![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CELLULAR AND MOLECULAR
Department of Medicine, Brown University School of Medicine, Providence, Rhode Island (S.Z.); Departments of Pharmaceutical Sciences (G.R.K., R.G.S.) and Surgery (Y.Y.), Medical University of South Carolina, Charleston, South Carolina; and Department of Immunology, the Scripps Research Institute, La Jolla, California (J.H.)
Received January 10, 2008; accepted March 12, 2008.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). In contrast, necrotic cell death is initiated by mitochondrial damage and ATP depletion, and it is associated with cellular swelling and progressive increases in plasma membrane permeability (Liu et al., 2004). The extent of exposure to an insult determines the nature of cell death: milder insults induce apoptosis, whereas more severe insults usually cause necrotic cell death. Both forms of cell death are observed in injured kidneys in animal models and in patients with acute renal failure (ARF) (Devarajan, 2005).
Necrotic cell death is often considered to be a passive process involving severe loss of ATP, release of endoplasmic reticulum Ca2+, increases in cytosolic-free Ca2+, influx of extracellular ions, and breakdown of the cytoskeletal-plasma membrane structure (Liu et al., 2004). Necrotic cell death is also regulated by the mitochondrial permeability transition (MPT) (Festjens et al., 2006).
The MPT is a process resulting in the permeabilization of the inner mitochondrial membrane that leads to loss of mitochondrial membrane potential (MMP), mitochondrial swelling, and rupture of the outer mitochondrial membrane (Brenner and Grimm, 2006). Although the molecular composition and regulation of MPT pores are being debated, it is thought that the MPT occurs after the opening of a channel, which is termed the permeability transition (PT) pore. This classically defined MPT pore putatively consists of the voltage-dependent anion channel, the adenine nucleotide translocator, CypD, and possibly other molecules (Baek et al., 2003). Cyclosporin A (CsA) binds to CypD and blocks MPT under many conditions; however, numerous reports show that MPT or the loss of MMP can occur in a CsA-insensitive manner (Kushnareva et al., 2001; Kinsey et al., 2007a). This CsA-insensitive, unregulated PT pore was first proposed by He and Lemasters (2002) to explain the finding that MPT can occur in a Ca2+-indepndent fashion that is CsA insensitive. This unregulated PT pore or increased inner membrane permeability can be activated after exposure of mitochondria to oxidants such as tert-butyl hydroperoxide (He and Lemasters, 2002). The formation and regulation of this membrane permeability remains unclear.
In response to reactive oxygen species (ROS) and other stimuli, mitogen-activated protein kinase (MAPK) pathways are activated (Zhuang and Schnellmann, 2006). MAPK pathways are composed of extracellular signal-regulated kinases (ERK)1/2, Jun N-terminal kinase, and p38 pathways. Among them, activation of ERK1/2 is generally thought to confer a survival advantage to cells (Zhuang and Schnellmann, 2006). However, increasing evidence suggests that the activation of ERK1/2 also contributes to cell death in some cell types and organs under certain conditions (Zhuang and Schnellmann, 2006). For example, ERK activation occurs in animal models of ischemia- and trauma-induced brain injury and cisplatin-induced renal injury, and inactivation of ERK reduces the extent of tissue damage (Zhuang and Schnellmann, 2006). ERK1/2 are also activated in neuronal and renal epithelial cells upon exposure to oxidative stress and toxicants or deprivation of growth factors, and inhibition of the ERK pathway blocks apoptosis (Zhuang and Schnellmann, 2006). ERK1/2 are typically located in the cytosol and mitochondria (Yoon and Seger, 2006). Whereas the role of cytosol ERK1/2 is well studied and involved in multiple cellular functions (Yoon and Seger, 2006), the role of mitochondrial ERK1/2 remains poorly understood. Nowak et al. (2006) have recently shown that activation of the ERK pathway precedes mitochondrial dysfunction and is associated with the decrease in oxygen consumption in a model of sublethal injury produced by tert-butylhydroperoxide. ERK1/2 are activated by ERK kinase1/2 [mitogen-activated protein kinase kinase (MEK)1/2] by phosphorylation of threonine and tyrosine residues (Wolf, 2005).
