Porcine Renal Epithelial Cell Line Research Articles

Identification of the Porcine Vascular Endothelial Cell-Specific Promoter ESAM1.0 Using Transcriptome Analysis.

The vascular endothelium of xenografted pig organs represents the initial site of rejection after exposure to recipient immune cells. In this study, we aimed to develop a promoter specific to porcine vascular endothelial cells as a step toward overcoming xenograft rejection. Transcriptome analysis was performed on porcine aortic endothelial cells (PAECs), ear skin fibroblasts isolated from GGTA knockout (GTKO) pigs, and the porcine renal epithelial cell line pk-15. RNA sequencing confirmed 243 differentially expressed genes with expression changes of more than 10-fold among the three cell types. Employing the Human Protein Atlas database as a reference, we identified 34 genes exclusive to GTKO PAECs. The endothelial cell-specific adhesion molecule (ESAM) was selected via qPCR validation and showed high endothelial cell specificity and stable expression across tissues. We selected 1.0 kb upstream sequences of the translation start site of the gene as the promoter ESAM1.0. A luciferase assay revealed that ESAM1.0 promoter transcriptional activity was significant in PAECs, leading to a 2.8-fold higher level of expression than that of the porcine intercellular adhesion molecule 2 (ICAM2) promoter, which is frequently used to target endothelial cells in transgenic pigs. Consequently, ESAM1.0 will enable the generation of genetically modified pigs with endothelium-specific target genes to reduce xenograft rejection.

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.

Heat Shock Protein 27 Is the Major Differentially Phosphorylated Protein Involved in Renal Epithelial Cellular Stress Response and Controls Focal Adhesion Organization and Apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Urotensin II is an autocrine/paracrine growth factor for the porcine renal epithelial cell line, LLCPK1.

Urotensin-II (UII), a cyclic dodecapeptide with potent cardiovascular effects, has recently been shown to be abundantly expressed in the human kidney and excreted in human urine. To investigate whether UII acts as an autocrine/paracrine growth factor for renal epithelial cells, we have studied the effects of human UII (hUII) on DNA synthesis, cytosolic free Ca(2+) concentration ([Ca(2+)](i)), ERK activation, and protooncogene (c-myc) expression in a porcine renal epithelial cell line (LLCPK1). hUII stimulated [(3)H]thymidine uptake into quiescent cells in a dose-dependent manner (10(-9) to 10(-7) M); this effect was inhibited by a protein kinase C inhibitor (GF109203X), a MAPK kinase inhibitor (PD98059), and a calcium channel blocker (nicardipine). Neither phosphatidyl inositol-3 kinase inhibitors (LY294002, wortmannin) nor p38 kinase inhibitor (SB203580) affected the hUII-induced DNA syntheses. hUII rapidly (within 5 min) and dose-dependently (10(-9) to 10(-7) M) increased [Ca(2+)](i) in fura-2-loaded cells. hUII also caused a rapid and transient activation of ERK1/2 and induction of c-myc. LLCPK1 cells expressed UII mRNA and its receptor GPR14 mRNA, as determined by RT-PCR, and released UII-like immunoreactivity into media. Neutralization of endogenous UII by anti-hUII antibody, but not nonimmune serum, significantly suppressed DNA synthesis. These data suggest that hUII is an autocrine/paracrine growth factor for renal epithelial cells via activation of both protein kinase C and ERK1/2 pathways as well as Ca(2+) influx via voltage-dependent Ca(2+) channels.

