Confluent monolayers of LLC-PK1 cells, an epithelial cell derived from a normal pig kidney, contain high levels of alkaline phosphatase and gamma-glutamyl transpeptidase activities. Inhibition studies show a close similarity between alkaline phosphatase and the so-called liver-bone-kidney isoenzyme. Nearly complete recovery of both activities in the microsomal fraction demonstrates the membrane-bound characteristics of these enzymes. Histochemical localization of the enzymes activities on the apical membrane of LLC-PK1 cells confirms this observation. The activity of both enzymes decreases to very low levels when the cells are in exponential growth. Confluent monolayers of LLC-PK1 cells plated at saturation density with cells that were in active growth show a progressive increase in the activity of alkaline phosphatase and gamma-glutamyl transpeptidase. This increase is dependent on the synthesis de novo of RNA and protein and supports the conclusion that the activity of both enzymes is regulated at the transcriptional level. The development of these enzymes is delayed with respect to the development of the occluding junctions. This delay and the direct appearance of alkaline phosphatase activity in the apical membrane indicate that the different components of this membrane are inserted after the limits of the membrane have been established by the synthesis and assembly of the occluding junctions. Alkaline phosphatase activity of confluent monolayers can be further induced by reducing the concentration of phosphate in the medium. When the junctions are dissociated by incubating the monolayers in Ca2+-free medium, the alkaline phosphatase activity migrates freely beyond the limits of the apical membrane until it covers the entire cell surface. Both enzymes are synthesized even in the absence of contact between the cells, suggesting that the occluding junctions in LLC-PK1 monolayers are involved in the polarized distribution, but not in the modulation, of the synthesis of these enzymes. In addition, the progressive decrease in enzyme synthesis that is obtained by reducing the cell-substratum adhesion supports the idea that it is the cell-to-substrate and not the cell-to-cell interaction which is involved in the modulation of these enzymatic markers of the apical membrane.

Total internal reflection (TIR) microfluorimetry was established as a method to measure continuously the volume of adherent cells and applied to measure membrane permeabilities in cells transfected with water channel homologs. Cytosol was labeled with the membrane-impermeant fluorophore calcein. Fluorescence was excited by the TIR evanescent field in a thin section of cytosol (approximately 150 nm) adjacent to the cell-substrate interface. Because cytosolic fluorophore number per cell remains constant, the TIR fluorescence signal should be inversely related to cell volume. For small volume changes in Sf-9 and LLC-PK1 cells, relative TIR fluorescence was nearly equal to inverse relative cell volume; deviations from the ideal were modeled theoretically. To measure plasma membrane osmotic water permeability, Pf, the time course of osmotically induced cell volume change was inferred from the TIR fluorescence signal. LLC-PK1 cells expressing the CHIP28 water channel had an HgCl2-sensitive, threefold increase in Pf compared to nontransfected cells (Pf = 0.0043 cm/s at 10 degrees C). Solute permeability was measured from the TIR fluorescence time course in response to solute gradients. Glycerol permeability in Sf-9 cells expressing the water channel homolog GLIP was (1.3 +/- 0.2) x 10(-5) cm/s (22 degrees C), greater than that of (0.36 +/- 0.04) x 10(-5) cm/s (n = 4, p < 0.05) for control cells, indicating functional expression of GLIP. Water and urea permeabilities were similar in GLIP-expressing and control cells. The TIR method should be applicable to the study of water and solute permeabilities and cell volume regulation in cells of arbitrary shape and size.

Alkaline titrations of different samples of poly(dG).poly(dC) and of the constituent homopolymers poly(dG) and poly(dC) have been performed in 0.15 M NaCl and their CD spectra followed. Sample I contained a slight excess of poly(dC) (52% C: 48% G) and showed a single reversible transition (pK = 11.9) due to the dissociation of double stranded poly(dG).poly(dC). Sample II, containing an excess of poly(dG) (43% C: 57% G), showed two transitions (pK1 = 11.4, PK2 = 11.9) the first one being only partially reversible. Examination of the CD spectra along the alkaline titrations indicated the presence of another hydrogen-bonded complex of higher G content. Mixing curves performed at pH 8 have confirmed the presence of a 2G: 1C complex, besides the double stranded complex. It can be formed in amounts up to 30% by mixing the two homopolymers, alkali treatment and heating. The CD spectra of the two complexes have been computed from the CD data of the mixing curves. This permitted the determination of the concentrations of both complexes and homopolymers in all samples. The ratio of triple to double stranded complex is not only dependent on the G/C ratio of the sample, but also a function of the previous physico-chemical conditions. These results explain the variability of many properties of different poly(dG).poly(dC) samples observed by other workers.

