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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3906-3914
Defective Recovery and Severe Renal Damage After Acute Hemolysis in
Hemopexin-Deficient Mice
By
Emanuela Tolosano,
Emilio Hirsch,
Enrico Patrucco,
Clara Camaschella,
Roberto Navone,
Lorenzo Silengo, and
Fiorella Altruda
From the Department of Genetics, Biology and Biochemistry, Department
of Clinical and Biological Sciences, and Department of Biomedical
Sciences and Human Oncology, University of Turin, Turin, Italy.
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ABSTRACT |
Hemopexin (Hx) is a plasma glycoprotein mainly expressed in liver
and, less abundantly, in the central and peripheral nervous systems. Hx
has a high binding affinity with heme and is considered to be a major
transport vehicle of heme into the liver, thus preventing both
heme-catalyzed oxidative damage and heme-bound iron loss. To determine
the physiologic relevance of heme-Hx complex formation, Hx-deficient
mice were generated by homologous recombination in embryonic stem (ES)
cells. The Hx-deficient mice were viable and fertile. Their plasma iron
level and blood parameters were comparable to those of control mice and
they showed no evidence of tissue lesions caused by oxidative damage or
abnormal iron deposits. Moreover, they were sensitive to acute
hemolysis, as are wild-type mice. Nevertheless, Hx-null mice recovered
more slowly after hemolysis and were seen to have more severe renal
damage than controls. After hemolytic stimulus, Hx-deficient mice
presented prolonged hemoglobinuria with a higher kidney iron load and
higher lipid peroxidation than control mice. Moreover, Hx-null mice
showed altered posthemolysis haptoglobin (Hp) turnover in as much as Hp
persisted in the circulation after hemolytic stimulus. These data
indicate that, although Hx is not crucial either for iron metabolism or
as a protection against oxidative stress under physiologic conditions,
it does play an important protective role after hemolytic processes.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMOPEXIN (Hx) is a 60-kD plasma
glycoprotein with a high binding affinity to heme in an equimolar
ratio.1,2 It is mainly expressed in liver and belongs to
the family of the acute-phase proteins whose synthesis may be induced
by several cytokines as a result of inflammatory
processes.3,4 Other than in the liver, Hx is expressed in
the CNS,5 in the retina,6,7 and in peripheral
nerves.8 Subsequent to heme binding, Hx undergoes a
conformational change that allows for interaction with a specific receptor mainly expressed on the hepatocyte membrane and its
internalization. In the cytosol, heme is catabolized into bilirubin,
biliverdin, and iron, and the Hx-Hx receptor complex is then
recycled.9 Receptors for heme-Hx are expressed not only by
liver parenchymal cells,10 but also by retinal pigment
epithelia cells,6 lymphocytes, and several cell
lines.11
Hx is the plasma protein that has the highest affinity for heme
(affinity constant [kd]< 1 pmol/L). Heme
is composed of protoporphyrin IX and iron forming a rigid hydrophobic
planar structure, which rapidly intercalates into lipid membranes and
other hydrophobic compartments when not associated with
proteins.12 As heme is able to intercalate into lipid
membranes and participate in the Fenton reaction in the production of
hydroxyl radicals, it is a potent catalyst for injury from hydrogen
peroxide, oxidized low-density lipoprotein, and activated
neutrophils.13,14 Under normal conditions, the presence of
heme in plasma is due to oxidation of hemoglobin, which is released
during the enucleation of erythroblasts or in states of intramedullary
or intravascular hemolysis. When this process produces a low hemoglobin
concentration, most of it dissociates in   dimers, which rapidly
bind to haptoglobin (Hp) and metabolize in the liver. The plasma
hemoglobin that remains unbound to Hp is quickly oxidized into
ferrihemoglobin that, in turn, dissociates into globin and ferriheme.
