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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3218-3225
Erythropoietin mRNA Expression in Human Fetal and Neonatal Tissue
By
Christof Dame,
Hubert Fahnenstich,
Patricia Freitag,
Dietmar Hofmann,
Thair Abdul-Nour,
Peter Bartmann, and
Joachim Fandrey
From the Department of Neonatology, University of Bonn, Bonn; the
Institute of Physiology, University of Bonn, Bonn; the Institute of
Pediatric Pathology, University of Bonn, Bonn; and the Institute of
Physiology, Medical University of Lübeck, Lübeck, Germany.
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ABSTRACT |
Based on animal experiments, a switch of the erythropoietin (EPO)
production site from the liver in the fetus to the kidneys in the adult
has been postulated. To study the switch in humans, we have quantitated
EPO mRNA expression in liver, kidney, spleen, and bone marrow of human
fetuses and neonates by means of a competitive polymerase chain
reaction (PCR). Tissue samples from 66 routine postmortem examinations
were obtained. EPO mRNA was expressed in 97% of the tissue specimen
derived from the liver (n = 66) and in 93% of those from the kidneys
(17 weeks of gestation until 18 months after birth; n = 59). For the
first time the EPO gene was found expressed in vivo in human spleen
(96% of 64 samples) and in fetal and neonatal bone marrow (81% of 21 samples). EPO mRNA expression in the kidneys increased significantly
beyond 30 weeks of gestation (P < .05). Although there was a
slight decrease in EPO mRNA content per g liver tissue towards birth,
the liver accounted for about 80% of the total body EPO mRNA. The
contribution of the spleen and bone marrow were minor compared with
liver and kidneys. Our results indicate that in humans the liver is the primary site of EPO gene expression not only in fetal, but also in
neonatal life. A significant increase of renal EPO mRNA expression after 30 weeks of gestation might indicate the beginning switch.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ERYTHROPOIETIN (EPO) stimulates the
proliferation and differentiation of erythroid precursors during fetal,
neonatal, and adult life.1 In adult rats, EPO is mainly
produced by the kidneys, as bilateral nephrectomy almost abolished the
production of the hormone.2 For humans the role of the
kidney in EPO production is obvious, because patients with chronic
renal failure suffer from an anemia caused by EPO
deficiency.3,4 Recombinant human EPO corrects this
anemia.4,5
From animal experiments it was concluded that during fetal life the
liver is the primary site of EPO synthesis.6,7 In humans,
normal or even elevated EPO concentrations in cord blood of neonates
with bilateral renal agenesis have been reported.8,9 Because in adults the kidneys are the site of EPO synthesis, a switch
of the production site from the liver in the fetus to the kidneys in
the adult has been postulated. After the switch hepatic EPO synthesis
appears to be too low to compensate for the loss of renal EPO
production in chronic renal failure.10
Animal studies on nephrectomized or hepatectomized fetuses or adult
animals of different mammalian species (rat, mouse, sheep) showed clear
differences in the onset and dynamics of the switch in relation to
gestational age and maturity.7,11-14 The characteristics of
fetal and neonatal hematopoiesis and EPO synthesis in sheep probably
most closely resemble the situation in humans15-17: in
sheep the switch of the EPO production site from the liver to the
kidneys appears to be genetically determined and begins in the last
trimester of pregnancy. The switch is obviously completed 6 weeks after
birth because the kidneys, like in the adult sheep, then contribute
about 85% and the liver about 15% to total EPO production.13,14
For human fetuses and newborns, the contribution of the different
organs to EPO production and the characteristics of the switch have not
yet been defined. In addition, elucidation of the change in the
production site may have clinical implications: besides a greater
clearance of EPO in human newborns, disturbances in the switch from the
liver to the kidneys have been made responsible for the low EPO
concentrations in the anemia of prematurity or the anemia during
replacement of fetal by adult hemoglobin in neonates born at
term.15,16 Because preformed EPO protein is not stored in
the tissue, but is de novo synthesized by expression of the EPO gene,
EPO mRNA levels in the tissue most likely reflect the contribution of
the different organs to total EPO production.1
We have first applied a sensitive reverse transcription-polymerase
chain reaction (RT-PCR) to detect EPO mRNA expression in samples from
different tissues such as liver, kidneys, spleen, and bone marrow of
human fetuses and infants. Second, by using a competitive PCR for EPO
mRNA, we have quantitated the levels of EPO mRNA in different
organs to obtain information about the contribution of the liver, the
kidneys, and the spleen to total EPO production during fetal life. Our
results allowed us to conclude on the proposed switch of EPO production
from the liver during fetal life to the kidneys in the adult.