Our and other studies have recently demonstrated that ERK mediates apoptosis of renal epithelial cells after exposure to oxidants or nephrotoxicants (Nowak, 2002; Zhuang et al., 2007). Although necrotic cell death also occurs in response to these stimuli, the role of ERK in necrotic cell death and the mechanistic basis of ERK in this process remain poorly understood. In this study, we investigated the role of the ERK pathway in mitochondrial dysfunction and necrotic cell death using the naturally occurring oxidant, H2O2. Herein, our studies reveal that H2O2 elicits renal proximal tubular cell (RPTC) necrotic cell death and mitochondrial dysfunction through ERK-dependent and -independent pathways.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Isolation and Culture of Renal Proximal Tubules and Experimental Protocols. Female New Zealand White rabbits (2–3 kg) were purchased from Myrtle's Rabbitry (Thompson Station, TN). RPTC were isolated using the iron oxide perfusion method and grown in six-well or 35-mm tissue culture dishes under improved conditions as described previously (Nowak and Schnellmann, 1996). The serum-free culture medium was a 1:1 mixture of Dulbecco's modified Eagle's medium/Ham's F-12 (without glucose, phenol red, or sodium pyruvate) supplemented with 15 mM HEPES buffer, 2.5 mM L-glutamine, 1 µM pyridoxine HCl, 15 mM sodium bicarbonate, and 6 mM lactate. Hydrocortisone (50 nM), selenium (5 ng/ml), human transferrin (5 µg/ml), bovine insulin (10 nM), and L-ascorbic acid-2-phosphate (50 µM) were added daily to fresh culture medium. RPTC were used in all experiments at approximately 60 to 70% confluence. When various pharmacological inhibitors were used, the same volume of dimethyl sulfoxide was added to the culture in control samples.
Replication-Deficient Adenovirus Infection. The construction and characterization of recombinant adenoviruses containing the coding regions of the active MEK1 (Ad-caMEK1) and dominant-negative mutant of MEK1 (Ad-dnMEK) driven by the cytomegalovirus immediate early promoter were provided by J. Han (Foschi et al., 1997). RPTC were infected with each virus at a multiplicity of infection (MOI) of 100 plaque-forming units (pfu) for 2 h at 37°C in a humidified, 5% CO2 incubator. Afterward, the cultures were placed in culture media for an additional 48 h and then exposed to oxidant injury for the time periods described in the figure legends. At an MOI of 100 pfu, approximately 100% of the cells showed expression of the viral gene insert as indicated by the X-gal assay (data not shown).
Assessment of Necrotic Cell Death. Necrotic cell death was assessed by the trypan blue exclusion assay and lactate dehydrogenase (LDH) release in the medium. At the end of protocol, 0.4% trypan blue was added into the culture dish. After 
5 min of equilibration, the cells in five fields (40x) were counted using a microscope, and the numbers of necrotic cells were expressed as the percentage of the total cell population. For LDH measurements, the cellular medium was collected, and the enzyme activity was determined spectrophotometrically using an assay kit according to the instructions provided by the manufacturer (Sigma-Aldrich). Data were normalized to solvent-treated cultures.
MTT Assay. An MTT assay was used to assess cell viability. MTT was added (final concentration of 0.5 mg/ml) to RPTC and incubated for 1 h, and tetrazolium was released by dimethyl sulfoxide. Optical density was determined with a spectrophotometer (570 nm), and data were normalized to solvent-treated cultures.
Measurement of Annexin V and Propidium Iodide Staining. Annexin and propidium iodide (PI) staining were determined using flow cytometry as described previously with modifications (Cummings and Schnellmann, 2002). In brief, media were removed, and RPTC were washed twice with phosphate-buffered saline (PBS) and incubated in binding buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2, pH 7.4) containing annexin V-fluorescein isothiocyanate (25 µg/ml) and PI (25 µg/ml) for 10 min. Cells were washed three times in binding buffer and then released from the monolayers using a rubber policeman. Staining was quantified using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). For each measurement, 10,000 events were counted.
