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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- (TNF-
) mRNA
production, protein bioactivity, and apoptosis. Results
demonstrate that 60 min of SI induced maximal TNF-
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-
mRNA production and
TNF-
bioactivity, and both p38 MAPK inhibition and TNF-
neutralization (anti-porcine TNF-
antibody) prevented
apoptosis after 60 min of SI. These results constitute the
initial demonstration that 1) renal tubular cells produce
TNF-
mRNA and biologically active TNF-
and undergo
apoptosis in response to SI, and 2) p38 MAPK
mediates renal tubular cell TNF-
production and TNF-
-dependent apoptosis after SI.
cytokine; necrosis; inflammation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- (TNF-
) is a
significant mediator of this process (2, 10). TNF-
, 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-
was originally described as a
lipopolysaccharide (LPS)-induced macrophage product, it has recently
become clear in animal models that renal tubular cells produce TNF-
in response to ischemia (9). The intracellular signaling cascade leading to renal cell TNF-
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-
B (NF-
B) activation (TNF-
transcription factor) and
subsequent TNF-
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-
production and
TNF-
-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-
mRNA production after renal
tubular cell-simulated ischemia, 3) the time course
of TNF-
bioactivity after renal tubular cell-simulated ischemia, 4) the time course of apoptosis
after renal tubular cell simulated ischemia, 5)
whether neutralization of TNF-
prevents ischemia-induced
apoptosis, and 6) whether p38 MAP kinase mediates TNF-
production and TNF-
-dependent apoptosis after renal
tubular cell ischemia.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- RT-PCR.
Semiquantitative reverse transcriptase-polymerase chain reaction
(RT-PCR) was used to assess TNF-
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-
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-
and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 60 pmol primer) were
performed. Thirty picomoles of each pig TNF-
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- 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-
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-
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- activity was neutralized by exposing the
cells in three additional wells to 2 µg/ml of goat anti-porcine
TNF-
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).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
Time course of TNF- mRNA induction with renal tubular
cell-simulated ischemia.
TNF-
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-
mRNA induction (Fig. 2,
A and B). However, TNF-
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-
mRNA induction approached
control levels. Densitometric analyses of TNF-
mRNA expressed as a
percentage of GAPDH mRNA are shown in Fig. 2B. After 60 min
and 2 h of simulated ischemia, the TNF-
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-
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).
|
Time course of TNF- bioactivity (cytotoxicity).
In parallel with TNF-
mRNA induction, TNF-
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-
bioactivity did
not increase significantly after 30 min of simulated ischemia
(0.08 ± 0.03 U/ml vs. control).
|
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).
|
|
Effect of TNF- neutralization.
TNF-
was neutralized by applying anti-porcine TNF-
antibody to
cells 24 h before initiating 60 min of simulated ischemia (time point of maximal TNF-
mRNA production and TNF-
bioactivity) and during substrate replacement. TNF-
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- mRNA production (48 ± 2% GAPDH mRNA, not significantly increased over control, Fig. 2, C and D) and TNF-
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of this study constitute the initial demonstration
that 1) renal tubular epithelial cells produce TNF- mRNA
and biologically active TNF-
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-
production and TNF-
-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- (15, 16, 33); however, the individual role of
these stimuli in ischemia-induced renal cell apoptosis
has not yet been determined.
TNF- 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-
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-
results in a high frequency of apoptotic cell death (49). The primary
source of TNF-
in renal disease has traditionally been considered
the infiltrating macrophage; however, it has recently become clear that
TNF-
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-
(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- mRNA induction and TNF-
bioactivity
were detected after 60 min of ischemia. This corroborates our
previous findings that TNF-
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-
production and concomitant apoptotic cell death.
The intracellular signaling cascade leading to TNF- 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-
B activation (TNF-
transcription factor) then ensues, leading to an increase in the cellular production of TNF-
(18, 31, 46, 53). The signaling cascade leading to ischemia-induced renal cell TNF-
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-
production and apoptosis during simulated ischemia.