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MATERIALS AND METHODS |
Patients
From January 1996 to June 1997, we collected tissue samples of 66 fetuses, neonates, and infants after written parental consent for
routine postmortem examination was obtained. The age ranged from 16 weeks of gestation to 18 months after birth. Tissue specimens were
obtained: (1) from 32 fetuses after elective termination of pregnancy
due to severe malformations or congenital disorders (gestational age,
16 to 24 weeks); (2) from 31 preterm and term neonates (22 to 41 weeks
of gestational age) with perinatal death (latest at day
9); and (3) from 2 term babies (death at 3 weeks and 7 months after
birth) clinically suffering from sudden infant death syndrome and from
1 18-month-old twin with diffuse lymph-hemangiomatosis.
Within these groups, the following diagnoses were made: spontaneous
abortion/intrauterine death (n = 5), amniotic infection syndrome
(n = 4), multiple pregnancy (one twin suffering from an acute
twin-twin transfusion syndrome, triplets suffering from a twin-twin
transfusion syndrome, quadruplets suffering from amniotic infection
syndrome; n = 8), malformations of the central nervous system
(Dandy-Walker malformation: n = 3, Arnold-Chiari malformation: n = 3, anencephalus: n = 2, complex malformation: n = 1), congenital renal
malformations (severe obstructive uropathy: n = 1, renal cysts (Potter
syndrome): n = 5, renal agenesis: n = 3), complex of multiple
malformations (n = 10), severe congenital heart failure (n = 1),
chromosomal abnormalities (trisomia 18: n = 2, trisomia 21: n = 5;
other: n = 2), death due to neonatal complications (sepsis,
disseminated intravascular coagulation, cardiopulmonary insufficiency,
shock, cerebral hemorrhage, pulmonary hemorrhage, nonimmunologic
hydrops fetalis) in preterm and term neonates (n = 6), sudden infant
death syndrome (n = 2, postmortem examination in one of them showed a
beginning pneumonia, acute splenitis and reduced erythropoiesis with
dominant myelopoiesis), osteogenesis imperfecta (n = 1), Ivemark
syndrome (n = 1), diffuse lymph-hemangiomatosis (n = 1).
Immediately after death, peripheral blood cell counts were determined
if possible. Blood was collected from a peripheral vein, from a cord
vessel, or the heart; serum was stored at  20°C.
Tissue biopsy specimens from liver, kidney, spleen, and bone marrow
from the vertebral body (without loss of the blood elements) were taken
during routine postmortem examinations at the Institute of Pediatric
Pathology. The time interval from death to biopsy ranged from 0 to 7 days (median, 3 days; mean, 3.1 days). To keep degradation as low as
possible, we have taken the following precautions: all deceased fetuses
and newborns were stored at +4°C until postmortem examination. All
tissue samples from a patient were taken immediately one after the
other to avoid exposure of the organs to room air and room temperature
after opening of the abdomen, which would possibly accelerate
autolysis. Because postmortem examinations were done at different time
points after death, it is of importance to assure the integrity of
mRNA. Tissue was snap frozen in liquid nitrogen immediately after
excision. Tissue of all organs was not available from every
patient. In 48 patients we obtained liver tissue
specimen from the left and the right lobe separately.
Laboratory Methods
Preparation of total RNA.
Frozen tissue samples were weighed and homogenized in guanidinium
thiocyanate solution (4 mol/L with 0.1 mol/L  -mercaptoethanol) using
a Polytron homogenizer (Kinematica GmbH, Lucerne, Switzerland) at
setting 10 for 20 seconds. Per gram tissue 10 mL guanidinium thiocyanate solution was added. Tissue homogenates were subsequently centrifuged at 4°C with 3,500 rpm for 5 minutes. The supernatant was frozen at 80°C until RNA isolation. For isolation of
total RNA, 700 µL of the supernatant was used to extract RNA with the acidic phenol-chloroform method.18 After redissolving the
RNA in diethyl pyrocarbonate-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.
RT of RNA into cDNA.
A total of 5 µg of total RNA was reverse transcribed into first
strand cDNA using oligo-dT15 as primer for the RT Moloney murine leukemia virus (M-MLRV) RT-superscript (GIBCO Life
Technologies, Eggenstein, Germany) in a reaction volume of 25 µL.
After initial denaturation (68°C for 10 minutes) RT was performed
at 42°C for 60 minutes and terminated by boiling the samples for 10 minutes. Until quantitation by competitive PCR, cDNA stocks were kept
at 20°C.
