Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0022116 (ischemia)
 91,303 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To further understand and investigate how ischemia affects the tight junction we have developed a 2-h model of rapidly reversible ATP depletion and cellular injury in confluent LLC-PK1 monolayers. ATP depletion was achieved utilizing substrate-free medium containing 0.1 microM antimycin A (AA). Cellular ATP levels dropped rapidly to less than 5% of control values, but recovery of ATP and cell morphology was possible even after 2 h of exposure to AA. Ruthenium red, an electron-dense marker of tight-junction integrity, was excluded from the tight junctions of control monolayers but penetrated cellular tight junctions during ATP depletion in a duration-dependent manner. Electrical resistance across the monolayers remained unchanged in control monolayers but decreased linearly during ATP depletion to 59% of control values. Transmonolayer movement of [3H]mannitol increased from a control level of 7 to 13.5% during ATP depletion. Recovery of tight-junction integrity was demonstrated by a slowing of [3H]mannitol transfer from the basolateral to the apical medium. The transfer rate in control monolayers was 0.0126%/min. During the initial 120 min of cellular recovery from 2 h of ATP depletion, the transfer rate was 0.0789%/min, but this decreased to 0.0045%/min between 2 and 4 h of recovery. In summary, physiology, biochemical, and morphological evidence indicates that reversible ATP depletion results in rapid opening of cellular tight junctions. After ATP-repletion physiological studies indicate a recovery of tight-junction integrity.
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PMID:Effect of reversible ATP depletion on tight-junction integrity in LLC-PK1 cells. 172 94

The aim of this study was to characterize injuries of LLC-PK1 and MDCK cells exposed to hypoxia and reoxygenation. Exposure of LLC-PK1 cells to hypoxia reduced the ATP contents and increased the leakage of lactate dehydrogenase (LDH), but MDCK cells had no such injuries. Hypoxia-reoxygenation of LLC-PK1 cells dramatically increased LDH leakage, which was suppressed by free radical scavengers, N,N'-diphenyl-p-phenylenediamine, superoxide dismutase and N,N'-dimethylthiourea. These results suggest that use of LLC-PK1 cells has advantages for the investigation of ischemia-reperfusion injury of the kidney as an in vitro model and that generation of oxygen radicals is involved in the cellular injury induced by hypoxia-reoxygenation.
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PMID:Hypoxia and reoxygenation-induced injury of renal epithelial cells: effect of free radical scavengers. 756 83

Extracellular acidosis has been shown to be protective during ischemia in renal tubule cells. However, the mechanism of protection remains unknown. Since ischemia leads to disruption and polymerization of the cortical actin cytoskeleton, we hypothesized acidosis may better preserve the actin cytoskeleton during ischemia. Therefore, the purpose of our studies was to examine the effect of pH on the integrity of the actin cytoskeleton during ATP depletion and ATP repletion. To do this, we used an in vitro model of reversible ATP depletion in LLC-PK1 cells at extracellular pH values (pHo) of 6.9, 7.4, and 7.9. Immunofluorescent studies with rhodamine-phalloidin demonstrated more marked redistribution and clumping of cortical actin at pHo 7.9 and 7.4 vs. 6.9 after 90 min of chemical anoxia. After 15 min of ATP depletion, G-actin, quantified by the deoxyribonuclease assay, decreased from 53.7 +/- 0.8 to 43.2 +/- 1.5 microgram/mg protein at pHo 6.9 vs. 37.6 +/- 1.8 microgram/mg protein at pHo 7.4 (P < 0.001). After 60 min, there still was significantly less conversion of G-actin to F-actin at pHo 6.9 vs. 7.4, with a decrease from 55.9 +/- 2.0 to 39.6 +/- 2.0 micrograms/mg protein at 6.9 vs. 35.8 +/- 2.4 at 7.4 micrograms/mg protein (P < 0.05). Furthermore, extracellular acidosis during the phase of ATP repletion resulted in more rapid normalization of cellular G-actin levels (95 +/- 3% of control vs. 82 +/- 2% for pH 6.9 vs. 7.4, respectively, P < 0.01). Together, these findings indicate the actin cytoskeleton is better preserved in an acidic environment during ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Extracellular acidosis minimizes actin cytoskeletal alterations during ATP depletion. 794 55