Determination of Mitochondrial Membrane Potential. The mitochondrial membrane potential (MMP) was assessed using JC-1, a lipophilic cation that can selectively enter into mitochondria. This mitochondrial dye, which normally exists in solution as a monomer emitting a green fluorescence, forms a dimer emitting red fluorescence in a reaction driven by the MMP. The change in fluorescence can be detected by flow cytometry with a decrease in the red fluorescence or the increase in green fluorescence intensity indicating mitochondrial depolarization. Cell staining was performed following the manufacturer's instructions. In brief, 1 x 106 cells were incubated with 10 mg/ml JC-1 for 10 min at 37°C and washed twice in ice-cold PBS, and staining was determined using flow cytometry.
Isolation of Renal Cortical Mitochondria. After euthanasia, rabbit kidneys were removed by blunt dissection, and cortical tissue was collected and placed in ice-cold mitochondrial isolation buffer containing the following: 270 mM sucrose, 5 mM Tris-HCl, and 1 mM EGTA, pH 7.4. Renal cortical mitochondria (RCM) were isolated by differential centrifugation as described previously by Kinsey et al. (2007a).
Mitochondrial Swelling. Isolated mitochondria were suspended at a concentration of 0.4 mg protein/ml in swelling buffer (150 mM KCl and 20 mM Tris-HCl, pH 7.4) in a 96-well plate and incubated with diluent, inhibitors, or antioxidants at room temperature for 10 min. Ferrous sulfate heptahydrate (Sigma-Aldrich) to achieve a final Fe2+ concentration of 10 µM or diluent (swelling buffer) was then added to initiate oxidative stress as described previously (Hunter et al., 1963; Kinsey et al., 2007a). Mitochondrial swelling was measured using a SpectraMax 190 spectrophotometric plate reader (Molecular Devices, Sunnyvale, CA) at 540 nm over time as described previously (Kinsey et al., 2007a).
Measurement of cis-Parinaric Acid Oxidation. Lipid peroxidation in isolated mitochondria was measured using the fluorescent lipid, cis-parinaric acid as described previously (Kinsey et al., 2007a). In brief, isolated mitochondria were suspended at a concentration of 1 mg protein/ml in swelling buffer and incubated on ice with cis-parinaric acid (6.4 µM) for 10 min. The mitochondria were centrifuged, the supernatant was discarded, and the mitochondria were resuspended (0.4 mg protein/ml) in deoxygenated swelling buffer. Mitochondria were added to a 96-well plate and incubated with diluent, inhibitors, or antioxidants at room temperature for 10 min then treated with 10 µMFe2+. Lipid peroxidation was measured as the loss of cis-parinaric acid fluorescence (excitation 320 nm, emission 405 nm) over time using a Fluoroskan Ascent fluorescent plate reader (Thermo Fisher Scientific, Waltham, MA).
Immunoblot Analysis. Cells were washed once with PBS without Ca2+ and Mg2+ and then suspended in the lysis buffer (0.25 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.1 mg/ml bromphenol blue, and 0.5% 2-mercaptoethanol). Mitochondria were harvested after various treatments and then suspended in the same lysis buffer. After sonication for 15 s, equal amounts of cellular protein lysates were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. After treatment with 5% skim milk at 4°C overnight, the membranes were probed with various antibodies for 1 h followed by appropriate horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Bound antibodies were visualized by chemiluminescence detection on autoradiographic film.
Statistical Analysis. Data are presented as means ± S.E. and were subjected to one-way analysis of variance. Multiple means were compared using Tukey's test. Differences between mean values were considered to be statistically significant at P < 0.05. Renal proximal tubules isolated from an individual rabbit represent a single experiment (n = 1) consisting of data obtained from three wells.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). To induce necrosis of RPTC, 60 to 70% confluent cells were treated with 1 mM H2O2 for 0 to 3 h, and necrotic cell death was monitored using established markers, trypan blue and propidium iodide uptake, and LDH release. Exposure of cells to H2O2 for 2 and 3 h resulted in an increase in trypan blue-positive cells (Fig. 1, A and B). Only 7 ± 1% of cells were necrotic in the control group. As shown in Fig. 1C, a 3-h H2O2 treatment resulted in a 2.3-fold increase in the level of LDH activity in the medium. Similar results were obtained by measuring PI staining using flow cytometry (Fig. 1D).
