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.

Quantitative imaging of subcellular calcium stores in mammalian LLC-PK1 epithelial cells undergoing mitosis by SIMS ion microscopy.

Quantitative 3-D total calcium gradients, representing subcellular stored calcium, were imaged with a CAMECA IMS-3f SIMS ion microscope in cryogenically prepared frozen freeze-dried LLC-PK1 cells captured in interphase and various stages of mitosis. 39K and 23Na concentrations were also measured in the same cells. Correlative optical (or SEM) and SIMS analysis of cells revealed a redistribution of the interphase Golgi calcium store in prophase and prometaphase cells. In metaphase cells, simultaneous SIMS imaging of total calcium in both the spindle and the non-spindle cytoplasm of individual cells revealed a gradual and dynamic alignment of calcium stores in both half-spindles prior to the onset of anaphase. The anaphase cells revealed the highest local total calcium concentrations in the spindle regions behind the daughter chromosomes and the lowest in the central spindle region. The pericentriolar material in telophase cells contained calcium stores. Quantitatively, a typical metaphase cell with well-aligned calcium stores in the spindle region contained 1.1 mM total calcium in each half-spindle, 0.8 mM total calcium in the non-spindle cytoplasm, and 0.5mM total calcium in the chromosomes. At the submicron scale, the distribution of total calcium was heterogeneous in the chromosomes, metaphase spindle, and non-spindle cytoplasm. An increased binding of calcium to chromosomes is not a physiological requirement for chromosomal condensation in mitosis, since interphase nuclei and mitotic chromosomes contained comparable total calcium concentrations measured per unit volume. A significant reduction of total calcium in the non-spindle cytoplasm was observed in the metaphase, anaphase, and telophase cells, which is indicative of the limited storage of the releasable calcium pool in these specific stages of mitosis. Direct total calcium measurements in subcellular regions confirmed that both the spindle and the non-spindle cytoplasm of metaphase cells contained inositol 1,4,5-trisphosphate (IP3)-sensitive calcium stores sensitive to arginine vasopressin, thapsigargin, and calcium ionophore A23187. The dynamic alignment of calcium stores in both half-spindles may be an integral part of the time-dependent process of a cell's overall preparation for exiting the metaphase stage in mammalian LLC-PK1 cells.

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.

Parathyroid hormone (PTH)/PTH-related peptide receptor density modulates activation of phospholipase C and phosphate transport by PTH in LLC-PK1 cells.

We showed previously that a single species of cloned PTH/PTH-related peptide (PTHrP) receptors, when stably expressed in LLC-PK1 kidney cells, couples to multiple second messenger signals and biological responses. To address the linkages of individual messenger signals to specific biological responses in these cells, we examined the relations among PTH/PTHrP receptor expression, PTH-activated phospholipase C (PLC) and adenylyl cyclase, and PTH-regulated phosphate transport in LLC-PK1 cells that stably express cloned rat PTH/PTHrP receptors. Among 18 such subclones, PTH stimulation of intracellular cAMP accumulation was nearly equivalent, despite differences in receptor density ranging from 20,000-400,000 sites/cell. In contrast, activation of PLC by PTH was directly and continuously dependent upon receptor density. PTH-stimulated phosphate uptake also was strongly dependent upon receptor expression, correlated well with PLC activity, was mimicked by active phorbol esters but not by cAMP analogs or forskolin, and was strikingly inhibited by the protein kinase C inhibitor, staurosporine. The peptide analog [Arg2]human PTH-(1-34), which significantly stimulated cAMP accumulation but failed to activate PLC, also did not increase phosphate uptake. We conclude that in LLC-PK1 cells, PTH-modulated PLC activation, unlike adenylyl cyclase activation, is strongly dependent upon PTH/PTHrP receptor density. This feature is reflected in the analogous relation between receptor density and PTH regulation of phosphate uptake, which appears to be mediated via a PKC-dependent pathway in these transfected cells. The results suggest that regulation of PTH/PTHrP receptor expression on target cells may provide a mechanism for altering the character as well as the magnitude of the signaling response to the hormone.

The molecular response to reductive stress in LLC-PK1 renal epithelial cells: coordinate transcriptional regulation of gadd153 and grp78 genes by thiols.

