p38 MAPK mediates renal tubular cell TNF-alpha production and TNF-alpha -dependent apoptosis during simulated ischemia

K. K. Meldrum, D. R. Meldrum, K. L. Hile, E. B. Yerkes, A. Ayala, M. P. Cain, R. C. Rink, A. J. Casale, and M. A. Kaefer

Department of Urology, Indiana University Medical Center, Indianapolis, Indiana 46202; Department of Surgery, Johns Hopkins University, Baltimore, Maryland 21287; and the Departments of Physiology and Immunology/Microbiology, Brown University School of Medicine, Providence, Rhode Island 02903


    ABSTRACT
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ABSTRACT
INTRODUCTION
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Ischemia causes renal tubular cell loss through apoptosis; however, the mechanisms of this process remain unclear. Using the renal tubular epithelial cell line LLC-PK1, we developed a model of simulated ischemia (SI) to investigate the role of p38 MAPK (mitogen-activated protein kinase) in renal cell tumor necrosis factor-alpha (TNF-alpha ) mRNA production, protein bioactivity, and apoptosis. Results demonstrate that 60 min of SI induced maximal TNF-alpha mRNA production and bioactivity. Furthermore, 60 min of ischemia induced renal tubular cell apoptosis at all substrate replacement time points examined, with peak apoptotic cell death occurring after either 24 or 48 h. p38 MAPK inhibition abolished TNF-alpha mRNA production and TNF-alpha bioactivity, and both p38 MAPK inhibition and TNF-alpha neutralization (anti-porcine TNF-alpha antibody) prevented apoptosis after 60 min of SI. These results constitute the initial demonstration that 1) renal tubular cells produce TNF-alpha mRNA and biologically active TNF-alpha and undergo apoptosis in response to SI, and 2) p38 MAPK mediates renal tubular cell TNF-alpha production and TNF-alpha -dependent apoptosis after SI.

cytokine; necrosis; inflammation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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CLINICALLY, RENAL ISCHEMIA may occur during renal transplantation (13, 55), trauma (21, 24), elective urologic surgery (11, 17), and cardiopulmonary bypass (35). Depending on the degree of ischemia, the ensuing acute tubular necrosis and renal failure may or may not be reversible. Renal tubular epithelial cells have been demonstrated to be the cell type most susceptible to renal ischemic injury (14, 34, 48). Generally, severe anoxic insults trigger a cascade of events that culminate in renal cell death by either an apoptotic or necrotic pathway (34, 48). Apoptosis is distinct from necrosis in that cells maintain membrane integrity and therefore do not incite as significant an inflammatory response (26, 51, 56). In addition, apoptosis is an energy-dependent, gene-driven process and therefore is uniquely suited to molecular modulation and possible therapeutic inhibition. Indeed, apoptosis is the primary mode of cell death during renal ischemic intervals <60 min (48).

While many different mechanisms of renal ischemic cell injury and death have been elucidated (22, 30, 41, 45, 60), recent evidence suggests that tumor necrosis factor-alpha (TNF-alpha ) is a significant mediator of this process (2, 10). TNF-alpha , a proinflammatory cytokine, induces apoptosis in many cells, including renal tubular epithelial cells, through an interaction with specific membrane-bound receptors [i.e., TNF receptor 1 (TNFR1); Refs. 7, 23, 49]. Although TNF-alpha was originally described as a lipopolysaccharide (LPS)-induced macrophage product, it has recently become clear in animal models that renal tubular cells produce TNF-alpha in response to ischemia (9). The intracellular signaling cascade leading to renal cell TNF-alpha production after ischemia is not well defined. p38 MAP (mitogen-activated protein) kinase, a stress-activated kinase, is induced with renal ischemia and has been shown to be important for nuclear factor-kappa B (NF-kappa B) activation (TNF-alpha transcription factor) and subsequent TNF-alpha production (19, 31, 32, 62). Yin and colleagues (62) demonstrated p38 MAP kinase activation after renal ischemic injury; however, its role in ischemia-induced renal tubular cell TNF-alpha production and TNF-alpha -dependent apoptosis has not been defined. Therefore, the purposes of this study were to determine 1) the activation of p38 MAP kinase with simulated ischemia, 2) the time course of TNF-alpha mRNA production after renal tubular cell-simulated ischemia, 3) the time course of TNF-alpha bioactivity after renal tubular cell-simulated ischemia, 4) the time course of apoptosis after renal tubular cell simulated ischemia, 5) whether neutralization of TNF-alpha prevents ischemia-induced apoptosis, and 6) whether p38 MAP kinase mediates TNF-alpha production and TNF-alpha -dependent apoptosis after renal tubular cell ischemia.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Cell culture. The renal tubular epithelial cell line LLC-PK1, established from pig renal cortex, was cultured in endotoxin-free medium 199-Earle's balanced salt solution (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS). The cells were passaged weekly by trypsinization (0.25% trypsin, 0.02% EDTA) after formation of a confluent monolayer and placed in serum-free media (0.25% FBS) 24 h before stimulation.