Ferriheme can then be bound by albumin (kd ~10 nmol/L),
transferred to Hx, and transported to the liver.9 Heme
concentration increases in plasma after hemolysis and this state, in
humans, is associated with several pathologic conditions such as
reperfusion injury and/or ischemia.9
The strength of the binding between heme and Hx, together with the
presence of specific receptors for the heme-Hx complex on liver
parenchyma cells, have led to the belief that Hx is mainly responsible
for the transportation of heme into the liver. Consequently, it may be
hypothesized that the function of Hx is that of preventing both
heme-bound iron loss and heme-catalyzed oxidative
damage.15,16 Such a hypothesis is supported by data on
cultured primary hepatocytes and hepatoma cell lines demonstrating
that, once the heme-Hx complex is bound and internalized by the Hx
receptor, several events involved in cellular response to stress take
place. These include (1) induction of heme oxygenase 1 (HO-1),
ferritin, and metallothionein 1 (MT-1) genes that function at an
intracellular level as antioxidants17,18; and (2) rapid
c-jun N-terminal kinase/stress-activated protein kinase
(JNK/SAPK) activation with phosphorylation of c-jun and nuclear
translocation of NFkB together with the induction and maintenance of
p21 and p53.19 These events may help cell survival, in as
much as the cell neutralizes the oxidative potential of heme and iron.
Therefore, it would logically follow that Hx would tend to target heme
towards a discrete set of tissues that express Hx receptor and are able
to catabolize heme and would also protect receptor-null cells,
including endothelial cells, by preventing the diffusion of free heme.
On the other hand, the pivotal role of Hx in scavenging free heme from
circulation was recently questioned by Taketani et al,20
who showed that primary rat hepatocytes are able to uptake heme from
culture medium directly from albumin, and that concentrations of Hx in
excess of heme inhibit both heme uptake and heme-mediated HO-1
induction. Moreover, cultured adult rat hepatocytes exhibit only about
2,000 Hx receptors per cell, which is not consistent with the magnitude
of the heme flux into hepatocytes.21 When these data are
considered together, it suggests that heme may be transferred from
albumin with the help of a plasma membrane heme transporter and that,
having crossed the hepatocyte membrane, it is then bound by cytosolic
heme-binding proteins.
In an effort to determine the physiologic relevance of the heme-Hx
complex formation in vivo, Hx-deficient mice were generated by
homologous recombination in embryonic stem (ES) cells. It was observed
that, under physiologic conditions, Hx depletion is not crucial for
viability, fertility, or heme-iron metabolism. Nevertheless, following
acute hemolysis, Hx-deficient mice recover less efficiently than
control mice and suffer major renal damage. Even several days after
hemolytic stimulus, they still present elevated hemoglobinuria and
accumulate renal iron causing oxidative damage. Moreover, Hp turnover
after hemolysis is altered in Hx-null mice, since Hp persisted in the
circulation for several days after hemolytic stimulus, suggesting the
existence of compensatory mechanisms between Hx and Hp in the
acute-phase reaction.
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MATERIALS AND METHODS |
Hx-targeting vector.
Genomic DNA from the Hx locus was isolated by a 129/SV genomic library
using a human Hx cDNA probe. A 12-kb ClaI-BamHI clone containing exons 1 to 4 of the Hx gene and approximately 10-kb upstream
sequences was used to construct a targeting vector. A restriction site
for SalI was inserted instead of the ATG start codon in the
first exon of the Hx gene using polymerase chain reaction (PCR). This
restriction site was used to insert a 5-kb lacZ-PGKneo
cassette.22
Generation of Hx  / mice.
Gene targeting in ES cells and the generation of Hx-mutant mice were
performed as previously described.23 Briefly, the targeting vector was linearized with NotI and introduced by
electroporation into approximately 1.5 × 107 R1 ES
cells. After 24 hours, the cells were placed under selection with 400 µg/mL G418 (GIBCO, Gaithersburg, MD) for 7 to 9 days. The genomic
DNAs of resistant clones were digested with EcoRV and analyzed
by Southern blotting with a 3' 1-kb external probe encompassing
exons 5 and 6 of the Hx gene. Three targeted ES cell clones were
injected into the blastocysts of C57B6 mice and transferred into
pseudopregnant females as described previously.23 Chimeric male offspring were bred to C57B6 or 129/SV females and F1
offspring were tested for transmission of the disrupted allele by
Southern blot analysis. Homozygous F2 mutant mice were
obtained by the heterozygous mating of the F1 mice.