|
Inhibition of the MEK/ERK Pathway Blocks H2O2-Induced RPTC Necrotic Cell Death. To determine whether the MEK/ERK pathway mediates necrotic cell death, RPTC were pretreated with either U0126 or PD98059, two MEK1/2-specific inhibitors (Alessi et al., 1995; Favata et al., 1998), and then exposed to 1 mM H2O2 for 3 h. H2O2-induced release of LDH was blocked by U0126 and PD98059 (Fig. 2, A and C). Likewise, pretreatment with U0126 or PD98059 increased cell viability in H2O2-treated RPTC as reflected by the MTT assay (Fig. 2, B and D). In contrast, pretreatment of cells with SB203580, a specific inhibitor of p38 MAPK, at concentrations of up 20 µM or SP600125, an inhibitor of Jun N-terminal kinase, at doses of 20 µM did not protect RPTC from death (data not shown). Thus, ERK is a critical mediator of H2O2-induced RPTC necrotic cell death.
|
|
; Zhuang et al., 2007). ERK1/2 phosphorylation levels increased and decreased in Ad-caMEK1- and Ad-dnMEK1-transfected RPTC, respectively, as demonstrated in our recent report (Zhuang et al., 2007). These results support the pharmacological inhibitor data that ERK1/2 are mediators of H2O2-induced RPTC.
|
H2O2 Treatment Results in the Loss of MMP and Is Blocked by Inhibition of the MEK1/ERK Pathway. Previous studies have shown that H2O2-induced cell death is associated with the loss of MMP (Zhou et al., 2006), and recent studies suggested that ERK is constitutively expressed in the mitochondria of RPTC (Nowak et al., 2006). To determine whether ERK plays a role in regulating H2O2-induced loss of MMP, we examined the effect of U0126 and PD98059 on the loss of MMP using flow cytometry after JC-1 staining. Mitochondrial depolarization was measured as an increase in green fluorescence intensity (Reers et al., 1995). Figure 5 illustrates that H2O2 treatment resulted in an increase in green fluorescence intensity, which was blocked by U0126 and decreased by PD98059. These results are consistent with the partial block of ERK1/2 phosphorylation by PD98059 (Fig. 3C). We suggest that ERK mediates, in part, the loss of MMP and, in turn, necrotic cell death after H2O2 exposure.
|
). In response to Fe2+, ERK1/2 were phosphorylated at 10 and 25 min. Pretreatment with U0126 (10 µM) completely blocked and PD98059 (100 µM) partially inhibited Fe2+-induced phosphorylation of ERK1/2 (Fig. 6). U0126 inhibited Fe2+-induced mitochondrial swelling in a concentration-dependent manner with 1 µM U0126 partially blocking and 10 µM U0126 completely blocking mitochondrial swelling (Fig. 7). PD98059 treatment led to a partial inhibition of mitochondrial swelling with equal effects at 50 and 100 µM. As a positive control, the antioxidant butylated hydroxyanisole (BHA) blocked Fe2+-induced mitochondrial swelling (Fig. 7). This result is consistent with our statement that the mitochondrial swelling induced by Fe2+ occurs through production of ROS (Kinsey et al., 2007a).
|
|
|
H2O2-Induced Necrotic Cell Death and Mitochondrial Swelling Are Not Sensitive to CsA. Recent studies indicated that the MPT plays an important role in necrotic cell death induced by Ca2+ and H2O2 in fibroblasts and hepatocytes (Festjens et al., 2006). CsA binds cyclophilin D and blocks the MPT (Kallen et al., 1991). The primary goal of this experiment was to determine whether the oxidant-induced necrotic cell death and mitochondrial dysfunction are mediated through the CsA-sensitive MPT. Treatment of RPTC with CsA at various concentrations (0.1–10 µM) did not block H2O2-induced LDH release nor loss of MMP at 3 h (Fig. 9, A and B). These data suggest that the oxidant-induced RPTC necrosis is not mediated by the classic CsA-sensitive MPT.
|
). We suggest that oxidant stress does not induce classic MPT but induces an unregulated CsA-insensitive form of the MPT or a nonspecific increase in inner membrane permeability.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Golstein and Kroemer, 2007). In this study, we demonstrate that inactivation of the MEK/ERK pathway blocked oxidant-induced necrotic cell death of RPTC, implying that ERK is a critical signaling molecule that drives the necrotic response in renal epithelial cells.
