Activation of ERK or inhibition of JNK ameliorates H2O2 cytotoxicity in mouse renal proximal tubule cells (original) (raw)
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MAPK activation determines renal epithelial cell survival during oxidative injury
American Journal of Physiology-Renal Physiology, 1999
Ischemia/reperfusion (I/R) injury induces both functional and morphological changes in the kidney. Necrosis, predominantly of the proximal tubule (PT), is the hallmark of this model of renal injury, whereas cells of the distal nephron survive, apparently intact. We examined whether differences in cellular outcome of the various regions of the nephron may be due to segmental variation in the activation of the mitogen-activated protein kinases (MAPKs) in response to I/R injury. Whereas c-Jun N-terminal kinase (JNK) is activated in both the cortex and inner stripe of the outer medulla, the extracellular regulated kinase (ERK) pathway is activated only in the inner stripe in which thick ascending limb (TAL) cells predominate. These studies are consistent with the notion that ERK activation is essential for survival. To test this hypothesis directly, we studied an in vitro system in which manipulation of these pathways and their effects on cellular survival could be examined. Oxidant inj...
American Journal of Physiology-Renal Physiology, 2006
Reactive oxygen species, including hydrogen peroxide (H2O2), are generated during ischemia-reperfusion and are critically involved in acute renal failure. The present studies examined the role of the extracellular signal-regulated kinase (ERK) pathway in H2O2-induced renal proximal tubular cells (RPTC) apoptosis. Exposure of RPTC to 1 mM H2O2resulted in apoptosis and activation of ERK1/2 and Akt. Pretreatment with the specific MEK inhibitors, U0126 and PD98059, or adenoviral infection with a construct that encodes a negative mutant of MEK1, protected cells against H2O2-induced apoptosis. In contrast, expression of constitutively active MEK1 enhanced H2O2-induced apoptosis. H2O2induced activation of caspase-3 and phosphorylation of histone H2B at serine 14, a posttranslational modification required for nuclear condensation, which also were blocked by ERK1/2 inhibition. Furthermore, blockade of ERK1/2 resulted in an increase in Akt phosphorylation and blockade of Akt potentiated apopt...
Biochemical Journal, 1998
The mammalian response to stress is complex, often involving multiple signalling pathways that act in concert to influence cell fate. To examine potential interactions between the signalling cascades, we have focused on the effects of a model oxidant stress in a single cell type through an examination of the relative influences of mitogen-activated protein kinases (MAPKs) as well as two proposed apoptosis regulators, nuclear factor κB (NF-κB) and Bcl-2, in determining cell survival. Treatment of HeLa cells with H2O2 resulted in a time- and dose-dependent induction of apoptosis accompanied by sustained activation of all three MAPK subfamilies: extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38. This H2O2-induced apoptosis was markedly enhanced when ERK2 activation was selectively inhibited by PD098059. Apoptosis decreased when JNK/SAPK activation was inhibited by expression of a dominant negative mutant form...
Toxicologic Pathology, 2003
Our earlier studies with S-(1,2-dichlorovinyl)-L -cysteine (DCVC) showed that prior administration of a low priming dose of 15 mg/kg, IP to mice, given 72 hours before administration of a normally lethal dose of DCVC (75 mg/kg, IP) led to renal tubule necrosis, however sustained renal tubule regeneration was observed and these mice recovered from renal failure and survived. The objective of the present study was to investigate the role of extracellular signal-regulated kinase (ERK) pathway in this autoprotection model. Following the priming dose of DCVC, IL-6 protein and mRNA increased markedly as early as 1 hour after dosing, peaking at 3 hours with a 1.5-fold increase in plasma. Immunocytochemistry on kidney sections using specific antibodies against TGF-α, HB-EGF, EGFr, IGF-1Rβ, Grb-2, and phospho-p44/42 MAP kinase (ERK1/2) revealed a significantly higher staining of these molecules 3 to 72 hours after dosing, indicating up regulation of the ERK pathway. Following a lethal dose of DCVC (75 mg/kg) the early increase in these signaling molecules was not sustained, being markedly reduced 24 and 36 hours after dosing, leading to inhibition of S-phase DNA synthesis, cell division and renal tubule repair. In contrast, prior treatment with a low dose of DCVC, followed by a high dose led to a sustained stimulation of the renal ERK pathway, renal tubule regeneration and recovery from acute renal failure. These results suggest that a sustained activation of the ERK1/2 pathway may be a key factor in enabling a continued renal tubule repair and hence protection from the progressive phase of DCVC-induced acute renal tubular necrosis in the mouse.