Using pyridinyl-imidazole p38 MAP kinase inhibitors, we effectively
inhibited TNF-
mRNA production, TNF-
bioactivity, and
apoptosis after 60 min of simulated ischemia. Furthermore, we demonstrated that TNF-
neutralization prevented ischemia-induced apoptosis, suggesting that p38 MAP
kinase mediates renal tubular cell apoptosis through a
TNF-
-dependent mechanism. The activation of p38 MAP kinase,
production of TNF-
, and subsequent increase in TNF-
-dependent
apoptosis suggest that NF-
B is a proapoptotic signal in
this cellular model. Interestingly, NF-
B is largely considered to be
an antiapoptotic signal and has been demonstrated to inhibit
TNF-
-dependent apoptosis in a number of cells (4, 27,
29, 44, 50, 52, 58). Most likely, NF-
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-
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ayala, A,
Perrin MM,
Meldrum DR,
Ertel W,
and
Chaudry IH.
Hemorrhage induces an increase in serum TNF which is not associated with elevated levels of endotoxin.
Cytokine
2:
170-174,
1990[Medline].
2.
Azuma, H,
Nadeau K,
Takada M,
Mackenzie HS,
and
Tilney NL.
Cellular and molecular predictors of chronic renal dysfunction after initial ischemia/reperfusion injury of a single kidney.
Transplantation
64:
190-197,
1997[ISI][Medline].
3.
Baud, L,
Oudinet JP,
Bens M,
Noe L,
Peraldi MN,
Rondeau E,
Etienne J,
and
Ardaillou R.
Production of tumor necrosis factor by rat mesangial cells in response to bacterial lipopolysaccharide.
Kidney Int
35:
1111,
1989[ISI][Medline].
4.
Beg, A,
and
Baltimore D.
An essential role for NF-B in preventing TNF-
-induced cell death.
Science
274:
782,
1996
5.
Brady, HR,
Spertini O,
Jimenez W,
Brenner BM,
Marsden PA,
and
Tedder TF.
Neutrophils, monocytes, and lymphocytes bind to cytokine-activated kidney glomerular endothelial cells through L-selectin (LAM-1) in vitro.
J Immunol
149:
2437-2444,
1992
6.
Brennan, DC,
Yui MA,
Wuthrich RP,
and
Kelley VF.
Tumor necrosis factor and interleukin 1 in New Zealand black/white mice. Enhanced gene expression and acceleration of renal injury.
J Immunol
143:
3470-3475,
1989
7.
Chinnaiyan, AM,
O'Rourke K,
Tewari M,
and
Dixit VM.
FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis.
Cell
81:
505-512,
1995[ISI][Medline].
8.
Daemen, MA,
van't Veer C,
Denecker G,
Heemskerk VH,
Wolfs TG,
Clauss M,
Vandenabeele P,
and
Buurman WA.
Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation.
J Clin Invest
104:
541-549,
1999
9.
Donnahoo, KK,
Meng X,
Ao L,
Ayala A,
Shames B,
Cain MP,
Harken AH,
and
Meldrum DR.
Differential cellular immunolocalization of renal TNF- production during ischemia versus endotoxemia.
Immunology
102:
53-58,
2001[ISI][Medline].
10.
Donnahoo, KK,
Meng X,
Ayala A,
Cain MP,
Harken AH,
and
Meldrum DR.
Early kidney TNF- expression mediates neutrophil infiltration and injury following isolated renal ischemia-reperfusion.
Am J Physiol Regulatory Integrative Comp Physiol
277:
R922-R929,
1999
11.
Donnahoo, KK,
Shames BD,
Harken AH,
and
Meldrum DR.
The role of tumor necrosis factor in renal ischemia-reperfusion injury.
J Urol
162:
196-203,
1999[ISI][Medline].
12.
Fouqueray, B,
Philippe C,
Herbelin A,
Perez J,
Ardaillou R,
and
Baud L.
Cytokine formation within rat glomeruli during experimental endotoxemia.
J Am Soc Nephrol
3:
1783-1791,
1993[Abstract].
13.
Garcia-Criado, FJ,
Eleno N,
Santos-Benito F,
Valdunciel JJ,
Reverte M,
Lozano-Sanchez FS,
Ludena MD,
Gomez-Alonso A,
and
Lopez-Novoa JM.
Protective effect of exogenous nitric oxide on the renal function and inflammatory response in a model of ischemia-reperfusion.