Organic thiols are toxic to eukaryotic cells. Treatment of cells with thiols activates expression of grp78, but it is not known if, like other forms of stress, there is a battery of stress response genes that are induced by thiols. In LLC-PK1 renal epithelial cells, mRNAs for both grp78 and gadd153 were induced by thiols with similar time, concentration and structure-activity dependence. Dithiothreitol (DTT) was the most potent reductant and inducer of gene expression among the thiols tested. Nuclear run-on assays demonstrated that DTT activated both grp78 and gadd153 genes transcriptionally. A hamster gadd153 promoter construct which contains enhancer elements necessary for gadd153 activation was stably integrated into the LLC-PK1 cell genome and was activated by DTT. Although auto-oxidation of thiols can generate active oxygen species, transcriptional activation of the gadd153 promoter was not due to formation of hydrogen peroxide or superoxide since neither catalase nor superoxide dismutase prevented activation of the gadd153 promoter by DTT. The concentration dependence for activation of the gadd153 promoter correlated with inhibition of dome formation and protein synthesis, two toxic effects of DTT in LLC-PK1 cells. Thus, both grp78 and gadd153 are members of a gene battery which is responsive to reductive stress. There appears to be considerable, but not complete, overlap between the upstream signaling pathways for activation of both genes.

Culturing cells without serum: lessons learnt using molecules of plant origin.

To successfully grow cells in serum-free medium is an interesting challenge to cell biology. The use of such media for in vitro cell culture work would be a key contribution to the 3Rs concept, enabling the avoidance of the use of animals and animal products at all stages of the experiment. In addition, numerous problems related to virus infections transmitted by animal serum would be avoided, thus increasing reproducibility. Prolifix is a new reagent of plant origin. It contains a molecule (GCR 1003) that has an activity similar to that of the mitogenic molecules in serum and could be suitable to substitute serum in culture medium. Two epithelial cell lines, LLC-PK1 and Caco-2, were progressively adapted to a special culture medium containing 10% Prolifix in the absence of serum. After adaptation, cell cultures were characterised. We found that these reagents of plant origin could be promising alternatives to serum. However, more work and effort is needed to improve cell adaptation, cell attachment, growth rates as well as freezing/thawing protocols.

Tyrosine phosphorylation in DNA damage and cell death in hypoxic injury to LLC-PK1 cells.

Hypoxia is classically considered to result in a necrotic form of cell injury. We have recently demonstrated a role of endonuclease activation, considered a feature of apoptosis, in DNA damage and cell death in chemical hypoxic injury to renal tubular epithelial cells (LLC-PK1 cells). Tyrosine phosphorylation has been implicated to be involved in cell signaling pathway leading to cell growth, proliferation, and apoptotic death. However, a role of tyrosine phosphorylation as a signal transduction pathway involved in DNA damage and cell death has not been previously examined in hypoxic injury in any tissue. In the present study, we have demonstrated that chemical hypoxia with a combination of antimycin A, a mitochondrial respiration inhibitor, and substrate deprivation resulted in rapid increase in protein tyrosine kinases activity and protein tyrosine phosphorylation prior to any evidence of cell death in LLC-PK1 cells. The inhibitors of protein tyrosine kinases, genistein, lavendustin A, tyrphostin, and herbimycin A provided a marked protection against chemical hypoxia-induced DNA damage (as measured by alkaline unwinding assay) and cell death (as measured by trypan blue exclusion assay). In a separate study, we confirmed the ability of the inhibitors, lavendustin A and herbimycin A to prevent chemical hypoxia-induced increase in protein tyrosine kinases activity and protein tyrosine phosphorylation. In addition, the inhibitors used did not affect ATP depletion induced by antimycin A, suggesting that the inhibitors do not alter cellular uptake of antimycin A. Taken together, our data provide a strong evidence that tyrosine phosphorylation plays as important role in DNA damage and cell death in chemical hypoxic injury to renal tubular epithelial cells.

Regulation of HSP70 by PTH: a model of gene regulation not mediated by changes in cAMP levels.

