1 University of Texas Medical Branch at Galveston, Galveston, Texas 77555-0562; and 2 Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605
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ABSTRACT |
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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 injury was induced in mouse PT and TAL cells in culture by the catabolism of hypoxanthine by xanthine oxidase. PT cells were found to be more sensitive than TAL cells to oxidative stress as assessed by cell counting, light microscopy, propidium iodide uptake, and fluorescence-activated cell sorting (FACS) analysis. Immunoprecipitation/kinase analysis revealed that JNK activation occurred in both cell types, whereas ERK activation occurred only in TAL cells. We then examined the effect of PD-098059, a MAP kinase kinase (MEK)-1 inhibitor of the ERK pathway, on PT and TAL survival. In TAL cells, ERK inhibition reduced cell survival nearly fourfold (P < 0.001) after oxidant exposure. In PT cells, activation of the ERK pathway by insulin-like growth factor I (IGF-I) increased survival by threefold (P < 0.001), and this IGF-I-enhanced cell survival was inhibited by PD-098059. These results indicate that cell survival in the kidney after ischemia may be dependent on ERK activation, suggesting that this pathway may be a target for therapeutic treatment in I/R injury.
oxidant stress; acute renal failure; cell death; mitogen-activated protein kinases
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INTRODUCTION |
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RENAL ISCHEMIA/REPERFUSION (I/R) injury in rodents results in both functional and morphological changes which, although reversible, cause a serious decline in renal function. Morphologically, proximal tubules (PTs) undergo necrosis predominantly in their S3 portion, while the thick ascending limb (TAL), distal convoluted tubule, and collecting duct remain intact (4, 31, 37). Evidence to date would indicate that, although the distal nephron remains intact, it is not indifferent to ischemia. Cells of the TAL especially respond to ischemia at the molecular level. Transcriptional downregulation of epidermal growth factor (EGF), as well as the activation of the immediate early gene response, characterizes this segment's response to such injury (25, 35).
Because of these differences in reaction to injury, we speculate that the difference in survival seen between the PT and TAL may be due to a difference in the molecular response of these cells and the determinants of that molecular response. We and others have previously demonstrated that I/R injury induces the activation of the c-Jun N-terminal kinases (JNKs) (9, 33). Furthermore, inhibition of the JNKs during ischemia ameliorates renal failure (9), a finding consistent with other studies that demonstrate the cytoreductive nature of JNK activation (14, 20, 21, 34, 38, 39, 41). Similarly, studies in several cellular systems indicate that the activation of extracellular regulated kinases (ERKs) during JNK activation can block the deleterious nature of the JNK response and lead to cellular survival (6, 24, 40). Thus cell fate may be determined by the balance between ERK and JNK kinase activity in particular cells.
Here we present evidence demonstrating that mitogen-activated protein kinase (MAPK) activation is regionally distributed in the postischemic kidney. The activation of JNK kinases occurred in both the cortex, where PTs prevail, and inner stripe of the outer medulla; whereas ERK activation was found to be limited to the inner stripe, where TALs predominate. Using an in vitro system of oxidant stress in epithelial cells derived from PTs and TALs, we have established a causal link between the activation of the ERK kinases and cellular survival. These data suggest that the activation of the ERK kinases during ischemic insult may be essential for cellular survival.
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METHODS |
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Animal preparation. Male
Sprague-Dawley rats were anesthetized with 50 mg/ml of ketamine, and a
50-min period of ischemia was induced by bilateral renal hilum
clamping. The clamps were removed and kidneys harvested at various
times after reestablishing perfusion. Control animals were manipulated
as above, but clamping of the renal hilum was excluded.
Protein isolation and kinase assays were then performed as previously
described (9). Briefly, kidneys were placed into ice-cold PBS and then
sectioned sagittally. The cortex and the inner stripe of the outer
medulla were isolated and homogenized using a Teflon Dounce homogenizer
in Triton lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 25 mM -glycerophosphate, 1 mM sodium
orthovanadate, 2 mM sodium pyrophosphate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/ml leupeptin)
and incubated for 4 h at 4°C on an orbital shaker, the minimal time
necessary for complete cell lysis. The lysate was cleared by
centrifugation (10 min at 15,000 g),
and protein concentration was determined using the Micro BCA protein
assay kit (Pierce, Rockford, IL).