Transplantation
66:
982-990,
1998[ISI][Medline].
14.
Gobe, G,
Willgoss D,
Hogg N,
Schoch E,
and
Endre Z.
Cell survival or death in renal tubular epithelium after ischemia-reperfusion injury.
Kidney Int
56:
1299-1304,
1999[ISI][Medline].
15.
Gobe, G,
Zhang XJ,
Cuttle L,
Pat B,
Willgoss D,
Hancock J,
Barnard R,
and
Endre RB.
Bcl-2 genes and growth factors in the pathology of ischaemic acute renal failure.
Immunol Cell Biol
77:
279-286,
1999[ISI][Medline].
16.
Gobe, G,
Zhang XJ,
Willgoss DA,
Schoch E,
Hogg NA,
and
Endre ZH.
Relationship between expression of Bcl-2 genes and growth factors in ischemic acute renal failure in the rat.
J Am Soc Nephrol
11:
454-467,
2000
17.
Gschwend, J,
de Petriconi R,
Maier S,
Kleinschmidt K,
and
Hautmann R.
Continuous in situ cold perfusion with histidine tryptophan ketoglutarate solution in nephron sparing surgery for renal tumors.
J Urol
154:
1307-1311,
1995[ISI][Medline].
18.
Han, H,
Lee JD,
Tobias PS,
and
Ulevitch RJ.
Endotoxin induces rapid tyrosine phosphorylation in 70Z/3 cells expressing CD14.
J Biol Chem
268:
25009,
1993
19.
Han, J,
Richter B,
Li Z,
Kravchenko V,
and
Ulevitch RJ.
Molecular cloning of the p38 MAP kinase.
Biochim Biophys Acta
16:
224-227,
1995.
20.
Henry, P,
Popescu A,
Puceat M,
Hinescu ME,
and
Escande D.
Acute simulated ischaemia produces both inhibition and activation of K+ currents in isolated ventricular myocytes.
Cardiovasc Res
32:
930-939,
1996[ISI][Medline].
21.
Hoch, RC,
Rodriguez R,
and
Manning T.
Effects of accidental trauma on cytokine and endotoxin production.
Crit Care Med
21:
839-845,
1993[ISI][Medline].
22.
Hourmant, M,
Vasse N,
le Mauff B,
and
Soulillou JP.
The role of adhesion molecules in ischaemia-reperfusion injury of renal transplants.
Nephrol Dial Transplant
12:
2485-2487,
1997
23.
Hsu, H,
Xiong J,
and
Goeddel DV.
The TNF receptor 1-associated protein TRADD signals cell death and NF-B activation.
Cell
81:
495-504,
1995[ISI][Medline].
24.
Ichimura, T,
Bonventre JV,
Bailly V,
Wei H,
Hession CA,
Cate RL,
and
Sanicola M.
Kidney injury molecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury.
J Biol Chem
273:
4135-4142,
1998
25.
Kelly, KJ,
Williams WW, Jr,
Colvin RB,
Meehan SM,
Springer TA,
Gutierrez-Ramos JC,
and
Bonventre JV.
Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury.
J Clin Invest
97:
1056-1063,
1996
26.
Kerr, JF,
Wyllie AH,
and
Currie AR.
Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.
Br J Cancer
26:
239-257,
1972[ISI][Medline].
27.
Kirshenbaum, LA.
Bcl-2 intersects the NF-B signaling pathway and suppresses apoptosis in ventricular myocytes.
Clin Invest Med
23:
322-330,
2000[ISI][Medline].
28.
Kita, T,
Tanaka N,
and
Nagano T.
The immunocytochemical localization of tumor necrosis factor and leukotriene in the rat kidney after treatment with lipopolysaccharide.
Int J Exp Pathol
74:
471-479,
1993[ISI][Medline].
29.
Lang, A,
Schoonhoven R,
Tuvia S,
Brenner DA,
and
Rippe RA.
Nuclear factor-B in proliferation, activation, and apoptosis in rat hepatic stellate cells.
J Hepatol
33:
49-58,
2000[ISI][Medline].
30.
Lauriat, S,
and
Linas SL.
The role of neutrophils in acute renal failure.