Simulated ischemia. The cells were grown to confluence. After formation of a confluent epithelial sheet, the cells were washed twice with PBS, and the monolayer was immersed in mineral oil to simulate ischemic conditions (20, 59).

Western blot analysis. Western blot analysis was performed on control samples and samples exposed to varying lengths of simulated ischemia. Each 100-mm confluent plate of cells was washed twice with PBS, scraped, and centrifuged. The supernatant was removed, and the cell pellet was lysed using a 2% SDS solution in protease inhibitor buffer (20 mM Tris, pH 7.5, 1 mM EGTA, 20 mM p-nitrophenyl phosphate, 50 mM sodium fluoride, and 50 µM sodium orthovanadate) supplemented with aprotinin (5 µg/ml), leupeptin (1 µg/ml), and Pefabloc (1 mM). After filtration through a QIAshredder (Qiagen,Valencia, CA), the protein extracts (200 µg/lane) were electrophoresed on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoblotting was performed by incubating each membrane in 5% dry milk for 1 h, followed by incubation with a rabbit polyclonal antibody to activated dual phosphorylated (Thr 180/Tyr 182) p38 (1:1,000, Cell Signaling Technologies, Beverly, CA) for 2 h. After washing twice in Tris-buffered saline-Tween 20, each membrane was incubated for 2 h with a peroxidase-conjugated secondary antibody (1:10,000, StressGen, British Columbia, Canada). The membranes were then developed using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).

TNF-alpha RT-PCR. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess TNF-alpha gene expression. The cells were seeded at 2.0 × 105 on 100-mm culture plates and grown to confluence. The cells were exposed to either 0 (control), 30 min, 1 h, 1 h + 1 µmol/l SB-203580 [Calbiochem, Berkeley, CA; pyridinyl-imidazole inhibitor of p38 MAP kinase (31, 57) was applied to the cells 24 h before the onset of ischemia; dose was previously shown to inhibit TNF-alpha production by cardiac myocytes (42)], 2 h, or 4 h of simulated ischemia. Total RNA was extracted from each culture dish of cells (3 culture dishes per time point) using Tripure (Boehringer Mannheim, Indianapolis, IN) after washing the cells twice with PBS. The RNA was then isolated by precipitation with chloroform and isopropanol. Two micrograms of the isolated RNA were subjected to RT-PCR with reverse transcriptase, using random hexaoligonucleotides as primers (Promega, Madison, WI). The samples were incubated for 10 min at 70°C, chilled for 5 min, and after the addition of SuperScript II RT (Life Technologies, Gaithersburg, MD), were incubated at 37°C for 1 h. PCR was performed by adding 2 µl of RT product to PCR SuperMix containing Taq DNA polymerase (GIBCO BRL, Gaithersburg, MD). For each RT sample, PCRs for TNF-alpha and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 60 pmol primer) were performed. Thirty picomoles of each pig TNF-alpha primer sequence (sense, 5'-TTC CTC ACT CAC ACC ATC AGC C-3'; antisense, 5'-TGC CCA GAT TCA GCA AAG TCC-3') were used, yielding a 224-bp product. After demonstrating that the intensity of the amplicons was linear throughout the number of PCR cycles performed, the samples were loaded in a thermocycler and run for 3 min at 94°C, then 34 cycles of 94°C for 1 min, 55°C for 2 min, and 72°C for 2 min. The samples were run for an additional 7 min at 72°C and held at 4°C until loaded onto the gel. The amplified products were separated in a 2% agarose gel containing 0.5 × Tris-borate-EDTA, pH 8.3. PCR amplification products were quantified by staining the gel with ethidium bromide and determining the density of each band using the BioRad gel documentation system (Hercules, CA). The data are presented as the ratio of the densitometric units of the TNF mRNA band to the densitometric units of the GAPDH mRNA band.

