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Query: UMLS:C0432222 (SEM)
 47,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reactive oxygen metabolites have been postulated to play an important role in both toxic and ischemic forms of acute renal tubular epithelial injury. In the present study, we examined the effect of enzymatically generated hydrogen peroxide on LLC-PK1 cells, a renal proximal tubule cell line. Exposure of LLC-PK1 cells to glucose and glucose oxidase (GO; which generates hydrogen peroxide) resulted in cytotoxicity (as measured by trypan blue exclusion) which was dose dependent and increased linearly over time to 81 +/- 5% at 180 minutes (8 +/- 1% at time 0; mean +/- SEM, N = 3 to 7). Catalase (which decomposes hydrogen peroxide) completely prevented the cytotoxicity, confirming that the toxicity was due to hydrogen peroxide production. To assess whether the hydrogen peroxide toxicity was a direct effect or mediated by other toxic oxygen metabolites, several scavengers of reactive oxygen metabolites and iron chelators were used. Superoxide dismutase (a scavenger of superoxide) had no effect. Deferoxamine (DFO), an iron chelator, provided marked protection (GO alone 45.9 +/- 4.4%; GO + DFO 13.0 +/- 2.0%; control 7.1 +/- 1.2%; N = 15 to 17, P less than 0.001). Pretreatment with DFO (1 hr, then 2 washes to remove DFO before GO addition) also markedly inhibited the cytotoxicity, suggesting that DFO's effect was due to iron chelation. Two other metal chelators (dihydroxybenzoic acid and 1,10-phenanthroline) also significantly decreased the GO-induced cytotoxicity. However, three of four hydroxyl radical scavengers used (mannitol, dimethyl sulfoxide, sodium benzoate) did not significantly decrease cell death. Only dimethylthiourea provided protection.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hydrogen peroxide cytotoxicity in LLC-PK1 cells: a role for iron. 166 14

A surface preparation method for the SEM based on cryofixation is presented suitable for the demonstration of membrane particles on whole cells. LLC-PK1 cells (a renal epithelial cell line in culture) were fast frozen, freeze substituted, critical point dried, shadowed with 2 nm of platinum carbon and stabilized with a carbon backing layer. Membrane bound particles are visualized by the material dependent backscattered electron image mainly originating from the platinum shadow. The surface of the LLC-PK1 cells is almost free of precipitated material indicating that the culture medium is removed during freeze substitution or critical point drying. The apical plasma membrane with microvilli and ciliae is well preserved and differences in particle density can be detected. The feasibility of the coating technique for backscattered electron imaging was tested on the well known hexagonally arranged intramembranous particles of fractured and partially freeze dried yeast. This 16.5 nm periodic structure is clearly demonstrable on the bulk SEM-specimen stablized with the carbon backing layer. Without a carbon layer severe shrinking artifacts occurred.
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PMID:Improved representation of cell surface structures by freeze substitution and backscattered electron imaging. 269 69

