From Medical Services, Massachusetts General
Hospital, Charlestown, Massachusetts 02129-2060, the Department of
Medicine, Harvard Medical School, Boston, Massachusetts 02114, and
§ Harvard-Massachusetts Institute of Technology Division of
Health Sciences and Technology, Boston, Massachusetts 02115-6092 and Cambridge, Massachusetts 02139-4307
Received for publication, August 17, 2000, and in revised form, January 5, 2001
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ABSTRACT |
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MAPK activities, including JNK, p38, and
ERK, are markedly enhanced after ischemia in vivo
and chemical anoxia in vitro. The relative extent of
JNK, p38, or ERK activation has been proposed to determine cell
fate after injury. A mouse model was established in which prior
exposure to ischemia protected against a second ischemic insult imposed
8 or 15 days later. In contrast to what was observed after 30 min of
bilateral ischemia, when a second period of ischemia of 30- or 35-min
duration was imposed 8 days later, there was no subsequent increase in
plasma creatinine, decrease in glomerular filtration rate, or increase
in fractional excretion of sodium. A shorter period of prior ischemia
(15 min) was partially protective against subsequent ischemic injury 8 days later. Unilateral ischemia was also protective against a subsequent ischemic insult to the same kidney, revealing that systemic
uremia is not necessary for protection. The ischemia-related activation
of JNK and p38 and outer medullary vascular congestion were
markedly mitigated by prior exposure to ischemia, whereas preconditioning had no effect on post-ischemic activation of ERK1/2. The phosphorylation of MKK7, MKK4, and MKK3/6, upstream activators of
JNK and p38, was markedly reduced by ischemic preconditioning, whereas
the post-ischemic phosphorylation of MEK1/2, the upstream activator of
ERK1/2, was unaffected by preconditioning. Pre- and post-ischemic
HSP-25 levels were much higher in the preconditioned kidney. In
summary, post-ischemic JNK and p38 (but not ERK1/2) activation was
markedly reduced in a model of kidney ischemic preconditioning that was
established in the mouse. The reduction in JNK and p38 activation can
be accounted for by reduced activation of upstream MAPK kinases. The
post-ischemic activation patterns of MAPKs may explain the remarkable
protection against ischemic injury observed in this model.
Ischemic injury to brain, heart, and kidney is associated with
high morbidity and mortality. Improving the ability of these organs to
tolerate ischemic injury would have important implications. Ischemic
insults are often recurrent in patients. In the setting of loss of
renal blood flow autoregulation that characterizes the post-ischemic
kidney (1), it might be expected that the post-ischemic patient would
be more susceptible to a second insult. It has been reported in
animals, however, that prior acute renal failure induced by toxins can
confer resistance to subsequent insults, although this is not a
universal finding (2). Zager et al. (3) have focused on
early time points after an initial ischemic exposure and have concluded
that the rat is not more susceptible to a second ischemic episode when
timed at or near the peak of functional deficit after the first. The
authors further concluded that "a modicum of protection appears to
exist, possibly due to renal failure-induced increments in solute
load." Proximal tubules isolated 24 h (but not 15 min or 4 h) after an ischemic episode are protected against hypoxia, reactive
oxygen species, or calcium ionophore in vitro (4, 5). The
lack of protection at 4 h and the induction of protection by
inducing azotemia in vivo or adding urine to the tubules
in vitro led the authors to conclude that the protection is
due to the uremic environment. Subsequently protection was found in
tubules obtained from obstructed kidneys even when the tubules had not
been exposed to a uremic environment (6). In a recent study (7), four
cycles of 8 min (but not 4 or 6 min) of ischemia, separated by 5 min of
reperfusion, conferred protection against a subsequent 45-min ischemic
exposure immediately after the four cycles. In another recent study,
four cycles of 4 min of ischemia, separated by 11 min of reperfusion, followed 5 min later by 45 min of ischemia, resulted in fewer dead
renal cells, but no difference in creatinine at 6 h post-ischemia with this preconditioning protocol (8). It had previously been shown
that there were no morphological differences with this preconditioning protocol if the time delay between the cycles and the prolonged ischemia was 30 min (9). Thus, controversy remains as to the conditions
under which preconditioning occurs in the kidney in response to prior
ischemia. In addition, the reported experiments are limited to the
study of short intervals of time between the preconditioning protocol
and the tested period of subsequent ischemia.
One approach to the design of therapies to protect the kidney against
ischemic injury is to establish a model in which endogenous mechanisms
result in protection and then identify those processes. A model in mice
would be particularly useful given the plethora of transgenic models
that exist, which would facilitate exploration of the role of a
particular protein in the protection. Furthermore, we wanted to
establish a model in which the acute cellular and molecular response to
the initial event had subsided, reducing the "background" genetic
response so that it would be more possible to identify an endogenous
protective factor or factors.