TNF-alpha bioactivity assay. After simulated ischemia, the cells in each culture dish were washed twice with PBS, lysed with distilled water, and suspended in a lysis buffer solution (in mM: 10 HEPES, 10 KCl, 0.1 EDTA, 0.1 EGTA, 1 dithiothreitol, and 0.5 phenylmethylsulfonyl fluoride). TNF-alpha bioactivity was then determined after cell exposure to 0 (control), 30 min, 1 h, 1 h + 1 µmol/l SB-203580, 2 h, or 4 h of simulated ischemia (4 culture dishes per time point) using the WEHI-164 clone cytotoxicity assay as previously described (1, 37-40). Final results were expressed as units of TNF-alpha cytotoxicity per milliliter.

Quantitation of apoptosis. Cells were plated in four-well chamber slides at 1.5 × 104 cells per well and grown in 1 ml of medium 199 supplemented with 10% FBS until a confluent monolayer was obtained. The cells were then washed twice in PBS, and the monolayer was immersed in 1 ml of mineral oil. The cells were exposed to either media alone (control), media supplemented with 100 µl of mineral oil (control), or 1 h of simulated ischemia followed by 12, 24, or 48 h of substrate replacement (reimmersion in media) using three wells per time point. TNF-alpha activity was neutralized by exposing the cells in three additional wells to 2 µg/ml of goat anti-porcine TNF-alpha antibody [R&D systems, Minneapolis, MN; concentration based on neutralizing dose (ND50) for this antibody as defined by the manufacturer] 24 h before the onset of ischemia and during 24 h of substrate replacement. The p38 inhibitors SB-203580 and SB-202190 (AG Scientific, San Diego, CA) were also each applied to three wells at a concentration of 1 µmol/l 24 h before the onset of ischemia and during 24 h of substrate replacement. Apoptosis in each well was quantified using a kit from Boehringer Mannheim (Indianapolis, IN). The kit is based on terminal deoxynucleotidyl transferase incorporation of fluorescein-dUTP to detect DNA strand breaks in the nuclei of cells undergoing apoptosis. The cell nuclei in each well were then counterstained with bisbenzimide (10 µg/ml in PBS) for 30 s to ensure a constant cell density across all treatment groups examined. The number of apoptotic nuclei were counted in three nonoverlapping 200× microscope fields/well and averaged. Furthermore, the characteristic morphological features of apoptosis (i.e., nuclear condensation) were observed among cells in each well examined. The entire protocol was then repeated in triplicate.

Cell viability. Cells were seeded at 2.0 × 105 and grown in 3 ml of media in 60-mm petri dishes until a confluent monolayer was obtained. Using the treatment groups described above, a cell suspension in PBS was prepared from each culture dish. Trypan blue (0.4%) was added to each cell suspension, and the mixture was allowed to stand at room temperature for 5 min. The number of viable cells were counted in each sample and expressed as a percentage of the total cell count. Viability as measured by trypan blue exclusion is based on a lack of cell membrane integrity; therefore, only necrotic cells (not apoptotic cells) are identified as nonviable.

Statistical analysis. Data are presented as means ± SE. Differences at the 95% confidence level were considered significant. The experimental groups were compared using ANOVA with post hoc Bonferroni-Dunn (StatView 4.5, Berkeley, CA).


