![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 291, Issue 2, 911-919, November 1999
-phenylnitrone)-Mediated
Suprainduction of Heme Oxygenase-1 in Kidney Ischemia/Reperfusion
Model: Role of the Oxygenase in Protection against Oxidative
Injury1
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
In mammals the rate-limiting step in heme catabolism is the heme
oxygenase (HO) system. Two isozymes, HO-1 and HO-2, oxidatively cleave
the substrate to form biliverdin, and the potential cellular messenger,
CO; the chelated iron is released as the result of the tetrapyrrole
ring opening. Biliverdin is subsequently reduced to bilirubin, an
antioxidant, by biliverdin reductase. The aim of the present study was
to investigate the involvement of HO-1, a heat shock/stress protein, in
protection offered by the spin trap agent,
N-tert-butyl-
-phenyl-nitrone (PBN), against kidney ischemia/reperfusion injury. For this, HO-1 expression and assessment of the parameters associated with tissue-oxidative injury were compared
in the presence or absence of PBN pretreatment of rats (100 mg/kg i.p.,
30 min) before the onset of 30-min ischemia. Twenty-four hours after
reperfusion, Northern blot analysis showed an unprecedented ~37-fold
increase in 1.8-kb HO-1 mRNA in PBN pretreated rat kidney; HO-2 mRNA
levels did not increase. At 48 h, the levels of HO-1 mRNA remained
nearly 14-fold higher than the control value. In the absence of PBN,
the levels measured approximately 5- and 2-fold higher than control
values at the 24- and 48-h intervals, respectively. PBN pretreatment
also resulted in a most impressive increase in the levels of HO-1
protein as judged by Western blot analysis and measurement of enzyme
activity at the 24-h time point. As detected by immunohistochemical
analysis, PBN pretreatment caused an increase in HO-1 and biliverdin
reductase-immunoreactive proteins in the cortex and in the outer stripe
of the outer medulla. In the absence of PBN pretreatment, there was an
intense immunostaining for HO-1 in the medullary rays, which
corresponded with iron and lipid peroxidation staining of the region;
these observations were not made with PBN-pretreated kidneys.
Collectively, the findings are consistent with the likelihood that
suprainduction of HO-1 gene expression protects the kidney from free
radical-mediated injury by increasing the capacity to produce the
potent cellular antioxidant bilirubin. We also suggest spin
trap-mediated protection against ischemia/reperfusion injury is likely
due to a sustained elevation of HO-1 gene expression by formation of
stable radicals.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
In
mammals the heme molecule (hemin, Fe-protoporphyrin-IX) is cleaved at
the -meso carbon bridge by the microsomal heme oxygenase (HO) system to form the open tetrapyrrole ring, biliverdin. The carbon bridge atom is converted to CO and iron is released in the
course of the pyrrole ring cleavage. Two catalytically active isozymes
of the oxygenase, HO-1 and HO-2, have been characterized (Maines et
al., 1986
), and are derived from different genes (Cruse and Maines,
1988). The third form, HO-3, which in its predicted primary structure
resembles HO-2, is marginally active (McCoubrey et al., 1997).
HO-1 and HO-2 have been fully characterized. They differ in their
molecular and biochemical characteristics, as well as how they are
regulated (reviewed by Maines, 1992, 1997). HO-1, also known as heat
shock/stress protein (HSP)32 (Shibahara et al., 1987; Keyse and
Tyrrell, 1989; Ewing and Maines, 1991), is induced by a host of stimuli
that have in common the ability to produce oxidative stress (Smith et
al., 1995; Guyton et al., 1996; Goodman et al., 1997). In contrast,
HO-2 is a constitutive form and to date only the adrenal
glucocorticoids have been identified as the inducers of its gene
expression (Weber et al., 1994; Raju et al., 1997).
Under normal conditions HO-1 is present at low levels in all organs but
the spleen. Its expression, however, is rapidly and robustly
accelerated not only in response to exogenous agents, but also in
response to pathophysiological conditions, such as those of renal
ischemia/reperfusion and cellular transformation (Maines et al., 1993;
Raju and Maines, 1996; Maines and Abrahamsson, 1996; Goodman et al.,
1997). Unlike larger HSPs, for example the HSP70 and HSP90 families of
proteins, that have chaperonin function in the cell, HSP32 proteins do
not appear to function in this capacity. Induction of HSPs is
customarily considered as a component of defense mechanisms of the cell
against injurious insults.