Semin Nephrol
18:
498-504,
1998[ISI][Medline].
31.
Lee, JC,
Laydon JT,
McDonnel PC,
Gallagher TF,
Kumar S,
Green D,
McNulty D,
Blumenthal MJ,
Heys JR,
Landvatter SW,
Strickler JE,
McLaughlin MM,
Siemens IR,
Fisher SM,
Livi GP,
White JR,
Adams JL,
and
Young PR.
A protein kinase involved in the regulation of inflammatory cytokine biosynthesis.
Nature
372:
739-746,
1994[ISI][Medline].
32.
Lee, JC,
and
Young PR.
Role of CSB/p38/RK stress response kinase in LPS and cytokine signaling mechanisms.
J Leukoc Biol
59:
152-157,
1996[Abstract].
33.
Lieberthal, W,
Koh JS,
and
Levine JS.
Necrosis and apoptosis in acute renal failure.
Semin Nephrol
18:
505-518,
1998[ISI][Medline].
34.
Lieberthal, W,
and
Levine JS.
Mechanisms of apoptosis and its potential role in renal tubular epithelial cell injury.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F477-F488,
1996
35.
Mangano, CM,
Diamondstone LS,
Ramsay JG,
Aggarwal A,
Herskowitz A,
and
Mangano DT.
Renal dysfunction after myocardial revascularization: risk factors, adverse outcomes, and hospital resource utilization. The Multicenter Study of Perioperative Ischemia Research Group.
Ann Intern Med
128:
194-203,
1998
36.
Meldrum, DR.
Tumor necrosis factor in the heart.
Am J Physiol Regulatory Integrative Comp Physiol
274:
R577-R595,
1998
37.
Meldrum, DR,
Ayala A,
and
Chaudry IH.
Energetics of defective macrophage antigen presentation following hemorrhage.
Surgery
112:
150-158,
1992[ISI][Medline].
38.
Meldrum, DR,
Ayala A,
and
Chaudry IH.
Mechanism of diltiazem's immunomodulatory effects following hemorrhage and resuscitation.
Am J Physiol Cell Physiol
265:
C412-C421,
1993
39.
Meldrum, DR,
Ayala A,
Wang P,
Ertel W,
and
Chaudry IH.
Association between decreased splenic ATP levels and immunodepression.
Am J Physiol Regulatory Integrative Comp Physiol
261:
R351-R357,
1991
40.
Meldrum, DR,
Cain BS,
Cleveland JC,
Meng X,
Ayala A,
Banerjee A,
and
Harken AH.
Adenosine decreases post-ischemic myocardial TNF- production: anti-inflammatory implications for preconditioning and transplantation.
Immunology
92:
472-477,
1997[ISI][Medline].
41.
Meldrum, DR,
Cleveland JC,
Sheridan BC,
Rowland RT,
Banerjee A,
and
Harken AH.
Cardiac surgical implications of calcium dyshomeostasis in the heart.
Ann Thorac Surg
61:
1273-1280,
1996
42.
Meldrum, DR,
Dinarello CA,
Meng X,
Shapiro L,
and
Harken AH.
p38 MAP kinase-mediated myocardial TNF- production contributes to post-ischemic cardiac dysfunction.
Circulation
96:
I-556,
1997.
43.
Meldrum, DR,
Meng X,
Dinarello CA,
Ayala A,
Cain BS,
Shames BD,
Ao L,
Banerjee A,
and
Harken AH.
Human myocardial tissue TNF- expression following acute global ischemia in vivo.
J Mol Cell Cardiol
30:
1683-1689,
1998[ISI][Medline].
44.
Mustapha, S,
Kirshner A,
De Moissac D,
and
Kirshenbaum LA.
A direct requirement of nuclear factor-B for suppression of apoptosis in ventricular myocytes.
Am J Physiol Heart Circ Physiol
279:
H939-H945,
2000
45.
Rabb, H,
O'Meara YM,
Maderna P,
Coleman P,
and
Brady HR.
Leukocytes, cell adhesion molecules and ischemic acute renal failure.
Kidney Int
51:
1463-1468,
1997[ISI][Medline].
46.