We have previously demonstrated using immunocytochemical, histochemical, and biochemical techniques that ischemia in vivo and ATP depletion in vitro result in dissociation of Na(+)-K(+)-adenosinetriphosphatase (ATPase) from the actin cytoskeleton and redistribution to the apical domain in renal proximal tubule cells. To directly evaluate whether apical Na(+)-K(+)-ATPase retained Na+ pumping activity, a rapidly reversible model of cellular ATP depletion in confluent LLC-PK1 cells grown on semipermeable membranes was utilized. Tight-junction integrity, monitored by electrical resistance, was lost during ATP depletion and reestablished during 2 h of ATP repletion. Total cellular Na(+)-K(+)-ATPase activity and total surface membrane [3H]ouabain binding remained constant, but specific apical [3H]ouabain binding increased (7 vs. 26 fmol/filter, P < 0.01). Apical [3H]ouabain binding returned to baseline during 5 h of ATP repletion. Apically applied ouabain was then used to selectively inhibit apical Na(+)-K(+)-ATPase. It had no effect on apical-to-basolateral Na+ flux under physiological conditions (1.3 +/- 0.61 vs. 1.27 +/- 0.46 meq.filter-1.30 min-1), but it increased the apical-to-basolateral flux in ATP-depleted and then repleted monolayers (0.39 +/- 0.12 vs. 0.83 +/- 0.27 meq.filter-1.30 min-1, P < 0.01), implying that apical Na(+)-K(+)-ATPase retained Na+ pumping activity. Together, these data imply that ATP depletion induces loss of surface membrane polarity resulting in redistribution of functional proteins to the alternate domain.
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PMID:Na(+)-K(+)-ATPase that redistributes to apical membrane during ATP depletion remains functional. 823 49

We have identified a new putative transcription factor from the rat kidney, termed Kid-1 (for kidney, ischemia and developmentally regulated gene 1). Kid-1 belongs to the C2H2 class of zinc finger genes. Its mRNA accumulates with age in postnatal renal development and is detected predominantly in the kidney. Kid-1 mRNA levels decline after renal injury secondary to ischemia or folic acid administration, two insults which result in epithelial cell dedifferentiation, followed by regenerative hyperplasia and differentiation. The low expression of Kid-1 early in postnatal development, and when renal tissue is recovering after injury, suggests that the gene product is involved in establishment of a differentiated phenotype and/or regulation of the proliferative response. The deduced protein contains 13 C2H2 zinc fingers at the COOH end in groups of 4 and 9 separated by a 32-amino-acid spacer. There are consensus sites for phosphorylation in the NH2 terminus non-zinc finger region as well as in the spacer region between zinc fingers 4 and 5. A region of the deduced protein shares extensive homology with a catalytic region of Raf kinases, a feature shared only with TFIIE among transcription factors. To determine whether Kid-1 can modulate transcription, a chimeric construct encoding the Kid-1 non-zinc finger region (sense or antisense) and the DNA-binding region of GAL4 was transfected into COS and LLC-PK1 cells together with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 binding sites, driven by either a minimal promoter or a simian virus 40 enhancer. CAT activity was markedly inhibited in cells transfected with the sense construct compared with the activity in cells transfected with the antisense construct. To our knowledge, this pattern of developmental regulation, kidney expression, and regulation of transcription is unique among the C2H2 class of zinc finger-containing DNA-binding proteins.
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PMID:Kid-1, a putative renal transcription factor: regulation during ontogeny and in response to ischemia and toxic injury. 838 78