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p38 activation. Western blot analysis confirmed p38 MAP kinase activation after simulated ischemia. In contrast to control cells and cells exposed to 10 min of simulated ischemia, samples exposed to 20 or 30 min of ischemia demonstrated a marked increase in phosphorylated (activated) p38 MAP kinase expression (Fig. 1).


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Fig. 1.   Western blot analysis demonstrating p38 mitogen-activated protein kinase (MAPK) activation in response to 10, 20, or 30 min of ischemia. While control cells (C) and cells exposed to 10 min of simulated ischemia exhibited minimal expression of phosphorylated (activated) p38 MAPK, cells exposed to 20 or 30 min of simulated ischemia demonstrated a significant increase in phosphorylated p38 MAPK expression.

Time course of TNF-alpha mRNA induction with renal tubular cell-simulated ischemia. TNF-alpha mRNA production was assessed after a time course of graded renal tubular cell-simulated ischemia. Control cells and cells exposed to 30 min of simulated ischemia demonstrated very little TNF-alpha mRNA induction (Fig. 2, A and B). However, TNF-alpha mRNA induction increased after 60 min of simulated ischemia and remained elevated, although to a lesser degree, after 2 h of simulated ischemia. After 4 h, TNF-alpha mRNA induction approached control levels. Densitometric analyses of TNF-alpha mRNA expressed as a percentage of GAPDH mRNA are shown in Fig. 2B. After 60 min and 2 h of simulated ischemia, the TNF-alpha mRNA expressed represented 106 ± 12% and 79 ± 1.5% of GAPDH mRNA [P < 0.05 vs. control (36 ± 4.7% of GAPDH mRNA)], respectively. TNF-alpha mRNA expression after 30 min and 4 h of simulated ischemia was not significantly different from the control (49 ± 10.6% and 46 ± 3.3% of GAPDH mRNA, respectively).


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Fig. 2.   Time course depicting renal tubular cell tumor necrosis factor-alpha (TNF-alpha ) mRNA production after simulated ischemia. A: gel photograph depicting TNF-alpha and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA bands at various time points (30 and 60 min; 2 and 4 h) of simulated ischemia. C, control. B: densitometric analysis of TNF-alpha mRNA bands in A at corresponding time points, represented as the TNF-alpha mRNA percentage of GAPDH mRNA. C: gel photograph depicting TNF-alpha and GAPDH mRNA bands after 60 min of ischemia with (60I) and without (60) p38 MAPK inhibitor. C, control. D: densitometric analysis of TNF-alpha mRNA bands in C at corresponding time points, represented as the TNF-alpha mRNA percentage of GAPDH mRNA. TNF-alpha mRNA induction peaks after 60 min of simulated ischemia. TNF-alpha mRNA induction remains elevated after 2 h of simulated ischemia but returns to control levels after 4 h of ischemia. The p38 MAPK inhibitor SB-203580 prevented the observed increase in TNF-alpha mRNA production after 60 min of ischemia.

Time course of TNF-alpha bioactivity (cytotoxicity). In parallel with TNF-alpha mRNA induction, TNF-alpha bioactivity (cytotoxicity) increased significantly after 60 min (0.31 ± 0.12 U/ml), 2 h (0.13 ± 0.02 U/ml), and 4 h (0.18 ± 0.05 U/ml) of simulated ischemia [P < 0.05 vs. control (0.003 ± 0.002 U/ml)] as shown in Fig. 3A. TNF-alpha bioactivity did not increase significantly after 30 min of simulated ischemia (0.08 ± 0.03 U/ml vs. control).


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Fig. 3.   Time course depicting TNF-alpha bioactivity in renal tubular cells after simulated ischemia. A: TNF-alpha bioactivity after renal tubular cell exposure to graded ischemia. B: TNF-alpha bioactivity in renal tubular cells after 60 min of ischemia with and without p38 MAPK inhibitor. Sixty minutes of simulated ischemia induced peak TNF-alpha bioactivity. TNF-alpha bioactivity remained elevated, although to a lesser degree, after 2 and 4 h of simulated ischemia. The p38 MAPK inhibitor SB-203580 prevented the observed increase in TNF-alpha bioactivity after 60 min of ischemia.