Parathyroid hormone (PTH) activates both adenylate cyclase and phospholipase C in target cells, and cloned PTH/PTH-related protein (PTHrP) receptor can mediate both responses when expressed in host cells such as LLC-PK1 renal epithelial cells. Because calcitonin (CT) is known to augment 70-kDa heat shock protein (HSP70) mRNA by an adenosine 3',5'-cyclic monophosphate (cAMP)-independent mechanism in LLC-PK1 cells, we examined regulation of HSP70 transcription by PTH in these cells. Like CT, human PTH-(1-34) [hPTH-(1-34); 10(-10) to 10(-7) M)] increased porcine HSP70 mRNA and human HSP70 promoter-chloramphenicol acetyltransferase (CAT) expression within 4 h in LLC-PK1 cells that stably express > or = 100,000 PTH/PTHrP receptors per cell. The effect of PTH on HSP70 mRNA was not mimicked by cAMP analogues, forskolin, phorbol esters, Ca2+ ionophores, or alpha-thrombin; was insensitive to pertussis toxin; and was not due to increased mRNA stability. The upregulation of HSP70 gene transcription by hPTH (and CT) was clearly observed even after deletion of the functional heat shock consensus element in the promoter region of the human HSP70/CAT reporter. Upregulation of HSP70 transcription via endogenous PTH receptors also was observed in the osteoblastic cell lines SaOS-2 and ROS 17/2.8. Regulation of HSP70 gene transcription by PTH may be a common cellular response to the hormone, which, in some cells, may not be mediated by activation of adenylate cyclase or protein kinase C.

Partial characterization of apoptotic factor in Alzheimer plasma.

We have previously demonstrated that a plasma natriuretic factor is present in Alzheimer's disease (AD), but not in multi-infarct dementia (MID) or normal controls (C). We postulated that the natriuretic factor might induce the increased cytosolic calcium reported in AD by inhibiting the sodium-calcium antiporter, thereby activating the apoptotic pathway. To test for a factor in AD plasma that induces apoptosis, we exposed nonconfluent cultured LLC-PK1 cells to plasma from AD, MID, and C for 2 h and performed a terminal transferase-dUTP-nick-end labeling (TUNEL) assay. The plasma from AD increased apoptosis nearly fourfold compared with MID and C. The effect was dose dependent and the peak effect was attained after a 2-h exposure. Additionally, apoptotic morphology was detected by electron microscopy, and internucleosomal DNA cleavage was found. We inhibited apoptosis by removing calcium from the medium, inhibiting protein synthesis with cycloheximide, alternately boiling or freezing and thawing the plasma, and digesting a partially purified fraction with trypsin. Heating AD plasma to 56 degrees C did not deactivate the apoptotic factor. These results demonstrate the presence of an apoptotic factor in the plasma of patients with AD.

Na(+)-coupled alanine transport in LLC-PK1 cells.

Transport of alanine (Ala) was characterized in LLC-PK1 renal epithelial cells. Transport capability for Ala falls by 75% in postconfluent cultures, while Na(+)-coupled alpha-methylglucoside (AMG) transport rises more than fourfold during the same interval. The kinetics of Ala transport were characterized in ATP-depleted cells to allow experimental imposition of changes in Na+ gradient and control of membrane potential across the plasma membrane. At 100 microM Ala and 135 mM Na+, > 98% of the unidirectional Ala influx is dependent on the presence of Na+ in cells from postconfluent cultures. Li+ is only 1% as effective as Na+, and other monovalent cations are ineffective in supporting Ala uptake. alpha-(Methylamino)isobutyric acid (MeAIB; 5 mM) causes only a small inhibition (approximately 10%) of 100 microM Ala influx. The low selectivity for Li+; low sensitivity to competition by MeAIB or aminoisobutyric acid; pronounced inhibition by serine, homoserine, cysteine, homocysteine and threonine; moderate inhibition by valine, isoleucine, proline and histidine; and lack of inhibition by lysine, arginine, and aspartate are more consistent with those characteristics reported for entry via the ASC amino acid transport system rather than those associated with the A system. Alanine influx exhibits a hyperbolic relationship with increasing Ala or Na+ concentration. Kinetic analysis suggests a single transport pathway with a Michaelis constant (Km) for alanine of 380 microM (when Na+ is 135 mM), apparent Km for Na+ of 32 mM (with 100 microM Ala), and a maximum velocity of 7 nmol.min-1.mg cell protein-1. An interior-negative diffusion potential induces a similar enhancement of [14C]alanine or [14C]tetraphenylphosphonium influx (approximately 40%). In contrast, AMG influx is enhanced by a factor of 2.2 under the same conditions. AMG uptake also shows a sigmoidal relationship with Na+ concentration. Hill coefficients are 1.56 for AMG and 1.0 for alanine. Direct measurement of Na(+)-Ala coupling stoichiometry yields a value of 1.01 +/- 0.07. Under the same conditions, Na(+)-AMG coupling stoichiometry is 2.1 +/- 0.25. The difference in coupling stoichiometries provides an explanation for differences in intensity of interaction between Na(+)-coupled transport systems for sugars and amino acids.