The Plasma Membrane-associated Protein RS1 Decreases Transcription of the Transporter SGLT1 in Confluent LLC-PK1 Cells

Previously we cloned RS1, a 67-kDa polypeptide that is associated with the intracellular side of the plasma membrane. Upon co-expression in Xenopus laevis oocytes, human RS1 decreased the concentration of the Na(+)-D-glucose co-transporter hSGLT1 in the plasma membrane (Valentin, M., Kühlkamp, T., Wagner, K., Krohne, G., Arndt, P., Baumgarten, K., Weber, W.-M., Segal, A., Veyhl, M., and Koepsell, H. (2000) Biochim. Biophys. Acta 1468, 367-380). Here, the porcine renal epithelial cell line LLC-PK1 was used to investigate whether porcine RS1 (pRS1) plays a role in transcriptional up-regulation of SGLT1 after confluence and in down-regulation of SGLT1 by high extracellular D-glucose concentrations. Western blots indicated a dramatic decrease of endogenous pRS1 protein at the plasma membrane after confluence but no significant effect of D-glucose. In confluent LLC-PK1 cells overexpressing pRS1, SGLT1 mRNA, protein, and methyl-alpha-D-glucopyranoside uptakes were drastically decreased; however, the reduction of methyl-alpha-D-glucopyranoside uptake after cultivation with 25 mm D-glucose remained. In confluent pRS1 antisense cells, the expression of SGLT1 mRNA and protein was strongly increased, whereas the reduction of SGLT1 expression during cultivation with high D-glucose was not influenced. Nuclear run-on assays showed that the transcription of SGLT1 was 10-fold increased in the pRS1 antisense cells. The data suggest that RS1 participates in transcriptional up-regulation of SGLT1 after confluence but not in down-regulation by D-glucose.

Uptake properties of lamivudine (3TC) by a continuous renal epithelial cell line

The purpose of this study was to characterize the renal uptake properties of the cytidine analog and antiretroviral agent 3TC. The uptake of radiolabelled 3TC was measured at 37 degrees C in a continuous porcine renal epithelial cell line (i.e., LLC-PK1 cells) grown as a monolayer on an impermeable support. 3TC (5 microM) uptake (37 degrees C) by the monolayer cells was saturable (Km = 1.2 +/- 0.2 mM) but not significantly altered by various dideoxynucleoside analog drugs, nucleosides, and nucleoside transport inhibitors, suggesting that a nucleoside transporter is not involved in 3TC uptake. A number of endogenous organic cation probes and inhibitors significantly reduced 3TC uptake by the monolayer cells. Quinine, trimethoprim (TMP), and tetraethylammonium (TEA) inhibited 3TC uptake in a dose dependent manner with IC50 values of 0.6 mM, 0.63 mM, and 1.9 mM, respectively. In turn, the uptake of the typical organic cation substrate TEA was inhibited by high concentrations of 3TC. An outwardly directed proton gradient significantly increased the uptake of 3TC by the monolayer cells, suggesting the involvement of a proton exchange process. Conversely, in the presence of monensin, a Na+/H+ ionophore, the uptake of 3TC was significantly reduced. These results suggest that the uptake of 3TC by a cultured renal epithelium may be mediated by an organic cation-proton exchanger. The observed clinical interaction between 3TC and trimethoprim may be explained by competition for a common renal organic cation tubular transporter.

Uptake properties of lamivudine (3TC) by a continuous renal epithelial cell line

The purpose of this study was to characterize the renal uptake properties of the cytidine analog and antiretroviral agent 3TC. The uptake of radiolabelled 3TC was measured at 37 degrees C in a continuous porcine renal epithelial cell line (i.e., LLC-PK1 cells) grown as a monolayer on an impermeable support. 3TC (5 microM) uptake (37 degrees C) by the monolayer cells was saturable (Km = 1.2 +/- 0.2 mM) but not significantly altered by various dideoxynucleoside analog drugs, nucleosides, and nucleoside transport inhibitors, suggesting that a nucleoside transporter is not involved in 3TC uptake. A number of endogenous organic cation probes and inhibitors significantly reduced 3TC uptake by the monolayer cells. Quinine, trimethoprim (TMP), and tetraethylammonium (TEA) inhibited 3TC uptake in a dose dependent manner with IC50 values of 0.6 mM, 0.63 mM, and 1.9 mM, respectively. In turn, the uptake of the typical organic cation substrate TEA was inhibited by high concentrations of 3TC. An outwardly directed proton gradient significantly increased the uptake of 3TC by the monolayer cells, suggesting the involvement of a proton exchange process. Conversely, in the presence of monensin, a Na+/H+ ionophore, the uptake of 3TC was significantly reduced. These results suggest that the uptake of 3TC by a cultured renal epithelium may be mediated by an organic cation-proton exchanger. The observed clinical interaction between 3TC and trimethoprim may be explained by competition for a common renal organic cation tubular transporter.