This study investigates ischemia-induced degradation of the spectrin-based cytoskeleton in rat brain, heart, and kidney. Spectrin, in conjunction with ankyrin, structurally supports the plasma membrane and sequesters integral membrane proteins. After 60 and 120 min of ischemia, brain tissue displayed both spectrin and ankyrin breakdown. The spectrin fragmentation pattern is similar to previously reported ischemia-induced calpain I proteolysis of spectrin in N-methyl-D-aspartate receptor-containing neurons. Ischemic heart tissue displayed no spectrin or ankyrin degradation. Ischemic renal tissue showed minimal breakdown of spectrin but a major loss of ankyrin (25%/30 min of ischemia) that was essentially complete after 120 min of ischemia. Interestingly, this profound loss of ankyrin in the intact ischemic kidney was not mimicked in three renal cell lines (MDCK, LLC-PK1, and JTC cell lines) exposed to chemical anoxia. Immunocytochemistry showed ankyrin was concentrated in thick ascending limb (cTAL) cells and, although delayed by 30 min, was lost at the same rate as measured by immunoblot analysis. Spectrin and Na(+)-K(+)-ATPase, which complex with ankyrin, were essentially unaffected by ischemia. Ankyrin degradation in cTAL cells correlated with the loss of basal infolding organization. In conclusion, the spectrin-based cytoskeleton is differentially targeted by ischemia-induced degradative processes in different in vivo tissues.
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PMID:Degradation of spectrin and ankyrin in the ischemic rat kidney. 838 46

The transcription factors controlling the complex genetic response to ischemia and their modes of regulation are poorly understood. We found that ATF-2 and c-Jun DNA binding activity is markedly enhanced in post-ischemic kidney or in LLC-PK1 renal tubular epithelial cells exposed to reversible ATP depletion. After 40 min of renal ischemia followed by reperfusion for as little as 5 min, binding of ATF-2 and c-Jun, but not ATF-3 or CREB (cAMP response element binding protein), to oligonucleotides containing either an ATF/cAMP response element (ATF/CRE) or the jun2TRE from the c-jun promoter, was significantly increased. Binding to jun2TRE and ATF/CRE oligonucleotides occurred with an identical time course. In contrast, nuclear protein binding to an oligonucleotide containing a canonical AP-1 element was not detected until 40 min of reperfusion, and although c-Jun was present in the complex, ATF-2 was not. Incubating nuclear extracts from reperfused kidney with protein phosphatase 2A markedly reduced binding to both the ATF/CRE and jun2TRE oligonucleotides, compatible with regulation by an ATF-2 kinase. An ATF-2 kinase, which phosphorylated both the transactivation and DNA binding domains of ATF-2, was activated by reversible ATP depletion. This kinase coeluted on Mono Q column chromatography with a c-Jun amino-terminal kinase and with the peak of stress-activated protein kinase, but not p38, immunoreactivity. In conclusion, DNA binding activity of ATF-2 directed at both ATF/CRE and jun2TRE motifs is modulated in response to the extreme cellular stress of ischemia and reperfusion or reversible ATP depletion. Phosphorylation-dependent activation of the DNA binding activity of ATF-2, which appears to be regulated by the stress-activated protein kinases, may play an important role in the earliest stages of the genetic response to ischemia/reperfusion by targeting ATF-2 and c-Jun to specific promoters, including the c-jun promoter and those containing ATF/CREs.
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PMID:Ischemia and reperfusion enhance ATF-2 and c-Jun binding to cAMP response elements and to an AP-1 binding site from the c-jun promoter. 853 Apr 13