Cell culture. We have obtained both PT cells (a gift from Elsa Bello-Reuss, University of Texas Medical Branch, Galveston, TX) and TAL cells (a gift from Glenn Nagami, West Los Angeles VA Medical Center) from murine kidneys. The PT cells have been characterized (11) and express P-glycoprotein, have a brush border, and demonstrate a conserved epithelial morphology. Similarly, the TAL cells acidify upon the addition of ammonia, a unique response for TAL cells (18). TAL and PT cells were grown in DMEM + Ham's F-12 media supplemented with 5 µU/ml of insulin and 10% FBS.
Induction of free radical injury. Cells were seeded and grown as described above to 80% confluence, and quiescence was induced by serum starvation for 48 h. Cells were incubated with varying concentrations of hypoxanthine and 200 µU/ml of xanthine oxidase at 37°C for 1 h. The cells were then washed and incubated further in fresh media containing 10% FBS without xanthine oxidase or hypoxanthine. Parallel control cultures were grown as above with the omission of the xanthine oxidase treatment.
Immunoprecipitation/kinase assays.
Pansorbin, 20 µl, was precipitated in an Eppendorf tube and washed
twice with 100 µl of lysis buffer. The Pansorbin was then resuspended
in 500 µl of lysis buffer, to which 2 µg of antibody is added and
allowed to bind at 24°C for 2 h with gentle agitation. The anti-ERK
antibody was purchased from Upstate Biotechnologies (Lake Placid, NY), and the anti-JNK antibody was provided by Roger Davis (Howard Hughes
Institute) (34). One hundred micrograms of the lysate was mixed with
antibody-bound Pansorbin in a total volume of 1 ml. Immunoprecipitation
was carried out at 4°C for 2 h on a rocker plate. The
immunoprecipitate was spun at 5,000 g
for 5 min in the cold, washed twice with lysis buffer, and then two
additional times with kinase buffer (25 mM HEPES, pH 7.4, 25 mM
magnesium chloride, 25 mM -glycerophosphate, 0.1 mM sodium
orthovanadate, and 2 mM dithiothreitol) and resuspended in 45 µl of
kinase buffer containing 1 µM ATP, 10 µCi of
[
-32P]ATP, and 1 µg of substrate (GST-c-Jun for JNK, GST-ATF-2 for p38, and myelin
basic protein for ERK). The kinase reaction was carried out for 20 min
at 30°C, whereupon the immunocomplex was separated from the
supernatant by centrifugation (5 min at 5,000 g), and the supernatant was added to
an equal volume of 2× Laemmli buffer. The samples were then
heated at 100°C for 2 min, and proteins were resolved by SDS-PAGE.
The gel was dried, and substrate phosphorylation was assessed by
autoradiography and densitometry. The pellet was also resuspended in
1× Laemmli buffer and heated at 100°C for 2 min for Western
blotting to determine the amount of enzyme present in the reaction mixture.
Cell viability. Cell viability was determined using propidium iodide exclusion and cell counting. For cell counting, 20,000 cells were plated per well in a 96-well plate. The cells were grown to 80% confluence (~90,000 cells/ml), serum starved for 48 h, and then treated with various concentrations of hypoxanthine and 200 µU of xanthine oxidase for 1 h. The media was then changed, and the cells were allowed to grow for 24 h. The cells were then trypsinized and counted using a hemocytometer. For viability assayed by propidium iodide exclusion, cells were grown and treated as described above. Twenty-four hours after treatment, the cells were trypsinized and washed with PBS. The cells were then resuspended in PBS containing 40 µM of propidium iodide for 30 min, and propidium iodide uptake was analyzed by fluorescence-activated cell sorting (FACS) analysis. Viability was defined as cells excluding propidium iodide and maintaining a high forward scatter (8).