Identification of clusterin sequences mediating renal tubular cell interactions.

Expression of the glycoprotein clusterin is markedly increased following tissue injury. One function of clusterin is to promote cell interactions which are perturbed in these pathologic settings. Clusterin causes cell aggregation and adhesion in vitro yet the molecular mechanism for this effect is not known. In order to identify the active site(s) of clusterin, 34 peptides, each 15 amino acid residues in length, were synthesized from hydrophilic regions of human clusterin. When studied individually, none of the peptides caused aggregation of LLC-PK1 cells, a porcine renal epithelial cell line. However, two out of the 34 peptides inhibited clusterin-induced cell aggregation in a dose-dependent manner. Scrambled versions of these two 'active' peptides did not inhibit cell aggregation. Seven peptides promoted cell adhesion. In conclusion, these findings provide evidence for novel amino acid sequences mediating clusterin-induced renal cell interactions.

Cadmium (Cd2+) disrupts E-cadherin-dependent cell-cell junctions in MDCK cells.

Previous studies from our laboratory have shown that Cd2+ can selectively disrupt E-cadherin-dependent cell-cell junctions in the porcine renal epithelial cell line, LLC-PK1. The objective of the present studies was to determine whether or not Cd2+ could produce similar effects in Madin-Darby canine kidney (MDCK) cells, an immortal epithelial cell line derived from dog kidney. This is an important issue because MDCK cells have been used extensively as a model system to study the basic mechanisms of E-cadherin-dependent cell-cell adhesion. MDCK cells on permeable membrane supports were exposed to Cd2+ by adding CdCl2 to either the apical or the basolateral compartment. The integrity of cell-cell junctions was assessed by morphologic observation of the cells and by monitoring the transepithelial electrical resistance. The results showed that exposure to 10-40 microM Cd2+ for 15 min-4 h caused the cells to separate from each other without detaching from the growing surface. The separation of the cells was accompanied by a marked drop in the transepithelial electrical resistance, a loss of E-cadherin from the cell-cell contacts, and a reorganization of the actin cytoskeleton. These effects were much more pronounced when Cd2+ was added basolaterally than when it was added apically. Moreover, the effects of Cd2+ were qualitatively similar to those observed when the cells were incubated in Ca(2+)-free medium. These results show that Cd2+ can disrupt E-cadherin-dependent cell-cell junctions in MDCK cells, and they indicate that this cell line would be an appropriate model for further mechanistic studies in this area.

Cloning of the dopamine-1A (D1A) receptor gene expressed in porcine renal epithelial cells.

We sought to determine the molecular identify of the dopamine-1 (D1) receptor expressed in the porcine renal epithelial cell line LLC-PK1. We first isolated a partial cDNA by the reverse transcription-polymerase chain reaction procedure and then used the partial cDNA to isolate positive overlapping clones from a porcine genomic DNA library. Sequence analysis of the gene revealed that the longest open-reading frame encoded a 446 amino acid protein that was 95% identical to the human D1A receptor. Expression studies in mammalian cells were also consistent with the clones encoding a D1 receptor. Northern blot hybridizations with LLC-PK1 poly (A+) RNA were strongly positive. The porcine D1A gene has two exons and a short intron in the 5' untranslated region. The 5' flanking region lacks a TATA and CAAT box but is high in GC content (68%) and contains multiple Sp1 binding sites. The 5' flanking region also contains numerous other cis-acting elements for transcription factors. These results indicate that the D1A receptor is the major D1 receptor expressed in LLC-PK1 cells and further suggest that LLC-PK1 cells may be a useful model to study the regulation of renal D1A receptor gene transcription.

