Endocrinology Vol. 140, No. 2 641-645
Copyright © 1999 by The Endocrine Society
Effects of Pro- and Antioxidative Compounds on Renal Production of Erythropoietin1
Imke Neumcke,
Benjamin Schneider,
Joachim Fandrey and
Horst Pagel
Institute of Physiology, Medical University, Luebeck, Germany
Address all correspondence and requests for reprints to: Horst Pagel, Ph.D., Institute of Physiology, Medical University, Ratzeburger Allee 160, 23538 Luebeck, Germany. E-mail: pagel{at}physio.mu-luebeck.de
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Abstract
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The most important stimulus for the enhanced synthesis of
erythropoietin (Epo) is a lowered O2 tension in the tissue.
However, the mechanism by which an impaired O2 supply is
transduced into appropriate Epo production is still not fully
understood. Recently, studies in human hepatoma cells (line HepG2)
indicate that reactive O2 species are involved in the
signal transduction from the cellular O2 sensor to the Epo
gene. To clarify the role of reactive O2 species in the
regulation of Epo synthesis in the kidney, the principal Epo-producing
organ in vivo, we investigated the influence of potent
pro- and antioxidants on Epo production in isolated perfused rat
kidneys. Under normoxic conditions, the iron chelator desferrioxamine
and the antioxidant vitamin A increased renal Epo production, mimicking
hypoxic induction. In contrast, supplementation of the perfusion medium
of hypoxically perfused kidneys with the prooxidant compounds
H2O2 or pyrogallol caused a significant
reduction of Epo synthesis. The inhibition of Epo formation by reactive
O2 species could be completely antagonized by
desferrioxamine and the hydroxyl radical-(OH.)-scavenger
tetramethylthiourea. Vitamin A also antagonized the
H2O2-dependent inhibition of hypoxically
induced Epo synthesis. Interestingly, the addition of the antioxidant
vitamin A to hypoxically perfused kidneys also induced Epo production
significantly. Our data strongly support the idea that reactive
O2 species, especially H2O2, are
part of the signaling chain of the cellular O2-sensing
mechanism regulating the renal synthesis of Epo.
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Introduction
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THE GLYCOPROTEIN hormone erythropoietin
(Epo) is the principal regulator of the proliferation and
differentiation of erythroid progenitors in bone marrow. In adults, Epo
is mainly produced in the kidneys. The most important stimulus for an
enhanced production of Epo is a lowered oxygen (O2) supply
to the tissue as a result of anemia or hypoxia. However, the mechanism
by which changes in O2 tension are transduced into an
appropriate renal Epo production is still not fully understood.
Specific hemoproteins have been proposed as the O2-binding
part of the O2 sensor (1). Previous in vitro
studies in cultures of the human hepatoma cell line HepG2 revealed an
important role of membrane-bound and/or microsomal b-type
cytochromes (2, 3). Similar to the NADPH-oxidase from phagocytes (4),
the cytochromes generate low, nondamaging amounts of reactive
O2 species dependening on the cellular O2
tension. These O2 species may oxidize sulfhydryl groups of
regulatory peptides and therefore serve as signaling molecules between
the O2 sensor and the transcriptional machinery of the Epo
gene. Referring to this, the small, noncharged, and thus freely
diffusible hydrogen peroxide (H2O2) is a very
attractive candidate. In addition, H2O2 has a
relatively long biological half-life and is far less cytotoxic than
other reactive O2 species (5, 6).
From the studies in HepG2 cells the hypothesis was put forward that
high cellular levels of H2O2 under normoxia
suppress Epo synthesis, whereas low levels under hypoxia allow full
scale Epo gene expression (7, 8). Hence, H2O2
could act as a second messenger, possibly influencing cytosolic
transcription factors that bind to regulatory sequences of the Epo
gene. In fact, hypoxia-induced expression of the Epo gene is critically
dependent on the activation of an enhancer element located 3' of the
Epo gene. To this enhancer binds a protein complex termed
"hypoxia-inducible factor-1" (HIF-1), which is composed of the two
subunits HIF-1
and HIF-1ß (9, 10). Only recently, it has been
found that H2O2 reduces HIF-1 DNA-binding
activity under hypoxic conditions by destabilizing the -subunit
(11). Strong reducing agents, however, increase HIF-1 levels by
inhibiting the proteasome-dependent degradation of the -subunit
(12).