Our experiments demonstrate that 30 min of bilateral renal ischemia,
resulting in significant increases in blood urea nitrogen and
creatinine, leads to protection of the mouse kidney against a
subsequent ischemic insult 8 or 15 days later, even when the second
ischemic period is extended to 35 min. Graded levels of time of initial
ischemia resulted in graded levels of protection 8 days later. This
protection is unrelated to systemic effects of transient uremia since
unilateral ischemia is also associated with protection under conditions
in which there is very little increase in systemic blood urea nitrogen
or creatinine. Preconditioning results in prevention of medullary
congestion. With varying times of initial ischemia, the protection
afforded 8 days later correlates with the level of sustained elevations
in HSP-251 (HSP-27 in rat)
protein levels prior to the second ischemic period, but not superoxide
dismutase or HSP-72 levels.
The MAPKs have been implicated in post-ischemia/reperfusion cell
survival, necrosis, and apoptosis (10-12). MAPKs mediate the response
of cells to a wide variety of physiological and stress-related stimuli,
including ultraviolet light, heat shock, ischemia, oxygen free
radicals, and hyperosmolality. It has been proposed that activation of
JNK and p38 kinases contributes to cell death, whereas activation of
ERK1/2 contributes to protection against cell injury in multiple organs
(11, 13). We examined whether the effect of ischemic preconditioning to
protect the kidney is associated with corresponding changes in stress
kinases and ERKs as well as their upstream activating kinases, which
might account for the functional protection observed. Post-ischemic
activation of JNK and p38 is markedly mitigated in the preconditioned
kidneys, as is the post-ischemic activation of the MAPK kinases MKK7,
MKK4, and MKK3/6, upstream activators of JNK and p38. By contrast, the post-ischemic activation of MKK3/4, the MAPK kinases responsible for
activating ERK1/2, is unaltered by preconditioning.
Animal Preparation--
All experiments were performed with male
BALB/c mice (Charles River Laboratories) weighing 20-25 g. Mice
were allowed free access to water and standard mice chow. Blood was
drawn, and base-line levels of serum creatinine and blood urea nitrogen
were determined 2 days before surgery. Animals were anesthetized with
pentobarbital sodium (50 mg/kg intraperitoneally) and administered 1 ml
of 0.9% NaCl (37 °C) on the day of surgery (day 0). Body
temperature was maintained at 36.0-37.5 °C. Animals were divided
into 13 (I-XIII) groups (Table I).
Kidneys were exposed through flank incisions. Mice in Groups II-IV,
VI, and VII were subjected to bilateral renal ischemia by
clamping both renal pedicles with nontraumatic microaneurysm clamps
(Roboz Surgical Instrument Co., Inc.). Animals in Groups I, V, and VIII
underwent sham surgery. The incisions were temporarily closed during
ischemia or sham surgery. After 5 (Group II), 15 (Group III), or 30 (Groups IV, VI, and VII) min, the clamps were removed, and reperfusion
of the kidneys was visually confirmed. Animals were exposed to 30 (Groups I-IV) or 35 (Groups V and VI) min of bilateral ischemia on day
8 or 30 min of ischemia on day 15 (Groups VII and VIII).
Mice in Groups IX-XIII underwent unilateral ischemia for 30 min. In
Group IX, the contralateral kidney was removed on day 2, whereas in
Group X, the contralateral kidney was removed on day 8, at the time of
the second ischemia. In Group XI, the left kidney was made ischemic on
day 0; the contralateral kidney was removed on day 8; and the left
kidney was made ischemic again on day 15. In Group XII, the left kidney
was made ischemic for 30 min on day 0. On day 8, that kidney was
removed, and the right kidney was made ischemic on day 15. In Group
XIII, ischemia to one kidney on day 0 was followed by ischemia to the
contralateral kidney on day 8, and bilateral ischemia was imposed on
day 15.
Kidneys of experimental groups were harvested on day 1, 2, or 8 after
the first surgery or 24 or 48 h after the second ischemic period.
Kidneys were snap-frozen in liquid nitrogen and subsequently used for
Western analysis or were rinsed in phosphate-buffered saline and fixed
in 4% paraformaldehyde for histological analysis.
Renal Functional Parameters--
Seventy microliters of blood
were taken from the retrobulbar vein plexus at the times indicated in
the figures. Plasma and urine sodium, creatinine, and blood urea
nitrogen concentrations were measured using a flame photometer, a
Beckman Creatinine Analyzer II, or a spectrophotometer (blood urea
nitrogen). To determine glomerular filtration rate and fractional
excretion of sodium, urine was collected for 24 h.
Western Blot Analysis--
Proteins were extracted from kidneys
as previously described (10). Protein samples were separated on either
a 10 or 12% SDS-polyacrylamide gel and then transferred to an
Immobilon membrane. Membranes were incubated with antibodies against
active phospho-JNK(Thr183/Tyr185), active
phospho-p38(Thr180/Tyr182), active
phospho-ERK1/2(Thr202/Tyr204), phospho-SEK1/MKK4,
phospho-MEK1/2, MEK1/2, phospho-MKK3/6, and MKK3 (Cell
Signaling); JNK1, ERK1/2, and p38 (Santa Cruz Biotechnology); phospho-MKK7 (provided by A. Nelsbach, Cell Signaling); HSP-72 (Stressgen Biotech Corp.); HSP-27 (Upstate Biotechnology, Inc.); and
superoxide dismutase (Chemicon International, Inc.). Secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology) were detected by the ECL system (Amersham Pharmacia Biotech).