There has been much debate about the physiological significance of HO-1
induction. Good arguments have been made both for beneficial as well as
deleterious effects of HO-1 induction (e.g., Poss and Tonegawa, 1997;
Dwyer et al., 1998). The arguments are supported by the fact that
induction of HO-1 accelerates catalysis of heme and generates a most
potent pro-oxidant, iron, and an antioxidant precursor, biliverdin. The
reduction product of biliverdin, bilirubin (Huang et al., 1989), is an
effective antioxidant and scavenger of free radicals (Stocker et al.,
1987). Oxygen-derived free radicals are commonly suspected to underlie
injury caused by reperfusion of an ischemic organ (Baker et al., 1985).
H2O2, which is generated by
univalent reduction of molecular oxygen to superoxide
(O2·
), can interact with
Fe2+ to form the hydroxy radical (·OH) (Floyd,
1997). This oxygen species is the most reactive oxygen radical and can
interact with cellular proteins, lipids, and chromatin material.
Peroxidation of membrane lipids by hydroxy radicals, commonly defined
as lipid peroxidation, has injurious effects on the cell by alternating membrane structure and function (Beckman and Koppenol, 1996).
A number of organic spin trap agents have been designed specifically to
form stable adducts with free radicals to reduce the vulnerability of
organs to ischemia/reperfusion injury and protect renal functions when
subjected to this injury (Pedraza-Chaverri et al., 1992; Sen and
Phillis, 1993; Floyd, 1997). Spin trap agents are usually either
nitroso compounds or nitrones (Evans, 1979). A nitrone, which has been
most frequently used in experimental settings to trap radicals produced
in the course of reperfusion injury, is
N-tert-butyl--phenylnitrone (PBN). PBN
interacts not only with oxygen radicals but also with carbon- or
nitrogen-centered radicals such as nitric oxide (Phillis, 1997).
Production of nitric oxide is increased in ischemic/reperfusion injury
largely due to inducible nitric oxide synthase (Hensley et al., 1997).
Nitric oxide interacts with superoxide to produce potent redox
congeners, such as peroxynitrites (Lipton et al., 1994; Beckman and
Koppenol, 1996). Targets for nitric oxide congeners include all kinds
of cellular constituents, acting sites, and prosthetic moieties
of enzymes and nuclear material (Stuehr, 1997).
The overall goal of this investigation was to further examine the role of HO-1 in cellular defense mechanisms, and specifically to investigate whether induction of HO-1 gene expression has a bearing on the beneficial effects of spin trap agents in ameliorating ischemic/reperfusion injury. The findings of this investigation has identified the spin trap agent, PBN, when used under oxidative stress conditions, as the most potent modulator of HO-1 expression described to date, and strongly suggest the beneficial effects of HO-1 induction in defense against free radical-mediated tissue injury.
| |
Experimental Procedures |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Materials.
Oligo(dT) cellulose, DNase I, salmon testes DNA,
PBN, and cofactors were obtained from Sigma Chemical Co. (St. Louis,
MO). Restriction enzymes, Rediprime random-primer DNA labeling system, and Taq polymerase were purchased from USB Corporation
(Cleveland, OH). Horseradish peroxidase conjugated goat anti-rabbit IgG
was purchased from Organon Teknika Corporation (Westchester, PA). Nytran membranes and nitrocellulose membranes (0.2-µm pore size) were
obtained from Schleicher & Schuell (Keene, NH). All chemicals used were
of the highest purity commercially available. [-32P]
dCTP (3000 Ci/mmol) was purchased from DuPont-NEN (Boston, MA). Male
Sprague-Dawley rats (290-370 g) were purchased from Harlan Industries
(Madison, WI).
Induction of Renal Ischemia and Tissue Preparation.
All
animal treatments were performed in strict accordance with the National
Institutes of Health Guide for the Care and Use of Laboratory
Animals as approved by the University Committee for Animal
Resources. Rats were subjected to pentobarbital anesthesia (40 mg/kg
i.p.). Experimental group, 30 min before induction of ischemia was
injected with PBN (100 mg/kg i.p.) in 100 µl of dimethyl sulfoxide
(DMSO), control rats were injected (i.p.) with 100 µl of DMSO.