Chronic kidney disease (CKD) is a worldwide public health concern with limited treatment options. The incidence of CDK is increasing and the disease is associated with a poor quality of life and a high financial cost of treatment. Shen-Kang (SK), a traditional Chinese herbal medicine, has been used clinically in the treatment of renal diseases for decades. This study was carried out to validate the therapeutic effects of SK on renal injury induced by 5/6 nephrectomy, as well as its effects on the apoptosis of proximal tubule epithelial cells (HK-2 cells), in an aim to elucidate its mechanisms of action. For this purpose, an animal model of renal injury was created by subjecting rats to a 5/6 nephrectomy. The rats in the sham-operated and model groups received distilled water, while the rats in the SK and enalapril (EN) groups were treated with SK or EN. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were measured. Kidney tissues obtained from the rats were stained with hematoxylin and eosin. HK-2 cells were employed to investigate the effects of SK on the apoptosis of renal proximal tubule epithelial cells induced by treatment with hydrogen peroxide (H 2 O 2). In addition, cell viability was measured by MTT assay. Apoptotic events were monitored by western blot analysis, flow cytometric analysis and nuclear morphological anlaysis. The levels of intracellular reactive oxygen species (ROS) were measured by flow cytometric analysis with dihydroethidium staining. The results revealed that the administration of SK to 5/6 nephrectomized rats for 1 week significantly decreased the levels of SCr and BUN. The morphological observations of the kidneys also indicated the amelioration of damage to renal tissue. Treatment of the HK-2 cells with SK significantly protected the cells from H 2 O 2-induced apoptosis, as indicated by an increase in cell viability, the decrease in the cleavage of poly(ADP-ribose) polymerase (PARP) and fewer condensed nuclei. H 2 O 2-induced ROS production was also attenuated by treatment with SK. Of note, the increase in the levels of phosphorylated extracel-lular signal-regulated kinase (ERK) and phosphorylated p38 which occurred in response to exposure to H 2 O 2 was inhibited by treatment with SK. No changes were observed in the levels of phosphorylated JNK under the same treatment conditions. Thus, the mitogen-activated protein kinase (MAPK) signaling pathways play an essential role in the development of CKD. SK alleviated renal injury in rats induced by 5/6 nephrectomy and prevented the H 2 O 2-induced apoptosis of HK-2 cells through the MAPK signaling pathways.