The present investigation was performed to determine whether this
hypothesis also fits the Epo production in the kidney, the predominant
site of Epo synthesis after birth. The experiments were performed with
isolated serum-free perfused rat kidneys. Levels of secreted Epo and
Epo messenger RNA (mRNA) levels in kidney tissue were measured during
normoxic and hypoxic perfusion after the addition of
H2O2 and/or
H2O2-generating and -scavenging compounds.
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Materials and Methods
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Kidney perfusion
Right kidneys from adult male Sprague-Dawley rats were perfused
as described in detail previously (13). In brief, the kidneys were
perfused at constant pressure (100 mm Hg) in a recirculation system for
3 h. The perfusion medium was a substrate- and amino acid-enriched
Krebs-Henseleit buffer supplemented with predialyzed (three periods of
3 h each) BSA (60 g/liter; Biomol, Hamburg, Germany) and freshly
drawn and washed human erythrocytes (5%). To ensure stable organ
function during the experiments, the perfusion medium was dialyzed
against a 25-fold volume of a protein- and cell-free solution. The
dialyzer also served for oxygenation of the perfusion medium by
bubbling prewarmed and moistened gas mixtures through the dialysate.
The arterial pO2 was 149.0 ± 1.8 mm Hg during
normoxic and 26.3 ± 0.9 mm Hg during hypoxic perfusion. The
arterial pH was 7.4 throughout. During the different experimental
series one of the following agents or a combination of them were added
to the perfusion medium before starting the isolated perfusion (the
final concentrations are given in parentheses): hydrogen
peroxide (H2O2; 100 µmol/liter), pyrogallol
(1,2,3-trihydroxybenzene; 100 µmol/liter), desferrioxamine mesylate
(DFO; 100 µmol/liter), 1,1,3,3-tetramethyl-2-thiourea (TMTU; 1
mmol/liter), and vitamin A (retinol acetate; 0.5 mg/liter). The
concentrations were chosen according to redox experiments in
HepG2/Hep3B cell culture systems (8, 14). All substances were obtained
from Sigma Chemical Co. (Deisenhofen, Germany), except
TMTU, which was obtained from Fluka (Neu-Ulm, Germany).
Determination of the fractional sodium reabsorption
The sodium concentrations in urine and perfusate were determined
by flame photometry (AFM 5051, Eppendorf, Hamburg,
Germany). The fractional sodium reabsorption was calculated as the
ratio of the sodium reabsorption rate to the sodium filtration
rate.
Epo measurements
Epo was measured in duplicate by an enzyme-linked immunoassay in
samples of the perfusion medium. The assay was performed according to
the instructions of the manufacturer (Medac, Hamburg, Germany), except
that rat serum Epo previously calibrated by bioassay was used as the
standard instead of human Epo.
RNA extraction and Epo mRNA quantitation
At the end of the perfusion period, kidneys were weighed,
snap-frozen in liquid nitrogen, and subsequently homogenized in
guanidinium thiocyanate solution (4 mol/liter with 0.1 mol/liter
ß-mercaptoethanol) using a Polytron homogenizer (Kinematica, Luzern,
Switzerland) at setting 10 for 20 s. From 700 µl of the
homogenate, total RNA was extracted using the acidic phenol-chloroform
method (15). After redissolving the RNA in diethylpyrocarbonate-treated
water, the concentration was determined by measuring the absorbance at
260 nm. To check the integrity of the RNA, aliquots were run on a 1.1%
formaldehyde/agarose gel. Five micrograms of total RNA were reverse
transcribed into first strand complementary DNA (cDNA) using
oligo(deoxythymidine)15 as primer for the reverse
transcriptase Moloney murine leukemia virus (Promega Corp., Heidelberg, Germany). The total volume of the reaction
was 25 µl. RT was performed at 42 C for 60 min after an initial
denaturation step at 68 C for 10 min. The reaction was terminated by
boiling the samples for 10 min. Until quantitation by competitive PCR,
cDNA stocks were kept at -20 C. All RNA samples were run in one RT
reaction to minimize differences in RT efficiency, which was less than
or equal to 5%, as determined previously (16).