The last decade has seen successful clinical application of polymer-protein conjugates (e.g. Oncaspar, Neulasta) and promising results in clinical trials with polymer-anticancer drug conjugates. This, together with the realisation that nanomedicines may play an important future role in cancer diagnosis and treatment, has increased interest in this emerging field. More than 10 anticancer conjugates have now entered clinical development. Phase I/II clinical trials involving N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (PK1; FCE28068) showed a four- to fivefold reduction in anthracycline-related toxicity, and, despite cumulative doses up to 1680 mg/m2 (doxorubicin equivalent), no cardiotoxicity was observed. Antitumour activity in chemotherapy-resistant/refractory patients (including breast cancer) was also seen at doxorubicin doses of 80-320 mg/m2, consistent with tumour targeting by the enhanced permeability (EPR) effect. Hints, preclinical and clinical, that polymer anthracycline conjugation can bypass multidrug resistance (MDR) reinforce our hope that polymer drugs will prove useful in improving treatment of endocrine-related cancers. These promising early clinical results open the possibility of using the water-soluble polymers as platforms for delivery of a cocktail of pendant drugs. In particular, we have recently described the first conjugates to combine endocrine therapy and chemotherapy. Their markedly enhanced in vitro activity encourages further development of such novel, polymer-based combination therapies. This review briefly describes the current status of polymer therapeutics as anticancer agents, and discusses the opportunities for design of second-generation, polymer-based combination therapy, including the cocktail of agents that will be needed to treat resistant metastatic cancer.

To investigate renal tubular epithelial cell injury mediated by reactive oxygen molecules and to explore the relative susceptibility of epithelial cells and endothelial cells to oxidant injury, we determined cell injury in human umbilical vein endothelial cells and in four renal tubular epithelial cell lines including LLC-PK1, MDCK, OK and normal human kidney cortical epithelial cells (NHK-C). Cells were exposed to reactive oxygen molecules including superoxide anion, hydrogen peroxide and hydroxyl radical generated by xanthine oxidase and hypoxanthine. We determined early sublethal injury with efflux of 3H-adenine metabolites and a decline in ATP levels, while late lytic injury and cell detachment were determined by release of 51chromium. When the cells were exposed to 25, 50, and 100 mU/ml xanthine oxidase with 5.0 mM hypoxanthine, ATP levels were significantly lower (P less than 0.001) in LLC-PK1, NHK-C and OK cells compared to MDCK cells while ATP levels were significantly lower (P less than 0.01) in endothelial cells compared to all tubular cell lines. A similar pattern of injury was seen with efflux of 3H-adenine metabolites. When the cells were exposed to 50 mU/ml xanthine oxidase with 5.0 mM hypoxanthine for five hours, total 51chromium release was significantly (P less than 0.001) greater in LLC-PK1, NHK-C and OK cells compared to MDCK cells, while total 51chromium release was significantly (P less than 0.001) greater in endothelial cells compared to all tubular cells. However, lytic injury was the greatest in LLC-PK1 cells and NHK-C cells while cell detachment was the greatest in endothelial cells. MDCK cells were remarkably resistant to oxidant-mediated cell detachment and cell lysis. In addition, we determined ATP levels, 3H-adenine release and 51chromium release in LLC-PK1, NHK-C and endothelial cells in the presence of superoxide dismutase to dismute superoxide anion, catalase to metabolize hydrogen peroxide, DMPO to trap hydroxyl radical and DMTU to scavenge hydrogen peroxide and hydroxyl radical. We found that catalase and DMTU (scavengers of hydrogen peroxide) provided significant protection from ATP depletion, prevented efflux of 3H-adenine metabolites and cell detachment while DMPO (scavenger of hydroxyl radical) prevented lytic injury. In addition, we found that the membrane-permeable iron chelator, phenanthroline, and preincubation with deferoxamine prevented cell detachment and cell lysis, confirming the role of hydroxyl radical in cell injury.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND

Peroxidation of renal cells is a critical event in the nucleation and formation of calcium oxalate crystals under hyperoxaluric conditions. We previously demonstrated that oxalate-induced peroxidative injury is one of the major mechanisms in promoting crystal attachment to renal epithelial cells.