F2-isoprostanes are the newly identified reactive oxygen species-catalyzed peroxidation products of arachidonate. The infusion of these prostaglandin F2-like prostanaoids into the rat kidney induces profound parallel reductions in RBF and GFR, suggesting that these metabolites may be partly responsible for the hemodynamic alterations seen in free radical-linked acute renal injury models. The present study examined directly in renal proximal tubular (LLC-PK1) cells whether hydrogen peroxide, a reactive oxygen species implicated in many models of acute renal injury, induces F2-isoprostane production and whether its production can be inhibited by the recently synthesized lipid peroxidation inhibitor 21-aminosteroid (lazaroid U-74389G). The incubation of LLC-PK1 cell layers with hydrogen peroxide for 3 h resulted in a dose-related six-fold increase in F2-isoprostane production, measured by the gas chromatographic-mass spectroscopic method. The preincubation of cells with 21-aminosteroid prevented hydrogen peroxide-induced F2-isoprostane production, a finding also demonstrable with other lipid peroxidation inhibitors, e.g., 2-methyl aminochroman (U-83836E) and diphenyl-p-phenylenediamine. Besides inhibiting isoprostane production, 21-aminosteroid reduced hydrogen peroxide-induced lipid degradation and peroxidation, and protected the cells against hydrogen peroxide-induced cytolysis. The novel finding that hydrogen peroxide induces 21-aminosteroid-inhibitable F2-isoprostane production in renal epithelial cells supports the in vivo report that its levels are elevated in reactive oxygen species-linked renal injury models such as ischemia-reperfusion. Besides direct cell injury, lipid peroxidation by generating F2-isoprostanes may further contribute to renal dysfunction through a vasoconstrictive mechanism. Thus, the inhibition of excess F2-isoprostane production may be one of the additional mechanisms, besides cytoprotection, by which antioxidants ameliorate renal dysfunction in experimental models of acute renal injury.
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PMID:Hydrogen peroxide induces 21-aminosteroid-inhibitable F2-isoprostane production and cytolysis in renal tubular epithelial cells. 858 1

Sublethal heat shock has been shown to produce tolerance in cells and tissues subsequently exposed to heat or ischemia/ATP depletion. We tested whether heating LLC-PK1 cells for 2 h at 42 degrees C induced heat shock protein-70 (HSP-70) gene expression and conferred tolerance against subsequent cyclosporine A (CyA) toxicity. HSP-70 mRNA was increased immediately after heat shock, returning to baseline by 4 h. HSP-70 protein increased by 1 h after heat shock and declined thereafter, approaching baseline after 72 h. Cells heat shocked at 4 and 24 h prior to CyA exposure were significantly more viable than controls, at CyA concentrations near the median lethal dose (LD50). Cytoprotection declined with time after heat shock, concurrent with declining HSP-70 protein levels. Sublethal CyA exposure (50 micrograms/ml) for 24 h produced upregulation of HSP-70 mRNA and protein. Pretreatment with 50 micrograms/ml CyA for 24 h followed by exposure to a toxic concentration of CyA (200 micrograms/ml) produced significant cytoprotection compared with untreated controls. In conclusion, HSP-70 protein induction by sublethal heat shock or CyA exposure was associated with tolerance against subsequent lethal CyA exposure.
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PMID:Sublethal heat shock and cyclosporine exposure produce tolerance against subsequent cyclosporine toxicity. 885 18

Ischemia in vivo or ATP depletion in vitro result in disruption and cellular redistribution of the cortical F-actin cytoskeleton in epithelial cells. However, little is known regarding the effect of these two maneuvers on other components of the actin cytoskeleton. Because the spectrin (fodrin in epithelial cells)-based network links the actin cytoskeleton to the surface membrane, we have utilized a reversible model of ATP depletion in LLC-PK1 cells to study the effect of ATP depletion on fodrin and ankyrin. Under physiological conditions, both ankyrin and fodrin were largely Triton X-100 insoluble and colocalized immunofluorescently along the lateral membranes of LLC-PK1 cells. After ATP depletion, there was a rapid and duration-dependent increase in Triton X-100 solubility of both proteins. This was not true for villin and myosin 1, as Triton X-100 solubility was unaffected and reduced by ATP depletion, respectively. The increase in fodrin and ankyrin detergent solubility during ATP depletion was associated with cytosolic redistribution of the proteins, as determined using immunofluorescent techniques. Sucrose gradient fractionation and Western blot analysis of the Triton X-100-soluble fraction following ATP depletion revealed lack of association between fodrin and ankyrin. Furthermore, dual-label digital confocal immunofluorescent studies revealed lack of association of cytoplasmic ankyrin and fodrin following ATP depletion. Taken together, these data indicate that ATP depletion in LLC-PK1 cells leads to dissociation of both ankyrin and fodrin from the actin cytoskeleton. Furthermore, the two proteins dissociate from each other and redistribute throughout the cytoplasm.
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PMID:Cellular ATP depletion induces disruption of the spectrin cytoskeletal network. 889 8


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