Time course of apoptosis. Cells exposed to media alone or media supplemented with 100 µl of mineral oil did not undergo apoptosis [0.1 ± 0.1 apoptotic nuclei/high-powered field (hpf) and 0.5 ± 0.3 apoptotic nuclei/hpf, respectively]. In contrast, 1 h of simulated ischemia induced apoptosis at each substrate replacement time point examined. Apoptosis was minimal after 12 h of substrate replacement (4.1 ± 1.1 apoptotic nuclei/hpf, P < 0.05 vs. control, Fig. 4); however, a marked increase in the number of apoptotic nuclei was noted after both 24 h (Fig. 5B) and 48 h of substrate replacement (156 ± 18 and 161 ± 22 apoptotic nuclei/hpf, respectively, P < 0.05 vs. control). Cells exposed to media alone and media supplemented with 100 µl of mineral oil demonstrated 88 ± 0.9% and 84 ± 2.8% viability, respectively, while cells exposed to 1 h of simulated ischemia and 12, 24, or 48 h of substrate replacement demonstrated 91 ± 2.2%, 81 ± 5.3%, and 80 ± 3.8% viability, respectively (not significantly different from controls).


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Fig. 4.   Number of apoptotic nuclei per high-powered field (hpf; 200×) in each treatment group examined. In contrast to controls, 1 h of simulated ischemia induced a significant degree of apoptosis after either 24 or 48 h of substrate replacement. Cell exposure to either SB-203580, SB-202190, or anti-TNF-alpha antibody (Ab) prevented simulated ischemia-induced apoptosis.



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Fig. 5.   Photographs (magnification, ×200) demonstrating renal tubular cell apoptosis [TdT-mediated dUTP nick end labeling (TUNEL) assay] and the effects of SB-203580 and anti-TNF-alpha antibody on 60 min of simulated ischemia. A: control cells. B: cells exposed to 60 min of simulated ischemia and 24 h of substrate replacement. C: cells exposed to 60 min of simulated ischemia and 24 h of substrate replacement in the presence of SB-203580. D: cells exposed to 60 min of simulated ischemia and 24 h of substrate replacement in the presence of anti-TNF-alpha antibody. Sixty minutes of simulated ischemia induced a significant degree of apoptosis, while few apoptotic nuclei were visualized in control cells and cells exposed to 60 min of ischemia and 24 h of substrate replacement in the presence of either SB-203580 or anti-TNF-alpha antibody.

Effect of TNF-alpha neutralization. TNF-alpha was neutralized by applying anti-porcine TNF-alpha antibody to cells 24 h before initiating 60 min of simulated ischemia (time point of maximal TNF-alpha mRNA production and TNF-alpha bioactivity) and during substrate replacement. TNF-alpha neutralization successfully inhibited ischemia-induced apoptosis after 24 h of substrate replacement (8.3 ± 3.5 apoptotic nuclei/hpf; not significantly increased over control; Figs. 4 and 5D), with cell viability not significantly different from controls (87 ± 4.3%).

Role of p38 MAP kinase. The p38 MAP kinase inhibitor SB-203580 was applied to cells 24 h before initiating 60 min of simulated ischemia and during substrate replacement. SB-203580 successfully blocked TNF-alpha mRNA production (48 ± 2% GAPDH mRNA, not significantly increased over control, Fig. 2, C and D) and TNF-alpha bioactivity (0.02 ± 0.01 U/ml, not significantly increased over control, Fig. 3B). Both SB-203580 and SB-202190 blocked ischemia-induced apoptosis after 24 h of substrate replacement (9.3 ± 5.4 and 14 ± 8.3 apoptotic nuclei/hpf, respectively; not significantly increased over controls; Figs. 4 and 5C), with cell viability 78 ± 6% and 79 ± 3.4%, respectively (not significantly different from controls).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of this study constitute the initial demonstration that 1) renal tubular epithelial cells produce TNF-alpha mRNA and biologically active TNF-alpha in response to simulated ischemia, 2) renal tubular epithelial cells undergo apoptosis in response to simulated ischemia, and 3) p38 MAP kinase mediates renal tubular cell TNF-alpha production and TNF-alpha -dependent apoptosis during simulated ischemia.