Quantitation of cDNA was achieved by two PCR methods. First, the
TaqMan system (PE Applied Biosystems, Weiterstadt,
Germany) was used according to the manufacturer’s instructions.
Primers were selected by the software provided by PE Applied Biosystems for TaqMan and had the following sequences:
upstream, 5'-CTCCGAACACTCACAGCGG-3'; downstream,
5'-GGTCACCTGTCCCCTCTCCT-3'; and internal hybridization oligo,
5'-CGGGTCTACTCCAACTTCCTCCGGG-3'. The reaction temperatures were
95 and 55 C.
Second, a competitive PCR was performed as described previously (16).
Each sample was checked for possible DNA contamination. Competitive PCR
led to the same mean values, albeit a higher SE due to the
lower resolution of this method.
Statistics
The data were normalized to 1 g kidney wet weight (based on the
weight of the left, nonperfused kidney) and are given as the mean
± SEM. The results of Epo mRNA quantitation are expressed
as the percentage of Epo mRNA in hypoxically perfused rat kidneys and
are the mean ± SEM of the data obtained with the
TaqMan system. To compare Epo production during hypoxia with that
during normoxia, a two-way ANOVA was performed. Within the factor time,
Helmert contrasts were calculated (multiple ANOVA in SPSS-X).
Dunnett’s test was applied to compare a control mean with several
treatment means. P < 0.05 was set as the significance
level.
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Results
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Figure 1
shows that Epo production
in the isolated rat kidney continuously increased during the 3 h
of normoxic perfusion up to 198 ± 28 mU/g kidney (n = 5).
Hypoxic perfusion led to significantly increased Epo production of up
to 481 ± 38 mU/g kidney (n = 9).
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Figure 1. Epo production rates in the isolated perfused rat
kidney during normoxic (open circles; n = 5) and
hypoxic (filled circles; n = 9) perfusion. The
mean ± SEM are shown. *, P <
0.05 for the Epo time courses (by ANOVA with repeated measurements;
first factor, pO2; second factor, time).
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The addition of the antioxidants DFO and vitamin A increased Epo
production under normoxia (Fig. 2) almost
to levels achieved by hypoxic perfusion (see Fig. 3). Both substances roughly doubled the
normoxic renal Epo production (controls, 198 ± 28; DFO, 400
± 31; vitamin A, 374 ± 38 mU/g kidney; n = 6 each; Fig. 2).
Under these normoxic conditions, H2O2
significantly diminished Epo production from 198 ± 28 mU/g kidney
(controls; n = 5) to 90 ± 11 mU/g kidney
(H2O2 treated; n = 6; not shown)
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Figure 2. Effects of DFO (n = 6) and vitamin A (Vit.A;
n = 6) on the 3-h Epo production rates during normoxic perfusion
of the isolated rat kidney. The mean ± SEM are shown.
*, P < 0.05 vs. controls (by
Dunnett’s test; n = 5).
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Figure 3. Inhibition by hydrogen peroxide
(H2O2; n = 5) and pyrogallol (Pyro; n
= 5) of the hypoxically induced 3-h production rates of Epo in the
isolated perfused rat kidney. DFO (H2O2 plus
DFO; n = 4) and TMTU (H2O2 plus TMTU;
n = 5) antagonized the inhibition caused by
H2O2. The mean ± SEM are
shown. *, P < 0.05; n.s., P >
0.05 (vs. controls, by Dunnett’s test; n = 9).