Mechanism of sodium nitroprusside-mediated inhibition of aromatic amino acid decarboxylase activity.

The effects of sodium nitroprusside (SNP) on dopamine synthesis in a porcine renal epithelial cell line (LLC-PK1) were evaluated. Subsequent studies examined the actions of the degradation products of SNP (cyanide, ferrous ion and nitric oxide) on aromatic amino acid decarboxylase (AAAD) activity in tissue supernatants from LLC-PK1 cells and rat renal cortex. SNP (10-500 mumol/l) significantly inhibited dopamine production in LLC-PK1 cells in a dose-related manner. The activation of guanylate cyclase by nitric oxide was not found to be the mechanism whereby SNP inhibited dopamine synthesis in LLC-PK1 nor did the antioxidant glutathione attenuate the actions of SNP. Ferrous sulfate (0.5 mmol/l) and SNP (0.5 mmol/l) were found to inhibit dopamine synthesis in LLC-PK1 cells and to directly inhibit cytosolic AAAD activity from LLC-PK1 cells. A series of studies were conducted using AAAD from rat renal cortex and confirmed that SNP could directly inhibit the conversion of L-dopa to dopamine by AAAD. Furthermore, potassium ferricyanide (1 mmol/l) and potassium cyanide (1 mmol/l) could produce greater than 80% reductions in AAAD activity. Iron (0.5-1 mmol/l) was found to increase rat kidney AAAD activity. Kinetic analysis revealed that potassium cyanide was a potent (Ki = 40-50 mumol/l) noncompetitive/mixed noncompetitive inhibitor of AAAD. SNP was also found to be a noncompetitive inhibitor of AAAD with a Ki of approximately 300-500 mumol/l. In contrast, ferrous sulfate (0.5 mmol/l) was a competitive inhibitor (Ki = approximately 650 mumol/l) that actually increased the Vmax of AAAD. The results of these studies support that cyanide released from SNP can potently inhibit AAAD, although SNP has somewhat more complex interactions with AAAD due to the presence of ferrous ion.

Studies on the renal kinetics of growth hormone (GH) and on the GH receptor and related effects in animals.

Growth hormone (GH) is filtered through the kidney, and may exert effects on renal function when presented via the circulation. Investigations on kidney-related aspects of GH are increasing in number. Using in vitro and in vivo approaches, the present study attempted to provide answers to a number of unresolved or debated issues. In vitro, we detected both GH and type 1 IGF receptors (R) in a porcine renal epithelial cell line. The saturation and down regulation kinetics of the GH-R indicate that it has the properties of a classical GH-R. Furthermore, the simultaneous presence of GH-R and IGF-R on a phenotypically homogeneous cell line suggests the presence of GH-induced auto-/paracrine IGF-1 bioactivity in the kidney. Experiments with isolated proximal rabbit tubules incubated with physiological concentrations of 125I-GH demonstrated a time-and dose-dependent increase in unlabelled GH-displaceable cell-associated radioactivity, lending support to the concept of GH mediating its renal effects via proximal tubular GH-R. Short term administration of GH to rats and humans elicited electrolyte and water retention that may cause edema in adults. In the present study, long term administration of GH to rats caused only a minor increase in serum phosphate levels, with no changes observed in the renal electrolyte clearance. During the first 4 days of GH treatment in rats, no change in plasma renin activity was detected and we were thus unable to confirm the hypothesis that the renin-angiotensin system is responsible for the early phase of GH-associated fluid retention. Pharmacokinetically, when GH was administered to rats with functional disconnection of the kidneys as a model of renal insufficiency, the whole body clearance of GH decreased by ca. two thirds, and was reflected by an increase in the mean residence time and AUCplasma for GH. The plasma half-life, however, was not significantly affected, suggesting that the volume of distribution (Vd) had decreased for the GH administered to the renally compromised animals. A renal contribution to the Vd was visualized as intense radioactive staining in the kidney region on whole body autoradiographs (WBA) of rats dosed with 125I-labelled hGH. The liver region was also intensely stained. Kidney-associated radioactivity was found to be related not only to glomerular filtration, but also to peritubular uptake, since the renal clearance of free GH was found to exceed the GFR.(ABSTRACT TRUNCATED AT 400 WORDS)