Immunoprecipitation of JNK and Immune Complex Kinase
Assays--
Supernatants from mouse kidney lysates were matched for
protein concentration prior to immunoprecipitation with a polyclonal antiserum against JNK expressed as a glutathione
S-transferase fusion protein in Escherichia coli.
Immune complexes were collected with protein A-Sepharose beads, and
kinase assays were performed as described (10) with glutathione
S-transferase-c-Jun-(1-135) as substrate. After 20 min at
30 °C, the kinase reactions were stopped with Laemmli sample buffer.
Samples were resolved by SDS-polyacrylamide gel electrophoresis,
stained with Coomassie Blue, destained, dried, and subjected to
autoradiography. The bands corresponding to glutathione S-transferase-c-Jun-(1-135) were cut out of the gel.
Radioactivity was measured by liquid scintillation counting.
Histology--
Kidneys were perfused via the left ventricle with
phosphate-buffered saline at 37 °C and then with paraformaldehyde
lysine periodate (14) for 10 min. In some cases, the tissue was
embedded in paraffin, sectioned to 6-µm thickness, and stained with
hematoxylin and eosin.
Statistics--
All results are expressed as means ± S.E.
A p value <0.05 was taken as statistically significant.
Each group consisted of 4-10 animals as indicated in Table I.
Effects of Prior Ischemia on Renal Function after Subsequent
Ischemia/Reperfusion--
Animals were exposed to 0 (Group I) or 5, 15, or 30 min (Groups II-IV) of bilateral renal ischemia (Fig.
1A). Plasma levels of
creatinine increased 5-8-fold over base-line levels 24 h
post-ischemia only when the ischemic period was 30 min. By day 5 after
ischemia, creatinine levels were indistinguishable from those at base
line in all groups. On day 8 after the initial ischemia or sham
procedure, a subsequent 30 min of bilateral ischemia was induced. The
increase in creatinine measured 24 h later was inversely
correlated with the length of time of the prior ischemia. There was no
significant increase in serum creatinine at 24 and 48 h after the
second ischemic period in animals that were previously exposed to 30 min of ischemia. The patterns of change in blood urea nitrogen after
the first and second ischemic periods closely paralleled those in
creatinine in these and all subsequent experiments (Fig.
1B). The glomerular filtration rate was markedly decreased
during the first day after 30 min of ischemia, but returned to normal
within 7 days of reperfusion (Fig. 1C). Likewise, fractional
excretion of sodium was markedly increased during the first day of
reperfusion, but returned to normal levels by 7 days of reperfusion
(Fig. 1D). A subsequent second 30-min ischemic period
resulted in no decrease in glomerular filtration rate or increase in
fractional excretion of sodium.
Necrosis was found in kidneys from Group IV 48 h after 30 min of
bilateral ischemia. Neutrophils were found in the outer medulla. The
kidneys of sham-operated animals were normal. On day 8, the repair of
injured tubules was almost complete. Regenerated tubules were seen, and
some tubules were dilated and cystic. When kidneys were examined
48 h after the second ischemia, the "preconditioned" kidneys
(Group IV) had no significant outer medullary congestion, whereas there
was very significant congestion in the outer medulla of sham-pretreated
animals examined after their first exposure to ischemia (Group I) (Fig.
2A). Microscopic examination
revealed much less necrosis of proximal tubule cells, obstruction, and red cell trapping in the outer medulla of ischemia-pretreated preconditioned animals (Group IV) compared with Group I controls (Fig.
2, B and C).
When the first ischemic period was 30 min and the second was extended
to 35 min (Group VI), serum creatinine did not increase after the
initiation of reperfusion (Fig. 3). When
the length of time between bilateral ischemic exposures was lengthened
to 15 days (Group VII), serum creatinine increased by 24 h after the second ischemic insult (Fig. 4);
however, the increase was significantly lower than that observed in
animals that had been previously sham-operated on day 0 (Group
VIII).
Unilateral Renal Ischemia Followed 8 Days Later by
Ischemia/Reperfusion--
To evaluate whether ischemia itself was
protective independent of systemic effects of uremia, animals were
exposed to sham surgery or unilateral left kidney ischemia (Groups I,
IX, and X) for 30 min on day 0. We considered that the presence of a
normal contralateral kidney might influence mechanisms contributing to protection against a subsequent ischemic exposure to a kidney rendered
unilaterally ischemic (15). Two (Group IX) or 8 (Group X) days later,
the contralateral right kidney was removed; and at 8 days, the left
kidney was again rendered ischemic for 30 min (Fig.
5). Prior unilateral ischemia protected
that kidney against subsequent ischemia even in the absence of an
increase in serum creatinine (Fig. 5). If the contralateral kidney was left in for 8 days, however, the protection was less than was seen when
the contralateral kidney was removed at 2 days post-ischemia.
In Group XIII, the left kidney was rendered ischemic on day 0; the
right kidney was made ischemic for 30 min on day 8; and both kidneys
were made ischemic on day 15. There was protection against ischemic
injury under these conditions, with no increase in serum creatinine
24 h after the bilateral ischemia (Fig.