In animals models, exposure of the brain, heart, or kidneys to sublethal ischemia induces tolerance for subsequent ischemia. However, the ability of human renal cells to undergo hypoxic preconditioning has not been evaluated. In addition, it is unclear if renal ischemic preconditioning induces resistance at the cellular level, or if preconditioning is a result of altered postischemic hemodynamics or the azotemic environment. In this study, we tested the ability of cultured human proximal tubular epithelial cells (PTEC) to undergo hypoxic preconditioning at the cellular level. Hypoxia was induced by incubating cells in an anaerobic incubator in glucose-free buffer (combined oxygen-glucose deprivation; COGD). Cell injury was assessed by lactate dehydrogenase (LDH) efflux, release of arachidonic acid metabolites, and light microscopy. PTEC preconditioned with 12 h of COGD and a 24-h recovery period had less LDH efflux than control PTEC after subsequent exposure to 20 h of COGD (15.0 +/- 2.5% vs. 44.0 +/- 3.4%, p < 0.05). Preconditioned PTEC also retained relatively normal morphology and had less release of arachidonic acid metabolites than control PTEC. Because renal ischemia is characterized predominately by tubular injury with relative sparing of the glomerulus, we determined if PTEC are more susceptible to hypoxic injury than glomerular cells. For further comparison, we also assessed the susceptibility to hypoxia of the porcine tubular epithelial cell line LLC-PK1. After exposure to 18 h of COGD, LDH efflux from PTEC (25.5 +/- 3.3%, mean +/- SEM) was lower than from LLC-PK1 cells (47.6 +/- 4.0%; p < 0.01), but not mesangial cells (22.7 +/- 5.0%) or glomerular endothelial cells (38.2 +/- 6.2%). In conclusion, we have demonstrated that cultured PTEC are as resistant to hypoxic injury as glomerular cells, and that PTEC attain cytoresistance after hypoxic preconditioning. Characterization of the molecular changes that occur in human PTEC after hypoxic preconditioning may reveal innate survival mechanisms that can be manipulated to promote protection from renal ischemia in patients.
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PMID:Susceptibility of human proximal tubular cells to hypoxia: effect of hypoxic preconditioning and comparison to glomerular cells. 904 51

Cyclosporine A (CsA), a neutral, highly hydrophobic cyclic peptide with 11 amino acids, is currently the most widely used immunosuppressive drug for preventing graft rejection and autoimmune diseases. Despite its efficacy, the use of CsA is limited by severe side effects, mainly nephrotoxicity and arterial hypertension. Single cell microfluorimetry was used to evaluate the role of CsA on Ca(2+) signaling pathway in intact cells of the porcine proximal tubule-like cell line LLC-PK1; the assay of the in vitro activity of the plasma membrane Ca(2+) pump (PMCA) was carried out through the preparation and isolation of membranes. The addition of CsA to incubation medium at doses ranging from 0.1 to 2 microM did not change the basal level of intracellular calcium ([Ca(2+)](i)), whereas it affected the [Ca(2+)](i) response to thapsigargin (TG), a powerful inhibitor of microsomal Ca(2+) pump. In control studies, 5 microM TG produced a biphasic response: [Ca(2+)](i) peaked with a 60-s lag, and it then declined to a plateau of elevated [Ca(2+)](i), which remains above basal. However, it became evident that CsA strengthened the Ca(2+) response to TG because the addition of 5 microM TG to cells exposed to 400 nM CsA did not affect the peak response to TG, but it markedly affected the subsequent sustained phase ([Ca(2+)](i) = 156 +/- 4.84 versus 130 +/- 3.28 nmol, mean +/- SEM, n = 6, P < 0.001). In membrane preparations, 200 nM CsA brought about, in the presence of 10 microM calmodulin (CaM), a significant decrease of plasma membrane Ca(2+) pump (PMCA) activity (46.96 +/- 0.26 versus 53.48 +/- 1.96 nmol x mg of protein(-1) x min(-1), n = 6, P < 0.02), a value similar to that obtained in the presence of equimolar amounts of cyclosporine H (CsH), a non-immunosuppressive analogue of CsA. These findings suggest that in this cell line CsA affects the Ca(2+) export pathway through the reduction of the PMCA activity with consequent amplification and strengthening of [Ca(2+)](i) response after exposure to agents that trigger intracellular Ca(2+) release. The increased cell sensitivity during Ca(2+) signaling events ensuing from the impairment of this "defense system" may be regarded as one of the basic mechanisms involved in the development of the side effects induced by CsA.
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PMID:Cyclosporine A amplifies Ca2+ signaling pathway in LLC-PK1 cells through the inhibition of plasma membrane Ca2+ pump. 1276 Dec 43

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.
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PMID:Quantitative imaging of subcellular calcium stores in mammalian LLC-PK1 epithelial cells undergoing mitosis by SIMS ion microscopy. 1621 91