Role of polymeric Tamm-Horsfall protein in cast formation: oligosaccharide and tubular fluid ions.

BACKGROUND: In acute tubular necrosis (ATN), distal tubules are obstructed by casts formed by tubular debris, cells, and Tamm-Horsfall protein (THP). Since there are Arginine-Glycine-Aspartate (RGD) and Leucine-Aspartate-Valine (LDV) adhesive sequences in human THP, there may be direct integrin-mediated binding of tubular cells to THP. Alternatively, polymerization of THP may result in entrapment of the cells in its gel. METHODS: Adhesion of LLC-PK(1) cells to THP-coated wells was directly measured. THP concentrate was dissolved in solutions which mimic urine from ATN (ATN-S), distal convoluted tubule (DCT-S), collecting duct (CD-S), and monomeric buffer (M buffer). THP was also denatured by either boiling or N-glycanase digestion. Gel formation of THP was then measured. Inhibition of LLC-PK(1) cell adhesion to collagen type I was measured with each solution, as well as after the collagen was pretreated with either monomeric (mTHP) or polymeric (pTHP) THP. The effect of pTHP on the settling rate of LLC-PK(1) cells in suspension was also measured. RESULTS: LLC-PK(1) cells did not directly adhere to THP, a finding against integrin-mediated binding as a mechanism for in vivo tubular cell/THP cast formation. The high electrolyte concentration of ATN-S and CD-S, however, was associated with pTHP gel formation. Moreover, cells suspended in pTHP remained in suspension. In cell adhesion studies, mTHP attenuated cell adhesion by binding to the matrix, whereas pTHP attenuated cell adhesion by trapping cells in its gel. An active process was involved since both pTHP gel formation and attenuation of cell adhesion were abolished by boiling or oligosaccharide removal with N-glycanase digestion. CONCLUSIONS: With renal ischemia and proximal tubule cell shedding, ATN and collecting duct fluid composition enhance THP gel formation and thus favor tubular cast formation and obstruction. The present in vitro results indicate the importance of oligosaccharide residues in mediating the effect of the THP on gel formation and potential cast formation in ATN.

Regulation of the human Na(+)-glucose cotransporter gene, SGLT1, by HNF-1 and Sp1.

The Na(+)-glucose cotransporter (SGLT1) is expressed primarily by small intestinal epithelial cells and transports the monosaccharides glucose and galactose across the apical membrane. Here we describe the isolation and characterization of 5.3 kb of the 5'-flanking region of the SGLT1 gene by transiently transfecting reporter constructs into a variety of epithelial cell lines. A fragment (nt -235 to +22) of the promoter showed strong activity in the intestinal cell line Caco-2 but was inactive in a nonintestinal epithelial cell line (Chinese hamster ovary). Within this region, three cis-elements, a hepatocyte nuclear factor-1 (HNF-1) and two GC box sites are critical for maintaining the gene's basal level of expression. The two GC boxes bind to several members of the Sp1 family of transcription factors and, in the presence of HNF-1, synergistically upregulate transactivation of the promoter. A novel 16-bp element just downstream of one GC box was also shown to influence the interaction of Sp1 to its binding site. In summary, we report the identification and characterization of the human SGLT1 minimal promoter and the critical role that HNF-1 and Sp1-multigene members have in enhancing the basal level of its transcription in Caco-2 cells.

cAMP motifs regulating transcription in the aquaporin 2 gene.