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Hypoxia-induced Epo production during the 3-h perfusion period was
significantly reduced from 481 ± 38 mU/g kidney (controls; n
= 9) to 342 ± 49 mU/g kidney (n = 5) by the addition of
H2O2 or stimulation of endogenous
H2O2- formation by the superoxide anion
(O2-·)-generating substance pyrogallol
(391 ± 32 mU/g kidney; n = 5; Fig. 3
). The
H2O2-induced inhibition of Epo formation was
completely antagonized by the iron chelator DFO
(H2O2 plus DFO, 490 ± 20 mU/g kidney;
n = 4) and the hydroxyl radical (OH·) scavenger
tetramethylthiourea (H2O2 plus TMTU, 475
± 44 mU/g kidney; n = 5; Fig. 3). As expected, DFO alone had no
effect on Epo production of the isolated hypoxically perfused rat
kidney (464 ± 12 mU/g kidney; n = 4; not shown). The effects
on Epo protein were the result of an altered Epo gene expression, as
the addition of H2O2 significantly reduced Epo
mRNA levels that were antagonized by DFO and TMTU (Table 1).
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Table 1. Effects of H2O2 alone,
H2O2 plus desferrioxamine (DFO), and
H2O2 plus tetramethylthiourea (TMTU) on the Epo
mRNA levels in hypoxically perfused rat kidneys
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Interestingly, treatment with the antioxidant vitamin A significantly
increased hypoxia-induced Epo production to 682 ± 12 mU/g kidney
(n = 8; Fig. 4). Vitamin A was also
able to antagonize the inhibition of Epo production induced by
H2O2 (474 ± 82 mU/g kidney; n = 6;
Fig. 4).
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Figure 4. Effects of vitamin A (Vit.A; n = 8) and Vit.A
plus H2O2 (n = 6) on the hypoxically
induced 3-h production rates of Epo in the isolated perfused rat
kidney. The mean ± SEM are shown. *,
P < 0.05; n.s., P > 0.05
(vs. controls, by Dunnett’s test; n = 9).
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As judged by the fractional renal sodium reabsorption, the exocrine
function of the isolated perfused rat kidney was not influenced by the
addition of pro- or antioxidative compounds to the perfusion medium
(normoxic controls, 92 ± 1%; treatment groups, 89–93%; hypoxic
controls, 60 ± 3%; treatment groups, 52–63%; not shown).
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Discussion
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Isolated rat kidneys perfused with a serum-free perfusion medium
produce Epo in a pO2-dependent manner. Together with
previous results from our laboratory (13, 14) and others (17, 18), this
experimental set-up appears to be suitable to study the mechanisms of
pO2-dependent Epo synthesis.
The addition of 5% erythrocytes to the perfusion medium improves
kidney function (19), but increasing the hematocrit of more than 5%
shows no further improvement (20). It is important to note that the Epo
production rate in the isolated perfused kidney is independent of the
concentration of erythrocytes in the perfusion medium (13).
To check a possible nephrotoxicity of the used pro- and antioxidative
compounds in the chosen concentrations, the fractional renal sodium
reabsorption, a very sensitive marker for an intact renal metabolism,
was determined. Under both normoxic and hypoxic conditions, the
fractional sodium reabsorption of the treatment groups was not
statistically different from that of the respective controls,
indicating a normal function of the isolated perfused rat kidney.
The aim of the present study was to get more insights into the
mechanisms by which a variation in the renal O2 supply is
transduced into an appropriate Epo production. Recently, reactive
O2 species at low, nontoxic concentrations have been
recognized as intracellular signaling molecules (21). The human
hepatoma cell line HepG2 produces H2O2
depending on the pericellular pO2. The highest amounts of
secreted Epo in the culture supernatant were found at pO2
values where H2O2 production was lowest (7).
However, as in adult life the kidneys are the primary site for Epo
synthesis, we studied the effect of redox-modifying agents of Epo
production in isolated perfused rat kidneys.