METHODS

In this study we have demonstrated that the mechanism of oxalate-induced peroxidative injury is through the induction of TGF-beta1 and glutathione (GSH) redox imbalance in LLC-PK1 cells.

RESULTS

LLC-PK1, renal epithelial cells exposed to oxalate had significantly higher reactive oxygen species (ROS) production; higher TGF-beta1 levels, as measured by ELISA (1.89 +/- 0.035 fold increase) or Western blot (1.65 +/- 0.01 fold increase); increased malondialdehyde formation; increased LDH release, and loss of cell viability. In addition, oxalate exposure significantly decreased GSH content, glutathione reductase, glucose-6-phosphate dehydrogenase activities, and increased oxidized GSH content. Treatment with vitamin E, neutralizing anti-TGF-beta antibody, or diphenylene iodium, an inhibitor of NAD(P)H oxidase, significantly inhibited oxalate-induced ROS production and prevented peroxidative injury and cytolysis. Vitamin E, catalase, or desferoxamine treatment also significantly restored the oxalate-induced cellular GSH redox status toward the control level, and vitamin E treatment significantly attenuated the oxalate-mediated increase in TGF-beta1 protein in cultured LLC-PK1 cells.

CONCLUSIONS

This is the first study to demonstrate that the mechanism of oxalate-induced free radical production in renal tubular epithelial cells is through the activation of NAD(P)H oxidase via cytokine TGF-beta1 induction. These results also provide direct evidence that antioxidant therapy might prevent calcium oxalate nucleation and kidney stone formation by preventing oxalate-mediated peroxidative injury and GSH redox imbalance.

The prevalence of Toxoplasma gondii in 84 free-range chickens (34 from the northern Pará state, and 50 from Rio Grande do Sul, the southern state) from Brazil, South America was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 39 (46.4%) of 84 chickens with titers of 1:10 in one, 1:20 in two, 1:40 in four, 1:80 in seven, 1:160 in five, 1:320 in six, 1:640 in eight and>> or =1:1280 in six. Hearts and brains of 45 chickens with titers of 1:20 or less were pooled and fed to two T. gondii-free cats. Hearts and brains of 39 chickens with titers of 1:10 or higher were bioassayed in mice. Feces of cats were examined for oocysts. One cat fed tissues from 31 chickens with titers of less than 1:10 from Rio Grande do Sul shed T. gondii oocysts. T. gondii was isolated by bioassay in mice from 33 chickens with MAT titers of 1:20 or higher. All infected mice from 10 isolates died of toxoplasmosis. All 34 isolates (15 from Pará, 19 from Rio Grande do Sul) were genotyped using 11 genetic markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, a new SAG2 and Apico. Eleven genotypes were revealed for Pará isolates and seven genotypes for Rio Grande do Sul. No genotype was shared between the two geographical locations. These data suggest that T. gondii isolates are highly diverse and genetically distinct between the two different regions in Brazil that are 3500 km apart.

The synthesis of new capped silica mesoporous nanoparticles for on-command delivery applications is described. The gate-like functional hybrid systems consisted of nanoscopic MCM-41-based materials functionalized on the pore outlets with different "saccharide" derivatives and a dye contained in the mesopores. A series of hydrolyzed starch products as saccharides were selected. The mesoporous silica nanoparticles S1, S2, and S3 containing the grafted starch derivatives Glucidex 47, Gludicex 39, and Glucidex 29 were synthesized. Additionally, for comparative purposes solid S4 containing lactose was prepared. Delivery studies in pure water in the presence of pancreatin or β-d-galactosidase were carried out for S1-S3 and S4, respectively. S1, S2, and especially S3 showed very low release in the absence of enzyme, but displayed cargo delivery in the presence of the corresponding enzyme. Moreover, nanoparticles of S1 were used to study the controlled release of the dye in intracellular media. Cell viability assays using HeLa and LLC-PK1 cells indicated that S1 nanoparticles were devoid of unspecific cell toxicity. The endocytosis process for S1 nanoparticle internalization in HeLa cells was confirmed, and the anchored starch was degraded by the lysosomal enzymes. Furthermore, a new mesoporous silica nanoparticle functionalized with Glucidex 47 and loaded with a cytotoxic, S1-DOX, was developed. The cell viability with S1-DOX decreased due to the internalization of the nanoparticle, enzyme-dependent opening of the saccharide molecular gate and the consequent release of the cytotoxic agent. As far as the authors know, this is the first example of enzyme-induced in-cell delivery using capped silica mesoporous nanoparticles.