Because mitochondrial dysfunction has been identified as a key mechanism underlying both necrotic cell death and apoptosis (Kim et al., 2003), we examined the role of ERK in the regulation of mitochondrial dysfunction after oxidant exposure. In cultured RPTC, we showed that the MEK inhibitors, U0126 and PD98059, blocked and partially inhibited H2O2-induced decreases in MMP, respectively (Fig. 5). In isolated RCM, we demonstrated that inhibition of the MEK/ERK pathway partially blocked the mitochondrial swelling (Fig. 7). We suggest that ERK is functionally linked to mitochondrial dysfunction by promoting the loss of MMP. In support of this observation, Nowak et al. (2006) recently showed that ERK is constitutively expressed in the mitochondria of RPTC, and activation of the ERK pathway precedes mitochondrial dysfunction (measured by decreases in basal, uncoupled and state 3 respirations, and ATP production) in a model of sublethal injury produced by tert-butylhydroperoxide. Furthermore, the decrease in oxygen consumption was partially reversed by PD98059 and U0126 (Nowak et al., 2006). Because mitochondrial dysfunction and loss of MMP lead to ATP depletion and initiate a necrotic response, ERK-mediated loss of MMP would represent a novel mechanism of renal tubular epithelial cell death.
The mechanism and mitochondrial target(s) of ERK that directly function to promote loss of MMP remain unclear. However, it does not appear that the decreased MMP is mediated through the CsA-sensitive MPT pore because CsA did not prevent the loss of MMP in RPTC nor mitochondrial swelling in isolated RCM after oxidant stress (Fig. 9, B and C). In line with our observations, the loss of MMP was also not prevented by CsA in response to some other stimuli such as free fatty acids and thyroxine (Malkevitch et al., 1997; Sultan and Sokolove, 2001). Because CsA regulates the MPT pore through CypD, one of the primary components of the MPT pore (Festjens et al., 2006), our results also suggest that CypD is not involved in regulating the loss of MMP in renal epithelial cells exposed to H2O2. Similar to our observation, CypD is not required for necrotic cell death induced by staurosporine, tumor necrosis factor 
, and adenovirus-mediated Bax overexpression (Baines et al., 2005). In contrast, CypD has been reported to mediate necrosis of hepatocytes in response to this oxidant (Festjens et al., 2006). On this basis, we suggest that ERK mediates loss of MMP independent of CyD (CsA-resistant) in RPTC.
At this time, the molecular composition and regulation of the opening of the CsA-insensitive MPT pores or nonspecific inner membrane permeability remain unknown. He and Lemasters (2002) have proposed that an "unregulated" PT pore is formed when oxidant and other stresses result in the formation of large amphipathic protein aggregates, the size and number of which exceed the mitochondrial chaperone capacity that normally regulates the PT pore.
In isolated mitochondria, we observed that 10 µM U0126 partially inhibited Fe2+-induced lipid peroxidation, whereas 1 µM U0126 and 100 µM PD98059 had no effect (Fig. 8). These inconsistent results may be attributed to the different capacities of these two inhibitors. U0126 inhibits active and inactive MEK1/2, whereas PD98059 inhibits inactive MEK1/2, and U0126 is more potent than PD98059 (Favata et al., 1998). In support of this notion, our data revealed that pretreatment with U0126 (10 µM) completely blocked and PD98059 (100 µM) partially inhibited Fe2+-induced phosphorylation of mitochondrial ERK1/2 (Fig. 6).
Alternatively, U0126 may have more than one function. It has been reported that U0126 is able to inhibit formation of ROS induced by amyloid beta peptide in human neutrophil granulocytes (Andersen et al., 2003). Our observation that 10 µM U0126 blocks Fe2+-induced lipid peroxidation in isolated mitochondria (Fig. 8) is consistent with the study of Andersen et al. (2003) and provides evidence that U0126 may act as an antioxidant at higher concentrations. The observation that 1 µM U0126 and 50 and 100 µM PD98059, which had no effect on lipid peroxidation (Fig. 8), partially blocked Fe2+-induced mitochondrial swelling/injury supports the hypothesis that oxidant-induced loss of MMP occurs through ERK1/2-sensitive and -insensitive pathways. These results are consistent with the cellular data in that PD98059 partially blocked the loss of MMP produced by H2O2 and 10 µM U0126 completely blocked the loss of MMP (Fig. 3, B and C).