Northern blot.
Total RNA was extracted from the liver with the RNeasy Mini Kit
(Qiagen, Chatsworth, CA). A 10-µg quantity of RNA was electrophoresed on a 1% agarose gel under denaturing conditions, transferred to a
nylon membrane (Hybond N+; Amersham, Buckinghamshire, UK)
and hybridized with a 32P-labeled probe corresponding to a
fragment of murine Hx cDNA. The entire human Hp cDNA and the mouse
-actin cDNA were also used as probes. Quantitation of the band
intensity was performed by densitometry using a scanner connected to a
personal computer.
Western blot.
For Western blot analysis, mice were bled from the tail vein and 1 µL
of plasma was separated on 6% sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE), blotted onto nitrocellulose filter
(Amersham), and probed with a goat antiserum against human Hx (AES-217;
Harlan Sera-Lab, Crawley Down, Sussex, UK) or a goat antiserum against
human Hp (Sigma H5015; Sigma, St Louis, MO) that also cross-reacts with
mouse Hx or Hp, respectively. Filters were then incubated with
horseradish peroxidase-conjugated rabbit antigoat IgG (Southern
Biotechnology Associates, Birmingham, AL) and developed
with an ECL detection system (Amersham).
Phenylhydrazine treatment.
Phenylhydrazine hydrochloride (Sigma P6926) was dissolved in
phosphate-buffered saline (PBS) at 20 mg/mL and pH was adjusted to 7.4 with NaOH. Age- and sex-matched 5- to 7-week-old mice were injected
intraperitoneally with freshly prepared phenylhydrazine ranging from
0.2 to 0.25 mg/g body weight.
Histology and histochemistry.
Tissues were dissected, fixed in 10% formalin or Bouin's fluid for 24 hours, and embedded in paraffin. Microtome sections, 7 to 10 µm
thick, were mounted onto TESPA (3-aminopropyl-triethoxysilane; Sigma)-treated slides and stained with hematoxylin and eosin or Prussian blue to detect ferric iron according standard
procedures.24 Measurement of differences in ferric iron
deposition was performed by counting on a microscope at high
magnification (1,000×, immersion oil) the number of discrete blue
spots per cell. Blood smears were obtained from tail vein and stained
with May-Grünwald Giemsa as described previously.24
For -galactosidase histochemistry, dissected tissues were
snap-frozen in 10% vol/vol embedding medium (Bio-Optica, Milan,
Italy)/PBS on the surface of liquid nitrogen and sectioned at 15 µm
on a cryostat at 20°C to 25°C. Sections were
fixed in 2.5% glutaraldehyde in PBS (vol/vol) in a microwave oven at
maximum power for 10 seconds and processed for -galactosidase activity detection according standard procedures.5
Hematological parameters.
Blood was obtained by retroorbital sampling from anesthetized mice and
blood cell counts were determined using an automatic cell counter.
Iron, albumin, total protein, and bilirubin in serum were determined by
colorimetric detection systems (Sigma) according to standard procedures
on an automatic analyzer.
Analysis of oxidative damage.
Lipid peroxidation from tissue extracts was measured using the
colorimetric assay kit Bioxytech LPO-586 from Oxis International (Portland, OR) according to the manufacturer's instructions. Briefly, tissue samples were homogenated (20% to 30% wt/vol) in ice-cold 20 mmol/L Tris-HCl pH 7.4 containing 5 mmol/L butylated hydroxytoluene and
protein content was determined using the Biorad protein assay system
(Biorad, München, Germany). A 200-µL quantity of sample was
assayed for malonaldehyde (MDA) content in hydrochloride using the
chromogenic reagent N-methyl-2-phenylindole. Absorbance was measured at
586 nm and the results were expressed as nanomoles of MDA per milligram
of protein using a molar extinction coefficient of 110.
