Critical role of peroxiredoxin 6 in the repair of peroxidized cell membranes following oxidative stress.

Phospholipids are a major structural component of all cell membranes; their peroxidation represents a severe threat to cellular integrity and their repair is important to prevent cell death. Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activity, plays a critical role in antioxidant defense of the lung and other organs. We investigated the role of Prdx6 in the repair of peroxidized cell membranes in pulmonary microvascular endothelial cells (PMVEC) and isolated mouse lungs treated with tert-butyl hydroperoxide and lungs from mice exposed to hyperoxia (100% O(2)). Lipid peroxidation was evaluated by measurement of thiobarbituric acid reactive substances, oxidation of diphenyl-1-pyrenylphosphine, or ferrous xylenol orange assay. The exposure dose was varied to give a similar degree of lipid peroxidation at the end of exposure in the different models. Values for lipid peroxidation returned to control levels within 2 h after oxidant removal in wild-type PMVEC and perfused lungs but were unchanged in Pxdx6 null preparations. An intermediate degree of repair was observed with PMVEC and lungs that expressed only C47S or D140A mutant Prdx6; the former mutant does not have peroxidase activity, while the latter loses its PLA(2) activity. Prdx6 null mice showed markedly delayed recovery from lipid peroxidation during 20 h observation following exposure to hyperoxia. Thus, Prdx6 plays a critical role in the repair of peroxidized phospholipids in cell membranes and the recovery of lung cells from peroxidative stress; the peroxidase and PLA(2) activity each contribute to the recovery process.

Inhibition of the phospholipase A2 activity of peroxiredoxin 6 prevents lung damage with exposure to hyperoxia.

Lung injury associated with hyperoxia reflects in part the secondary effects of pulmonary inflammation and the associated production of reactive oxygen species due to activation of NADPH oxidase, type 2 (NOX2). Activation of NOX2 requires the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6). Therefore, we evaluated whether blocking Prdx6 PLA2 activity using the inhibitor MJ33 would be protective in a mouse model of acute lung injury resulting from hyperoxic exposure. Mice were treated with an intraperitoneal injection of MJ33 (2.5nmol/g body weight) at the start of exposure (zero time) and at 48h during continuous exposure to 100% O2 for 80h. Treatment with MJ33 reduced the number of neutrophils and the protein content in the fluid obtained by bronchoalveolar lavage, inhibited the increase in lipid peroxidation products in lung tissue, decreased the number of apoptotic cells in the lung, and decreased the perivascular edema associated with the 80h exposure to hyperoxia. Thus, blocking Prdx6 PLA2 activity by MJ33 significantly protected lungs against damage from hyperoxia, presumably by preventing the activation of NOX2 and the amplification of lung injury associated with inflammation. These findings demonstrate that MJ33, a potent inhibitor of Prdx6 PLA2 activity, can protect mouse lungs against the manifestations of acute lung injury due to oxidative stress.

Multigenerational study of chemically induced cytotoxicity and proliferation in cultures of human proximal tubular cells.

Primary cultures of human proximal tubular (hPT) cells are a useful experimental model to study transport, metabolism, cytotoxicity, and effects on gene expression of a diverse array of drugs and environmental chemicals because they are derived directly from the in vivo human kidney. To extend the model to investigate longer-term processes, primary cultures (P0) were passaged for up to four generations (P1-P4). hPT cells retained epithelial morphology and stained positively for cytokeratins through P4, although cell growth and proliferation successively slowed with each passage. Necrotic cell death due to the model oxidants tert-butyl hydroperoxide (tBH) and methyl vinyl ketone (MVK) increased with increasing passage number, whereas that due to the selective nephrotoxicant S-(1,2-dichlorovinyl)-l-cysteine (DCVC) was modest and did not change with passage number. Mitochondrial activity was lower in P2-P4 cells than in either P0 or P1 cells. P1 and P2 cells were most sensitive to DCVC-induced apoptosis. DCVC also increased cell proliferation most prominently in P1 and P2 cells. Modest differences with respect to passage number and response to DCVC exposure were observed in expression of three key proteins (Hsp27, GADD153, p53) involved in stress response. Hence, although there are some modest differences in function with passage, these results support the use of multiple generations of hPT cells as an experimental model.

Modulation of mitochondrial glutathione status and cellular energetics in primary cultures of proximal tubular cells from remnant kidney of uninephrectomized rats.

Compensatory renal hypertrophy following reduction in renal mass leads to a hypermetabolic state and increases in basal mitochondrial oxidative stress and susceptibility to several nephrotoxicants. Previous studies provide conflicting data on whether renal mitochondria after reduction in renal mass undergo proliferation or hypertrophy or both. In the present study, our goal was to determine whether mitochondria of hypertrophied kidney undergo hypertrophy or proliferation after uninephrectomy using the uninephrectomized (NPX) rat model. Renal proximal tubular (PT) cells from NPX rats exhibited increased mitochondrial density, membrane potential and protein but no significant difference in mitochondrial DNA, as compared to PT cells from control rats. Our previous studies showed that overexpression of two mitochondrial anion transporters, the dicarboxylate (DIC, Slc25a10) and oxoglutarate (OGC, Slc25a11) carriers, in NRK-52E cells resulted in increased mitochondrial uptake of glutathione (GSH) and protection from chemically induced apoptosis. In the present study, we overexpressed DIC- and OGC-cDNA plasmids to assess their function in renal PT cells after compensatory renal hypertrophy. PT cells from NPX rats that were first preincubated with GSH were protected from cytotoxicity due to the mitochondrial inhibitor antimycin A by overexpression of either of the two mitochondrial GSH transporters. Our present results provide further evidence that compensatory renal hypertrophy is associated primarily with mitochondrial hypertrophy and hyperpolarization and that manipulation of mitochondrial GSH transporters in PT cells of hypertrophied kidney can alter susceptibility to chemically induced injury under appropriate conditions and may be a suitable therapeutic approach.

Influence of renal compensatory hypertrophy on mitochondrial energetics and redox status.

A reduction in functional renal mass is common in numerous renal diseases and aging. The remaining functional renal tissue undergoes compensatory growth primarily due to hypertrophy. This is associated with a series of physiological, morphological and biochemical changes similar to those observed after uninephrectomy. Previous work showed that compensatory renal cellular hypertrophy resulted in an increase in susceptibility to several drugs and environmental chemicals and appeared to be associated with oxidative stress. Compensatory renal cellular hypertrophy was also associated with increases in mitochondrial metabolic activity, uptake of glutathione (GSH) across renal plasma and mitochondrial inner membranes, and intracellular GSH concentrations. Based on these observations, we hypothesize that the morphological, physiological and biochemical changes in the hypertrophied kidney are associated with marked alterations in renal cellular energetics, redox status and renal function in vivo. In this study, we used a uninephrectomized (NPX) rat model to induce compensatory renal growth. Our results show alterations in renal physiological parameters consistent with modest renal injury, altered renal cellular energetics, upregulation of certain renal plasma membrane transporters, including some that have been observed to transport GSH, and evidence of increased oxidative stress in mitochondria from the remnant kidney of NPX rats. These studies provide additional insight into the molecular changes that occur in compensatory renal hypertrophy and should help in the development of novel therapeutic approaches for patients with reduced renal mass.