ERK may contribute to MMP through a mechanism involving lipid peroxidation. It has been reported that ERK can directly activate cytosolic phospholipase A2 (cPLA2) by phosphorylation at serine 505 (Lin et al., 1993). cPLA2 activation results in hydrolysis of membrane phospholipids and release of free fatty acids including arachidonic acid, which may alter MMP (Higuchi and Yoshimoto, 2002). Alternatively, our laboratory has shown that RPTC and RCM possess Ca2+-independent phospholipase A2
(iPLA2). In contrast to cPLA2, iPLA2 protects against oxidant-induced lipid peroxidation, mitochondrial dysfunction, and necrotic cell death (Cummings et al., 2002). Protein kinase C
-mediated phosphorylation seems to activate iPLA2, but the effect of ERK activation on RCM iPLA2 activity has not been examined. It is possible that ERK-mediated phosphorylation of iPLA2 decreases its activity or inhibits its repair functions, leading to increased lipid peroxidation. A MAPK consensus motif has recently been identified in iPLA2 (Tanaka et al., 2000), and additional studies are required to determine the effect of ERK activation on iPLA2 activity in RPTC.
ERK activation has been implicated in tissue injury and inflammation in animal models with ARF and other renal diseases. Jo et al. (2005) showed that cisplatin-induced ARF is accompanied by activation of ERK1/2 and that inhibition of ERK1/2 with U0126 resulted in functional and histological protection in mice. Alderliesten et al. (2007) demonstrated that ERK1/2 are activated in a single-kidney rat model of ischemia/reperfusion-induced acute kidney injury, and inhibition of the ERK pathway with U0126 attenuated pathological damage to the kidney. Furthermore, Gong et al. (2006) showed that adenovirus-mediated antisense ERK2 gene therapy attenuated chronic allograft nephropathy in a rat model of renal transplantation. In these studies, ERK inhibition not only attenuated renal damage, but it also decreased inflammatory responses, suggesting a role for ERK in mediating inflammatory pathways. Although it is not clear whether ERK activation stimulates the release of inflammatory factors in renal epithelial cells, or other cell types in the kidney after oxidant injury, it is possible that ERK triggers the inflammatory response through induction of necrotic cell death. Necrotic cells can initiate proinflammatory signaling cascades by actively releasing inflammatory cytokines and by releasing their contents when they lyse (Proskuryakov et al., 2003).
In summary, our results demonstrate that ERK mediates mitochondrial dysfunction and necrotic cell death of renal epithelial cells after oxidant injury. Furthermore, ERK is functionally coupled to MMP through activation of a CsA-insensitive MPT pore or a nonspecific inner membrane permeability. This study, in conjunction with the proapoptotic role of ERK in RPTC, suggests that ERK is a critical mediator of renal epithelial cell death. Because ROS are generated in renal injury after ischemia/reperfusion or exposure to various insults, and both apoptosis and necrosis contribute to the pathogenesis of ARF, a better understanding of the mechanism by which ERK mediates necrotic cell death will help develop strategies to interrupt the cell death cascade and thereby abrogate renal injury.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ARF, acute renal failure; MPT, mitochondrial permeability transition; MMP, mitochondrial membrane potential; PT, permeability transition; CsA, cyclosporin A; ROS, reactive oxygen species; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase kinase; RPTC, renal proximal tubular cells; U0126, 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]-butadiene; SB203580, 4-(4-fluorophenyl)-2-(4 methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; SP600125, anthra[1,9-cd]pyrazol-6(2H)-one; PD98059, 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one; Ad-caMEK1, active MEK1; Ad-dnMEK, dominant-negative mutant of MEK1; MOI, multiplicity of infection; pfu, plaque-forming units; LDH, lactate dehydrogenase; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PBS, phosphate-buffered saline; PI, propidium iodide; MMP, matrix metalloproteinase; RCM, renal cortical mitochondria; BHA, antioxidant butylated hydroxyanisole; cPLA2, cytosolic phospholipase A2; iPLA2, independent phospholipase A2.