Hemoglobinuria.
Syringes (1 mL) were used to collect urine samples from spontaneous
urination or directly from the bladder of anesthetized mice by needle
aspiration. The values were then determined with Bayer Multistix 10 SG
(Milano, Italy).
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RESULTS |
Generation of Hx-null mice.
A targeting vector containing a lacZ-PGKneo cassette on the ATG start
codon located in the first exon of the Hx gene was designed to disrupt
the Hx gene. This targeting vector had a total of 12 kb of homologous
genomic sequence flanking the cassette, with 10 kb upstream and 2 kb
downstream (Fig 1A). The construct was linearized and introduced into R1 ES cells by electroporation. After
selection with G418, resistant clones were screened for homologous
recombination by Southern blotting with a 3' external probe.
Eleven of 200 screened clones contained the 8-kb EcoRV band
diagnostic of homologous recombination in one allele in addition to the
10-kb fragment from the wild-type allele (Fig 1B). Three targeted ES
clones were injected into the blastocysts of C57B6 mice. All of the
chimeric males showed germ-line transmission of the disrupted allele.
F1 offspring from chimeras were independently intercrossed
to generate F2 homozygous mice of 129/SV and hybrid strain
backgrounds. All of the mice that were heterozygous and homozygous for
the Hx gene disruption appeared healthy, grew, reproduced normally, and
produced homozygous offspring in the expected number. Northern blot
analysis on total RNA extracted from the liver of wild-type,
heterozygous, and homozygous littermates was performed to confirm that
Hx / mice did not express the Hx gene product. Hx mRNA was
absent in the liver of Hx / mice and was about half
normal in +/ mice, indicating that there was no compensation for
the reduced gene dosage of Hx in heterozygous mice (Fig 1C).
-Galactosidase staining on histologic sections was used to
demonstrate that the reporter gene was correctly expressed in the
hepatocytes (Fig 1D). Apart from the liver, the only other site in
which -galactosidase was detectable was the CNS, where staining was
present in the ependymal cells and choroid plexi (not shown).
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| Fig 1.
Targeted disruption of the Hx gene. (A) Structure of the
Hx gene (top), the targeting vector (middle) containing a LacZ-PGKneo
cassette in the first exon of the Hx gene, and the predicted structure
of the disrupted allele after homologous recombination (bottom). Only
the relevant restriction sites are shown: C, ClaI site; E,
EcoRV site; B, BamHI site. Solid boxes represent exons
1-6. The position of the 3' external probe is indicated. (B)
Southern blot analysis of EcoRV-digested genomic DNA from ES
clones. Filter was hybridized with the 3' external probe shown in (A).
The wild-type and mutant alleles are indicated by 10- and 8-kb
EcoRV fragments, respectively. (C) Northern blot analysis of
total RNA extracted from the liver of a wild-type, an Hx +/, and
an Hx / mouse. Filter was hybridized sequentially with an Hx
probe and a -actin probe. Hx transcript was reduced in Hx +/
liver and absent in Hx / liver. (D) -Galactosidase staining of
liver sections from a wild-type and an Hx / mouse. The majority
of hepatocytes were labeled in Hx knockouts. Bar, 10 µm.
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Analysis of Hx-deficient mice under physiologic conditions.
To test the possibility that Hx acts in vivo by scavenging free heme
from the plasma, preventing oxidative damage, and contributing to the
conservation of body iron, iron stores in the plasma and in the tissues
were analyzed. As an altered iron status may affect hematopoiesis,
peripheral blood was also analyzed and a histologic assessment of
hematopoietic organs was made.