It has been found that S-allylcysteine (SAC), a garlic-derived compound, has in vivo and in vitro antioxidant properties. In addition, it is known that SAC is able to scavenge different reactive oxygen or nitrogen species including superoxide anion (O(2)(-)), hydrogen peroxide (H(2)O(2)), hydroxyl radical (OH()), and peroxynitrite anion (ONOO(-)) although the IC(5O) values for each reactive species has not been calculated and the potential ability of SAC to scavenge singlet oxygen ((1)O(2)) and hypochlorous acid (HOCl) has not been explored. The purposes of this work was (a) to explore the potential ability of SAC to scavenge (1)O(2) and HOCl, (b) to further characterize the O(2)(-), H(2)O(2), OH(), and ONOO(-) scavenging ability of SAC by measuring the IC(50) values using in vitro assays, and (c) to explore the potential ability of SAC to ameliorate the potassium dichromate (K(2)Cr(2)O(7))-induced cytotoxicity in LLC-PK1 cells in which oxidative stress is involved. The scavenging activity was compared against the following reference compounds: N-acetylcysteine for O(2)(-), sodium pyruvate for H(2)O(2), dimethylthiourea for OH(), lipoic acid and glutathione for (1)O(2), lipoic acid for HOCl, and penicillamine for ONOO(-). It was found that SAC was able to scavenge concentration-dependently all the species assayed with the following IC(5O) (mean+/-SEM, mM): O(2)(-) (14.49+/-1.67), H(2)O(2) (68+/-1.92), OH() (0.68+/-0.06), (1)O(2) (1.93+/-0.27), HOCl (2.86+/-0.15), and ONOO(-) (0.80+/-0.05). When the ability of SAC to scavenge these species was compared to those of the reference compounds it was found that the efficacy of SAC (a) to scavenge O(2)(-), H(2)O(2), OH(), and ONOO(-) was lower, (b) to scavenge HOCl was similar, and (c) to scavenge (1)O(2) was higher. In addition, it was found that SAC was able to prevent K(2)Cr(2)O(7)-induced toxicity in LLC-PK1 cells in culture. It was showed for the first time that SAC is able to scavenge (1)O(2) and HOCl and to ameliorate the K(2)Cr(2)O(7)-induced toxicity.
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PMID:S-allylcysteine scavenges singlet oxygen and hypochlorous acid and protects LLC-PK(1) cells of potassium dichromate-induced toxicity. 1757 34

Bile salts are potent detergents and can disrupt cellular membranes, which causes cholestasis and hepatocellular injury. However, the mechanism for the resistance of the canalicular membrane against bile salts is not clear. Phosphatidylethanolamine (PE) is converted to phosphatidylcholine (PC) in the liver by phosphatidylethanolamine N-methyltransferase (PEMT). In this study, to investigate the effect of PEMT expression on the resistance to bile salts, we established an LLC-PK1 cell line stably expressing PEMT. By using enzymatic assays, we showed that the expression of PEMT increased the cellular PC content, lowered the PE content, but had no effect on the sphingomyelin content. Consequently, PEMT expression led to reductions in PE/PC and sphingomyelin/PC ratios. Mass spectrometry demonstrated that PEMT expression increased the levels of PC species containing longer acyl chains and almost all ether-linked PC species. PEMT expression enhanced the resistance to duramycin and lysenin, suggesting decreased ratios of PE and sphingomyelin in the apical membrane, respectively. In addition, SEM revealed that PEMT expression increased the diameter of microvilli. The expression of PEMT resulted in reduced resistance to unconjugated bile salts, but surprisingly in increased resistance to conjugated bile salts, which might be attributable to modifications of the phospholipid composition and/or structure in the apical membrane. Because most bile salts exist as conjugated forms in the bile canaliculi, PEMT may be important in the protection of hepatocytes from bile salts and in cholestatic liver injury.
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PMID:Effects of phosphatidylethanolamine N-methyltransferase on phospholipid composition, microvillus formation and bile salt resistance in LLC-PK1 cells. 2195 70