Significant renal dysfunction and cellular degeneration can occur after short periods of renal ischemia (25, 33, 54, 55). Despite an increased understanding of the mechanisms of cell injury and death during acute renal failure, patient death rates from this disease continue to be high (61). It has been well established that lethal degrees of anoxia result in renal cell death by either an apoptotic or necrotic pathway. The relative contribution of apoptosis or necrosis to total renal cell loss depends in part on the degree of initial ischemia. Prolonged renal ischemia generally results in overwhelming cellular ATP depletion and necrotic cell death. Less severe degrees of ischemia, however, trigger renal cell death by apoptosis, a process by which cells use their own energy sources and proteins to undergo nonnecrotic "suicide" (26, 48, 51, 56). Indeed, in animal models, apoptosis is the primary mode of cell death for renal ischemic intervals <60 min (48). Importantly, most clinical renal ischemic situations involve ischemia times of 60 min or less. During apoptosis, cell deletion occurs while plasma membrane integrity is maintained (14, 34). While this results in less surrounding tissue inflammation than necrosis, apoptosis alone has been demonstrated to be a significant initiator of ischemia-induced renal inflammation and tissue injury (8). Many potential triggers of apoptotic cell death have been identified, including cellular exposure to cytotoxic events, a deficiency in renal growth factors, loss of cell-cell interactions, and exposure to receptor-mediated cytotoxic agents such as TNF-alpha (15, 16, 33); however, the individual role of these stimuli in ischemia-induced renal cell apoptosis has not yet been determined.

TNF-alpha is a proinflammatory cytokine recently discovered to be a significant mediator of ischemiainduced renal cell injury (2, 10). In addition to stimulating the production of other inflammatory mediators (i.e., nitric oxide, platelet-activating factor, and eicosanoids) and recruiting and stimulating various cells in the immune system (5, 6, 11, 36, 47), TNF-alpha is directly cytotoxic and will induce apoptosis in many cells through interactions with the membrane-bound receptors, TNFR1 and Fas (7, 23, 49). Indeed, direct exposure of the renal tubular epithelial cell line LLC-PK1 to TNF-alpha results in a high frequency of apoptotic cell death (49). The primary source of TNF-alpha in renal disease has traditionally been considered the infiltrating macrophage; however, it has recently become clear that TNF-alpha is also an important locally produced mediator of cellular injury (3, 9, 12, 28, 43). In the kidney, we have demonstrated that ischemia induces renal tubular cell production of TNF-alpha (9), an observation that may be related to the increased susceptibility of renal tubular epithelium to ischemic conditions (14, 34, 48).

To specifically evaluate the effects of ischemia on renal tubular epithelial cells, an in vitro model of simulated ischemia was developed on the basis of prior work by Vanheel and colleagues (59) and Henry and colleagues (20). These authors demonstrated that myocyte immersion in mineral oil results in nutrient deprivation and a restriction in metabolite washout. Using the renal tubular epithelial cell line LLC-PK1, ischemia was simulated by immersing an entire cellular monolayer in mineral oil for variable lengths of time. Maximal TNF-alpha mRNA induction and TNF-alpha bioactivity were detected after 60 min of ischemia. This corroborates our previous findings that TNF-alpha production localizes primarily to renal tubular epithelium after renal ischemic injury (9). Furthermore, 60 min of ischemia induced apoptosis in nearly all cells after 24 or 48 h of reperfusion. While these time points may not be directly comparable to in vivo models of renal ischemia, it is clear from these data that simulated ischemia induces renal tubular cell TNF-alpha production and concomitant apoptotic cell death.