6). To evaluate whether unilateral
ischemia confers protection on the contralateral kidney, in Group XII,
the left kidney was made ischemic for 30 min on day 0 and subsequently
removed on day 8, and the right kidney was made ischemic for 30 min on
day 15. Twenty-four hours later, the mean serum creatinine was markedly
elevated (Fig. 6), indicating that unilateral ischemia did not confer
protection on the contralateral kidney.
Effect of Ischemic Preconditioning on the Levels of HSPs and
Superoxide Dismutase--
We evaluated whether HSPs or superoxide
dismutase protein levels correlate with the development of resistance
to subsequent ischemia. The levels of HSP-25 at 8 days post-ischemia
increased with time of ischemia (Fig.
7A), correlating well with the
extent of protection against functional deficits after the second
ischemic period (Fig. 1). After the second ischemic period, HSP-25
levels markedly increased to higher levels than in the preconditioned kidneys (Fig. 7B), whereas HSP-72 levels were no different
whether the first procedure was ischemia or sham surgery (Fig.
7A). The tissue protein levels of superoxide dismutase
(15-kDa subunit) were not markedly changed at 8 days post-ischemia
(data not shown).
Effect of Ischemic Preconditioning on Activity of MAPKs and MAPK
Kinases--
The JNK family of MAPKs (also known as SAPKs) is markedly
activated as early as 5 min after ischemia/reperfusion, peaks at 20 min, but remains at relatively high levels up to 2 h post-ischemia (10). In these studies, we have found that JNK activity remained above
control levels at 48 h of reperfusion (Fig.
8A). In animals previously
exposed to ischemia, subsequent ischemia on day 8 or 15 resulted in
much less activation of JNKs than was seen in post-ischemic kidneys not
previously exposed to ischemia (Fig. 8A). When activation was determined by immunoblotting with the anti-phospho-JNK antibody, a
similar pattern was seen, with marked reduction of the ischemia-induced increase in phospho-JNK at 0.5 and 1.5 h post-ischemia in the animals exposed to ischemia 8 days previously (Fig. 8A,
lower panel). Likewise, post-ischemic p38 activation was
markedly reduced in kidneys previously exposed to ischemia (Fig.
8B). ERK1/2 activation persisted 8 days after the initial
ischemic exposure; but in contrast to JNK and p38, there was no effect
of prior ischemia on post-ischemic ERK phosphorylation on day 8 (Fig.
8C).
To evaluate whether activation of JNK, p38, and ERK1/2 is potentially
explained by changes in activation patterns of upstream regulators of
these kinases, we compared the response to ischemia of MKK7 (which
activates JNK), MKK4 (which activates JNK and possibly p38), MKK3/6
(which activate p38), and MEK1/2 (which activate ERK1/2) in kidneys
previously exposed to ischemia or sham surgery (Fig.
9). The post-ischemic increase in
phosphorylation of MKK7, MKK4, and MKK3/6 was much less in the
preconditioned kidney than in the non-preconditioned kidney. By
contrast, post-ischemic phosphorylation of MEK1/2 was not decreased by
preconditioning. Whereas phosphorylation of MKK7 peaked at 1.5 h
after ischemia, phosphorylation of MKK4, MKK3/6, and MEK1/2 peaked at
0.5 h.
Ischemic preconditioning may lead to important insight into
how an organ uses endogenous mechanisms to protect itself against injury. To utilize the powerful genomic and proteomic technologies for
identification of proteins that might be implicated in this protection,
it is necessary to establish a mouse model in which that protection is
very reproducible and robust. We purposely sought to establish a model
that minimizes the number of residual nonspecific responses to the
initial intervention at the time when we study the mechanisms of
protection. This minimizes the problems associated with sorting through
the functional significance of up-regulation of hundreds of genes that
occurs within days of ischemia. Using RNA profiling techniques, we have
found many fewer genes altered in expression at 8 days post-ischemia
(data not shown). Understanding mechanisms of protection afforded by tissue responses to injury may lead to strategies for mimicking these
responses therapeutically to prevent ischemic injury.
Whether ischemic preconditioning exists in the kidney has been
somewhat controversial. Zager et al. (3) reported that 40 min of bilateral ischemia in the rat did not confer enhanced
predisposition to a second ischemic insult imposed at the peak of
functional deficit after the first ischemia. In a subsequent paper
(16), this group reported that 25 min of renal ischemia "can
significantly decrease renal resistance to a subsequent ischemic
event." Renal proximal tubule epithelial cells isolated from rats
24 h after 35 min of ischemia were protected against hypoxic
injury as manifested by lactate dehydrogenase release (5).
Proximal tubules were not protected if they were isolated from rats
sustaining 15 min of bilateral ischemia when uremia was not present.
Furthermore, the presence of a normal contralateral kidney markedly
attenuated cytoprotection after unilateral ischemia, a condition in
which uremia did not develop. Islam et al. (9) found
that brief cycles of renal artery occlusion followed by reperfusion did
not afford functional or morphological protection of rat kidney from a
subsequent ischemic insult 30 min later. Cochrane et al.