Rats were also subjected to renal ischemia or sham operation, without
PBN pretreatment. All surgeries were performed under normothermic conditions with rats kept on the homeothermic blanket with rectal temperature monitoring throughout surgery and the first 2 h after induction of reperfusion. Renal ischemia was induced by means of
occlusion of both renal arteries for 30 min using Yasargil vascular
clips (Aesculap, San Francisco, CA) with a closing force of 0.95 N. Reperfusion was confirmed in every case. After removal of the clips,
immediate reperfusion was assessed by visual examination of the kidney
surface, which recovered the usual color within 15 to 20 s, as
assessed with the help of an MZ-8 Leica stereomicroscope. Similar
criteria for establishment of reperfusion has been reported previously
(Kiyama et al., 1995; Terzi et al., 1997). At time points
indicated in appropriate figure legends, rats were sacrificed and
kidneys were removed and processed for microsomal isolation or frozen
at 
80°C for RNA isolation. The number of animals used
for biochemical experiments was six to seven rats per group and, for
histochemical analysis, four rats per group were used except for
sham-operated, which are based on three animals per group.
Probes.
An HO-1 cDNA corresponding to HO-1 nucleotides +71
to +833 reported by Shibahara et al. (1985) was generated using
polymerase chain reaction and cloned into PBS(+) vector as described
before (Sun et al., 1990). A full-length (1300 base pair) HO-2
cDNA isolated from a rat testis DNA library (Rotenberg and Maines,
1990) was used as an HO-2 hybridization probe. All probes, including a
full-length human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA
were labeled with [32P]dCTP by the random primers DNA
labeling system (USB Corp., Cleveland, OH) according to manufacturer's
instructions and further purified by spin column chromatography.
Microsomal Isolation and Measurement of HO Activity.
The
kidney was homogenized in 5 volumes of buffer containing 0.25 M sucrose
and 0.01 M Tris-HCl (pH 7.4). The microsomes were prepared and used for
HO activity measurement. The activity was measured in the presence of
purified biliverdin reductase (Huang et al., 1989) as described before
(Raju and Maines, 1996). The enzyme activity was expressed as nmoles of
bilirubin produced per hour per milligram protein. Proteins were
determined using Pierce BCA reagent (Pierce, Rockford, IL).
Isolation and Analysis of RNA.
Total RNA and
poly(A)+ RNA was isolated from rat kidney by oligo
(dT)-cellulose chromatography and the formaldehyde denatured RNA was
fractionated on 1.2% (w/v) agarose gel and subsequently transferred to
Nytran membrane. Prehybridization and hybridization of the membranes
with the appropriate 32P-labeled cDNA were performed
essentially as described previously (Raju and Maines, 1996). The
membranes were exposed at 70°C to Kodak X-OMAT film with
intensifying screens, and autoradiographs were quantified using BioRad
model GS-700 imaging densitometer and Molecular Analyst v.1.5 software.
Antibody Production and Western Blot Analysis.
Rat liver
biliverdin reductase and HO-1 were purified as described previously and
used for preparation of antibodies in New Zealand White rabbit as
described earlier (Huang et al., 1989; Maines et al., 1986). Protein
samples were fractionated by SDS-polyacrylamide gel electrophoresis and
transferred to a nitrocellulose membrane using an LKB2005 Transphor
Apparatus. Antigen-antibody complexes were immunochemically visualized
using horseradish peroxidase-conjugated goat anti-rabbit antibody.
Immunocytochemical Protocols. Twenty-four hours after induction of reperfusion, rats were given an overdose of pentobarbital (100 mg/kg i.p.) and perfused transcardially with heparinized saline, followed by chilled solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After overnight postfixation in 4% paraformaldehyde at 4-6°C, kidney was transferred into the cryoprotection solution of 30% ethylene glycol and 20% sucrose in 0.1 M phosphate buffer (pH 7.4) at 4-6°C for 2 to 3 days. Both kidneys were then frozen on crushed dry ice and cut serially in 25-µm-thick sections using a sliding microtome (Microm 400; Carl Zeiss, Thornwood, NY). Staining of kidney from control and treated rats was carried out under identical conditions using the same reagents and solutions. For all immunocytochemical and histochemical protocols, longitudinally cut specimens were used due to a larger surface area available for analysis.