A P-glycoprotein (P-gp) IC₅₀ working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC₅₀ determinations. Each laboratory followed its in-house protocol to determine in vitro IC₅₀ values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC₅₀ values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC₅₀ values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC₅₀ values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC₅₀ values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC₅₀ values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC₅₀ determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.

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.

OBJECTIVE

Formation of calcium oxalate (CaOx) kidney stones was investigated using three approaches.

METHODS

Pathogenesis of crystalluria and crystal deposition in the kidneys was examined in vivo by inducing hyperoxaluria in rats. Cultures of LLC-PK1 and MDCK cells were exposed to oxalate (Ox) and CaOx crystals to examine the effect on cells in various sections of the renal tubules. The nucleation potential of various substrates was examined by incubating them in metastable solutions of CaOx.

RESULTS

Calcium oxalate crystals and nonphysiologic levels of Ox were injurious to renal epithelial cells. To combat these nephrotoxins, renal epithelial cells produce a variety of macromolecules such as bikunin and osteopontin, which are modulators of crystallization and may also be involved in crystal attachment to the cells. Sustained hyperoxaluria in association with CaOx crystals induced apoptosis as well as necrosis. Cellular degradation products induced heterogeneous nucleation of crystals at lower and physiologic levels of oxalate. They also promoted aggregation.

CONCLUSIONS

Crystals begin to form in earlier segments of the nephron with a chance to grow and be retained in the collecting ducts after aggregation with other large crystals. Crystal deposits in the collecting ducts near the papillary surface, when exposed to the renal pelvic urine, become nidi for the development of kidney stones.

1. Human 5-HT1B (h5-HT1B) and human 5-HT1D (h5-HT1D) receptors show remarkably similar pharmacology with few compounds discriminating the receptors. We report here on a novel compound, SB-224289 (1'-Methyl-5-[[2'-methyl-4'-(5-methyl- 1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydro spiro [furo [2,3-f]indole-3,4'-piperidine] oxalate), which has high affinity for h5-HT1B receptors (pK1=8.16+/-0.06) and displays over 75 fold selectivity for the h5-HT1B receptor over all other 5-HT receptors including the h5-HT1D receptor and all other receptors tested thus far. 2. Functional activity of SB-224289 was measured in a [15S]GTPgammaS binding assay on recombinant h5-HT1B and h5-HT1D receptors expressed in Chinese Hamster Ovary (CHO) cells. SB-224289 displayed negative intrinsic activity at both receptors with higher potency at h5-HT1B receptors. SB-224289 caused a rightward shift of agonist concentration response curves consistent with competitive antagonism and generated affinities comparable with those obtained from competition radioligand receptor binding studies. 3. SB-224289 potentiated [3H]5-HT release from electrically stimulated guinea-pig cerebral cortical slices to the same extent as as the non-selective 5-HT1 antagonist methiothepin. SB-224289 also fully reversed the inhibitory effect of exogenously superfused 5-HT on electrically stimulated release. 4. Using SB-224289 as a tool compound, we confirm that in guinea-pig cerebral cortex the terminal 5-HT autoreceptor is of the 5-HT1B subtype.