The intracellular signaling cascade leading to TNF-alpha production has been well defined in the macrophage after LPS stimulation. After LPS binding to CD-14, a series of protein kinases, including p38 MAP kinase, are activated. NF-kappa B activation (TNF-alpha transcription factor) then ensues, leading to an increase in the cellular production of TNF-alpha (18, 31, 46, 53). The signaling cascade leading to ischemia-induced renal cell TNF-alpha production has been less well defined. Yin and colleagues (62) have demonstrated that p38 MAP kinase is also activated during renal ischemia and, furthermore, that p38 MAP kinase activation is associated with apoptotic renal cell death. In light of their findings, we investigated the role of p38 MAP kinase in renal tubular cell TNF-alpha production and apoptosis during simulated ischemia. Using pyridinyl-imidazole p38 MAP kinase inhibitors, we effectively inhibited TNF-alpha mRNA production, TNF-alpha bioactivity, and apoptosis after 60 min of simulated ischemia. Furthermore, we demonstrated that TNF-alpha neutralization prevented ischemia-induced apoptosis, suggesting that p38 MAP kinase mediates renal tubular cell apoptosis through a TNF-alpha -dependent mechanism. The activation of p38 MAP kinase, production of TNF-alpha , and subsequent increase in TNF-alpha -dependent apoptosis suggest that NF-kappa B is a proapoptotic signal in this cellular model. Interestingly, NF-kappa B is largely considered to be an antiapoptotic signal and has been demonstrated to inhibit TNF-alpha -dependent apoptosis in a number of cells (4, 27, 29, 44, 50, 52, 58). Most likely, NF-kappa B is an indiscriminate intracellular signal, triggering mechanisms of cellular protection or cell death depending on the surrounding milieu.

These results should be interpreted with several important caveats. First, in comparing our results to in vivo investigations of renal ischemia-reperfusion injury, it appears that cultured renal tubular cells undergo apoptosis to a greater degree after simulated ischemia than do in vivo tubular cells exposed to renal ischemia-reperfusion injury. Schumer et al. (48) examined the effect of renal ischemia-reperfusion injury on apoptosis and determined that while apoptotic bodies are absent in control and sham-operated animals, apoptosis becomes detectable after 30 min of ischemia and 12 h of reperfusion (2-3 apoptotic bodies per section) and peaks after 30 min of ischemia and 24 or 48 h of reperfusion (20 apoptotic bodies per section). Interestingly, while apoptosis appears to be more pronounced in cultured renal cells after simulated ischemia (4 apoptotic nuclei at 12 h; 156 apoptotic nuclei at 24 h), the cells require exposure to greater lengths of simulated ischemia (60 min) to induce a similar degree of injury. This model of simulated ischemia is not directly comparable to in vivo models of renal ischemiareperfusion injury; however, the time course of TNF-alpha mRNA production (10), a marker of cell injury, and ischemia-induced apoptosis is remarkably similar to that detected in animal models and suggests that this model of simulated ischemia does indeed reflect the biology of ischemic renal tubular cell injury. Second, the purpose of this study was to clarify the role of p38 MAP kinase in renal tubular cell ischemia and apoptosis. To eliminate confounding variables, we conducted the experiments in an established renal tubular epithelial cell line. Numerous other factors are involved in renal ischemic injury, and we did not attempt to account for these during the investigation.

Ischemia-induced acute renal failure is associated with significant morbidity and mortality. While prolonged ischemic renal insults cause cell death by necrosis, shorter durations of ischemia induce apoptosis, a process uniquely suited to molecular modulation and possible inhibition. As we develop a greater understanding of mediators of ischemia-induced renal cell apoptosis, such as p38 MAP kinase, effective therapeutic strategies that limit or prevent ischemia-induced renal damage may be realized.


    ACKNOWLEDGEMENTS

The authors express their sincere thanks to Drs. T. Gardner, C. Dinarello, and A. Burnett for technical instruction and constructive comments during the course of this work.


    FOOTNOTES

This research was supported by the Urology Research Fund, Department of Urology, Indiana University, Indianapolis, IN.

Address for reprint requests and other correspondence: K. K. Meldrum, Johns Hopkins Hospital, 600 N. Wolfe St., Marburg 414, Baltimore, MD 21287 (E-mail: kkmeldrum{at}earthlink.net).

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. Section 1734 solely to indicate this fact.

Received 1 December 2000; accepted in final form 27 March 2001.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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