HO-1 was detected using 3B8C8 monoclonal antibody developed in collaboration with StressGen (Vancouver, Canada). After 60 min of blocking in a solution of 5% normal horse serum in Tris-buffered saline (TBS) followed by a wash in 0.25% Triton X-100 solution in TBS, all specimens (in Costar net wells) were transferred into primary HO-1 antibody that had been diluted 1:1000 in carrier solution (0.1 M TBS containing 0.5% horse serum and 0.25% Triton X-100), and incubated for 48 h at 4-6°C. The specimens were then rinsed 5× 10 min with 0.1 M TBS containing 0.25% Triton and placed into biotinylated secondary antibody and avidin-biotin reagent according to manufacturer's recommendations for peroxidase detection (Vectastain Elite mouse, IgG kit) (Vector Labs, Burlingame, CA). After consecutive 10-min rinses in TBS and Tris-HCl, the sections were placed for 4 to 5 min into a filtered solution of 0.04% 3', 3'-diaminobenzidine (DAB), and 0.06% H2O2 in 0.1 M Tris buffer. An MZ-8 Leica stereomicroscope was used for detection of the reaction product during development. Sections were then rinsed in phosphate buffer, mounted on Superfrost-coated slides, dehydrated serially in 95 and 100% alcohol, incubated in histological grade xylene, and coverslipped with Permount (Fisher Scientific). Biliverdin reductase was detected using the above indicated polyclonal antibody. After blocking in a solution of 5% normal goat serum, specimens were transferred into primary antibiliverdin reductase antibody (1:5000 dilution) and incubated for 24 h at 4-6°C. The specimens were then placed into biotinylated anti-rabbit secondary antibody reagent and incubated for 90 min in the avidin-biotin reagent prepared in 0.1 M PBS (ABC Solution; Vector Labs, Burlingame, CA). Sections were rinsed and stained as above. Rabbit anti-ferritin antibody was purchased from Zymed (San Francisco, CA) and used at 1:250 dilution. Peroxidase detection (Vectastain Elite, rabbit IgG kit), with DAB as the chromogen was utilized to demonstrate expression of ferritin at the protein level. Vimentin, an intermediate early gene, was detected using a monoclonal IgM anti-vimentin antibody (Chemicon Int., Temecula, CA) in dilutions of 1:200. Expression of early genes (e.g., vimentin), which function as transcription regulators, is increased in response to tissue injury. In the case of vimentin, its expression is increased in the regeneration phase of ischemic renal injury (Witzgall et al., 1993; Terzi et
al., 1997). PBN-treated, vehicle-treated, and control kidneys were
immunostained with vimentin using peroxidase detection (Vectostain
Elite, mouse IgM kit) and 3,3'-DAB as a chromogen. To delineate nuclear
morphology of the epithelial cells of the tubules and glomeruli, select
specimens were counterstained with Nuclear Fast Red (Vector Labs).
Histochemical Detection of Iron and Lipid Peroxidation.
Iron
was detected by Perl's reaction followed by DAB enhancement (Hill and
Switzer, 1984). Perl's reaction is based on the formation of ferric
ferrocyanide (Prussian Blue) when a ferric ion, released from
iron-containing compounds by HCl, reacts with potassium ferrocyanide.
The ferric ferrocyanide then catalyzes the oxidation of DAB with the
formation of a brown precipitate (Smith et al., 1995). Under normal
conditions, iron is found primarily within the proximal tubules of the kidney.
based on detection of free aldehyde and
carbonyl functions formed after peroxidative breakdown of unsaturated
fatty acids. Free floating kidney specimens were incubated for 45 min
in the dark at room temperature in Schiff's reagent (filtered
pararosaniline base:thionyl chloride). The sections were rinsed in
three changes of sulfide water (1:1, 10%
K2S2O5/1 N HCl) before being mounted, dehydrated in alcohol, cleared in xylene,
and coverslipped using Permount.
Statistical Analysis.
Data is expressed as the mean ± S.D. of up to six determinations where one rat was used for each
determination. The data were analyzed using the unpaired Student's
t test and ANOVA. A value of p 
.05 was considered to denote statistical significance.
| |
Results |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Comparative Levels of HO-1 mRNA in Ischemic/Reperfused Kidney in
the Presence or Absence of PBN Pretreatment.
The effect of PBN
pretreatment on HO isozymes' transcript levels in kidney subjected to
30-min ischemia followed by 24-h reperfusion was examined by Northern
blot analysis. The levels of mRNA were normalized to that of GAPDH mRNA
for calculation of the relative levels of transcripts. As shown in Fig.