Genomic clones including the 5' flanking regions of the AQP2 (aquaporin 2) gene were isolated, and the promoter region was examined by transiently transfecting a promoter-luciferase reporter fusion gene into renal cultured epithelial cells. An orientation specific promoter for the AQP2 gene was found within the proximal 3 kb of 5'-flanking region. Minimal basal promoter activity of the AQP2 gene was found within 198 bp upstream from the transcription start site by deletion analysis. Sequencing the transcriptionally active region revealed a typical TATA box, adenosine 3',5'-cyclic monophosphate (cAMP) responsive element (CRE) and three putative CCAAT boxes in the proximal 1.2-kb region. Significantly, a GATA motif, AP1, AP2, and SP1 transcriptional factor consensus sites were also found in this region. Exposure to cAMP-enhancing agents (1 nM vasopressin or 20 mM forskolin and 250 mM 3-isobutyl-1-methylxanthine) showed that these agents increased luciferase activity in a parallel fashion, suggesting that vasopressin-induced AQP2 gene transcription is mediated through increases in intracellular cAMP in at least one renal cell type, the LLC-PK1 cells. The mechanism of cAMP responsiveness of AQP2 gene transcription was further studied using a series of deletion mutants in renal epithelial cells and other cell types. The cAMP regulatory motifs were shown to exist in a 50-bp sequence between -340 and -290 (containing CRE) and a 65-bp sequence (containing an AP2 site) between -150 and the ATG start site in LLC-PK1 cells. In rat inner medullary collecting duct (IMCD) cells, the cAMP regulatory motifs also exist in a 50-bp sequence between -340 and -290 (containing CRE) and in a 10-bp sequence between -160 and -150 (containing an SP1 site). These separate regions may cooperate to confer full cAMP inducibility to the AQP2 gene in a cell-specific manner.

A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells.

Although polarized epithelial cells are well known to maintain distinct apical and basolateral plasma membrane domains, the mechanisms responsible for targeting membrane proteins to the apical or basolateral surfaces have remained elusive. We have identified a novel form of the AP-1 clathrin adaptor complex that contains as one of its subunits mu1B, an epithelial cell-specific homolog of the ubiquitously expressed mu1A. LLC-PK1 kidney epithelial cells do not express mu1B and missort many basolateral proteins to the apical surface. Stable expression of mu1B selectively restored basolateral targeting, improved the overall organization of LLC-PK1 monolayers, and had no effect on apical targeting. We conclude that basolateral sorting is mediated by an epithelial cell-specific version of the AP-1 complex containing mu1B.

Tissue remodeling during tumor necrosis factor-induced apoptosis in LLC-PK1 renal epithelial cells.

The cytokine tumor necrosis factor-alpha (TNF) increases the frequency of apoptosis in confluent renal epithelial LLC-PK1 cells, an effect that can be blocked by an anti-TNFR1 monoclonal antibody. However, there were no visible "holes" in the cell sheet as a result of TNF-induced apoptosis. Instead a striking tissue remodeling occurred in response to the TNF-induced apoptosis. Apoptotic cells became surrounded and engulfed by repositioned neighboring cells distributed in a distinct "rosette" pattern. The cadherin-catenin cell-cell adhesion molecules, the tight junction-associated protein ZO-1, and actin accumulated at the sites of contact between apoptotic and neighboring cells. Pretreatment with cytochalasin B prevented the accumulation of cadherins-catenins and ZO-1 at the sites of apoptosis and resulted in microscopic holes in the TNF-treated cell sheet. Our results indicate that a renal epithelium can accommodate an increased frequency of apoptosis and still maintain its integrity by mechanisms of tissue remodeling involving the cadherin-catenin adhesion molecules, tight junctional proteins, and actin filaments.

Cisplatin induces apoptosis in LLC-PK1 cells via activation of mitochondrial pathways.