A68930 is a potent, full agonist at dopamine1 (D1) receptors in renal epithelial LLC‐PK1 cells

The selective dopamine1 (D1) receptor agonists SK&F 82526 (fenoldopam) and A68930 and the mixed D1/D2 agonist SK&F 85174 were tested for their ability to stimulate adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in the porcine renal epithelial cell line, LLC-PK1. SK&F 82526 and SK&F 85174 were potent stimulators of cyclic AMP accumulation (EC50s 21.4 and 14.5 nM, respectively), but only partial agonists (intrinsic activities 31% and 46% of dopamine respectively). In contrast, A68930 was a potent, full agonist (EC50 12.7 nM, intrinsic activity 102% of dopamine). The stimulatory effects of A68930 and dopamine on cyclic AMP accumulation were not additive, and the stimulation of cyclic AMP accumulation by A68930 was blocked by the D1-selective antagonist, SCH 23390. These properties of A68930 suggest that it may be a useful D1-selective agonist to study renal D1 receptor mechanisms in vitro and in vivo.

Cadmium (Cd 2+) disrupts Ca 2+-dependent cell-cell junctions and alters the pattern of E-cadherin immunofluorescence in LLC-PK 1 cells

Recent findings from our laboratories have shown that Cd 2+ has relatively specific damaging effects on the adhering and occluding junctions in the established porcine renal epithelial cell line, LLC-PK 1. Results of the present studies show that the junction-perturbing effects of Cd 2+ in LLC-PK 1 cells are more pronounced when Cd 2+ is applied to the basolateral cell surface than when it is applied to the apical surface, and that the severity of the effects is inversely related to the concentration of Ca 2+ in the medium. Additional results show that exposure to sublethal concentrations of Cd 2+ decreases the amount of E-cadherin that is associated with cell-cell contacts. These results suggest that Cd 2+ damages Ca 2+-dependent cell-cell junctions in LLC-PK 1 cells by interacting with E-cadherin or a similar Ca 2+-sensitive site that is oriented toward the basolateral cell surface.

Cadmium (Cd 2+) disrupts intercellular junctions and actin filaments in LLC-PK 1 cells

Studies reported in the literature suggest that cadmium (Cd 2+) may disrupt the junctions between cells in some tissues and cell culture systems. In order to examine this possibility in more detail, we have studied the effects of Cd 2+ on the integrity of intercellular junctions in the established porcine renal epithelial cell line, LLC-PK 1. Junctional integrity was assessed by monitoring the collapse of domes and by measuring changes in the transepithelial electrical resistance in confluent cell monolayers. Exposure to Cd 2+ caused a rapid decrease in transepithelial resistance and the concomitant collapse of domes. These effects occurred at Cd 2+ concentrations (20–60 μ m) and durations of exposure (as little as 1 hr) that did not alter levels of ATP or kill the cells. Electron microscopic studies showed that Cd 2+ caused time-dependent changes in adhering and occluding junctional complexes, which eventually resulted in the complete separation of the cells. Additional studies, in which rhodamine-coupled phalloidin was used to visualize F-actin, showed that Cd 2+ altered the structure of actin filaments in the cells; there was a significant reduction in the amount of junction-associated F-actin and in the number of stress fibers. These results indicate that Cd 2+ has relatively specific damaging effects on the adhering and occluding junctions between LLC-PK 1 cells and that these effects may involve the disruption of cytoskeletal actin filaments.