1, top, at this time point the levels of
~1.8-kb transcript for HO-1 were increased by approximately 5-fold in
ischemic/reperfused kidney when compared with those of sham-operated
animals (lanes 2 and 3 versus lane 1). Pretreatment of rats with PBN 30 min before ischemia/reperfusion markedly augmented induction of HO-1
mRNA, when compared with sham-operated rats receiving PBN alone (lane 5 versus lane 4). At this time, an astonishing increase of nearly 37-fold
in HO-1 mRNA levels was detected, an increase we have not experienced
before. It is notable that administration of PBN alone to sham-operated
animals did not significantly increase the HO-1 mRNA levels at 24 h (lane 4 versus lane 1). Unlike HO-1, the levels of HO-2 homologous
~1.3- and ~1.9-kb transcripts were not noticeably increased by PBN
pretreatments (middle).
|
|
Comparative Induction of HO-1 Protein and Activity in
Ischemic/Reperfused Kidney in the Presence or Absence of PBN
Pretreatment.
The induction of HO-1 transcript in the kidney was
reflected in an increased heme oxidation activity and in HO-1
immunoreactive protein in kidney (Fig. 3,
a and b). The activity data are shown in (a). Consistent with our
previous observation (Maines et al., 1993), a 3-fold increase in the
microsomal HO activity was observed 24 h after reperfusion.
Administration of PBN 30 min before ischemia/reperfusion, however,
resulted in the levels of HO activity, which exceeded that of the
control by more than 6-fold. PBN pretreatment alone caused a modest,
but not statistically significant, increase in HO activity when
compared with controls. The increase in levels of tissue heme degrading
capacity in PBN-pretreated rats subjected to ischemia/reperfusion was
likely due to elevation of the HO-1 isozyme in the kidney, as indicated
by Western blot analysis for HO-1 protein. As shown in Fig. 3b, the
intensity of immunoreactive HO-1 in PBN-pretreated ischemic/reperfused
kidney was most impressive (lanes 4 and 5). In fact, in extensive
experience with the system, we have never before encountered such a
tissue response. It should be noted that the amount of loaded protein
was only 30% of the amount of protein loaded in lanes that contained
samples from ischemic/reperfused kidney without PBN pretreatment (lanes
2 and 3) or the sham-operated rat kidney (lane 1). As noted, the
magnitude of increase in HO-1 gene expression exceeded the increase in
HO activity. The basis for this difference may be factors such as the
relative sensitivity of assays, inactivation of the enzyme in the
course of microsomal preparation, and/or decreased HO-2-dependent heme
oxidation activity.
|
Rats Treated with PBN Do Not Exhibit Increased Staining for Iron
and Lipid Peroxidation.
Figure 4
shows the overall pattern of iron (a and b) and lipid peroxidation (c
and d) of kidney subjected to ischemia/reperfusion in the presence of
PBN pretreatment (a and c) or its absence (b and d). Labeling for iron
in tissue of PBN pretreated rats, subjected to 30 min of ischemia
followed by 24 h of reperfusion, was confined to the cortex.
Staining for iron in untreated rats was not restricted to this region,
but also was prominently present in the medullary rays.
|
). Comparative expression of vimentin
in the presence and absence of PBN pretreatment of rats is shown in
Fig. 4, e and f. As noted, only low level vimentin staining together
with intact tissue morphology is observed in the presence of PBN
pretreatment in ischemic/reperfused kidney, and increase in vimentin
staining is observed in the absence of PBN pretreatment. Also noted is
disturbed morphology of the tissue under these conditions. Additional
support for the morphological differences is provided in Fig. 5.
Effect of PBN Pretreatment on Tissue Levels of Ferritin, HO-1, and
Iron in Ischemic/Reperfused Organ.
As shown in Fig.
5, there was a marked difference in
immunohistochemical staining for ferritin (a and b), HO-1 (c and d), and iron (e and f) in the renal cortex of rats subjected to
ischemia/reperfusion in the presence or absence of PBN pretreatment. As
shown, the pattern of staining for these three constituents was similar
in both groups and was restricted to renal tubules and not prominent within the glomeruli.
|
PBN Pretreatment Increases Expression of Biliverdin Reductase after
Ischemic/Reperfused Kidney.
The cellular basis by which HO-1
induction enhances tissue defense against ischemia/reperfusion, in
context of the ability to produce bilirubin, was examined by comparing
its pattern of immunostaining in the outer stripe of the outer medulla
(Fig. 6, a and b) to that of biliverdin
reductase (Fig. 6, c and d). The outer stripe of the outer medulla is
an area selectively vulnerable to ischemic-reperfusion injury (Kiyama
et al., 1995). A similar pattern of immunostaining was detected for the
oxygenase and the reductase. Treatment of rats with PBN alone did not
affect BVR and HO-1 levels or staining of tissues for iron.