(18) observed a lower serum creatinine level after 24 h of reflow
in rats pretreated with ischemia for 2 min or three successive 2-min
periods of ischemia (separated by 5 min each), but not three periods of
ischemia of 5-min duration.
Our experiments indicate that mice that had recovered from 30 min of
bilateral renal ischemia were protected against subsequent ischemia
imposed 8 or 15 days after the first ischemic challenge. The protection
against functional injury 8 days after ischemia was quite profound. As
a very sensitive measure of tubular injury, fractional excretion of
sodium was unaffected by 30 min of ischemia. By contrast, it increased
to ~7% in post-ischemic kidneys not previously exposed to ischemia.
The preconditioning protocol described is the most effective way to
protect the mouse kidney against ischemic injury.
Protection is not dependent on the presence of a uremic systemic
environment since 15 min of initial ischemia resulted in protection
against subsequent ischemia without causing an increase in blood urea
nitrogen. A local effect within the kidney is likely responsible for
protection against subsequent ischemia since protection was observed in
the same kidney that sustained unilateral ischemia, but not in the
contralateral kidney. Our finding that the protection was greater when
the contralateral kidney was removed on day 2 compared with day 8 is
compatible with prior observations of the adverse effects of the
presence of the contralateral kidney on the functional recovery of a
unilaterally ischemic kidney (15).
In the heart, although preconditioning is a very effective way to
reduce post-ischemic infarct size, the protective effects of
preconditioning are transient and initially last only for a short
period of time, i.e. <2 h. A so-called "second window of protection" has been observed in some species (19), occurring 24 h after the preconditioning stimulus in neurons and cardiomyocytes. In
our study, the protection in the mouse kidney after ischemia was seen
up to 15 days after the initial ischemic period, when most, but not
all, of the metabolic and stress responses associated with ischemia
have been resolved.
The degree of protection against the second ischemic insult after a
first ischemic period of varying time duration correlated positively
with levels of heat shock protein expression at 8 days post-ischemia.
HSPs may decrease production of cytokines (20), reducing
leukocyte-endothelial interactions and mitigating congestion in the
outer medulla, resulting in less hypoxic injury to the outer medullary
tubules. It has been suggested that HSP-25 protects against oxidative
or heat stress by stabilizing the actin cytoskeleton (21). The actin
cytoskeleton is markedly altered in the proximal tubule after ischemia
(22, 23). Another potential important role of HSP-25 is that of a
molecular chaperone efficiently trapping unfolding proteins in a
folding-competent state, allowing refolding after restoration of
physiological conditions post-ischemia (24). HSP-25 can also increase
cellular levels of glutathione (25), which can protect the cell against
oxidative stress, reduce stress kinase activation (13), and inhibit
apoptosis (26). HSP-25 levels increase in the cortical and outer
medullary proximal tubules of rat kidneys post-ischemia (27). By
contrast to HSP levels, changes in manganese-superoxide dismutase
protein levels, implicated in the delayed phase of preconditioning in
the dog heart (28), did not correlate with protection of the kidney in
our experiments.
Our demonstration of increases in JNK, p38, and ERK1/2 activation
with ischemia is consistent with previous published results from our
laboratory (10) and others (29) in the kidney and other organs. In the
heart, some have argued that activation of p38 is important for
preconditioning (30), whereas others have argued that activation is
detrimental and that a reduced level of activation is important for
preconditioning (31).
It was previously not known how the upstream kinases responsible for
activation of the MAPKs are affected by kidney ischemia. Our data
indicate that ischemia/reperfusion results in the activation of MKK3/6
and MKK4. Both are activators of p38, although the physiological relevance of MKK4 as an activator of p38 is unclear. This
ischemia-induced activation is reduced in kidneys previously exposed to
ischemia in a pattern that parallels that of p38 activation. JNK is
activated by dual phosphorylation on threonine and tyrosine by MKK4 and MKK7 (32). Ischemia/reperfusion results in MKK4 and MKK7 activation. This post-ischemic activation is reduced in kidneys previously exposed
to ischemia in a pattern that parallels that of p38 and JNK activation.
Interestingly, MKK7 activation peaks at 1.5 h after ischemia and
persists longer than JNK activation. This suggests that post-ischemic
JNK activation may be more dependent upon MKK4 than MKK7 activation. In
contrast to the upstream activators of the stress kinases,
post-ischemic MEK1/2 activation is not reduced in the preconditioned
kidney. The increase in MEK1/2 with a resultant increase in ERK1/2
activation post-ischemia/reperfusion in the setting of reduced stress
kinase activation may explain the protection seen with preconditioning
in the kidney. It has been proposed in neurons (11) and proximal tubule
cells (33) that the relative extent of JNK, p38, and ERK activation may
determine cell fate, with JNK activation associated with cell death and
ERK activity protective. JNK has recently been implicated in the
mitochondrial death pathway (34), and activation of the JNK cascade has
been reported to be important for cardiomyocyte death in response to simulated ischemia (35).