Plasma levels of bilirubin, iron, total proteins, and albumin were
unaffected by the Hx mutation, as were all blood cell lineages (Table
1).
Histologic analysis of liver, kidney, heart, brain, spleen, and bone
marrow showed no evident abnormalities or tissue lesions due to
oxidative damage in Hx / mice, nor did Prussian blue staining of tissue sections show any abnormal iron deposition (not
shown). These data indicate that, under physiologic conditions, Hx does
not protect from oxidative stress and is not crucial for iron metabolism.
Susceptibility of Hx-deficient mice to acute hemolysis.
To investigate the capability of Hx in the prevention of oxidative
stress, Hx-deficient and wild-type mice were subjected to acute
hemolysis by administration of a single dose of phenylhydrazine ( hyd) ranging from 0.2 mg/g to 0.25 mg/g body weight. Hemolysis was
evident on day 1 by a marked depression in hematocrit level (Fig
2A) and dark brown coloration of plasma and
urine, indicating hemoglobinemia and hemoglobinuria, respectively.
Susceptibility to hemolysis did not differ in wild-type and Hx-null
mice: after an injection of 0.23 mg/g of hyd, 80% of mice from both
genotypes began to die from day 1 to day 4 (Fig 2B).
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| Fig 2.
Effect of phenylhydrazine injection. (A) Mean hematocrit ± SD after a single dose of phenylhydrazine of 0.2 mg /g. A total of
4 mice for each genotype was used. (B) Survival after phenylhydrazine
treatment. A single dose of phenylhydrazine of 0.23 mg/g was
intraperitoneally injected and mice were monitored for 7 days; 20 animals per genotype were used.
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Spleens of both wild-type and Hx-deficient mice, after hemolysis, were
3 to 4 times normal size due to the hyperplasia of the
reticuloendothelial system. Histologic analysis of several organs
(liver, kidney, spleen, heart, and brain) from dying mice showed
hydropic degeneration as evidenced by numerous cytoplasmic vacuoles in
the liver and kidney of both +/+ and / mice. Prussian blue staining showed considerable nonheme iron deposits, in the form of
hemosiderin and ferritin, in renal cortical tubules and in splenic
macrophages and, to a less extent, in the Kupffer cells of the liver in
all the mice. Other organs, such as heart and brain, showed no signs of
degeneration or evident iron deposits.
On the basis of hemoglobinuria and histologic data, renal failure was
assumed to be the cause of death of hyd-treated mice.
Recovery of Hx-deficient mice following acute hemolysis.
To further assess the protective role of Hx after hemolysis, the
recovery after hemolytic stimulus in wild-type and Hx-deficient mice
was analyzed by administering a sublethal dose of hyd of 0.2 mg/g
and killing them after 7 days. This dose of hyd was effective in
producing hemolysis as indicated by the reduced hematocrit, hemoglobinemia, and hemoglobinuria. Seven days after injection, blood
parameters were within normal limits in both wild-type and Hx-null mice
(Fig 2A). Moreover, blood parameters and blood smears obtained from Hx
/ and controls 7 days after the hemolytic stress were
morphologically identical (data not shown).
Hp turnover.
As a depletion of Hp in the circulation is a clinical marker of a
hemolytic status, follow-up evaluation of recovery was made by
monitoring Hp plasma levels after hemolysis. Surprisingly, while in
control mice Hp levels decreased 3 days after the hyd injection,
they remained elevated for 7 days after treatment in Hx-null mice (Fig
3A). In wild-type mice, Hx increased 1 day
after the hyd injection and decreased slowly to the seventh day (Fig 3A). Therefore, to assess whether the difference in plasma Hp content
was due to a different transcriptional activation of the Hp gene in Hx
/ mice, Northern blot was used to analyze Hp mRNA expression in the liver. As shown in Fig 3B, there was no difference in
Hp mRNA induction and turnover after the hyd injection in either
wild-type or Hx-null mice: Hp mRNA level increased 1 day after the
hyd injection and remained elevated for the following 4 days. These
data indicated that, after hemolysis, the Hx mutation altered Hp
turnover at the protein level.