Light microscopy. Cell morphology was determined by light microscopy to ascertain the effect of oxidative injury on cell outcome. Cells were seeded in 6-well plates and treated with xanthine oxidase as previously described. The monolayers were examined for morphological changes that would be indicative of survival or cell death. Survival was determined by retention of an intact monolayer and no change in cell morphology, whereas cell death was assigned to monolayers that detached in sheets or when a significant amount of individual cells detached from the plate.
Inhibition of ERK activation. Cells were grown to 80% confluence and serum starved as described. The cells were incubated with various concentrations of PD-098059 (New England Biolabs), a specific inhibitor of MAP kinase kinase (MEK)-1, for 1 h and then treated with xanthine oxidase and hypoxanthine as described above. Immunoprecipitation/kinase analysis was performed on protein isolated from cells 30 min after xanthine oxidase treatment to determine MAPK activation as described above. Parallel cultures were grown in which the inhibitor, oxidative injury, or both were omitted and served as control treatment. Cell viability was determined by cell counting, and propidium iodide uptake 24 h after treatment and FACS analysis were performed to determine DNA content at various times after oxidative injury (see below).
IGF-I-induced ERK activation. TAL and
PT cells were seeded, and quiescence was induced by serum starvation as
before. Insulin-like growth factor I (IGF-I) was then added at
108 to
10
11 M for 30 min, at which
point the cells were harvested and lysed in Triton lysis buffer. One
hundred micrograms total protein was then precipitated with an anti-ERK
antibody, and ERK activation was assessed by immunoprecipitation/kinase
analysis as described above.
Effect of IGF-I and PD-098059
cotreatment. PT cells were made quiescent by serum
starvation as described above. Cells were then incubated in 50 µM
PD-098059 for 1 h. IGF-I (108 M) was then added, and the
cells were incubated for an additional hour. Oxidative injury was
induced as described above in the presence of 200 µM hypoxanthine and
200 µU of xanthine oxidase for 1 h. The media was removed and
replaced with fresh media containing 10% FBS. Parallel control plates
were incubated in which oxidative injury, IGF-I, or the inhibitor was
excluded. Cell viability was assessed by cell counting 24 h after the
addition of fresh media.
DNA analysis. Cellular DNA content was measured by propidium iodide staining, performed in the following manner: Cells were trypsinized and pelleted by gentle centrifugation and washed with a PBS/0.1% azide solution. The cells are then resuspended in 1 ml of low-salt buffer containing 3% polyethylene glycol (PEG) 8000, 0.05 mg/ml of propidium iodide, 180 U/ml RNase A, and 0.1% Triton X-100 in 4 mM sodium citrate buffer and incubated for 20 min at 37°C with gentle mixing every 5 min. The nuclei are then equilibrated by the addition of 1 ml of buffer containing 3% PEG 8000, 0.05 mg/ml of propidium iodide, 0.1% Triton X-100 in 400 mM sodium chloride, and the mix was incubated overnight at 4°C. DNA content was then analyzed by FACS analysis on a Becton-Dickinson FACS-SCAN Analyzer.
Cellular proliferation analysis.
[3H]thymidine
incorporation was employed to measure to what extent proliferation
contributed to increased cell number. PT cells were seeded and grown as
described above for oxidative injury in the presence and absence of
108 M IGF-I. The cells were
pulsed with 5 µCi/ml of
[3H]thymidine for 1 h
prior to the time of harvest, collected at 6, 12, 18, and 24 h after
oxidant injury by trypsinization, and DNA was isolated in the following
manner: Cells were resuspended in 0.5 ml of "L"
buffer [100 mM NaCl, 10 mM Tris · HCl, pH 8.0, 25 mM EDTA, 0.5% SDS, and 0.1 mg/ml proteinase K (Boehringer Mannheim Biochemicals, Indianapolis, IN)] and incubated at
55°C for 18 h. The aqueous phase was then removed after
phenol-chloroform separation, and the nucleic acids were precipitated
with ethanol. The pellets are dried and RNase treated in 300 µl of TE
buffer (10 mM Tris, 1 mM EDTA, pH 8) containing 100 U
each of T1 and pancreatic RNase (Sigma Biochemicals, St. Louis, MO) and
incubated at 37°C for 1 h. Nucleic acids were isolated from protein
via phenol-chloroform treatment and the DNA pelleted by
ethanol precipitation. The pellet was then resuspended in 200 µl of
TE and DNA content determined by spectrophotometry.