The functional protection we observed is associated with a
marked reduction in outer medullary congestion post-ischemia, which may
be related to decreased cytokine-induced leukocyte-endothelial interactions associated with the decrease in activation of JNK/SAPK and
p38. JNK and p38 activation enhances the expression of adhesion molecules (36) and cytokine production (17, 37, 38), which, in turn,
can enhance leukocyte-endothelial adhesion interactions in the small
vessels of the outer medulla with associated platelet activation and
resultant obstruction, leading to S3 segment injury. The dramatic
reduction in post-ischemic outer medullary congestion in the kidney
previously exposed to ischemia argues for an important effect of
preconditioning to prevent small vessel leukocyte- and perhaps
platelet-endothelial interactions.
In summary, we have demonstrated that the mouse kidney is profoundly
protected against ischemia/reperfusion injury up to 15 days after an
initial ischemic insult. This observation points to powerful endogenous
mechanisms that can be evoked by the kidney to protect itself against
ischemic injury. This protection is associated with prevention of outer
medullary vascular congestion and mitigation of the increase normally
seen in JNK and p38 activity, with no effect on ERK1/2 activation. The
effects of remote ischemic pre-exposure on MAPK kinase activation can
explain the changes observed in MAPK activation patterns. Since
protection is present long after many of the initial consequences of
the ischemia/reperfusion have subsided, it is likely that a genomic or
proteomic approach to mechanisms, involving identification of
"protective" proteins that are up-regulated or "detrimental"
proteins that are down-regulated, will be tractable. This will then
hopefully lead to therapies that will be effective in preventing and/or
treating ischemic acute renal failure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Animal groups and treatments
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of prior ischemia of varying durations
on serum creatinine (A), blood urea nitrogen
(BUN; B), glomerular filtration rate
(GFR; creatinine clearance) (C), and
fractional excretion of sodium (FENa;
D) after a second period of ischemia. Animals
were subjected to either sham surgery (S; Group I) or 5, 15, or 30 min of ischemia (I; Groups II-IV). Eight days after
the first surgery, animals were subjected to bilateral ischemia for 30 min. Values are expressed as means ± S.E. Arrows
indicate the day of ischemia or sham surgery. p < 0.001 (*) and p < 0.05 (#) versus sham
ischemia. BW, body weight.
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Fig. 2.
A, hemisected kidney
perfusion-fixed after sham surgery (Sham) or ischemia
(Isch.) (day 0), ischemia (day 8), and 48 h of
reperfusion. B and C, light microscopy of kidneys
treated as described for A. There was significant greater
necrosis of tubule cells, tubular obstruction, and vascular congestion
in the outer medulla in Group I animals (B) compared with
Group IV animals (C). Bar = 100 µm.
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Fig. 3.
Effect of prior ischemia for 30 min on serum
creatinine after 35 min of a second period of ischemia on day 8. On day 0, animals were subjected to either sham surgery (S;
Group V) or 30 min of ischemia (I; Group VI). Eight days
after the first surgery, animals were subjected to bilateral ischemia
for 35 min. Arrows indicate the day of ischemia
or sham surgery. *, p < 0.001 versus sham
ischemia.
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Fig. 4.
Effect of prior ischemia for 30 min on serum
creatinine after 30 min of a second period of ischemia on day 15. On day 0, animals were subjected to either surgery sham (S;
Group VIII) or 30 min of ischemia (I; Group VII). Fifteen
days after the first surgery, animals were subjected to bilateral
ischemia for 30 min. Arrows indicate the day of ischemia or
sham surgery. p < 0.001 (*) and p < 0.05 (#) versus sham ischemia.
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Fig. 5.
Effect of prior unilateral ischemia
(I) to the left (L) kidney, followed
by contralateral nephrectomy (N) of the right
(R) kidney, on day 2 (Group IX) or day 8 (Group X), on
serum creatinine after 30 min of a second period of unilateral
ischemia. Results are compared with creatinine values of mice
exposed to sham (S) ischemia on day 0, followed by bilateral
ischemia on day 8 (Group I). Arrows indicate the day of
ischemia or sham surgery. p < 0.001 (*) and
p < 0.05 (#) versus sham ischemia.
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Fig. 6.
Effect on serum creatinine of prior
unilateral ischemia (I) to the left
(L) kidney, followed by contralateral (Group XI) or
ipsilateral (Group XII) nephrectomy (N) on day 8 and
ischemia to the remaining kidney on day 15. Comparisons are made
with values obtained from animals subjected to left kidney ischemia on
day 0, followed by right (R) kidney ischemia on day 8 and
then bilateral ischemia on day 15 (Group XIII). Arrows
indicate the day of ischemia or sham surgery. p < 0.001 (*) and p < 0.05 (#) compared with data from
Group XII.
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Fig. 7.
Western blot analysis of HSP-25 and HSP-72 in
mouse kidneys exposed to ischemia. A, mouse kidneys
were exposed to various periods of ischemia on day 0. In some cases, a
30-min period of ischemia was imposed on day 8. Kidneys were removed
either on day 8 prior to any second period of ischemia or on day 9, 24 h after ischemia. B, mouse kidneys were exposed to
30 min of ischemia (I) on day 0 and, in some cases, exposed
to 30 min of ischemia on day 8. Kidneys were harvested at the indicated
times after the ischemic period. Densities of Western blot bands were
quantified by the NIH Image program. Data are presented as the mean
from three separate experiments. S, sham surgery.