Competitive PCR.
For quantitation of EPO cDNA, a competitive PCR was performed as
described earlier.19 The use of the internal standard
allows determination of the absolute amount of EPO cDNA. PCR products of the internal standard generated from full-length EPO genomic DNA
could be distinguished from cDNA-derived PCR products because of
different restriction fragments after cutting with Acc I and HindIII.19 Known molecular quantities of the
standard DNA were spiked into a series of PCR reaction tubes containing
equal amounts of EPO cDNA. PCR was performed in PCR buffer (50 mmol/L
Tris/HCl pH 8.3, 1.5 mmol/L MgCl2, 0.001% mass/vol
gelatine), 200 µmol/L of each deoxynucleotide (dNTP),
300 nmol/L of each 5 primer (5-CTG CTC CAC TCC GAA CAA
TCA C-3) and 3 primer (5-CTG GAG TGT CCA TGG GAC
AG-3) and 2 U/mL of Taq polymerase (GIBCO) in a final volume of
50 µL. PCR was run for 30 to 35 cycles after an initial denaturation
step at 94°C for 3 minutes with an amplification profile of each
cycle consisting of denaturation for 1 minute at 94°C, primer
annealing for 1.5 minutes at 58°C, and elongation for 3 minutes at
72°C. PCR products were run on a 3% agarose gel and made visible
by ethidium bromide (0.5 µg/mL) staining. Calculation of EPO
mRNA/µg total RNA was performed exactly as described.19 Each sample was checked for possible contamination by genomic DNA or
PCR products from previous amplifications. The lower detection limit of
the quantitative PCR was 0.03 attomol (amol) (amol = 10 18 mol) of competitor.
In addition, a qualitative PCR for
glyceraldehyde-phoshate-dehydrogenase (GAPDH) was performed using a
5-primer (5-ATC ATC CCT GCC TCT ACT GG-3) and a
3-primer (5-TGG GTG TCG CTG TTG AAG TC-3) under
the same conditions as descibed above for 30 cycles at an annealing
temperature of 55°C.
Measurement of hemoglobin and EPO concentrations and blood cell
counts.
EPO concentrations were measured with a commercial enzyme-linked
immunoassay (EPO-ELISA; Medac, Hamburg, Germany). Extinction was read
at 405 nm (DYNATECH MR 5000; Dynatech Laboratories, Denkendorf, Germany; reference wavelength for the measurements: 630 nm). EPO concentrations were calculated (BioLinx 2.20; Dynatech Laboratories) from a calibration curve based on EPO standard concentrations (1.25, 2.5, 10, 40, 80, 160 U/L recombinant human (rHu) EPO
prepared according the Third International Standard, World Health
Organization [WHO], 1990).
Hemoglobin concentration and erythrocyte count were determined with an
automatic cell counter Celldyn 1500; Abott, Wiesbaden, Germany).
Reticulocytes were stained by Brilliantkresyl and counted.
Statistical calculations.
From the amount of total RNA isolated, we calculated the total amount
of RNA in the respective tissue samples. This result was related to the
weight of the total organ, expressed as total RNA in micrograms per
gram of tissue. Taking into consideration the efficiency of our RT
reaction,19 we calculated the amount of EPO mRNA in
amol per gram of tissue. From the organ weights, the
amount of EPO mRNA per organ and the respective contribution of this
organ to total body EPO mRNA were calculated.
Data are presented as median and range. Statistical analysis was
performed by the SPSS 6.1.3 software program for Windows (SPSS,
Inc, Munich, Germany). We applied the Mann-Whitney-U test (2-tailed), Student's t-test or Wilcoxon matched-pairs signed ranks as indicated to evaluate statistical differences. A P
value  .05 was considered statistically significant.
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RESULTS |
Stability and Integrity of RNA
Total RNA was isolated from specimen obtained at routine postmortem
examination (Fig 1A). RNA appeared to be of
comparable quality in all organs that were subsequently analyzed for
quantitative EPO gene expression (Fig 1B). This is in agreement with
earlier reports of the successful isolation of mRNA from pancreatic
tissue notoriously rich in RNAses.20 Shorock et
al20 sampled tissue from human adults and
fetuses 48 hours after death at routine postmortem examinations, which
was of sufficient quality to show good in situ hybridization signals
for insulin. The investigators reported that "the degree of staining
in these cases (ie, the postmortem tissue) was comparable to that seen
in surgical material."20 Even if some degradatation of
28S RNA may be visible on a denaturing agarose gel stained with
ethidium bromide, mRNA was still intact to be translated into protein
in an in vitro system.21
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| Fig 1.