American Journal of Respiratory Cell and Molecular Biology, 2003
Therapy with high oxygen concentrations (hyperoxia) is often to hyperoxia, or 95% O 2 , is associated with an accumulation necessary to treat patients with respiratory failure. However, of ROS (3). Therapy with hyperoxia is often necessary to hyperoxia may exacerbate the development of acute lung intreat newborns, older children, and adults with respiratory jury, perhaps by increasing lung epithelial cell death. Therefore, failure. However, supplemental O 2 administered to patients interrupting lung epithelial cell death is an important protecwith respiratory failure can in itself add to the oxidative burtive and therapeutic strategy. In the present study, hyperoxia den already present in the inflamed lungs. Oxidant stress, (95% O 2) results in murine lung epithelium cell death by DNAsuch as hyperoxia, can lead to lung epithelial cell death, acute laddering, terminal deoxynucleotidyltransferase dUTP nick end lung injury, and eventually, respiratory failure. Although the labeling, and Annexin V-fluorescein isothiocyanate flow cytommechanisms by which hyperoxia mediates cell death are not etry assay. We show that hyperoxia increases superoxide production, as assessed by nicotinamide adenine dinucleotide phos-well defined, the mitogen-activated protein kinase (MAPK) phate reduced (NADPH) oxidase activity and flow cytometric and caspase pathways have been implicated in cell death assay, and increases phospho-extracellular signal-regulated that is induced by a variety of oxidant stresses (4, 5). kinase (ERK)1/2 by Western blot analysis. These processes are in-The MAPKs include extracellular signal-regulated kinase hibited by a reactive oxygen species inhibitor, diphenylene io-(ERK1/2), c-Jun N-terminal protein kinase (JNK1/2), and donium (DPI), and by an inhibitor of the mitogen-activated p38 kinase (6). Each MAPK is activated through dual phosprotein (MAP) or ERK kinase (MEK)/ERK1/2 pathway, PD98059. phorylation via a specific phosphorylation cascade. ERK1/2 ERK1/2 activation in hyperoxia is also inhibited by DPI. Hyperis generally considered to be a survival mediator involved in oxia-induced cell death is associated with cytochrome c release, the protective action of growth factors against cell death, but subsequent caspase 9 and 3 activation, and poly (ADP-ribosyl) polymerase cleavage, which can all be suppressed by DPI and it has also been reported that induction of cell death can be PD98059. However, the broad caspase inhibitor z-VAD-FMK mediated via ERK1/2 (7, 8). The other MAPKs (JNK1/2, protects cells from death without affecting superoxide genera-p38) are usually implicated in the induction of cell death and tion and ERK1/2 activation. Taken together, our data suggest inflammation after exposure to different agents; however, it that hyperoxia, by virtue of activating NADPH oxidase, generalso has been shown that p38 and JNK1/2 activation may ates reactive oxygen species (ROS), which mediates cell death protect against the induction of cell death (9, 10). of lung epithelium via ERK1/2 MAPK activation, and functions Caspases are a family of specific cysteine proteases, and upstream of caspase activation in lung epithelial cells.
CREB mediates ERK-induced survival of mouse renal tubular cells after oxidant stress
Kidney International, 2005
CREB mediates ERK-induced survival of mouse renal tubular cells after oxidant stress. Background. We showed that extracellular signal-regulated protein kinase (ERK) is prosurvival during oxidant stress both in the kidney and in cultured mouse proximal tubule (TKPTS) cells and demonstrated concomitant activation of ERK as well as the cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB), during survival in vitro. We now show that CREB is a necessary prosurvival target of ERK. Methods. Ischemia/reperfusion (I/R) injury was induced in 129Sv mice. Oxidant stress was induced by hydrogen peroxide (H 2 O 2) in TKPTS cells. Activation of CREB was determined by immunohistochemistry and Western blotting. Inhibition and activation of CREB was achieved by mutant or activated CREBcontaining adenoviruses in vitro. The effects of oxidant stress on cell survival, CREB binding, and CREB-mediated transcription was determined by cell counting, gelshift analysis, and luciferase assay, respectively. Results. I/R activates CREB in the surviving distal nephron segments of the kidney. Inhibition of ERK and CREB abrogates survival after 0.5 mmol/L H 2 O 2 treatment, while overexpression of