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| Fig 3.
Hp turnover after a single dose of phenylhydrazine of 0.2 mg/g. (A) Western blot: mice were monitored for 7 days after injection
and plasma from tail vein analyzed with an antibody against Hp (left)
and Hx (right). (B) Northern blot: mice were monitored for 7 days after
injection and total liver RNA analyzed with a probe for Hp (top) and
-actin (bottom). Diagrams refer to Hp turnover at the plasma (A) and
mRNA level (B).
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Hemoglobinuria.
Seven days after the hyd injection, Hx / mice had a
higher hemoglobinuria than did controls: 6 of 8 Hx /
presented high hemoglobin urine concentration while 6 of 8 wild-type
mice had no hemoglobin in the urine and only 2 of 8 animals had low
hemoglobin content in the urine (Table 2).
Histology.
When the organs of hyd-treated mice were examined for iron content,
it was observed that liver, spleen, bone marrow, and kidney of all mice
contained nonheme iron deposits, in the form of hemosiderin and
ferritin. Liver and spleen, where iron deposits were present in Kupffer
cells and macrophages, respectively, showed no differences between Hx
/ and wild-type mice. On the contrary, a significantly
higher iron load was found in the kidney of Hx-deficient mice than in
the control group: blue-stained hemosiderin was evident in proximal
tubules (Fig 4). To quantify the extent of
the renal damage, the number of blue-stained, Perls' positive
granules, detected by focusing on a microscope at high magnification,
was counted in 200 tubular cells for each animal. In Hx /
mice, we observed 6.58 ± 0.89 granules/cell, as compared with 3.59 ± 0.6 granules/cell in wild types (Fig
5A).
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| Fig 4.
Iron loading in Hx / tissues. Liver (A,B), spleen
(C,D) and kidney (E,F) sections from Hx / (A,C,E) and wild-type
(B,D,F) mice stained with Prussian blue for detection of ferric iron.
Liver and spleen show iron deposits in Kupffer cells and macrophages,
respectively, without differences between knockouts and controls.
Kidney of Hx / has much more iron loading compared with controls:
intense blue staining is evident in proximal cortical tubules. Bar, 20 µm.
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| Fig 5.
Analysis of renal damage after injection of 0.2 mg/g of
phenylhydrazione. (A) Measurement of iron loading in the kidney of Hx
/ and wild-type mice 7 days after phenylhydrazine treatment. Mean
number ± SD of Perls' positive granules per tubular cell. At least
200 cells for each animal were counted. A total of 6 mice for each
genotype was used (P < .001). (B) Lipid peroxidation
estimated as MDA levels of tissue homogenates of Hx / and
wild-type mice 7 days after phenylhydrazine treatment. Data represent
mean ± SEM from 5 mice for each genotype (P < .01). Kidney
from Hx / mice, which consistently shows iron loading, has
significantly greater oxidative damage than that from control mice.
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Analysis of oxidative stress.
To determine any oxidative stress that may have arisen as a result of
iron deposition, oxidized lipid levels were analyzed by measuring the
MDA content in the liver and kidney of hyd-treated mice. To avoid
new lipid peroxidation that can occur during homogenization and
detection of MDA, the antioxidant butylated-hydroxytoluene at a final
concentration of 5 mmol/L was used during all steps of the procedure.
The degree of lipid peroxidation in the kidney of Hx /
was higher than that in controls, whereas the MDA level in the liver
was similar (Fig 5B). These data indicated that Hx-deficient mice
presented an altered Hp turnover after hemolysis and suffered more
renal damage than did the control group.