Address correspondence to: Dr. Shougang Zhuang, Department of Medicine Rhode Island Hospital, Middle House 301, 593 Eddy Street, Providence, RI 02903. E-mail: szhuang{at}lifespan.org
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Alderliesten M, de Graauw M, Oldenampsen J, Qin Y, Pont C, van Buren L, and van de Water B (2007) Extracellular signal-regulated kinase activation during renal ischemia/reperfusion mediates focal adhesion dissolution and renal injury. Am J Pathol 171: 452-462.
Alessi DR, Cuenda A, Cohen P, Dudley DT, and Saltiel AR (1995) PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270: 27489-27494.
Andersen JM, Myhre O, Aarnes H, Vestad TA, and Fonnum F (2003) Identification of the hydroxyl radical and other reactive oxygen species in human neutrophil granulocytes exposed to a fragment of the amyloid beta peptide. Free Radic Res 37: 269-279.[CrossRef][Medline]
Baek SM, Kwon CH, Kim JH, Woo JS, Jung JS, and Kim YK (2003) Differential roles of hydrogen peroxide and hydroxyl radical in cisplatin-induced cell death in renal proximal tubular epithelial cells. J Lab Clin Med 142: 178-186.[CrossRef][Medline]
Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA, Brunskill EW, Sayen MR, Gottlieb RA, Dorn GW, et al. (2005) Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434: 658-662.[CrossRef][Medline]
Brenner C and Grimm S (2006) The permeability transition pore complex in cancer cell death. Oncogene 25: 4744-4756.[CrossRef][Medline]
Cummings BS, McHowat J, and Schnellmann RG (2002) Role of an endoplasmic reticulum Ca(2+)-independent phospholipase A(2) in oxidant-induced renal cell death. Am J Physiol Renal Physiol 283: F492-F498.
Cummings BS and Schnellmann RG (2002) Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways. J Pharmacol Exp Ther 302: 8-17.
Devarajan P (2005) Cellular and molecular derangements in acute tubular necrosis. Curr Opin Pediatr 17: 193-199.[CrossRef][Medline]
Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, et al. (1998) Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273: 18623-18632.
Festjens N, Vanden Berghe T, and Vandenabeele P (2006) Necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response. Biochim Biophys Acta 1757: 1371-1387.[Medline]
Foschi M, Chari S, Dunn MJ, and Sorokin A (1997) Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase. EMBO J 16: 6439-6451.[CrossRef][Medline]
Golstein P and Kroemer G (2007) Cell death by necrosis: towards a molecular definition. Trends Biochem Sci 32: 37-43.[CrossRef][Medline]
Gong N, Dong C, Chen Z, Chen X, Guo H, Zeng Z, Ming C, and Klaus Chen Z (2006) Adenovirus-mediated antisense-ERK2 gene therapy attenuates chronic allograft nephropathy. Transplant Proc 38: 3228-3230.[CrossRef][Medline]
He L and Lemasters JJ (2002) Regulated and unregulated mitochondrial permeability transition pores: a new paradigm of pore structure and function? FEBS Lett 512: 1-7.[CrossRef][Medline]
Higuchi Y and Yoshimoto T (2002) Arachidonic acid converts the glutathione depletion-induced apoptosis to necrosis by promoting lipid peroxidation and reducing caspase-3 activity in rat glioma cells. Arch Biochem Biophys 400: 133-140.[CrossRef][Medline]
Hunter FE Jr, Gebicki JM, Hoffsten PE, Weinstein J, and Scott A (1963) Swelling and lysis of rat liver mitochondria induced by ferrous ions. J Biol Chem 238: 828-835.
Jo SK, Cho WY, Sung SA, Kim HK, and Won NH (2005) MEK inhibitor, U0126, attenuates cisplatin-induced renal injury by decreasing inflammation and apoptosis. Kidney Int 67: 458-466.[CrossRef][Medline]
Kallen J, Spitzfaden C, Zurini MG, Wider G, Widmer H, Wuthrich K, and Walkinshaw MD (1991) Structure of human cyclophilin and its binding site for cyclosporin A determined by X-ray crystallography and NMR spectroscopy. Nature 353: 276-279.[CrossRef][Medline]
Kim JS, He L, and Lemasters JJ (2003) Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun 304: 463-470.[CrossRef][Medline]
Kinsey GR, McHowat J, Beckett CS, and Schnellmann RG (2007a) Identification of calcium-independent phospholipase A2 in mitochondria and its role in mitochondrial oxidative stress. Am J Physiol Renal Physiol 292: F853-F860.