[3H]thymidine
incorporation was determined by scintillation counting, and the degree
of incorporation determined as cpm per micrograms of DNA. Relative
[3H]thymidine
incorporation was then determined as the percentage of incorporation in
the experimental samples compared with control samples taken at the
same time points. Statistical analysis between means was performed by
analysis of variance with a Newman-Keuls multiple range test.
P < 0.05 was considered
statistically significant.
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RESULTS |
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Regional MAPK activation in the postischemic
kidney. Protein was isolated from the cortex and inner
stripe of the outer medulla to determine regional MAPK activation
during reperfusion injury. ERK and JNK activities in the postischemic
kidney are shown in Fig. 1. Whereas JNK
activation was demonstrated in both the cortex and inner stripe, ERK
activation was seen in the inner stripe only (368.1 ± 39.9%
increase compared with control, n = 3)
and not in the cortex (95.4 ± 3.0%, compared with control,
n = 3). The activation of these
kinases was not due to differences in expression as each kinase was
expressed at similar levels in the cortex and inner stripe (data not
shown).
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Effect of oxidative injury on cell
survival. I/R injury produces differential cell
survival in that the cells of the TAL survive the injury while cells of
the PT undergo necrosis (4, 31, 37). Since the production of reactive
oxygen species is increased during I/R injury and may be a mediator of
such injury (36), we examined the effect of oxidative injury on
cellular survival using the xanthine oxidase/hypoxanthine system. Cells
were prepared as described and then treated with 200 µU of xanthine
oxidase at varying concentrations of hypoxanthine. Such treatment
produced a dose-dependent decrease in cellular survival in both cell
types (Fig. 2). However, at hypoxanthine
concentrations greater than 200 µM, there was a marked decrease in PT
cell survival [28,000 ± 2,739 (200 µM) to 3,000 ± 707 cells/ml (500 µM)], whereas the TAL survival rate
was affected much less [86,500 ± 4,272 (200 µM) to
65,250 ± 5,950 cells/ml (500 µM)]. Thus the PT
cells had a lower survival rate during injury than TAL cells, a result
similar to the events occurring in the postischemic kidney.
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Cell cycle analysis was performed on PT and TAL cells treated with 200 µM xanthine oxidase to further characterize the mechanism of cell
death (Fig. 3). The prominent
G0/G1
peak seen for DNA in untreated PT and TAL cells is typical for
quiescent cells. Twenty-four hours after treatment the percentage of
DNA in S phase in both PT and TAL cells increased, indicating that
oxidative injury stimulated either DNA synthesis or repair in both cell types. However, although TAL cells returned to a quiescent profile by
48 h, the PT DNA increased in subdiploid content, indicating DNA
degradation.
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The effect of oxidative injury in vitro was further characterized by
light microscopy and propidium iodide exclusion. As shown in Fig.
4, oxidative injury had little effect on
the TAL cells as the morphology of the cell layer was almost entirely
intact and no morphological changes were apparent. In contrast, PT
cells lift off the dish in sheets, a pattern typical of cells dying by
necrosis. The loss in TAL cell number seen 24 h after xanthine oxidase
treatment, on the other hand, appeared to occur via individual cell
loss. PT and TAL cells were stained with propidium iodide and subjected
to FACS analysis. Cells demonstrating a 100-fold increase above control
fluorescence were considered permeable to the dye and thus nonviable.
TAL cells retained the ability to exclude the dye 24 h after xanthine
oxidase treatment (93.87 ± 1.0% vs. 87.14 ± 1.0% of the cells
excluding the dye, control vs. treated,
n = 3). However, PT cells
demonstrated increased permeability to the dye after xanthine oxidase
treatment (70.63 ± 5.3% vs. 45.40 ± 1.7%, of cells excluding
dye, control vs. treated, n = 3).
