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      Activation of Protein Kinase C Isozymes Protects LLCPK 1 Cells from H 2O 2 Induced Necrotic Cell Death

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          Background/Aims: We have previously reported that ischemia/reperfusion injury (IRI) to the kidney leads to induced expression of RACK1 and changes in the level of expression and subcellular distribution of PKC isozymes α, βII and ζ. In order to further define the role of PKC isozymes in IRI we investigated the effect of activation or inhibition of the isozymes on cytotoxicity mediated by H<sub>2</sub>O<sub>2</sub> in LLCPK<sub>1</sub> cells. Methods: Cytotoxicity was analyzed by Trypan blue assay and LDH release assay. Translocation of PKC isozymes postinjury in LLCPK1 cells was analyzed by immunostaining and Western blot analysis. Results: Western blot analysis showed that the expression of PKC-α was up-regulated in a triphasic pattern with the initial induction within the first 10 min of injury followed by higher levels of expression at 2 and 24 h postinjury. The expression of PKC-ζ was highly induced within the first 15 min of injury but its expression was down-regulated to that of normal levels by 30 min postinjury. Immunocytochemistry showed that both PKC-α and PKC-ζ translocated to the nucleus and perinuclear region during H<sub>2</sub>O<sub>2</sub> treatment. Following injury, PKC-α expression was localized to the nuclear membrane at earlier time points but a translocation to the nucleus occurred at later time points. PKC-ζ translocated to nucleus at 30 minutes post injury and relocated back to the nuclear membrane at later time points. Conclusion: These data suggest that activation of PKC-α and PKC-ζ is involved in the H<sub>2</sub>O<sub>2</sub> induced injury of LLCPK1 cells.

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          Most cited references 8

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          Prevention of kidney ischemia/reperfusion-induced functional injury and JNK, p38, and MAPK kinase activation by remote ischemic pretreatment.

          MAPK activities, including JNK, p38, and ERK, are markedly enhanced after ischemia in vivo and chemical anoxia in vitro. The relative extent of JNK, p38, or ERK activation has been proposed to determine cell fate after injury. A mouse model was established in which prior exposure to ischemia protected against a second ischemic insult imposed 8 or 15 days later. In contrast to what was observed after 30 min of bilateral ischemia, when a second period of ischemia of 30- or 35-min duration was imposed 8 days later, there was no subsequent increase in plasma creatinine, decrease in glomerular filtration rate, or increase in fractional excretion of sodium. A shorter period of prior ischemia (15 min) was partially protective against subsequent ischemic injury 8 days later. Unilateral ischemia was also protective against a subsequent ischemic insult to the same kidney, revealing that systemic uremia is not necessary for protection. The ischemia-related activation of JNK and p38 and outer medullary vascular congestion were markedly mitigated by prior exposure to ischemia, whereas preconditioning had no effect on post-ischemic activation of ERK1/2. The phosphorylation of MKK7, MKK4, and MKK3/6, upstream activators of JNK and p38, was markedly reduced by ischemic preconditioning, whereas the post-ischemic phosphorylation of MEK1/2, the upstream activator of ERK1/2, was unaffected by preconditioning. Pre- and post-ischemic HSP-25 levels were much higher in the preconditioned kidney. In summary, post-ischemic JNK and p38 (but not ERK1/2) activation was markedly reduced in a model of kidney ischemic preconditioning that was established in the mouse. The reduction in JNK and p38 activation can be accounted for by reduced activation of upstream MAPK kinases. The post-ischemic activation patterns of MAPKs may explain the remarkable protection against ischemic injury observed in this model.
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            Protein kinase C signaling and oxidative stress

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              Protein kinases C translocation responses to low concentrations of arachidonic acid.

              Arachidonic acid (AA) directly activates protein kinases C (PKC) and may thereby serve as a regulatory signal during cell stimulation. The effect, however, requires a > or =20 microm concentration of the fatty acid. We find that human polymorphonuclear neutrophils (PMN) equilibrated with a ligand for the diacylglycerol receptor on PKC, [(3)H]phorbol dibutyrate (PDB), increased binding of [(3)H]PDB within 15 s of exposure to > or =10-30 nm AA. Other unsaturated fatty acids, but not a saturated fatty acid, likewise stimulated PDB binding. These responses, similar to those caused by chemotactic factors, resulted from a rise in the number of diacylglycerol receptors that were plasma membrane-associated and therefore accessible to PDB. Unlike chemotactic factors, however, AA was fully active on cells overloaded with Ca(2+) chelators. The major metabolites of AA made by PMN, leukotriene B(4) and 5-hydroxyicosatetraenoate, did not mimic AA, and an AA antimetabolite did not block responses to AA. AA also induced PMN to translocate cytosolic PKCalpha, beta(II), and delta to membranes. This response paralleled PDB binding with respect to dose requirements, time, Ca(2+)-independence, resistance to an AA antimetabolite, and induction by another unsaturated fatty acid but not by a saturated fatty acid. Finally, HEK 293 cells transfected with vectors encoding PKCbeta(I) or PKCdelta fused to the reporter enhanced green fluorescent protein (EGFP) were studied. AA caused EGFP-PKCbeta translocation from cytosol to plasma membrane at > or =0.5 microm, and EGFP-PKCdelta translocation from cytosol to nuclear and, to a lesser extent, plasma membrane at as little as 30 nm. We conclude that AA induces PKC translocations to specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they occur in PMN, do not require changes in cell Ca(2+) or oxygenation of the fatty acid. AA seems more suited for signaling the movement than activation of PKC.

                Author and article information

                Am J Nephrol
                American Journal of Nephrology
                S. Karger AG
                December 2003
                21 November 2003
                : 23
                : 6
                : 380-389
                Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebr., USA
                73984 Am J Nephrol 2003;23:380–389
                © 2003 S. Karger AG, Basel

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                Figures: 10, References: 23, Pages: 10
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                Original Report: Laboratory Investigation


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