MAPK activation determines renal epithelial cell survival during oxidative injury

John F. di Mari1, Roger Davis2, and Robert L. Safirstein1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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 [gamma -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 10-8 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 (10-8 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 10-8 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Regional mitogen-activated protein kinase (MAPK) activation in the postischemic kidney. Immunoprecipitation/kinase analysis of 100 µg of protein isolated from control (con) and postischemic (Isch) rat cortex (C) and inner stripe (IS) of the outer medulla. MAPKs were precipitated using antibodies against c-Jun N-terminal kinase (JNK) or extracellular regulated kinase (ERK) and is indicated to the right of each row. Phosphorylation substrates were c-Jun (1-79) or myelin basic protein for the JNK and ERK kinases, respectively. Protein for the JNK kinase analysis was isolated 1 h postreperfusion, whereas ERK analysis was performed on samples isolated 10 min postreperfusion, since these time points demonstrated maximal activity. These are representative blots of proteins isolated from 3 groups of animals.

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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Fig. 2.   Effect of xanthine oxidase on proximal tubule (PT) and thick ascending limb (TAL) cell survival. Cell survival was analyzed by cell counting 24 h after treatment with 200 µU of xanthine oxidase and various concentrations of hypoxanthine. Cell number is given in number of cells per milliliter. Each data point represents the mean cell number from 5 parallel experiments with standard error bars given.

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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Fig. 3.   Effect of xanthine oxidase on the cell cycle. Fluorescence-activated cell sorting (FACS) analysis of cellular DNA from PT (top) and TAL (bottom) cells 24 (B and E) and 48 h (C and F) after xanthine oxidase treatment, stained with propidium iodide (A and D are controls). Cells were treated with 200 µU of xanthine oxidase and 200 µM hypoxanthine for 1 h as described in METHODS.

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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Fig. 4.   Effect of xanthine oxidase on PT and TAL cell monolayers. Light microscopic analysis was performed on PT and TAL cell monolayers in control and xanthine oxidase-treated samples. Cells were seeded, grown to 80% confluence, and serum starved for 48 h. Cells were then treated with 200 µU of xanthine oxidase and 200 µM hypoxanthine, refed with complete media, and examined at 4 and 24 h after treatment. Control cells received all of the above treatments except the xanthine oxidase-induced oxidative injury. Effect of xanthine oxidase on cells is shown in A and D (control), B and E (4 h postinjury), and C and F (24 h posttreatment), for the PT and TAL cells, respectively.

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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Fig. 5.   Effect of xanthine oxidase on MAPK activation in PT and TAL cells. Immunoprecipitation/kinase analysis was performed on 100 µg of protein isolated from control (C) cells and cells treated with 200 µU of xanthine oxidase and 200 µM hypoxanthine for 30 and 60 min. Immunoprecipitating antibody is designated above each column. Substrates used for the kinase reactions were c-Jun (1-79) and myelin basic protein for the JNK and ERK immunoprecipitation/kinase analysis, respectively.

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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Fig. 6.   Effect of the inhibition of ERK activation on TAL cell survival. Effect of the inhibitor PD-098059 on ERK and JNK activation (A) and cell survival (B) in TAL cells during oxidative injury was examined. Quantities of 100 µg of protein isolated from control cells, cells treated with PD-098059, and cells treated with 200 µU of xanthine oxidase, in presence (+) or absence (-) of the inhibitor, were used for immunoprecipitation/kinase analysis. Concentrations of PD-098059 (PD) are in µM. Cell survival was assessed by counting 5 parallel experiments of control cells and cells treated with 200 µU of xanthine oxidase and 200 µM hypoxanthine (XO) in presence or absence of 50 µM PD-098059 (PD) 24 h after XO treatment. Although no significant difference was seen between control and PD-098059-treated cells (P = 0.487), the inhibitor did significantly reduce cell survival after oxidant injury (P < 0.001).

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 10-8 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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Fig. 7.   Effect of activation of ERK on PT cell survival. Effect of insulin-like growth factor I (IGF-I) on ERK activation (A) and PT cell survival (B) was examined. Quantities of 100 µg of protein were isolated from control PT cells and cells treated with various concentrations of IGF-I, as indicated. Cell survival was analyzed by cell counting 24 h after treatment for control cells, cells treated with 10-8 M IGF-I, 200 µU of xanthine oxidase, or both IGF-I and xanthine oxidase as indicated (+/-). Twenty-four hours after treatment, IGF-I alone had no effect on cell number compared with control (P = 0.826, n = 5); however, it was found to significantly increase cell number after oxidant treatment compared with cells treated with xanthine oxidase alone (P < 0.001, n = 5).

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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Fig. 8.   Effect of IGF-I on DNA synthesis. DNA was isolated from cells treated with 10-8 M IGF-I (open boxes), 200 µU xanthine oxidase (hatched boxes) or both (solid boxes), 6, 12, 18, and 24 h after treatment. One hour prior to harvest, cells were incubated with [3H]thymidine, and scintillation counting and spectrophotometry were performed to determine cpm/µg DNA. The data are represented as percent incorporation compared with DNA isolated from control cells at the same time points; n = 4.

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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Fig. 9.   Effect of ERK inhibition on IGF-I-enhanced survival in PT cells. PT cells were seeded, grown to 80% confluence and serum starved for 48 h. Cells were then left untreated or treated with 50 µM of PD-098059 for 1 h. Cells from both groups were then incubated with 10-8 M IGF-I for 1 h, and then cells from all groups were subjected to oxidative injury using 200 µU xanthine oxidase. Cells were then washed and refed with complete media, and cell numbers were assessed 24 h posttreatment. Presence (+) or absence (-) of each of the treatments is indicated.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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