Specifically, in PBN-pretreated rats, both proteins were prominently
expressed in this region of the kidney (a and c). Immunoreactive
labeling for HO-1 and the reductase in the absence of PBN pretreatment was less intense in this region (b and d). The enlargement of the lumen
of tubules in the absence of PBN pretreatment is notable and is clearly
shown in (b) and (d) (versus a and c). In the absence of PBN
pretreatment, HO-1 immunoreactivity also was prominent in the tubular
constituents of the renal papilla (b). The pattern of reductase
immunostaining in renal papilla in the presence or absence of PBN
pretreatment is shown in (e) and (f). Prominent intracellular reductase
immunoreactivity was detected in the medullary apex region of
PBN-pretreated rat kidney (e). Also, noted is the intact tubular
morphology in the presence of PBN pretreatment. In the absence of the
pretreatment, immunoreactive aggregates were detected in the kidney
tubules, which exhibited distorted morphology. These aggregates could
represent damaged epithelial cells and/or their content, which was
likely exfoliated as the result of ischemia/reperfusion.
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Presently, we have identified the spin trap agent, PBN, in our experience, as the most effective stimulant for induction of HO-1 gene expression under ischemia/reperfusion conditions. And we have shown that suprainduction of HO-1, as evaluated by tissue lipid peroxidation and morphology, is beneficial to the kidney subjected to ischemia/reperfusion insult. These parameters are commonly associated with free radical-mediated tissue injury. Specifically: 1) at the tissue level, we have found that peroxidation of kidney tissue lipids is essentially absent in the presence of suprainduction of HO-1; 2) at histological levels, the tissue architecture was preserved in cortical and the outer strip of the outer medulla, a region that is most vulnerable to oxidative damage; and 3) these areas were the sites of high level expression of HO-1 and biliverdin reductase.
HO products all are biologically active molecules. The products that
are generated through catalytic transformation of the tetrapyrrole ring
of heme, i.e., biliverdin and CO, may be considered positive
contributors to cellular defense mechanisms and functions, whereas iron
is generally considered in a negative context. Bilirubin, the product
of biliverdin, is an effective antioxidant in vivo and in vitro
(Stocker et al., 1987). The endogenously produced CO is likely a signal
molecule (Marks et al., 1991; Maines, 1997; Snyder et al., 1998) and an
activator of guanylyl cyclase for the generation of cGMP in the
vascular system (Raju and Maines, 1996). As such, the gaseous molecule
has been suggested to act as a modulator of sinusoidal tone and
vascular perfusion (Suematsu et al., 1994) and arterial pressure
(Motterlini et al., 1998). On the basis of the present findings it is
difficult to differentiate the relative contribution of CO-mediated
effects and the antioxidant action of bilirubin to the apparent
enhanced tissue defense against ischemic/reperfusion injury.
Nonetheless, based on the findings with near undetectable levels of
tissue lipid peroxidation, and increased expression of biliverdin
reductase, it is reasonable to suggest the involvement of the
antioxidant component of HO activity in protection against
ischemia/reperfusion injury.
Because PBN is a scavenger of free radicals, the mechanism by which PBN
pretreatment causes suprainduction of HO-1 at a glance may appear
contradictory to the findings that HO-1 gene expression is induced by
free radicals. However, considering that the mechanism of PBN action as
a spin trap is to scavenge free radicals to give rise to relatively
stable free radicals (Evans, 1979; Phillis, 1997), two possible
mechanisms may be considered to explain the suprainduction phenomenon.
The first possibility would consider activity of stable radicals formed
through interaction of PBN with oxygen radicals, which, in turn, are
known to be formed in the course of the reperfusion component of
ischemia, in HO-1 gene regulation. According to this possibility,
stable free radicals with a prolonged half-life would be more effective
in modulating gene expression by the virtue of their continued presence
in the cell. HO-1 is among various genes whose expression is induced by
free radicals. The second possible mechanism for the suprainduction could involve the removal of cytotoxic free radical and reducing their
bioavailability for direct interaction with cellular components, including gene transcription machinery. Reactive oxygen species, including ·OH and O2· formed in
ischemic/reperfused organs, as well known, can cause nucleotide strand
breaks. Therefore, by trapping the powerful oxidizing radical species
the rate of decay in HO-1 mRNA could decrease. This possibility is
consistent with the sustained remarkable elevation of HO-1 mRNA levels
in PBN-pretreated rats. Normally, HO-1 mRNA is rather unstable in the
cell and its levels return to prestressed levels within a few hours
after induction (Ewing and Maines, 1991). In addition, as noted
earlier, production of nitric oxide is increased in ischemic/reperfused
tissue and oxygen radicals on interaction with nitric oxide form highly
reactive and toxic derivatives, e.g., peroxynitrite. Inactivation of
such derivatives, which if unchecked can cause inactivation/destruction of the cellular constituents, could likely protect HO-1 transcript and
protein. The possibility also exists that nitric oxide itself is
directly scavenged subsequent to its formation by the spin trap
agent. Clearly, the spin trap by itself in the absence of free
radicals is not an effective modulator of HO-1 gene expression, rather,
its interaction with free radicals, which are formed after resumption
of reperfusion, modulates HO-1 gene expression.