Until recently, Toxoplasma gondii was considered clonal with very little genetic variability. Recent studies indicate that T. gondii isolates from Brazil are genetically and biologically different from T. gondii isolates from USA and Europe. In the present study, we retyped 151 free range chicken isolates from Brazil including 117 newly isolated samples from 11 geographically areas (Alagoas, Bahia, Ceará, Maranhão, Paraná, Pernambuco, Rio de Janeiro, Rio Grande do Norte, São Paulo, Sergipe, and Rondonia) and 34 previously reported isolates from the very north (Pará) and the very south (Rio Grande do Sul). Ten PCR-RFLP markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico were used to genotype all isolates. Overall analysis of 151 T. gondii isolates revealed 58 genotypes. Half (29/58) of these genotypes had single isolate and the other half of the genotypes were characterized with two or more isolates. Only 1 of 151 isolates was clonal Type I strain and 5 were clonal Type III strains. Two isolates had mixed infections. Clonal Type II strain was absent. One strain was Type II at all loci, except BTUB. The results confirm high genetic diversity of T. gondii isolates from Brazil.

Epithelial cells accumulate distinct populations of membrane proteins at their two plasmalemmal domains. We have examined the molecular signals which specify the differential subcellular distributions of two closely related ion pumps. The Na,K-ATPase is normally restricted to the basolateral membranes of numerous epithelial cell types, whereas the H,K-ATPase is a component of the apical surfaces of the parietal cells of the gastric epithelium. We have expressed full length and chimeric H,K-ATPase/Na,K-ATPase cDNAs in polarized renal proximal tubular epithelial cells (LLC-PK1). We find that both the alpha and beta subunits of the H,K-ATPase encode independent signals that specify apical localization. Furthermore, the H,K-ATPase beta-subunit possesses a sequence which mediates its participation in the endocytic pathway. The interrelationship between epithelial sorting and endocytosis signals suggested by these studies supports the redefinition of apical and basolateral as functional, rather than simply topographic domains.

The pig kidney cell line LLC-PK1 cultured on a collagen coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by occluding junctions. A transepithelial electrical potential (PD) and short-circuit current (SCC) were dependent on the presence of Na and sugar in the apical bathing solution. In the presence of 5.5 mM D-glucose, a PD of 2.8 mV. apical surface negative a SCC of 13 microA cm-2 and transepithelial resistance of 211 ohm.cm2 were recorded. The SCC was promptly reduced by the addition of phlorizin to the apical bath but unaffected when placed in the basolateral bath. The effect on SCC of various sugars was compared by the concentrations required for half-maximal SCC: 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methyl-glucose. When [Na] was reduced, the concentration of D-glucose required for half-maximal SCC increase. Isotopically labeled 3H and 14C D-glucose were used to simultaneously determine bidirectional fluxes; a resultant net apical-to-basolateral transport was present and abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism of transported glucose. The pig kidney cell line, LLC-PK1, provides a cell culture model for the investigation of mechanisms of transepithelial glucose transport.

Na/K-ATPase in renal epithelium is expressed at the basolateral surface and thus is critical for vectorial solute transport. One potential mode of regulation of Na/K-ATPase involves the intracellular effector protein kinase C (PKC). In kidney cell lines, activation of PKC by the phorbol ester phorbol 12,13-dibutyrate (PDBu) (1 microM) inhibited Na/K-ATPase transport activity in OK cells (Vmax decreased 42%; p < 0.02), but not in LLC-PK1 cells. By immunoblot, both cell types expressed detectable levels of PKC alpha and PKC sigma. In response to PDBu, PKC alpha translocated from the cytosol to the membrane fractions of both cell lines. Phorbol ester treatment increased incorporation of 32PO4 in multiple substrates in both cell types, but a approximately 109-kDa substrate with neutral pI was detected only in the OK cell. Anti-LEAVE, directed against a highly conserved sequence in the H4-H5 loop of all known alpha isoforms of Na/K-ATPase, recognized a approximately 109-kDa membrane protein from both cell lines. Anti-LEAVE also identified a protein that comigrated with the large phosphoprotein which was only present in OK cells. Following 32PO4 loading and PDBu treatment, anti-LEAVE immunoprecipitated a approximately 109-kDa phosphoprotein in OK but not LLC-PK1 cells. These data support the notion that PKC is capable of phosphorylating the alpha subunit and inhibiting Na/K-ATPase transport activity in intact renal cells. Furthermore, they suggest that some forms of Na/K-ATPase in the kidney are not susceptible to PKC phosphorylation and that this heterogeneity may contribute to response diversity.