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Fig. 8.
JNK kinase activity and phosphorylation of
JNK, p38, and ERK1/2 in mouse kidneys exposed to ischemia or sham
surgery on day 0, followed in some cases by ischemia on day 8. A: upper panel, JNK activity was assayed in
kidneys removed 2, 8, or 15 days later or 1 h after an ischemic
period on day 8. In addition, JNK activity was measured in kidneys
removed on day 10, 2 days after ischemia on day 8. JNK activity was
also measured on day 17 in kidneys exposed to ischemia (I)
or sham surgery (S) on day 0 and ischemia on day 15. Lower panel, shown are the results from Western blot
analysis of phospho-JNK (p-JNK) and total JNK
(t-JNK) in kidneys exposed to sham or ischemic surgery on
day 0, followed on day 8 by sham surgery or ischemia. Kidneys were then
removed at either 0.5 or 1.5 h after the procedure on day 8. B, shown are the results from Western blot analysis of
phospho-p38 (p-p38) and total p38 (t-p38) in
kidneys exposed to sham or ischemic surgery on day 0, followed on day 8 by sham surgery or ischemia. Kidneys were then removed at 0.5, 1.5, or
24 h after the procedure on day 8. C, shown are the
results from Western blot analysis of phospho-ERK1 (p-ERK1),
phospho-ERK2 (p-ERK-2), total ERK1 (t-ERK1), and
total ERK2 (t-ERK2) in kidneys exposed to sham surgery or
ischemia on day 0, followed on day 8 by sham surgery or ischemia.
Kidneys were then removed on day 8 prior to, or 0.5 h after, sham
surgery or ischemic procedure on day 8.
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[in a new window]
Fig. 9.
Western blot analysis of phosphorylation of
MEK1/2, MKK3/6, MKK4, and MKK7 in mouse kidneys exposed to sham surgery
(S) or ischemia (I) on either day 0 or 8, followed by 0.5, 1.5, or 24 h of reperfusion. Total
MEK1/2, MKK3 are also presented. p, phospho; t,
total.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by National Institutes of Health Merit Awards DK39773, DK38452, and NS10828 (to J. V. B.).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.
¶ These authors contributed equally to this work.
To whom correspondence should be addressed: Massachusetts
General Hospital East, Suite 4002, 149 13th St., Charlestown, MA 02129-2060. Tel.: 617-726-3770; Fax: 617-726-4356; E-mail:
joseph_bonventre@hms.harvard.edu.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M007518200
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ABBREVIATIONS |
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The abbreviations used are: HSP, heat shock protein; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MKK, MAPK kinase; SEK, SAPK/ERK kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; SAPK, stress-activated protein kinase.
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REFERENCES |
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---|
1. | Conger, J. (1997) Adv. Renal Replace. Ther. 4 Suppl. 1, 25-37[Medline] [Order article via Infotrieve] |
2. | Honda, N., Hishida, A., Ikuma, K., and Yonemura, K. (1987) Kidney Int. 31, 1233-1238[Medline] [Order article via Infotrieve] |
3. | Zager, R. A., Baltes, L. A., Sharma, H. M., and Jurkowitz, M. S. (1984) Kidney Int. 26, 689-700[Medline] [Order article via Infotrieve] |
4. | Zager, R. A., Burkhart, K. M., and Gmur, D. J. (1995) Lab. Invest. 72, 592-600[Medline] [Order article via Infotrieve] |
5. | Zager, R. A., Iwata, M., Burkhart, K. M., and Schimpf, B. A. (1994) Kidney Int. 45, 1760-1768[Medline] [Order article via Infotrieve] |
6. | Zager, R. A. (1995) Kidney Int. 47, 628-637[Medline] [Order article via Infotrieve] |
7. |
Lee, H. T.,
and Emala, C. W.
(2000)
Am. J. Physiol. Renal Physiol.
278,
F380-F387 |
8. | Jefayri, M. K., Grace, P. A., and Mathie, R. T. (2000) Br. J. Urol. Int. 85, 1007-1013 |
9. | Islam, C. F., Mathie, R. T., Dinneen, M. D., Kiely, E. A., Peters, A. M., and Grace, P. A. (1997) Br. J. Urol. 79, 842-847[Medline] [Order article via Infotrieve] |
10. | Pombo, C. M., Bonventre, J. V., Avruch, J., Woodgett, J. R., Kyriakis, J. M., and Force, T. (1994) J. Biol. Chem. 269, 26545-26551 |
11. | Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., and Greenberg, M. E. (1995) Science 270, 1326-1331[Abstract] |
12. | Behrens, A., Sibilia, M., and Wagner, E. F. (1999) Nat. Genet. 21, 326-329[CrossRef][Medline] [Order article via Infotrieve] |
13. | Safirstein, R., DiMari, J., Megyesi, J., and Price, P. (1998) Semin. Nephrol. 18, 519-522[Medline] [Order article via Infotrieve] |
14. |
Abbate, M.,
Brown, D.,
and Bonventre, J. V.