Kinsey GR, McHowat J, Patrick KS, and Schnellmann RG (2007b) Role of Ca2+-independent phospholipase A2 in Ca2+-induced mitochondrial permeability transition. J Pharmacol Exp Ther 321: 707-715.
Kushnareva YE, Polster BM, Sokolove PM, Kinnally KW, and Fiskum G (2001) Mitochondrial precursor signal peptide induces a unique permeability transition and release of cytochrome c from liver and brain mitochondria. Arch Biochem Biophys 386: 251-260.[CrossRef][Medline]
Lin LL, Wartmann M, Lin AY, Knopf JL, Seth A, and Davis RJ (1993) cPLA2 is phosphorylated and activated by MAP kinase. Cell 72: 269-278.[CrossRef][Medline]
Liu X, Van Vleet T, and Schnellmann RG (2004) The role of calpain in oncotic cell death. Annu Rev Pharmacol Toxicol 44: 349-370.[CrossRef][Medline]
Malkevitch NV, Dedukhova VI, Simonian RA, Skulachev VP, and Starkov AA (1997) Thyroxine induces cyclosporin A-insensitive, Ca2+-dependent reversible permeability transition pore in rat liver mitochondria. FEBS Lett 412: 173-178.[CrossRef][Medline]
Nowak G (2002) Protein kinase C- and ERK1/2 mediate mitochondrial dysfunction, decreases in active Na+ transport, and cisplatin-induced apoptosis in renal cells. J Biol Chem 277: 43377-43388.
Nowak G, Clifton GL, Godwin ML, and Bakajsova D (2006) Activation of ERK1/2 pathway mediates oxidant-induced decreases in mitochondrial function in renal cells. Am J Physiol Renal Physiol 291: F840-F855.
Nowak G and Schnellmann RG (1996) L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture. Am J Physiol 271: C2072-C2080.[Medline]
Proskuryakov SY, Konoplyannikov AG, and Gabai VL (2003) Necrosis: a specific form of programmed cell death? Exp Cell Res 283: 1-16.[CrossRef][Medline]
Reers M, Smiley ST, Mottola-Hartshorn C, Chen A, Lin M, and Chen LB (1995) Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol 260: 406-417.[Medline]
Subramaniam S, Zirrgiebel U, von Bohlen Und Halbach O, Strelau J, Laliberte C, Kaplan DR, and Unsicker K (2004) ERK activation promotes neuronal degeneration predominantly through plasma membrane damage and independently of caspase-3. J Cell Biol 165: 357-369.
Sultan A and Sokolove PM (2001) Free fatty acid effects on mitochondrial permeability: an overview. Arch Biochem Biophys 386: 52-61.[CrossRef][Medline]
Tanaka H, Takeya R, and Sumimoto H (2000) A novel intracellular membrane-bound calcium-independent phospholipase A(2). Biochem Biophys Res Commun 272: 320-326.[CrossRef][Medline]
Wolf G (2005) Role of reactive oxygen species in angiotensin II-mediated renal growth, differentiation, and apoptosis. Antioxid Redox Signal 7: 1337-1345.[CrossRef][Medline]
Yoon S and Seger R (2006) The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 24: 21-44.[Medline]
Yu SP, Canzoniero LM, and Choi DW (2001) Ion homeostasis and apoptosis. Curr Opin Cell Biol 13: 405-411.[CrossRef][Medline]
Zhou Y, Wang Q, Mark Evers B, and Chung DH (2006) Oxidative stress-induced intestinal epithelial cell apoptosis is mediated by p38 MAPK. Biochem Biophys Res Commun 350: 860-865.[CrossRef][Medline]
Zhuang S and Schnellmann RG (2006) A death-promoting role for extracellular signal-regulated kinase. J Pharmacol Exp Ther 319: 991-997.
Zhuang S, Yan Y, Daubert RA, Han J, and Schnellmann RG (2007) ERK promotes hydrogen peroxide-induced apoptosis through caspase-3 activation and inhibition of Akt in renal epithelial cells. Am J Physiol Renal Physiol 292: F440-F447.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
