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.

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.

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.

Role of cytochrome P4502E1 activation in proximal tubular cell injury induced by hydrogen peroxide.

BACKGROUND: There is now good evidence to suggest that cytochrome P450 (CYP450) may act as an iron-donating catalyst for the production of hydroxyl ion (OH*), which contributes to proximal tubular cell injury. However, it remains unclear which isoform of CYP450 is involved in this process. Cytochrome P4502E1 (CYP2E1) is a highly labile isoform which is not only involved in free radical generation, but has also been shown to be a source of iron in cisplatin-induced renal injury. This study investigates the role of CYP2E1 in the proximal tubular cell injury induced by hydrogen peroxide (H2O2). METHODS: Porcine proximal tubular cells (LLC-PK1) were incubated with H2O2 (1 mM) for 4 h in the presence or absence of 0.1 mM of two CYP2E1 inhibitors; diallyl sulfide (DAS), or disulfiram (DSF), desferrioxamine (DFO) (0.1-0.4 mM), or catalase (CT) (78, 150, 300 U/mL). Cell death was determined by measuring LDH release. CYP2E1 activity was determined by p-nitrophenol hydroxylation after 2 h incubation with H2O2. RESULTS: Exposure of LLC-PKI to H2O2 significantly increased cell death. CT, DFO, DAS and DSF significantly reduced H2O2-mediated cell death. Incubation with H2O2 increased CYP2EI activation in time- and dose-dependent manner, which was significantly reduced by CT, DFO, DAS and DSF. CONCLUSION: We propose that CYP2E1 activation occurs possibly due to OH* and contributes to H2O2-mediated LLC-PK1 cell necrosis by acting as a source of iron and perpetuating the generation of OH* via the Fenton reaction. Inhibition of CYP2E1 may be a novel approach for the prevention of tubular injury caused by oxidative stress.

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.

Cyclic AMP reverses radiocontrast media-induced apoptosis in LLC-PK1 cells by activating A kinase/PI3 kinase.

BACKGROUND: Radiographic contrast material is one of agents that are prone to cause nephropathy, although little is known about cellular mechanisms underlying contrast media-induced renal failure. The present study was designed to determine the role of caspase in contrast media-induced renal injury. The modulation by cyclic adenosine monophosphate (cAMP) of cell injury was subsequently examined. METHODS: LLC-PK1 cells (a proximal renal tubular cell line of porcine origin) were exposed to diverse contrast media for 30 minutes followed by incubation for 24 hours in normal medium. Cell viability was assessed by mitochondrial enzyme activity and propidium iodide stain. Apoptosis was determined by DNA electrophoresis and annexin V stain. Caspase activity was measured fluorometrically. The mRNA for bax and bcl-2 was determined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Iodinated and magnetic resonance contrast media reduced cell viability due to apoptosis. The cell damage induced by a non-ionic contrast medium ioversol was inhibited by specific inhibitors for caspase-3 and -9 but not caspase-8. Ioversol enhanced the activities of caspase-3 and -9, but to a lesser extent, caspase-8. The bax mRNA was enhanced, while bcl-2 mRNA was reduced, after exposure to ioversol. All of these actions of ioversol were reversed by dibutyl cAMP in the manner sensitive to a protein kinase A inhibitor H89 and a phosphatidylinositol 3 (PI3) kinase inhibitor wortmannin. CONCLUSION: We demonstrated for the first time that cAMP reversed caspase-dependent apoptotic renal cell damage caused by contrast media. Both protein kinase A and PI3 kinase might be involved in protective effect of cAMP.

Effects of gentamicin, lipopolysaccharide, and contrast media on immortalized proximal tubular cells.

Aminoglycosides are widely used in the treatment of gram-negative bacterial infections. Gentamicin (GE) acts mainly in proximal tubular cells, where it is uptake via organic anion transport system and it induces a high incidence of nephrotoxicity, which is characterized by tubular necrosis [5] leading to acute renal failure in 10 to 50% of patients. Gram-negative bacteria has lipopolysaccharide (LPS) which is an endotoxin that cause renal damage. [1] Moreover, many patients are undergone exams using radiologic contrast, which is a risk factor to induce a hemodynamic change in the kidney and to develop acute renal failure. [6] Intracellular calcium [Ca2+]i is involved in renal cellular injury [7,3] and maybe mediate the effects provoked by these drugs. This study was performed to evaluate necrosis, apoptosis, and intracellular calcium levels ([Ca2+]i) in LLC-PK1 (epithelial cell line from pig kidney) induced by GE associated with LPS and a low-osmolality media, Hexabrix (HE).

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.

Effects of gentamicin, lipopolysaccharide, and contrast media on immortalized proximal tubular cells.

Aminoglycosides are widely used in the treatment of gram-negative bacterial infections. Gentamicin (GE) acts mainly in proximal tubular cells, where it is uptake via organic anion transport system and it induces a high incidence of nephrotoxicity, which is characterized by tubular necrosis leading to acute renal failure in 10 to 50% of patients. Gram-negative bacteria have lipopolysaccharide (LPS) which is an endotoxin that causes renal damage. Moreover, many patients are undergone exams using radiologic contrast, which is a risk factor to induce a hemodynamic change in the kidney and to develop acute renal failure. Intracellular calcium [Ca2+]i is involved in renal cellular injury and maybe mediate the effects provoked by these drugs. This study was performed to evaluate necrosis, apoptosis and intracellular calcium levels ([Ca2+]i) in LLC-PK1 (epithelial cell line from pig kidney) induced by GE associated with LPS and a low-osmolality media, Hexabrix (HE).

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.

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.

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.

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.

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.

Up- and down-expression of the dopamine transporter by plasmid DNA transfer in the rat brain.

The functional role of the dopamine transporter (DAT) in central dopaminergic neurotransmission was assessed further by investigating the consequences on dopamine (DA) turn-over of up- and down-regulation of this protein induced by a non-viral gene transfer approach. For this purpose, expression plasmids containing the sense or antisense coding sequence of DAT complexed with the cationic polymer, polyethylenimine (PEI), were injected into the rat substantia nigra, the brain region containing the majority of DA cell bodies. Before in vivo injection, the efficacies of the different DNA constructs were assessed by transfection studies into LLC-PK1 cells. Stereotaxic administration of the sense plasmid complexed to PEI induced, 3 days later, a significant increase in the immunoautoradiographic labelling by anti-DAT antibodies of the substantia nigra and various DA projection areas. These effects were associated with a significantly enhanced capacity of striatal synaptosomes to take up [3H]-DA and lasted up to 14 days postinjection. In contrast, 7 days after intranigral administration of the antisense plasmid complexed to PEI, we observed a significant decrease of DAT immunolabelling in the substantia nigra and [3H]-DA uptake by striatal synaptosomes. Whereas DA turnover in the striatum was unaltered 3 days after intranigral administration of the sense plasmid, it was increased 7 days after intranigral administration of the antisense construct. These data indicate that non-viral transfer of the sense or antisense coding sequence of DAT can be used as a novel approach to induce long-term changes in central DA neurotransmission.

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.

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.

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.

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.