To date, although many known consensus sequences of known transcription
elements have been identified in the 5' region of the HO-1 gene
(Shibahara et al., 1987; Alam et al., 1989; Lavrovsky et al., 1993),
the mechanism by which free radicals transcriptionally regulate the
HO-1 gene is not known, although involvement of a number of
transcription factors such as AP-1 and NF-kB have been implicated in
this process. It remains to be established whether stable free
radicals mediate their effect on HO-1 gene expression through the same
mechanism as the transient radicals. Also, at this time we are not
clear as to the cellular basis for the targeted increase in HO-1
expression in the cortex and the outer strip of outer medulla by PBN
pretreatment and stable free radicals.
As judged by the overall tissue pattern of Schiff's reagent staining
for lipid peroxidation in the absence of PBN pretreatment of
ischemic/reperfused organ, which nearly mirrors that of iron staining,
it is reasonable to link the two parameters as causally related. Iron
is released from the heme molecule through the activity of HO. Iron,
however, is sequestered in ferritin, which is not effective in
catalyzing lipid peroxidation (Ponka et al., 1998). Because increased
HO-1 activity has been shown to mediate induction of ferritin
expression (Eisenstein et al., 1991), the presently observed similarity
of the pattern of staining for ferritin, HO-1, and iron suggests that
iron released by HO is sequestered in ferritin. The possibility also
must be considered that enhancement of tissue reperfusion caused by
increased CO production could facilitate removal of iron from the
tissue. As noted above, CO generated by HO activity has been implicated
in relaxation of vascular tone and tissue reperfusion. The absence of
Schiff's staining in the renal medulla of PBN-pretreated rats suggests
a decreased availability of free iron in this region. Furthermore, the
finding that in the presence of PBN pretreatment there is an increase
in HO-1 and biliverdin reductase staining in the cortex and the outer stripe of the outer medulla of the medullary rays would be consistent with the likelihood that degradation of heme would have occurred in
this region, leading to production of the antioxidant bilirubin. Involvement of additional factors relating to biological activity of CO
as it relates to cGMP-mediated cellular functions must also be
considered in the apparent production that is rendered by an increase
in HO-1 activity.
In closing, the data shown here provide good evidence for role of the
HO system in the cellular defense mechanism against free
radical-mediated tissue damage and are consistent with reports on the
cytoprotection offered to organs by heat shock and induction of HO-1
before organ transplantation (Emami et al., 1991; Woo et al., 1998).
The findings provide a biochemical basis for the protection offered by
spin trap agents against ischemic/reperfusion injury and identify these
agents as the most potent modulators of HO-1 expression when combined
with oxidative stress. The findings also allow stipulation on the
utility of HO modulation in clinical settings aimed at new approaches
to treatment of ischemia/reperfusion injury and allograft rejection.
| |
Acknowledgments |
|---|
We thank Suzanne Bono for preparation of the manuscript and Deana Amico for editorial assistance.
| |
Footnotes |
|---|
Accepted for publication July 26, 1999.
Received for publication April 29, 1999.
1 This study was supported by National Institute for Environmental Health Sciences Grant ES04066.
2 Current address: Department of Medicine, Cardiology Unit, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642-0001.
Send reprint requests to: Mahin D. Maines, Ph.D., Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642-0001. E-mail: MAHIN_MAINES{at}URMC.ROCHESTER.EDU
| |
Abbreviations |
|---|
HO, heme oxygenase;
HSP, heat shock/stress
protein;
PBN, N-tert-butyl--phenyl
nitrone;
DMSO, dimethyl sulfoxide;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
TBS, Tris-buffered saline;
DAB, diaminobenzidine.
| |
References |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
-phenyl-N-tert.-butylnitrone (PBN).