Cisplatin, a commonly used chemotherapeutic agent, has a major limitation because of its nephrotoxicity. Recent studies have shown that cisplatin causes apoptotic cell death in renal tubule cells, but the underlying molecular mechanisms remain to be elucidated. In this study, cisplatin was found to induce apoptosis in a dose- and duration-dependent manner in cultured proximal tubule (LLC-PK1) cells, as evidenced by DNA laddering and TdT-mediated dUTP nick end-labeling assay. Pretreatment with the specific caspase 9 inhibitor LEHD-CHO completely prevented the apoptosis, whereas the caspase 8 inhibitor IETD-fmk had no effect. Furthermore, the activity of caspase 9 was upregulated about sixfold by cisplatin in a dose-dependent manner. These results implicated the caspase 9-dependent mitochondrial apoptotic pathways. Indeed, cisplatin triggered a duration-dependent translocation of cytochrome c from the mitochondria to the cytosol, by immunofluorescence and Western blots. Cisplatin treatment also resulted in the duration-dependent activation and mitochondrial translocation of the pro-apoptotic molecule Bax, by immunofluorescence. Finally, cisplatin induced a duration-dependent onset of the mitochondrial permeability transition. Our results indicate that cisplatin induces apoptosis in LLC-PK1 cells via activation of mitochondrial signaling pathways. The sequence of events may be summarized as follows: activation of Bax induces mitochondrial permeability transition, leading to release of cytochrome c, activation of caspase 9, and entry into the execution phase of apoptosis. Inhibition of this specific pathway may provide a strategy to minimize cisplatin-induced nephrotoxicity.

Activation of peroxisome proliferator-activated receptor-gamma inhibits apoptosis induced by serum deprivation in LLC-PK1 cells.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) belongs to a superfamily of nuclear receptors, which plays important roles in lipid and glucose metabolism. However, expression of PPARgamma in extra-adipose tissues and stimulation of apoptosis by PPARgamma activators has been previously reported. We investigated the functions of PPARgamma using a clonal kidney cell line (LLC-PK1). RT-PCR revealed the expression of PPARgamma in LLC-PK1 cells. The cells accumulated fat droplets and increased beta-oxidation of free fatty acids in response to troglitazone, a ligand for PPARgamma. At physiological concentrations, ligands for PPARgamma including troglitazone, BRL49653, and 15-deoxy-delta-12,14-prostaglandin J(2) inhibited serum-deprivation-induced apoptosis of the cells. On the other hand, PPARalpha activators did not inhibit the apoptosis. Apoptosis of LLC-PK1 cells was determined by a cell viability assay, condensation of the nucleus on fluorescent and electron microscopy, and DNA fragmentation as indicated by the appearance of nucleosomal ladders on an agarose gel. Troglitazone also suppressed serum-deprivation-induced activation of Caspase 3. However, troglitazone did not suppress apoptosis induced by ATP deprivation. Anti-apoptotic effects of troglitazone were partially blocked by a phosphatidylinositol-3-kinase (PI3K) inhibitor, wortmannin, but not by other kinase inhibitors such as PD98059 and AG490. These results suggest that PPARgamma is functionally expressed in LLC-PK1 cells, and its activation inhibits apoptosis induced by serum deprivation, at least in part, through the PI3K pathway.

Direct targeting of neutral endopeptidase (EC 3.4.24.11) to the apical cell surface of transfected LLC-PK1 cells and unpolarized secretion of its soluble form.

LLC-PK1 cells were transfected with a cDNA encoding rabbit neutral endopeptidase (NEP; EC 3.4.24.11), an abundant enzyme of the kidney proximal brush border. Clones of cells expressing high levels of the protein were isolated. Selective biotinylation and radioimmunolabelling were used to determine that 85-95% of NEP was localized in the apical domain of filter-grown LLC-PK1 cells. Pulse-chase and selective biotinylation studies revealed that the majority (85%) of newly made NEP was directly targeted to the apical membrane. However, a soluble form of NEP was found to be secreted in approximately equal amounts from both sides of the monolayer when expressed in LLC-PK1 cells. Transfected pro-opiomelanocortin, a pituitary hormone precursor, was secreted almost exclusively into the basolateral medium, suggesting that the bulk flow is to the basolateral membrane. This behaviour contrasts with that observed in MDCK cells, where both the transmembrane and secreted forms of NEP are directly targeted to the apical membrane and where the secretion of pro-opiomelanocortin is unpolarized.

Calcitonin stimulates lysosomal enzyme release and uptake in LLC-PK1 cells.