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DISCUSSION |
Hx is the plasma protein that has the highest affinity for heme. Due to
the strength of the binding between heme and Hx and the presence of
specific receptors on hepatocyte membrane, Hx is believed to be the
major factor responsible for the scavenging of free heme from plasma,
thus preventing oxidative damage and contributing to the conservation
of body iron.9,15,16
Analysis of Hx-deficient mice showed that the presence of Hx was not
crucial for heme catabolism under physiologic conditions; indeed, the
plasma levels of iron and bilirubin, the major products of heme
catabolism, were unaffected by the Hx mutation. Hematopoietic organs
and blood parameters were normal. There was no abnormal iron deposition
or oxidative lesions at the tissue level. These data are consistent
with the absence of a known human inherited disorder caused by Hx
deficiency. On the contrary, HO-1 knockout mice25 with
ageing develop an anemia associated with abnormally low serum iron
content, and accumulate hepatic and renal iron that causes oxidative
damage, tissue injury, and chronic inflammation. Our data demonstrated
that Hx is not essential for the transport of heme into the hepatic
cells where HO-1 catabolizes it. In plasma, heme can be bound by
albumin, present at higher concentrations than Hx (30 to 55 mg/mL
v 0.5 to 1 mg/mL).9 Recently, Noyer et
al21 showed that heme can be transferred directly from the albumin to primary rat hepatocytes in culture and suggested the presence of a transmembrane heme transporter. A similar mechanism may
also take place in vivo. After crossing the hepatocyte membrane, heme
could then be bound by cytosolic heme-binding proteins. Several candidate heme-binding proteins have been suggested: fatty acid binding
protein,26 glutathione S-transferases,27 MSP23
or the rat homologous HBP 23,28-30 or even the recently
cloned p22 HBP.31
The role of Hx became evident after hyd-induced hemolysis. The
susceptibility of mice lacking Hx to hyd treatment was the same as
that of wild-type mice. On the other hand, mice lacking the Hp
gene32 showed increased susceptibility to hemolysis, ie,
55% of them died within 5 days of hyd treatment compared with 18%
of wild-type mice. These data indicate that Hp is more efficient than
is Hx in protecting from hemolytic stress. The slight difference in the
lethal dose between Hp and Hx knockouts (0.2 mg/g v 0.23 mg/g)
is probably due to a different hyd batch or to differences in the
genetic background of the mice. Other than susceptibility to hemolysis,
also recovery of blood parameters after hyd injection was unaffected
by Hx mutation; indeed, hematocrit normalized within 1 week in
knockouts, as in wild-type mice. Nevertheless, Hx-deficient mice
responded to hemolysis in a different way, since Hp persisted in the
circulation for several days after the hyd injection. The prolonged
presence of Hp in the plasma of Hx / after hyd could
be the consequence of a major Hp protein stability and/or of a major
rate of translation. This conclusion is supported by the fact that Hp
is not recycled after internalization of the Hp-hemoglobin complex and
that Hp mRNA induction and turnover in Hx / after hyd
is unaltered. Alternatively, Hx null mice may have an alteration in the
kinetic of internalization of the hemoglobin-Hp complex, thus
suggesting interactions between the Hp and Hx receptor systems. It
would, therefore, be interesting to analyze Hx turnover in Hp
/ mice after hemolysis. Considered together, the data on
the susceptibility to hemolysis in Hx and Hp-deficient mice suggest
that Hp is in the first line of defense against hemolytic stress,
followed by Hx, and that the 2 proteins cooperate with each other in
the response to hemolysis.
To the best of our knowledge, this is the first time that data have
been presented on a possible cross-talk between Hx and Hp in the
acute-phase response. This could be mediated by several cytokines such
as interleukin-6, which has cis-acting responsive elements in
the promoter of both Hx and Hp.33,34 However, an altered Hp
turnover cannot explain the lack of phenotype in Hx /
under physiologic conditions, since basal Hp plasma levels are
unaffected by the Hx mutation.