(A) RNA was extracted from tissue biopsy samples obtained
at the indicated time intervalls after death at routine postmortem
examinations. A total of 10 µg total RNA was separated on a 1.1%
agarose gel with formaldehyde and stained with ethidium bromide (0.5 µg/mL). Below the gel the values from EPO mRNA quantitation
(in amol per microgram of total RNA) by competitive
PCR are given. (B) RNA extracted from tissue samples of different
organs taken 4 days after death was run on an agarose gel as
described for (A).
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A representative agarose gel of a RT-PCR for EPO mRNA detected in the
different organs of one patient is shown in
Fig 2. EPO mRNA was detected in most tissue
specimen derived from liver, kidney, spleen, and bone marrow. The
percentage of positive samples ranged from 81% to 97% of the
respective total number of samples for each organ and spanned the time
from 16 weeks of gestation up to 18 months of life
(Table 1). No significant correlation was
found between the amount of extracted RNA per g tissue from liver,
kidney, spleen, or bone marrow or the amount of EPO mRNA per µg total
RNA and the time interval between death and biopsy. Failure to detect
EPO mRNA by PCR was confirmed two times for each sample, but only
occurred in a single tissue sample of one organ. Other tissue samples
from the same patient were positive for EPO mRNA. Samples negative for
EPO mRNA were positive for GAPDH mRNA. PCR signals for GAPDH mRNA
appeared equal after 30 cycles from samples of the different organs
examined even when postmortem analysis was performed 5 or 6 days after
death (Fig 3).
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| Fig 2.
Representative ethidium bromide stained 3% wt/vol
agarose gel showing RT-PCR signals for EPO mRNA in liver, kidney,
spleen, and bone marrow tissue. MWM, Molecular weight marker (100-bp
ladder); RT, negative control for RT reaction (all RT reagents, but
no RNA); H2O, negative control for PCR reaction (all PCR
reagents, but no cDNA).
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| Fig 3.
DNA fragments of a qualitative PCR for GAPDH separated on
an ethidium bromide stained 3% wt/vol agarose gel. Tissue samples were
from routine postmortem examinations obtained at 5 or 6 days after
death.
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EPO mRNA expression in the liver.
In 49 cases tissue biopsy specimens were obtained both from left and
right liver lobe. EPO mRNA was detected in 41 (83%) of the samples
from the left lobe and 46 (93%) of the samples from the right lobe. In
only 1 patient PCR for EPO mRNA was negative in both lobes. For none of
the calculated parameters (calculated as the median [and range]) for
total RNA extracted in micrograms per gram of tissue (left: 2,171 µg/g [675 to 4,771]; right: 2,185 [556 to 3,900]), EPO mRNA
amol per microgram of total RNA tissue (left: 2.90 amol/mg
total RNA [0.12 to 290.00]; right: 2.90 amol/µg total RNA [0.12 to
145.00]), or EPO mRNA/g tissue (left: 8,932 amol/g [43 to 119,605];
right: 7,094 amol/g [234 to 387,357]) no significant difference was
found between the right and the left liver lobe using Wilcoxon matched
pairs signed ranks. For further statistical analysis with these cases,
we calculated the mean value of EPO mRNA and total RNA from both liver
lobes. The amount of EPO mRNA did not correlate with weeks of gestation
or postnatal age, EPO serum concentration, hemoglobin concentration, or
the time interval between death and autopsy.
EPO mRNA expression in the kidneys.
The amount of EPO mRNA expression per microgram of total RNA and per
gram of renal tissue was significantly lower (P = .001) than in
the liver. There was no significant difference between the kidneys and
the spleen or the kidneys and the bone marrow (Fig 4). In the kidneys, the amount of EPO
mRNA per microgram of total RNA and per gram of tissue increased with
gestational age. It was significantly lower in fetuses with a
gestational age 30 weeks when compared with older fetuses, neonates
or infants (Table 2). EPO mRNA expression
showed no relation to other parameters such as EPO serum concentration,
hemoglobin concentration, or age.
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| Fig 4.
EPO mRNA (amol per microgram of total RN)
in the liver, kidney, spleen, and bone marrow tissue samples. Data are
presented as boxplots with the median and the 25th and 75th percentile
defining the box. The error bars indicate the 10th and 90th percentile,
respectively. Single data points ( ) with their respective values
that lie outside the 10th and 90th percentile are also
shown. Additional values (not shown) were for the liver
218, 45, and 19 amol/µg total RNA and for the kidneys 731 and 290 amol/µg total RNA.