The primary cilium is a ubiquitous, non-motile microtubular organelle lacking the central pair of microtubules found in motile cilia. Primary cilia are surrounded by a membrane, which has a unique complement of membrane proteins, and may thus be functionally different from the plasma membrane. The function of the primary cilium remains largely unknown. However, primary cilia have important sensory transducer properties, including the response of renal epithelial cells to fluid flow or mechanical stimulation. Recently, renal cystic diseases have been associated with dysfunctional ciliary proteins. Although the sensory properties of renal epithelial primary cilia may be associated with functional channel activity in the organelle, information in this regard is still lacking. This may be related to the inherent difficulties in assessing electrical activity in this rather small and narrow organelle. In the present study, we provide the first direct electrophysiological evidence for the presence of single channel currents from isolated primary cilia of LLC-PK1 renal epithelial cells. Several channel phenotypes were observed, and addition of vasopressin increased cation channel activity, which suggests the regulation, by the cAMP pathway of ciliary conductance. Ion channel reconstitution of ciliary versus plasma membranes indicated a much higher channel density in cilia. At least three channel proteins, polycystin-2, TRPC1, and interestingly, the alpha-epithelial sodium channel, were immunodetected in this organelle. Ion channel activity in the primary cilium of renal cells may be an important component of its role as a sensory transducer.

1. Previous studies have shown that suramin and FPL 66301 are competitive antagonists at the P2X-purinoceptor in the rabbit ear artery. Those studies employed alpha,beta-methylene ATP, a poorly hydrolysable ATP analogue, as the agonist. In this study these compounds have been tested using ATP as the agonist. 2. Suramin, in the concentration range 30-1000 microM, potentiated the contractile effects of ATP, producing a 3-fold leftward shift of the ATP E/[A] curves. FPL 66301, in the concentration range 100-1000 microM, produced a significant but small (approximately 3-fold) rightward shift of the ATP curves. These results are in marked contrast with previous studies using alpha,beta-methylene ATP in which 30-fold rightward shifts were achieved using the same concentration ranges of suramin and FPL 66301. 3. Suramin and FPL 66301 were tested as ecto-ATPase inhibitors in a human blood cell assay. Suramin inhibited the enzyme with a pIC50 of 4.3, FPL 66301 with a pIC50 of 3.3. 4. The pharmacological data were analysed using a theoretical model describing the action of a compound with dual enzyme inhibitory and receptor antagonistic properties on the effects of an agonist susceptible to enzymatic degradation. The model was found to fit the data well using the known pKB estimates for suramin and FPL 66301 and similar relative (but not absolute) pK1 estimates to those obtained for the compounds in the enzyme assay. 5. From this analysis it was concluded that the limited shifts of ATP E/[A] curves produced by suramin and FPL 66301 were the result of 'self-cancellation' of the potentiating (enzyme inhibitory) and rightward-shifting (receptor antagonistic) properties.6. The analysis also indicated that the presence of ecto-ATPase activity in the rabbit ear artery preparation has a marked effect on the apparent potency of ATP. The experimental p[A50] was 3.4,whereas the 'true' value, that is the value which would be obtained in the absence of ecto-ATPase activity, was 6.0, some 400-fold higher.7 Two conclusions are drawn from this study. Firstly, caution must be exercised in the use of suramin and FPL 66301 as tools for receptor classification. Absence of overt antagonism by these compounds when metabolically unstable agonists are used could lead to erroneous claims for receptor subtypes.Secondly, the agonist potency order currently used to designate P2X- purinoceptors may require modification.

