in Renal Proximal-Tubule-Like OK Cells 129 November 2016 | Experimental Biology and Medicine, Vol. 227, No. 8Sodium-Phosphate Symport by Aplysia Californica GutZoological Science, Vol. 19, No. 2Renal and Skeletal Actions of Parathyroid Hormone (PTH) and PTH-Related ProteinAmino acids involved in sodium interaction of murine type II Na + ‐P i cotransporters expressed in Xenopus oocytes1 March 2001 | The Journal of Physiology, Vol. 531, No. 2Evolution of the Na-Pi cotransport systemsAndreas Werner, and Rolf K. H. Kinne1 February 2001 | American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, Vol. 280, No. 2PLC-γ1 Signaling Pathway and Villin Activation Are Involved In Actin Cytoskeleton Reorganization Induced by Na+/Pi Cotransport Up-regulation1 April 2000 | Molecular Medicine, Vol. 6, No. 4Proximal Tubular Phosphate Reabsorption: Molecular MechanismsHeini Murer, Nati Hernando, Ian Forster, and Jürg Biber10 January 2000 | Physiological Reviews, Vol. 80, No. 4Adaptation to changes in dietary phosphorus intake in health and in renal failureJournal of Laboratory and Clinical Medicine, Vol. 129, No. 2Immunodetection and characterization of proteins implicated in renal sodium/phosphate cotransportBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1190, No. 2Inhibition by cyclic AMP and phorbol esters of sodium-dependent uptake of phosphate by rat hepatocytesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1190, No. 2Expression of Na/Pi cotransport from opossum kidney cells in Xenopus laevis oocytesBiochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1178, No. 2Sodium-dependent transport of phosphate by rat liver plasma membrane vesiclesBiochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1143, No. 2Uptake of phosphate by rat hepatocytes in primary culture: a sodium-dependent system that is stimulated by insulinBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1148, No. 1Expression of rat renal sodium/phosphate contransporter in Xenopus laevis oocytesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1104, No. 1Molecular size of the renal sodium/phosphate symporter in native and reconstituted systemsBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1104, No. 1Inhibition of sodium-phosphate cotransport in renal brush-border membranes with the stilbenedisulfonate, H2-DIDSBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1069, No. 1Some characteristics of sodium-independent phosphate transport across renal basolateral membranesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1064, No. 2The stepwise hydrolysis of adenine nucleotides by ectoenzymes of rat renal brush-border membranesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1030, No. 1Different molecular sizes for Na+-dependent phosphonoformic acid binding and phosphate transport in renal brush border membrane vesiclesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1028, No. 2Neomycin inhibits PDGF-induced IP3 formation and DNA synthesis but not PDGF-stimulated uptake of inorganic phosphate in C3H/10T1/2 fibroblastsBiochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1054, No. 2Effect of parathyroid hormone and dietary phosphate on phosphate transport in renal outer cortical and outer medullary brush-border membrane vesiclesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1024, No. 1Increase in membrane fluidity modulates sodium-coupled uptakes and cyclic AMP synthesis by renal proximal tubular cells in primary cultureBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1022, No. 1Thiol redox and phosphate transport in renal brush-border membrane. Effect of nicotinamideBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1021, No. 1[29] Phosphate transport in established renal epithelial cell lines[37] Stimulus-secretion coupling: General models and specific aspects in epithelial cellsMechanisms of mammary gland ion transporComparative Biochemistry and Physiology Part A: Physiology, Vol. 97, No. 3Effect of phosphonoformic acid, dietary phosphate and the Hyp mutation on kinetically distinct phosphate transport processes in mouse kidneyBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 984, No. 2The Na+/Pi-cotransporter of OK cells: reaction and tentative identification with N-acetylimidazoleBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 981, No. 2[16] Cation-anion cotransportTransport of sulfate and phosphate in small intestine and renal proximal tubule: Methods and basic propertiesComparative Biochemistry and Physiology Part A: Physiology, Vol. 90, No. 4Colchicine blocks the action of parathyroid hormone but not nicotinamide on renal phosphate transportBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 905, No. 2Phosphate homeostasisMolecular Aspects of Medicine, Vol. 9, No. 4 More from this issue > Volume 66Issue 1January 1986Pages 36-70 Copyright & PermissionsCopyright © 1986 the American Physiological Societyhttps://doi.org/10.1152/physrev.1986.66.1.36PubMed3003769History Published online 1 January 1986 Published in print 1 January 1986 Metrics

Characterization of a Na∶K∶2Cl cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1)

86Rb uptake into LLC-PK1 cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na+ and Cl- for activity, and a "leak" component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of 86Rb, 22Na and 36Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na+ : K+ : Cl- cotransport system. The kinetic parameters for Na+ and K+ interaction have been analyzed under initial linear zero trans conditions. The following values were obtained KmNa+ = 0.42 +/- 0.05 mmol/liter, Vmax = 303 +/- 24 pmol/mg/6 sec; KmK+ = 11.9 +/- 1.0 mmol/liter, VmaxK+ = 307 +/- 27 pmol/mg/6 sec. For Cl- interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na+ : 1K+ :2Cl- can be calculated. It is concluded that the apical membrane of LLC-PK1 cells contains a Na+ : K+ : 2Cl- co-transport system with properties similar to those described for the thick ascending limb of the loop of Henle.

Absence of gap junctional complexes in two established renal epithelial cell lines (LLC-PK 1 and MDCK)

The type of junctions present in the membranes of two renal epithelial cell lines, LLC-PK 1 and MDCK, and of subcultured porcine aortic endothelial (PAE) cells have been studied by freeze-fracture. No gap junctions were observed in the two renal cell lines, while they were numerous in the endothelial cells. Tight junctions were abundant in LLC-PK 1 and MDCK cells and varied in numbers of ridges from one to ten. Only a few simple tight junctions unconnected with gap junctions were observed in PAE cells. The occurrence of gap junctions in these cells correlates with their ability to form intercellular communicating channels.

Deficiency in intercellular communication in two established renal epithelial cell lines (LLC-PK1 and MDCK).

We have studied the cell-to-cell passage of uridine nucleotides in two renal epithelial cell lines (LLC-PK1 and MDCK) and in porcine aortic endothelial cells (PAE). All three cell types incorporated tritiated uridine. After a 3 h incubation the radioactivity was predominantly in the form of acid-soluble compounds, mainly UTP. Prelabelled LLC-PK1 or MDCK cells were unable to transfer radioactivity to added adjacent, non-labelled cells, whereas PAE cells readily formed communicating intercellular junctions, as judged by autoradiographic analysis after a 3 h co-culture period. Cell-to-cell communication in either of the renal cell lines was not promoted by treatment with dibutyryl cyclic AMP and methylisobutylxanthine. Radioactivity incorporated into the acid-insoluble pool was not available for intercellular transfer, as assessed in experiments in which cells were prelabelled 24 h before co-culture.