The addition of the antioxidants DFO or vitamin A to the normoxic
perfusion medium increased renal Epo synthesis and almost completely
mimicked hypoxia. This would be in line with the hypothesis that
reactive O2 species, especially
H2O2, suppress Epo production under normoxic
conditions. Under hypoxia, when reduced endogenous production of
reactive O2 species allows full scale Epo production, both
exogenous H2O2 and the stimulation of
endogenous H2O2 formation by pyrogallol
significantly suppressed the rate of Epo production. The iron chelator
DFO or the hydroxyl scavenger TMTU completely antagonized the
H2O2-induced suppression of the Epo production
in the isolated perfused rat kidney. These Epo protein data reflected
the respective data of the Epo mRNA levels in kidney tissue.
Our data suggest that reactive O2 species control renal Epo
production and thus participate in the O2-sensing process
in the kidney. The results herein from experiments with isolated
kidneys corroborate similar data that have been obtained from cell
culture studies with HepG2 hepatoma cells (7). Thus,
H2O2 seems to be an attractive candidate for a
signaling molecule between the renal O2 sensor and the
transcriptional activator(s) of the Epo gene. As the suggested mode of
action of DFO is an interference with the decomposition of
H2O2 to OH. (Fenton reaction) (8),
it was important to see this antagonistic effect in the kidney as well.
Moreover, it appears reasonable to assume that the DFO-dependent
induction of Epo production under normoxic conditions is also due to
the inhibition of hydroxyl radical production from endogenously
produced H2O2. Thus, DFO may mimic hypoxia in
human hepatoma cells and rat kidneys without removing iron from
putative hemeproteins (1). In a very recent publication, Srinivas
et al. (22) reported that HIF-1 itself appears to be a
nonheme iron protein. At least within hepatoma cells, hydroxyl radicals
are preferentially detected in close vicinity of the nucleus (23), and
one may speculate that they are generated from
H2O2 in the presence of iron proteins such as
HIF-1. A local Fenton-type reaction would explain the reduced
stability of HIF-1-protein upon H2O2
treatment (11).
Therefore, the lack of effect of DFO under hypoxic conditions, when
little H2O2 is generated by the kidney, was
less surprising than the H2O2-dependent
inhibition of Epo production at perfusion with high pO2.
After our hypothesis even under these normoxic conditions one can
expect some HIF-1 activation, which was abolished by the addition of
H2O2.
Under hypoxic conditions the production of Epo in the isolated perfused
rat kidney significantly increased when vitamin A was added to the
perfusion medium. Carotenoids are considered antioxidants because of
their capacity to scavenge reactive O2 species (24). The
vitamin A-induced increase in Epo production disappeared after the
addition of H2O2. However, unlike DFO, vitamin
A also increased Epo synthesis under normoxic and hypoxic conditions.
This finding is in line with the results of corresponding experiments
in Hep3B and HepG2 cells (16, 25). Okano et al. have
proposed that vitamin A activates Epo gene transcription, because Epo
mRNA levels are elevated in vitamin A-treated HepG2 cell cultures (25).
As vitamin A, in addition to its ability to scavenge reactive
O2 species (24), also binds to retinoic acid receptors, one
may speculate that its action is mediated through binding to the
steroid-responsive element (DR-2 sequence) in the 3'-enhancer region of
the Epo gene (25).
Based on the findings presented herein together with the current
knowledge from the literature, it can be proposed that
H2O2 acts as a negative signaling molecule
connecting O2-sensitive hemeproteins with the Epo gene
transcription factor(s). The lack of H2O2 in
hypoxia allows for an increased Epo gene transcription. Possibly,
HIF-1 may be the oxygen sensor and/or the effector of
H2O2 function. Moreover, our data are in line
with previous reports of the important role of
H2O2 in other O2 sensor systems,
such as in preparations of the carotid body (26), pulmonary
neuroepithelial bodies (27), or pulmonary resistance vessels (28).
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Acknowledgments
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We acknowledge the excellent technical assistance of Mrs.
Urszula Frackowski. In addition, we like to thank PE Applied Biosystems
(Weiterstadt, Germany) for their support in setting up the TaqMan
system.
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Footnotes
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1 This work was supported by the Deutsche Forschungsgemeinschaft (SFB
367/C8). The paper presents parts of the M.D. theses of I.N. (protein
data) and B.S. (mRNA data). 
Received July 14, 1998.
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Abstract
Introduction
