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Native LDL-induced oxidative stress in human proximal tubular cells: multiple players involved.

Piccoli C, Quarato G, D'Aprile A, Montemurno E, Scrima R, Ripoli M, Gomaraschi M, Cirillo P, Boffoli D, Calabresi L, Gesualdo L, Capitanio N - J. Cell. Mol. Med. (2011)

Bottom Line: This study aimed to examine the effects of native non-oxidized LDL on cellular oxidative metabolism in cultured human proximal tubular cells.All the above LDL-induced effects were completely abrogated by chelating extracellular Ca(2+) as well as by inhibition of the Ca(2+) -activated cytoplasmic phospholipase A2, NADPH oxidase and mitochondrial permeability transition.This involves first oxidants production via the plasmamembrane NADPH oxidase and then propagates downstream to mitochondria eliciting redox- and Ca(2+) -dependent dysfunctions leading to cell-harming conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, University of Foggia, Foggia, Italy.

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Related in: MedlinePlus

Extracellular Ca2+ mediates the nLDL-induced cPLA2-linked ROS production. (A) HK-2 cells were incubated for 24 hrs with 100 μg/ml nLDL either alone or in the presence of 20 μM AACOCF3 or 0.5 mM EGTA or 30 μM verapamil and after that assessed for intracellular H2O2 production by DCF. Representative images of the LSCM analysis are show along with the statistical evaluation of the fluorescence intensity averaged ± S.E.M. from n= 3 for each condition. Bars inside all the micrographs: 30 μm. (B) Effect of AACOCF3 on the nLDL-mediated activation of the NADPH oxidase. The SOD inhibitable cytochrome c reduction values are averages ± S.E.M. from n= 3 for each condition; the statistical differences versus untreated HK-2 cells is also indicated when significant.
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fig06: Extracellular Ca2+ mediates the nLDL-induced cPLA2-linked ROS production. (A) HK-2 cells were incubated for 24 hrs with 100 μg/ml nLDL either alone or in the presence of 20 μM AACOCF3 or 0.5 mM EGTA or 30 μM verapamil and after that assessed for intracellular H2O2 production by DCF. Representative images of the LSCM analysis are show along with the statistical evaluation of the fluorescence intensity averaged ± S.E.M. from n= 3 for each condition. Bars inside all the micrographs: 30 μm. (B) Effect of AACOCF3 on the nLDL-mediated activation of the NADPH oxidase. The SOD inhibitable cytochrome c reduction values are averages ± S.E.M. from n= 3 for each condition; the statistical differences versus untreated HK-2 cells is also indicated when significant.

Mentions: Stimulation of the Ca2+-dependent cytoplasmic phospholipase A2 (cPLA2) with release of arachidonic acid (AA) is a process known to activate NOX [13, 21]. Therefore, we tested the effect of arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cPLA2, on the nLDL-induced ROS overproduction. Figure 6A clearly shows that AACOCF3 abrogated completely the DCF-fluorescence in nLDL-treated HK-2. Moreover, either chelation of the external Ca2+ by EGTA and treatment with the Ca2+-channel blocker vera-pamil caused, similarly, a depression of the nLDL-induced ROS production. Importantly AACOCF3 prevented the nLDL-dependent extra-cellular production of superoxide (Fig. 6B) supporting the occurrence of NOX activation. These results indicated that the pro-oxidant effect of nLDL on HK-2 was largely mediated by the reaction products of the cPLA2 whose activation was likely linked to stimulation of the inward current of external Ca2+ into the cell.


Native LDL-induced oxidative stress in human proximal tubular cells: multiple players involved.

Piccoli C, Quarato G, D'Aprile A, Montemurno E, Scrima R, Ripoli M, Gomaraschi M, Cirillo P, Boffoli D, Calabresi L, Gesualdo L, Capitanio N - J. Cell. Mol. Med. (2011)

Extracellular Ca2+ mediates the nLDL-induced cPLA2-linked ROS production. (A) HK-2 cells were incubated for 24 hrs with 100 μg/ml nLDL either alone or in the presence of 20 μM AACOCF3 or 0.5 mM EGTA or 30 μM verapamil and after that assessed for intracellular H2O2 production by DCF. Representative images of the LSCM analysis are show along with the statistical evaluation of the fluorescence intensity averaged ± S.E.M. from n= 3 for each condition. Bars inside all the micrographs: 30 μm. (B) Effect of AACOCF3 on the nLDL-mediated activation of the NADPH oxidase. The SOD inhibitable cytochrome c reduction values are averages ± S.E.M. from n= 3 for each condition; the statistical differences versus untreated HK-2 cells is also indicated when significant.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822803&req=5

fig06: Extracellular Ca2+ mediates the nLDL-induced cPLA2-linked ROS production. (A) HK-2 cells were incubated for 24 hrs with 100 μg/ml nLDL either alone or in the presence of 20 μM AACOCF3 or 0.5 mM EGTA or 30 μM verapamil and after that assessed for intracellular H2O2 production by DCF. Representative images of the LSCM analysis are show along with the statistical evaluation of the fluorescence intensity averaged ± S.E.M. from n= 3 for each condition. Bars inside all the micrographs: 30 μm. (B) Effect of AACOCF3 on the nLDL-mediated activation of the NADPH oxidase. The SOD inhibitable cytochrome c reduction values are averages ± S.E.M. from n= 3 for each condition; the statistical differences versus untreated HK-2 cells is also indicated when significant.
Mentions: Stimulation of the Ca2+-dependent cytoplasmic phospholipase A2 (cPLA2) with release of arachidonic acid (AA) is a process known to activate NOX [13, 21]. Therefore, we tested the effect of arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cPLA2, on the nLDL-induced ROS overproduction. Figure 6A clearly shows that AACOCF3 abrogated completely the DCF-fluorescence in nLDL-treated HK-2. Moreover, either chelation of the external Ca2+ by EGTA and treatment with the Ca2+-channel blocker vera-pamil caused, similarly, a depression of the nLDL-induced ROS production. Importantly AACOCF3 prevented the nLDL-dependent extra-cellular production of superoxide (Fig. 6B) supporting the occurrence of NOX activation. These results indicated that the pro-oxidant effect of nLDL on HK-2 was largely mediated by the reaction products of the cPLA2 whose activation was likely linked to stimulation of the inward current of external Ca2+ into the cell.

Bottom Line: This study aimed to examine the effects of native non-oxidized LDL on cellular oxidative metabolism in cultured human proximal tubular cells.All the above LDL-induced effects were completely abrogated by chelating extracellular Ca(2+) as well as by inhibition of the Ca(2+) -activated cytoplasmic phospholipase A2, NADPH oxidase and mitochondrial permeability transition.This involves first oxidants production via the plasmamembrane NADPH oxidase and then propagates downstream to mitochondria eliciting redox- and Ca(2+) -dependent dysfunctions leading to cell-harming conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, University of Foggia, Foggia, Italy.

Show MeSH
Related in: MedlinePlus