Renal tubular targeted hormones increase urinary excretion of a lysosomal enzyme, N-acetyl-beta-D-glucosaminidase (NAG). To elucidate the mechanism of this event, the calcitonin effect on NAG handling by LLC-PK1 cells was examined. Calcitonin (1 nM to 1 microM), phorbol myristate (10 nM to 1 microM), and ionomycin (1 to 10 microM) promoted NAG release without any increase in lactate dehydrogenase release or any reduction of mitochondrial dehydrogenase activity. Treatment with 100 nM calphostin C or 50 microM KN-93 partially reversed the calcitonin effect on NAG release. Calcitonin promoted secretion of fluorescence ceramide, a reporter of protein transport from Golgi apparatus to cell surface. Calcitonin-stimulated NAG release was partially inhibited by 10 microg/ml brefeldin A, a blocker of protein transport through the Golgi apparatus. Calcitonin accelerated cellular uptake of exogenous NAG, which was inhibited by low temperature, 0.1 mM monodansyl cadaverine (receptor-mediated endocytosis inhibitor), and 10 mM mannose-6-phosphate. Furthermore, calcitonin promoted progression of intracellular membranes stained by a fluorescence membrane marker, styryl pyridinium dye, from cell periphery to perinuclear regions (commonly referred to as recycling vesicles) and increased dye release from preloaded cells. Fluorescence release from the cells pre-loaded with FITC-labeled NAG or albumin was also stimulated by calcitonin. These calcitonin effects on endocytotic and re-exocytotic pathways were inhibited by 100 nM cytochalasin D, 100 nM nocodazole, 0.1 to 1 microM bafilomycin A1, or 0.1 mM monodansyl cadaverine. Increased urinary NAG excretion has been considered to reflect renal tubular damage. However, it was demonstrated here that stimulation of secretory and recycling pathways may be an alternative mechanism for calcitonin-induced enzymuria, which will become a new indicator of renal tubular response to this hormone.

Chronic exposure of LLC-PK1 epithelia to the phorbol ester TPA produces polyp-like foci with leaky tight junctions and altered protein kinase C-alpha expression and localization.

Acute exposure (up to 4 h) of LLC-PK1 epithelial cell sheets to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a rapid decrease of transepithelial electrical resistance (TER) to less than 15% of initial value. As the TPA exposure period is continued by chronically passaging cells in the presence of TPA, TER begins to recover. By 6 weeks of exposure, TER recovers to almost 50% of its initial value, suggesting that tight junctions (TJs) are recovering barrier function even in the continued presence of TPA. Between 6 and 8 weeks, TER then decreases a second time to approximately 20 to 40% of initial values, and TER values remain at this level for at least 18 weeks of exposure. Transepithelial (paracellular) fluxes of D-mannitol inversely correspond with TER changes. Across chronically treated cell sheets, rates are higher than those across control cell sheets, but lower than those across acutely treated cell sheets. The decrease of TER at 6-8 weeks coincides with the appearance of multilayered, polyp-like foci (PLFs) on the otherwise one cell layer thick epithelium. Electron microscopy shows that the electron-dense dye ruthenium red cannot penetrate across TJs of control cells but passes across all of the TJs of a cell sheet treated acutely with TPA. In chronically treated cultures, ruthenium red penetrates TJs between most cells of PLFs, but not TJs of adjacent morphologically normal epithelium. A clonal subline derived from cells of a PLF (clone PLF-A) is multilayered almost throughout and exhibits ruthenium red penetration across nearly all of its tight junctions, monolayer or multilayer. Acute exposure of control cell sheets to TPA induces activation, translocation, and down-regulation of protein kinase C-alpha (PKC-alpha). In chronically TPA-treated and clone PLF-A cells, total PKC-alpha levels are reduced even further and almost all remaining PKC-alpha is found in the membrane-associated and Triton-insoluble fractions. Immunofluorescence shows that PKC-alpha expression is restricted to the PLFs in chronically TPA-treated cells and is more homogeneously distributed in clone PLF-A cultures. In summary, the data show that chronic treatment of epithelial cells with a tumor promoter induces the formation of abnormal cell architecture (PLFs) associated with increased leakiness of TJs and membrane translocation of PKC-alpha. Recovery of barrier function in portions of chronically TPA-treated cultures does not correlate with up-regulation of PKC-alpha nor translocation back to the cytosolic compartment.