The cellular and biochemical events which transduce chemical insults into signals for increased expression of the stress-responsive gene gadd 153 were investigated using nephrotoxic cysteine conjugates. In LLC-PK1 cells, cysteine conjugate toxicity is initiated by covalent binding, but depletion of cellular thiols, an increase in cytosolic free calcium, and lipid peroxidation couple the binding to cell death (Chen, Q., Jones, T. W., Brown, P. C., and Stevens, J. L. (1990) J. Biol. Chem. 265, 21603-21611; Chen, Q., Jones, T. W., and Stevens, J. L. (1991) Toxicologist 11, 101, 1991). Three different toxic cysteine conjugates induced gadd 153 mRNA. With S-(1,2-dichlorovinyl)-L-cysteine (DCVC), the induction was both concentration and time-dependent. Preventing the metabolism of DCVC and covalent binding of DCVC-derived reactive metabolites to cellular macromolecules with the beta-lyase inhibitor (aminooxy)acetic acid blocked the induction. However, buffering free calcium with a cell permeable calcium chelator or blocking lipid peroxidation with an antioxidant did not affect the induction of gadd 153 mRNA by DCVC even though these treatments inhibit toxicity. These data suggest that covalent binding of reactive metabolites to cellular macromolecules may serve as a primary signal for the induction of gadd 153 mRNA by nephrotoxic cysteine conjugates. Interestingly, the sulfhydryl agent dithiothreitol, which was nontoxic and prevented the toxicity of DCVC, also induced an increase in gadd 153 mRNA. When both dithiothreitol and DCVC were added to cells, there were no inhibitory or additive effects on expression. Therefore, cellular thiol-disulfide status may also play a role in gadd 153 induction.

The canalicular multispecific organic anion transporter (cMOAT), also termed MRP2, is a recently identified ATP-binding cassette transporter. We previously established stable human cMOAT cDNA-transfected cells, LLC/cMOAT-1 from LLC-PK1 cells, and LLC/CMV cells that were transfected with an empty vector. We found that LLC/cMOAT-1 cells have increased resistance to vincristine (VCR), 7-ethyl-10-hydroxy-camptothecin, and cisplatin but not to etoposide. The multidrug resistance-reversing agents cyclosporin A (CsA) and 2-[4-(diphenylmethyl)-1-piperazinyl]-5-(trans-4,6-dimethyl-1,3, 2-dioxaphosphorinan-2-yl)-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P) almost completely reversed the resistance to VCR, 7-ethyl-10-hydroxy-camptothecin, and cisplatin of LLC/cMOAT-1 cells; and DL-buthionine-(S,R)-sulfoximine, (3'-oxo-4-butenyl-4-methyl-threonine(1), (valine(2)) cyclosporin (PSC833), and 3-([(3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl)-((3-dimethylamino-3- oxopropyl)-thio)-methyl]thio)propanoic acid (MK571) partially reversed the resistance to these drugs. CsA and PAK-104P at 10 microM enhanced the accumulation of VCR in LLC/cMOAT-1 cells almost to the level in LLC/CMV cells without the agents. The efflux of VCR from LLC/cMOAT-1 cells was enhanced compared with LLC/CMV cells and inhibited by CsA and PAK-104P. Transport of leukotriene C(4) (LTC(4)) and S-(2, 4-dinitrophenyl)glutathione also was studied with membrane vesicles prepared from these cells. LTC(4) and S-(2, 4-dinitrophenyl)glutathione were actively transported into membrane vesicles prepared from LLC/cMOAT-1 cells. The K(m) and V(max) values for the uptake of LTC(4) by the LLC/cMOAT-1 membrane vesicles were 0. 26 +/- 0.05 microM and 7.48 +/- 0.67 pmol/min/mg protein, respectively. LTC(4) transport was competitively inhibited by PAK-104P, CsA, MK571, and PSC833, with K(i) values of 3.7, 4.7, 13.1, and 28.9 microM, respectively. These findings demonstrate that cMOAT confers a novel drug-resistance phenotype. CsA and PAK-104P may be useful for reversing cMOAT-mediated drug resistance in tumors.

OBJECTIVE

The present studies assessed the possibility that high concentrations of oxalate may be toxic to renal epithelial cells.

METHODS

Subconfluent cultures of LLC-PK1 cells were exposed to oxalate, and the effects on cell morphology, membrane permeability to vital dyes, DNA integrity and cell density were assessed.

RESULTS

Oxalate exposure produced time- and concentration-dependent changes in the light microscopic appearance of LLC-PK1 cells with higher concentrations (>> 140 microM.) inducing marked cytosolic vacuolization and nuclear pyknosis. Exposure to oxalate also increased membrane permeability to vital dyes, promoted DNA fragmentation and, at high concentrations (350 microM. free oxalate), induced a net loss of LLC-PK1 cells.