These data, coupled with the FACS analysis and light microscopic appearance, suggest that PT cells undergo necrosis in vitro during oxidative injury, similar to the mechanism of PT cell death in vivo
during I/R injury (4, 31, 37).
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MAPK activation in vitro.
Immunoprecipitation/kinase analysis was performed to determine
activation of ERK and JNK in each cell type. Cells were grown and
treated as before and kinase activity evaluated at various times after
the addition of 200 µU xanthine oxidase/hypoxanthine. JNK was
activated as early as 30 min after the induction of oxidative stress in
both the PT and TAL cells, whereas ERK was activated only in the TAL
cells (Fig. 5). This was similar to the
assessment of regional ERK and JNK activity post I/R injury in vivo.
Thus both cellular survival and MAPK activation were similar in the two
forms of cellular injury.
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Inhibition of ERK activation in TAL
cells. The results so far suggested that renal
epithelial cell survival during oxidative injury may be dependent on
ERK activation. Therefore, we tested whether the inhibition of ERK
activation in TAL cells would diminish cell viability during oxidative
injury. Pretreating cells with PD-098059, a specific inhibitor of the
activation of the ERK kinases via MEK-1 and MEK-2 (1, 10), completely
inhibited ERK activation at 50 µM PD-098059 during oxidative injury
in TAL cells (Fig. 6A) and
reduced cell survival fourfold (388,000 ± 20,659 compared with
82,500 ± 13,149 cells/ml;
P < 0.001, n = 5) (Fig.
6B). Treatment of cells with the
inhibitor alone had no effect on cell number. These results support the
hypothesis that ERK activation enhances renal epithelial cell survival
during oxidant injury.
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Effect of IGF-I on PT survival. If ERK
activation is a determinant of renal epithelial cell survival, then
activation of ERK in PT cells would be expected to increase cell
survival during oxidant injury. Cells were treated with various
concentrations of IGF-I, and immunoprecipitation/kinase analysis was
performed to determine ERK activation (Fig.
7A).
IGF-I was selected, since both PT and TAL cells express IGF-I receptors
(27) and IGF-I activates ERK in a variety of cell types (22, 28).
Additionally, IGF-I has been shown to improve outcome after renal
ischemic injury (13, 27). ERK activation was detected at 1 and 10 nM
IGF-I compared with control cells (Fig.
7A). Cells treated with
108 IGF-I 1 h prior to and
during xanthine oxidase/hypoxanthine-induced oxidative injury showed a
nearly threefold increase in cellular survival compared with cells
treated with xanthine oxidase/hypoxanthine alone (Fig.
7B) (72,400 ± 7,846 compared with 200,000 ± 8,832 cells/ml;
P < 0.001, n = 5). Thus ERK activation in PT
cells enhanced cell survival.
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To determine whether the increase in PT cell number after IGF-I
pretreatment was due to proliferation, DNA content was examined from
control and IGF-I-treated cells with or without xanthine oxidase
treatment. IGF-I pretreatment of PT cells was found to increase the
percentage of cells entering G2/M
phase in PT cells compared with xanthine oxidase treatment alone by a
small amount (from 14.65% to 24.13%) when analyzed 24 h after
xanthine oxidase treatment. This result suggests that IGF-I induced an
increase in cell number, at least partially, by increasing
proliferation. To further examine the time course of the effect of
IGF-I pretreatment on PT DNA synthesis,
[3H]thymidine
incorporation was examined and the results summarized in Fig.
8. IGF-I treatment had no statistically
significant effect (P > 0.05) on DNA
synthesis compared with cells treated with xanthine oxidase alone up to
18 h after its addition. Twenty-four hours after treatment, an increase
in DNA synthesis was observed between cells treated with IGF-I alone
vs. cells treated with xanthine oxidase or xanthine oxidase + IGF-I
(P = 0.014, n = 4). However, no statistical
significance was seen in tritiated thymidine incorporation between the
xanthine oxidase and xanthine oxidase + IGF-I-treated groups
(P > 0.05, n = 4).