(1999)
Am. J. Physiol.
277,
F454-F463 |
15. | Finn, W. F., Fernandez-Repollet, E., Goldfarb, D., Iaina, A., and Eliahou, H. E. (1984) J. Lab. Clin. Med. 103, 193-203[Medline] [Order article via Infotrieve] |
16. | Zager, R. A., Jurkowitz, M. S., and Merola, A. J. (1985) Am. J. Physiol. 249, F148-F159[Medline] [Order article via Infotrieve] |
17. |
Holtmann, H.,
Winzen, R.,
Holland, P.,
Eickemeier, S.,
Hoffmann, E.,
Wallach, D.,
Malinin, N. L.,
Cooper, J. A.,
Resch, K.,
and Kracht, M.
(1999)
Mol. Cell. Biol.
19,
6742-6753 |
18. | Cochrane, J., Williams, B. T., Banerjee, A., Harken, A. H., Burke, T. J., Cairns, C. B., and Shapiro, J. I. (1999) Renal Failure 21, 135-145[Medline] [Order article via Infotrieve] |
19. | Schwarz, E. R., Whyte, W. S., and Kloner, R. A. (1997) Curr. Opin. Cardiol. 12, 475-481[Medline] [Order article via Infotrieve] |
20. |
Meng, X.,
Banerjee, A.,
Ao, L.,
Meldrum, D. R.,
Cain, B. S.,
Shames, B. D.,
and Harken, A. H.
(1999)
Ann. N. Y. Acad. Sci.
874,
69-82 |
21. | Huot, J., Houle, F., Spitz, D. R., and Landry, J. (1996) Cancer Res. 56, 273-279[Abstract] |
22. |
Molitoris, B. A.,
Dahl, R.,
and Geerdes, A.
(1992)
Am. J. Physiol.
263,
F488-F495 |
23. |
Brown, D.,
Lee, R.,
and Bonventre, J. V.
(1997)
Am. J. Physiol.
273,
F1003-F1012 |
24. |
Ehrnsperger, M.,
Graber, S.,
Gaestel, M.,
and Buchner, J.
(1997)
EMBO J.
16,
221-229 |
25. | Mehlen, P., Kretz-Remy, C., Preville, X., and Arrigo, A. P. (1996) EMBO J. 15, 2695-2706[Abstract] |
26. |
Mehlen, P.,
Schulze-Osthoff, K.,
and Arrigo, A. P.
(1996)
J. Biol. Chem.
271,
16510-16514 |
27. |
Smoyer, W. E.,
Ransom, R.,
Harris, R. C.,
Welsh, M. J.,
Lutsch, G.,
and Benndorf, R.
(2000)
J. Am. Soc. Nephrol.
11,
211-221 |
28. | Yamashita, N., Hoshida, S., Taniguchi, N., Kuzuya, T., and Hori, M. (1998) J. Mol. Cell. Cardiol. 30, 1181-1189[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Bogoyevitch, M.,
Gillespie-Brown, J.,
Ketterman, A.,
Fuller, S.,
Ben-Levy, R.,
Ashworth, A.,
Marshall, C.,
and Sugden, P.
(1996)
Circ. Res.
79,
162-173 |
30. |
Nakano, A.,
Baines, C. P.,
Kim, S. O.,
Pelech, S. L.,
Downey, J. M.,
Cohen, M. V.,
and Critz, S. D.
(2000)
Circ. Res.
86,
144-151 |
31. |
Saurin, A. T.,
Martin, J. L.,
Heads, R. J.,
Foley, C.,
Mockridge, J. W.,
Wright, M. J.,
Wang, Y.,
and Marber, M. S.
(2000)
FASEB J.
14,
2237-2246 |
32. | Davis, R. J. (2000) Cell 103, 239-252[Medline] [Order article via Infotrieve] |
33. | DiMari, J. F., Davis, R., and Safirstein, R. L. (1999) Am. J. Physiol. 277, F195-F203[Medline] [Order article via Infotrieve] |
34. |
Tournier, C.,
Hess, P.,
Yang, D. D.,
Xu, J.,
Turner, T. K.,
Nimnual, A.,
Bar-Sagi, D.,
Jones, S. N.,
Flavell, R. A.,
and Davis, R. J.
(2000)
Science
288,
870-874 |
35. | He, H., Li, H. L., Lin, A., and Gottlieb, R. A. (1999) Cell Death Differ. 6, 987-991[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Read, M. A.,
Whitley, M. Z.,
Gupta, S.,
Pierce, J. W.,
Best, J.,
Davis, R. J.,
and Collins, T.
(1997)
J. Biol. Chem.
272,
2753-2761 |
37. | Swantek, J. L., Cobb, M. H., and Geppert, T. D. (1997) Mol. Cell. Biol. 17, 6274-6282[Abstract] |
38. |
Srivastava, S.,
Weitzmann, M. N.,
Cenci, S.,
Ross, F. P.,
Adler, S.,
and Pacifici, R.
(1999)
J. Clin. Invest.
104,
503-513 |