CONCLUSIONS

Since high concentrations of oxalate can be toxic to renal epithelial cells, hyperoxaluria may contribute to several forms of renal disease including both calcium stone disease and end-stage renal disease.

Cells cultured on thin plastic (e.g. Formvar, Teflon, polycarbonate) membranes can be clearly imaged from the side in vivo by video microscopy. We have used this flexible-substratum technique to examine the behaviour and properties of primary cilia in confluent cultures of the kidney epithelial cell lines PtK1, PtK2, LLC-PK1, MDCK and BSC-40. In these cells primary cilia appear as rigid rods, up to 55 micron long, which project at various angles from the dorsal cell surface. The length distribution of primary cilia in confluent cultures is a distinct characteristic of each established kidney cell line examined, with LLC-PK1 exhibiting three distinct length populations. Primary cilia of kidney cell lines bend passively in response to flow but do not display propagated bending or vortical motions. Up to 26% of the cilia in the cell types examined possess one or more conspicuous swellings along the ciliary shaft. Treatment with 0.05% trypsin, which is sufficient to cause cell rounding, does not induce the resorption or shedding of the cilium. These direct observations demonstrate that kidney epithelial-cell primary cilia are non-motile and longer than previously thought, and suggest that their length represents a phenotypic marker for each cell line.

The most common cause of hereditary nephrogenic diabetes insipidus is a nonfunctional vasopressin (VP) receptor type 2 (V2R). Calcitonin, another ligand of G-protein-coupled receptors, has a VP-like effect on electrolytes and water reabsorption, suggesting that it may affect AQP2 trafficking. Here, calcitonin increased intracellular cAMP and stimulated the membrane accumulation of AQP2 in LLC-PK1 cells. Pharmacologic inhibition of protein kinase A (PKA) and deficiency of a critical PKA phosphorylation site on AQP2 both prevented calcitonin-induced membrane accumulation of AQP2. Fluorescence assays showed that calcitonin led to a 70% increase in exocytosis and a 20% decrease in endocytosis of AQP2. Immunostaining of rat kidney slices demonstrated that calcitonin induced a significant redistribution of AQP2 to the apical membrane of principal cells in cortical collecting ducts and connecting segments but not in the inner stripe or inner medulla. Calcitonin-treated VP-deficient Brattleboro rats had a reduced urine flow and two-fold higher urine osmolality during the first 12 hours of treatment compared with control groups. Although this VP-like effect of calcitonin diminished over the following 72 hours, the tachyphylaxis was reversible. Taken together, these data show that calcitonin induces cAMP-dependent AQP2 trafficking in cortical collecting and connecting tubules in parallel with an increase in urine concentration. This suggests that calcitonin has a potential therapeutic use in nephrogenic diabetes insipidus.

Gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubules and renal cell lines. Using LLC-PK1 cells, we have examined the concentration- and time-dependency of the effects exerted by gentamicin (1-3 mM; 0-3 days) on (i) lysosomal stability; (ii) activation of mitochondrial pathway; (iii) occurrence of apoptosis (concentrations larger than 3 mM caused extensive necrosis as assessed by the measurement of lactate dehydrogenase release). Within 2 h, gentamicin induced a partial relocalization [from lysosomes to cytosol] of the weak organic base acridine orange. We thereafter observed (a) a loss of mitochondrial membrane potential (as from 10 h, based on spectrophotometric and confocal microscopy using JC1 probe) and (b) the release of cytochrome c from granules to cytosol, and the activation of caspase-9 (as from 12 h; evidenced by Western blot analysis). Increase in caspase-3 activity (assayed with Ac-DEVD-AFC in the presence of z-VAD-fmk]) and appearance of fragmented nuclei (DAPI staining) was then detected as from 16 to 24 h together with nuclear fragmentation. Gentamicin produces a fast (within 4 h) release of calcein from negatively-charged liposomes at pH 5.4, which was slowed down by raising the pH to 7.4, or when phosphatidylinositol was replaced by cardiolipin (to mimic the inner mitochondrial membrane). The present data provide temporal evidence that gentamicin causes apoptosis in LLC-PK1 with successive alteration of the permeability of lysosomes, triggering of the mitochondrial pathway, and activation of caspase-3.