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Effect of ERK inhibition on IGF-I-enhanced cell
survival. To determine whether IGF-I enhanced cell
survival by activating ERK, we examined the effect of IGF-I
pretreatment in the presence of the MEK-1/2-specific inhibitor,
PD-098059. Cells were treated as described for IGF-I pretreatment,
however, the inhibitor was added 1 h prior to the addition of IGF-I.
The results demonstrate (Fig. 9) that
pretreatment of PT cells with PD-098059 blocked the enhanced cell
survival seen with IGF-I pretreatment alone during oxidative injury.
Although cells pretreated with IGF-I showed increased survival after
oxidative injury (290,000 ± 25,500 vs. 587,500 ± 13,500 cells/ml, without or with IGF-I, respectively; n = 4, P 0.01), cells pretreated with both
PD-098059 and IGF-I demonstrated no significant increase in cell
survival compared with cells treated with xanthine oxidase alone
(312,500 ± 11,000 vs. 290,000 ± 25,500 cells/ml,
pretreated vs. xanthine oxidase alone;
n = 4, P = 0.23). These results suggest that
the enhanced cell survival induced by IGF-I pretreatment during
oxidative injury is directly linked to the activation of ERK.
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DISCUSSION |
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I/R-induced acute renal failure (ARF) results in segmental differences in cell survival. Previous studies have demonstrated that this form of ARF produces proximal tubular necrosis in the S3 segment of the PT, whereas the distal nephron remains intact, although reports have demonstrated limited apoptosis in the TAL (29). Although the TAL remains intact, it is not indifferent to the effects of ischemia-induced ARF, as the changes in c-fos, c-jun, prepro-EGF, and Tamm-Horsfall mRNAs are restricted to these cells following I/R injury (25, 35). Thus the TAL is an active participant in the molecular response to ischemia.
The present studies now demonstrate that regional differences in MAPK activation correlate with differences in segmental survival. Analysis of the activation of MAPKs in the cortex, in which the PT predominates, reveals activation of the JNK kinases without activation of ERK during I/R injury. Immunoprecipitation/kinase analysis of the postischemic inner stripe of the outer medulla, which is made up predominantly of TAL cells, revealed activation of both the JNK and ERK kinases.
To examine whether similar signaling events could be reproduced in vitro, as well as provide the opportunity to manipulate them to examine mechanism, we developed an in vitro system in which the effect of injury on PT and TAL survival could be examined. We also tested the hypothesis that survival was dependent on MAPK activation. Injury was induced via oxygen free radical generation using a xanthine oxidase/hypoxanthine system, since the production of free oxygen species occurs during ischemic insult (32, 36, 42). The results clearly demonstrate that MAPK activation in vitro is similar to that seen in the postischemic kidney. Oxidative injury was found to induce both ERK and JNK kinases in the TAL cells, whereas only JNK was activated in the PT cells. Cell survival analysis revealed that TAL cells survived this injury better than PT cells (Fig. 2), similar to what is seen in the postischemic kidney. Inhibition of ERK activation during oxidative injury using PD-098059, a specific inhibitor of MEK-1, the upstream activator of ERK, reduced TAL cell survival to one-fourth of that seen for TAL cells treated with xanthine oxidase alone. Conversely, treatment of PT cells with IGF-I was found to activate ERK and increase cell survival during oxidative injury, and this increased survival was prevented by ERK inhibition. These results strongly suggest that renal epithelial cell survival during oxidative injury is dependent on the activation of the ERK kinases. However, these studies do not rule out possible alternative pathways through which IGF-I may enhance cell survival. IGF-I may enhance cell survival through the activation of other signaling, such as the activation of phosphatidylinositol 3-kinase. Future experiments are being designed to address this issue but are beyond the scope of this report.