Based on histologic data, hemoglobinemia, hemoglobinuria, and clinical
reports we assumed that renal failure was the cause of mortality after
hyd, as reported for Hp-null mice.32 The kidney is also
the organ that suffered the most damage after the sublethal dose of
hyd, as demonstrated by prolonged hemoglobinuria, high lipid
peroxidation, and iron loading. It is unlikely that prolonged
hemoglobinuria is due to a continued hemolysis in Hx /
mice since blood parameters normalized at the same rate as they did in
control mice. It may be due to a reduced capability of the Hp system to
remove hemoglobin from the circulation. Hemoglobin accumulates at the
tubular level and causes iron loading. The presence of ferric iron in
the kidney could then account for the higher lipid peroxidation
observed in Hx-deficient mice compared with controls. Consistent with
our results, Hp-null mice that recovered after hemolysis presented
impaired renal regeneration and repair.32 These data are in
agreement with those observed in acute renal tubular necrosis in
patients with a high degree of hemolysis.35
Iron loading was present exclusively in renal tubular cells and in
macrophages or Kupffer cells of the reticuloendothelial system. The
latter sites of deposition, where no difference was found between Hx
/ and wild-type mice, are to be expected due to the large
expansion of the reticuloendothelial system after hemolytic processes.
The absence of iron loading in hepatocytes was also predictable, unlike
HO-1 /, 2-microglobulin /,
and HFE / mice25,36,37 that
suffered from specific defects in their iron metabolism.
In conclusion, our data demonstrate that the most significant role of
Hx is that of protecting cells against heme toxicity, rather than
participating in iron metabolism. This is in agreement with previously
reported biochemical studies showing the protective role of Hx in
heme-catalyzed oxidations.38,39 The role of Hx becomes
evident after a hemolytic process, particularly in the kidney, which is
also the most compromised organ involved in human hemolytic
pathologies. These results are in agreement with those previously
reported for Hp knockout mice, which showed that the major function of
Hp is to protect renal tubules from hemoglobin-mediated oxidative
damage rather than to clear free plasma hemoglobin under normal
conditions.32 Therefore, when considering the data on Hp
and Hx knockouts, we may conclude that the response to hemolysis occurs
in 2 stages: first, Hp and Hx are rapidly induced to protect from
increased plasma hemoglobin and heme concentrations, then, once Hp
disappears from the circulation, the delayed presence of Hx in the
plasma takes on a relevant role in the protection against heme derived
from hemoglobin oxidation.
Although the data reported herein refer only to the "systemic"
physiologic role of Hx, it may well act locally at the level of
peripheral nerves after an injury. Indeed, in transected rat sciatic
nerves, Hx is expressed by fibroblasts, Schwann cells, and invading
blood macrophages, and is accumulated in the extracellular matrix, thus
suggesting that it may protect injured tissues from oxidative
damage.8,40,41 Moreover, Hx is synthesized in the retina,
which is isolated from the circulation by the blood-retinal barrier,
and the Hx receptor is expressed by the retinal pigment epithelia
cells.6,7 The binding between the heme-Hx complex and the
Hx receptor results in the increased expression of several proteins
that all interact in the degradation of heme, the storage of iron from
heme catabolism, and as intracellular antioxidants.42 Hx-deficient mice could be useful in characterizing other Hx functions, particularly at injury sites in the nervous system.
| |
ACKNOWLEDGMENT |
The authors thank Drs V. Poli, G. Pescarmona, F. Di Cunto, and G. Saglio for stimulating discussion, and Drs G. Topley and B. Wade for
critical reading of the manuscript. We would also like to thank Dr A. Gariboldi and Dr A. Grillo for performing hematologic analyses. The
technical help of L. Cavarretta and I. Carfora is gratefully acknowledged.
| |
FOOTNOTES |
Submitted May 26, 1999; accepted July 21, 1999.
Supported by grants from the National Research Council (Target Project
on Biotechnology) to F.A. and from the Ministry of University and
Scientific Research to F.A.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Fiorella Altruda, PhD, Department of
Genetics, Biology and Biochemistry, Via Santena 5bis, 10126, Turin,
Italy; e-mail: altruda{at}molinette.unito.it.
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