Renal Failure
14:
467-471[Medline].This article has been cited by other articles:
|
|
 |
|
  M. G. Salom, S. Nieto Ceron, F. Rodriguez, B. Lopez, I. Hernandez, J. Gil Martinez, A. Martinez Losa, and F. J. Fenoy Heme oxygenase-1 induction improves ischemic renal failure: role of nitric oxide and peroxynitrite Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3542 - H3549. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kirkby, C. Baylis, A. Agarwal, B. Croker, L. Archer, and C. Adin Intravenous bilirubin provides incomplete protection against renal ischemia-reperfusion injury in vivo Am J Physiol Renal Physiol, February 1, 2007; 292(2): F888 - F894. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mishra, T. Fujita, V. N. Lama, D. Nam, H. Liao, M. Okada, K. Minamoto, Y. Yoshikawa, H. Harada, and D. J. Pinsky Carbon monoxide rescues ischemic lungs by interrupting MAPK-driven expression of early growth response 1 gene and its downstream target genes PNAS, March 28, 2006; 103(13): 5191 - 5196. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Kirkby and C. A. Adin Products of heme oxygenase and their potential therapeutic applications Am J Physiol Renal Physiol, March 1, 2006; 290(3): F563 - F571. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Qiao, X. Chen, D. Wu, R. Ding, J. Wang, Q. Hong, S. Shi, J. Li, Y. Xie, Y. Lu, et al. Mitochondrial Pathway Is Responsible for Aging-Related Increase of Tubular Cell Apoptosis in Renal Ischemia/Reperfusion Injury J. Gerontol. A Biol. Sci. Med. Sci., June 1, 2005; 60(7): 830 - 839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Adin, B. P. Croker, and A. Agarwal Protective effects of exogenous bilirubin on ischemia-reperfusion injury in the isolated, perfused rat kidney Am J Physiol Renal Physiol, April 1, 2005; 288(4): F778 - F784. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Szabo, E. Gallyas, I. Bak, A. Rakotovao, F. Boucher, J. de Leiris, N. Nagy, E. Varga, and A. Tosaki Heme Oxygenase-1-Related Carbon Monoxide and Flavonoids in Ischemic/Reperfused Rat Retina Invest. Ophthalmol. Vis. Sci., October 1, 2004; 45(10): 3727 - 3732. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. D. Mayer, X. Wang, and M. D. Maines Nitric Oxide Inhibitor N{omega}-Nitro-L-arginine Methyl Ester Potentiates Induction of Heme Oxygenase-1 in Kidney Ischemia/Reperfusion Model: A Novel Mechanism for Regulation of the Oxygenase J. Pharmacol. Exp. Ther., July 1, 2003; 306(1): 43 - 50. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. D. Blydt-Hansen, M. Katori, C. Lassman, B. Ke, A. J. Coito, S. Iyer, R. Buelow, R. Ettenger, R. W. Busuttil, and J. W. Kupiec-Weglinski Gene Transfer-Induced Local Heme Oxygenase-1 Overexpression Protects Rat Kidney Transplants From Ischemia/Reperfusion Injury J. Am. Soc. Nephrol., March 1, 2003; 14(3): 745 - 754. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Vulapalli, Z. Chen, B. H. L. Chua, T. Wang, and C.-S. Liang Cardioselective overexpression of HO-1 prevents I/R-induced cardiac dysfunction and apoptosis Am J Physiol Heart Circ Physiol, August 1, 2002; 283(2): H688 - H694. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. A. Lapchak, D. M. Araujo, D. Song, J. Wei, R. Purdy, and J. A. Zivin Effects of the Spin Trap Agent Disodium- [tert-butylimino)methyl]benzene-1,3-disulfonate N-Oxide (Generic NXY-059) on Intracerebral Hemorrhage in a Rabbit Large Clot Embolic Stroke Model: Combination Studies With Tissue Plasminogen Activator Stroke, June 1, 2002; 33(6): 1665 - 1670. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Lapchak, D. F. Chapman, and J. A. Zivin Pharmacological Effects of the Spin Trap Agents N-t-Butyl-Phenylnitrone (PBN) and 2,2,6,6-Tetramethylpiperidine-N-Oxyl (TEMPO) in a Rabbit Thromboembolic Stroke Model : Combination Studies With the Thrombolytic Tissue Plasminogen Activator Stroke, January 1, 2001; 32(1): 147 - 153. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