To determine whether enhanced PT cell number after IGF-I pretreatment was a product of increased cell survival or proliferation, we examined the effect of such treatment on cellular DNA content as well as [3H]thymidine incorporation. IGF-I pretreatment was found to increase the number of PT cells in G2/M phase 24 h posttreatment but had no effect on [3H]thymidine incorporation at 6, 12, and 18 h after xanthine oxidase treatment compared with cells treated with xanthine oxidase alone (Fig. 8). Using the increase in DNA synthesis (~10%) and with the awareness that the cells were counted before they could complete the cell cycle, we assert that the increase in cell number induced by IGF-I after oxidative stress could not be due solely to proliferation. Taken together, the results of these experiments indicate that IGF-I limits the degree of cell death in PT cells induced by oxidative stress.
Although the extracellular oxygen tension should be the same for both cell types, we have not, as of yet, measured the intracellular oxygen levels during xanthine oxidase treatment. Therefore, the difference in cell survival could be due to differences in the level of intracellular oxygen. However, even if a difference in the level of intracellular oxygen exists, our results clearly demonstrate that ERK activation increases renal epithelial cell survival in both cell types. Whether the absence of ERK activation in PT cells is due to the level of intracellular oxygen or an inability of these cells to activate ERK during oxidative stress, our data still suggest that ERK activation leads to enhanced cellular survival in PT and TAL cells treated with xanthine oxidase.
In other cell types, the ability of cells to survive a variety of stresses including oxidant stress (2, 3, 23, 43) often depends on the balance between ERK and JNK induction. In some cells the unopposed activation of JNK alone leads to apoptosis (9-16). Several reports have demonstrated the benefits of growth factor treatment on the amelioration of ischemia-induced renal failure. Treatment with IGF-I, EGF, hepatocyte growth factor, and other growth factors demonstrated improved repair and retention of renal function following such treatment (7, 19, 27, 30). Our results suggest that such beneficial effects may be mediated by activation of ERK (Fig. 7B). Consistent with the notion that cell survival may be critically determined by the balance between ERK and JNK are the studies using N-acetyl-L-cysteine (NAC) to ameliorate postischemic renal failure (9). NAC was found to inhibit the activation of JNK completely in the postischemic kidney, and inhibition of JNK was accompanied by improved renal function and accelerated renal repair compared with saline-treated animals. Thus the beneficial effects of IGF-I and other growth factors may be due to shifting the balance between ERK and JNK activation. Increasing ERK activation via growth factors during I/R injury may have the same effect on renal outcome as decreasing JNK activation in the NAC-treated animals. ERK inhibition with PD-098059 in the presence of IGF-I strongly suggests that ERK activation enhances cell survival during oxidative injury.
The mechanism by which ERK activation enhanced cell survival in our system is unknown. ERK activation may increase the cellular repair capacity of renal epithelial cells by a variety of as yet undefined mechanisms. As suggested by Harris (16) growth factors may promote repair by increasing cellular protein and lipid biosynthesis (5, 15), stimulating nutrient uptake (41), and by increasing ATP production and activation of Na/H exchange (15), thereby establishing an intracellular environment better adapted for repair. Oxidative stress has been shown to disrupt normal epithelial cell attachment to the basement membrane via altering the function and distribution of integrin receptors and disrupting focal contacts (12). Thus ERK may also enhance cell survival by maintaining intercellular integrity or by enhancing cell attachment.
Although our studies demonstrate that ERK activation enhances renal epithelial cell survival, these studies do not exclude a role of other MAPKs in determining cell outcome. For example, recent studies demonstrate that inhibition of p38 activity leads to improved cellular outcome by preventing apoptosis in hepatic and neuronal cells (17, 26). Regardless of mechanism, it appears that members of the MAPK family are key regulators of cell outcome and potential targets by which to improve renal outcome after oxidative stress in ARF.
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ACKNOWLEDGEMENTS |
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Partial support for this study was provided by a National Kidney Foundation Matching Grant and a University of Texas Small Grant to J. F. Di Mari as well as a National Cancer Institute Grant RO-1-CA-68561 to R. J. Davis.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. F. Di Mari, Univ. of Texas Medical Branch at Galveston, 301 University Blvd., 4.200 John Sealy Annex, Route 0562, Galveston, TX 77555-0562 (E-mail: jdimari{at}utmb.edu).
Received 11 December 1998; accepted in final form 21 April 1999.
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