CREB ameliorates necrotic death caused by 1 mmol/L H 2 O 2. Inhibition of ERK also inhibited CREB activation. Binding of phosphorylated CREB to a CREB oligonucleotide was significantly increased after 0.5 mmol/L H 2 O 2 but decreased after 1 mmol/L H 2 O 2. Similarly, CREB-mediated transcription was significantly increased after 0.5 mmol/L H 2 O 2 treatment, while 1 mmol/L H 2 O 2 inhibited it. Interestingly, transcription from the CREB-driven bcl-2 promoter was unchanged after 0.5 mmol/L but decreased after 1 mmol/L H 2 O 2 treatment in agreement with Western blot studies. Conclusion. We show that survival during oxidant stress is mediated through CREB and identification of its downstream targets will reveal important survival pathways. Ischemia/reperfusion (I/R) injury is a major cause of acute renal failure [1]. In the rat kidney, survival of
2004
Cytotoxicity to renal tubular epithelial cells (RTE) is dependent on the relative response of cell survival and cell death signals triggered by the injury. Forkhead transcription factors, Bcl-2 family member Bad, and mitogen-activated protein kinases are regulated by phosphorylation that plays crucial roles in determining cell fate. We examined the role of phosphorylation of these proteins in regulation of H 2 O 2 -induced caspase activation in RTE. The phosphorylation of FKHR, FKHRL, and Bcl-2 family member Bad were markedly increased in response to oxidant injury, and this increase was associated with elevated levels of basal phosphorylation of Akt/protein kinase B. PI-3 kinase inhibitors abolished this phosphorylation and also decreased expression of antiapoptotic proteins Bcl-2 and BclxL. Inhibition of phosphorylation of forkhead proteins resulted in a marked increase in proapoptotic protein Bim. These downstream effects of PI-3 kinase inhibition promoted the oxidantinduced activation of caspase-3 and -9, but not caspase-8 and -1. The impact of enhanced activation of caspases by PI-3 kinase inhibition was reflected on accelerated oxidant-induced cell death.
Hypochlorous Acid Stimulation of the Mitogen-Activated Protein Kinase Pathway Enhances Cell Survival
Archives of Biochemistry and Biophysics, 2001
We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAP kinase), the extracellular regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), by the myeloperoxidase-derived oxidant HOCl, in human umbilical vein endothelial cells (HUVEC) and human skin fibroblasts. Treatment of fibroblasts with 10-30 M HOCl induced a dose-dependent increase in the tyrosine phosphorylation of several proteins. ERK1/2 was activated by exposure to sublethal concentrations of reagent HOCl or by HOCl generated by myeloperoxidase as shown by immune complex kinase assays. Maximum activation was seen at 20 M and peak activation occurred within 10 min. Western blot analysis demonstrated activation of p38 with 30 M HOCl, occurring at 15-30 min. No activation of JNK was detected in the concentration range investigated. These results show that HOCl is able to activate MAP kinases. Effective doses were considerably lower than with H 2 O 2 and the lack of JNK activation contrasts with the activation frequently seen with H 2 O 2. Exposure to HOCl caused a loss of viability in HUVEC that was markedly enhanced when ERK1/2 activation was inhibited by U0126. This suggests that the activation of ERK promotes cell survival in response to the oxidative challenge.
Kidney International, 2009
A is an immunosuppressant drug widely used in solid organ transplantation, but it has nephrotoxic properties that promote oxidative stress. The JAK2/STAT pathway has been implicated in both cell protection and cell injury; therefore, we determined a role of JAK2 in oxidative stressmediated renal cell injury using pathophysiologically relevant oxidative challenges. The AG490 JAK2 inhibitor and overexpression of a dominant negative JAK2 protein protected endothelial and renal epithelial cells in culture against peroxide, superoxide anion and cyclosporin A induced cell death while reducing intracellular oxidation in cells challenged with peroxide and cyclosporin A. The decrease in Bcl2 expression and caspase 3 activation, induced by oxidative stress, was prevented by AG490. In mouse models of ischemia/reperfusion and cyclosporin A nephrotoxicity, AG490 decreased peritubular capillary and tubular cell injury. Our study shows that JAK2 inhibition is a promising renoprotective strategy defending endothelial and tubular cells from cyclosporin A-and oxidative stress-induced death.