Sanghera, JS,
Weinstein SL,
Aluwalia M,
Girn J,
and
Pelech SL.
Activation of multiple proline-directed kinases by bacterial lipopolysaccharide in murine macrophages.
J Immunol
156:
4457-4465,
1996[Abstract].
47.
Satriano, JA,
Hora K,
Shan Z,
Stanley ER,
Mori T,
and
Schlondorff D.
Regulation of monocyte chemoattractant protein-1 and macrophage colony-stimulating factor-1 by IFN-, tumor necrosis factor-
, IgG aggregates, and cAMP in mouse mesangial cells.
J Immunol
150:
1971-1978,
1993
48.
Schumer, M,
Colombel MC,
Sawczuk IS,
Gobe G,
Connor J,
O'Toole KM,
Olsson CA,
Wise GJ,
and
Buttyan R.
Morphologic, biochemical, and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia.
Am J Pathol
140:
831-838,
1992[Abstract].
49.
Soler, PA,
Mullin JM,
Knudsen KA,
and
Marano CW.
Tissue remodeling during tumor necrosis factor-induced apoptosis in LLC-PK1 renal epithelial cells.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F869-F870,
1996
50.
Sonenshein, GE.
Rel/NF-B transcription factors and the control of apoptosis.
Semin Cancer Biol
8:
113-119,
1997[ISI][Medline].
51.
Steller, H.
Mechanisms and genes of cellular suicide.
Science
267:
1445-1449,
1995[ISI][Medline].
52.
Sumitomo, M,
Tachibana M,
Nakashima J,
Murai M,
Miyajima A,
Kimura F,
Hayakawa M,
and
Nakamura H.
An essential role for nuclear factor B in preventing TNF-
-induced cell death in prostate cancer cells.
J Urol
161:
674-679,
1999[ISI][Medline].
53.
Sweet, MJ,
and
Hume DA.
Endotoxin signal transduction in macrophages.
J Leukoc Biol
60:
8-26,
1996[Abstract].
54.
Takada, M,
Nadeau KC,
Shaw GD,
Marquette KA,
and
Tilney NL.
The cytokine-adhesion molecule cascade in ischemia/reperfusion injury of the rat kidney. Inhibition by a soluble P-selectin ligand.
J Clin Invest
99:
2682-2690,
1997
55.
Takada, M,
Nadeau KC,
Shaw GD,
and
Tilney NL.
Prevention of late renal changes after initial ischemia/reperfusion injury by blocking early selectin binding.
Transplantation
64:
1520-1525,
1997[ISI][Medline].
56.
Thompson, CB.
Apoptosis in the pathogenesis and treatment of disease.
Science
267:
1456-1462,
1995[ISI][Medline].
57.
Tong, L,
Pav S,
White DM,
Rogers S,
Crane KM,
Cywin CL,
Brown ML,
and
Pargellis CA.
A highly specific inhibitor of human p38 kinase binds in the ATP pocket.
Nat Struct Biol
4:
311-316,
1997[ISI][Medline].
58.
Van Antwerp, D,
Martin S,
Kafri T,
Green D,
and
Verma I.
Suppression of TNF--induced apoptosis by NF-
B.
Science
274:
787,
1996
59.
Vanheel, B,
Leybaert L,
De Hemptinne A,
and
Leusen I.
Simulated ischemia and intracellular pH in isolated ventricular muscle.
Am J Physiol Cell Physiol
257:
C365-C376,
1989
60.
Weight, SC,
Bell PR,
and
Nicholson ML.
Renal ischaemia-reperfusion injury.
Br J Surg
83:
162-170,
1996[ISI][Medline].
61.
Weisberg, LS,
Allgren RL,
Genter FC,
and
Kurnik BR.
Cause of acute tubular necrosis affects its prognosis. The Auriculin Anaritide Acute Renal Failure Study Group.
Arch Intern Med
157:
1833-1888,
1997[Abstract].
62.
Yin, T,
Sandhu G,
Wolfgang CD,
Burrier A,
Webb RL,
Rigel DF,
Hai T,
and
Whelan J.
Tissue-specific pattern of stress kinase activation in ischemic/reperfused heart and kidney.
J Biol Chem
272:
19943-19950,
1997