Ca2+ potentiates cAMP-dependent expression of urokinase-type plasminogen activator gene through a calmodulin- and protein kinase C-independent mechanism.

In the porcine renal epithelial cell line, LLC-PK1, activation of the cAMP-dependent signal transduction pathway induces the urokinase-type plasminogen activator (uPA) gene. We show here that the cAMP response is enhanced when the intracellular calcium concentration is increased. When LLC-PK1 cells were treated with the calcium ionophore ionomycin alone, there was no uPA mRNA accumulation. However, in the presence of ionomycin the dose-response of 8-bromo-cAMP (Br-cAMP) with respect to uPA mRNA accumulation was shifted toward the lower concentrations of Br-cAMP. A Northern blot analysis after the inhibition of RNA synthesis and nuclear run-on assays showed that the synergistic effect of Ca2+ could be attributed to increases in uPA gene transcription and mRNA stability. In the presence of cycloheximide, a protein synthesis inhibitor, uPA mRNA was stabilized, but the effect of ionomycin on Br-cAMP-induced mRNA accumulation was still maintained. The result suggests that the Ca2+, at least on transcription, does not require new protein synthesis. Ionomycin treatment did not modify the activity of the cAMP-dependent protein kinase, suggesting that Ca2+ either affects a step in the pathway between the kinase and the uPA gene, or acts independently of the cAMP-dependent protein kinase pathway. The effect of ionomycin was not suppressed by protein kinase C down-regulation nor by inhibitors of calmodulin. Synergism was also observed when Br-cAMP was replaced with calcitonin, a peptide hormone which is coupled to adenylate cyclase, and when ionomycin was replaced with another ionophore A23187, suggesting that the synergism is due to an interaction between cAMP-dependent and Ca2(+)-dependent signal transduction pathways.

Toxicity of Cyclosporine Metabolites

Eight cyclosporine (CsA) metabolites were isolated from the urine of renal transplant recipients. The structure and purity of the metabolites were characterized by fast atom bombardment/mass spectroscopy as well as by proton and 13C nuclear magnetic resonance. The in vitro toxicity of the metabolites were tested using a porcine renal epithelial cell line (LLC-PK1). None of the metabolites was as effective as CsA in inhibiting cell growth and DNA, RNA, or protein synthesis, with the majority of them exhibiting activity less than 10% of that of CsA when the IC50 (the concentration required for 50% inhibition of that particular metabolic function) values were compared. The exception to this was the demethylated metabolite M-21, which exhibited a potency of 17-50% of CsA for the various metabolic parameters examined. The results suggest that the immunosuppressive activity of metabolites may be dissociated from their toxicity. Morphologically, CsA and the metabolite M-21 resulted in changes consistent with the vacuolization seen in tubular cells exposed to CsA in vivo. In contrast, M-17 up to the maximum concentration tested (25,000 micrograms/L) was found not to cause such changes.

Cytokine-induced release of endothelin-1 from porcine renal epithelial cell line

To elucidate the mechanism by which endothelin-1 (ET-1) is released from renal epithelial cells, we have investigated the effects of several compounds on release of ET-1-like immuno-reactivity (LI) from LLCPK 1 cell line. Thrombin, transforming growth factor-β, cytokines (tumor necrotizing factor-α, interleukin-1β), and phorbol ester stimulated ET-1-LI release in a time- and dose-dependent manner. The cytokine-induced ET-1-LI release was not affected by indomethacin. Northern blot analysis using cDNA for porcine preproET-1 as a probe revealed a single major band corresponding to the size of ET-1 mRNA in LLCPK 1. These data indicate that the preproET-1 gene is also expressed in renal epithelial cells and the release of ET-1 from renal cells is regulated by the similar mechanism to that from endothelial cells.