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EPO mRNA expression in the spleen and bone marrow.
In the spleen, values for EPO mRNA per µg extracted total RNA and per
g of tissue were the lowest of all organs studied (Table 1).
Statistical analysis of EPO mRNA expression in the bone marrow was
limited, as it is impossible to determine the total weight of the bone
marrow. The total amount of RNA per gram of tissue was low, but the
median of EPO mRNA per microgram of extracted total RNA was not grossly
different from that in kidneys or spleen. For both the spleen and the
bone marrow, no correlation of EPO mRNA levels with gestational or
postnatal age was found.
EPO serum concentration.
EPO protein concentrations in the serum ranged from 4 to 12,336 mU/mL
(median, 16 mU/mL). As reference values for serum EPO concentrations in
early fetal gestation, only data from living fetuses (by fetal
umbilical cord blood sampling) and preterms are available. In this
group with hemoglobin concentrations in the normal range, EPO serum
concentrations of 40 mU/mL are considered normal.22,23
Herein, in all cases with gestational age less than 20 weeks EPO
concentrations were lower than 40 mU/mL. About 30% of the samples from
patients with a gestational age of 20 weeks or more had elevated EPO
concentrations. No correlation was found between total body EPO mRNA or
EPO mRNA content in the liver, kidneys, or spleen and EPO serum
concentrations. For this calculation, two cases with extremely high EPO
serum concentrations (case A: 3,586 and case B: 12,336 mU/mL) were
excluded. Both patients died after extensive, but unsuccessful,
attempts of resuscitation. Longlasting tissue hypoxia may have caused
the extremely high EPO concentrations, which were accompanied by high
EPO mRNA levels in the liver (case A: 217 amol EPO mRNA/µg total;
case B: 45 amol EPO mRNA/µg total RNA) and the kidney (case A: 290 amol EPO mRNA/µg total RNA; case B: 731 amol EPO mRNA/µg total
RNA). Therefore, these two cases were excluded to avoid
overinterpretation of the data by calculating a statistically
significant correlation between EPO mRNA and protein.
In addition, total body EPO mRNA did not correlate with reticulocyte
counts. No correlation was found between reticulocyte counts and EPO
concentrations. Because of the heterogeneity of diagnoses, we were not
able to determine any qualitative or quantitative changes in EPO gene
expression associated with a clinical or postmortem diagnosis.
Relative organ contribution to total body EPO mRNA content.
Considering the EPO mRNA contents (in amol per gram of
tissue) and the organ weights, the total body EPO mRNA content and the
fractional contribution of the liver, the kidneys, and the spleen to
this value were calculated. Results are shown in
Fig 5 where mean values and the 95%
confidence intervals of 55 cases are presented. By far, the liver was
the dominant organ with respect to EPO mRNA expression. The fraction of
the kidneys contributing to total EPO mRNA expression was significantly
higher in cases with a gestational age greater than 30 weeks (mean,
14.3% v. 6.0%; P = .04; Student's t-test).
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| Fig 5.
Mean and 95% confidence interval (in percent of the
total body EPO mRNA) for the fractional contribution of liver (),
kidneys ( ), and spleen ( ) for fetuses younger than 30 weeks of
gestation, between 31 and 40 weeks and older than 40 weeks.
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DISCUSSION |
During human fetal and neonatal development, erythropoiesis undergoes
substantial genetically determined changes, which are reflected by
different organ sites for red blood cell production, altered erythroid progenitor morphology, and functionally, as well as
structurally distinct types of hemoglobin. The glycoprotein hormone,
EPO, appears to play a dominant role for adequate embryonic and fetal
erythropoiesis in humans. Mice in which the EPO gene or the EPO
receptor gene have been knocked out die in utero with clear signs of
failure of erythropoiesis in fetal liver in early gestation.24 Erythroid precursor cells (burst-forming
unit-erythroid [BFU-E] and colony-forming unit-erythorid
[CFU-E]) in fetal mice express high numbers of EPO
receptors and need erythropoietin for maturation, differentiation, and
prevention of an early apoptotic cell death. Moreover, the sensitivity
of the erythrocytic progenitors to EPO in mice is higher in embryonic
or early fetal life than in older fetuses or adults.25
Nevertheless, as in the adult, EPO production is regulated by tissue
PO2 because EPO serum concentration is increased in anemic or hypoxic human fetuses.23,26 EPO is not intracellularly
stored, but de novo synthesized on hypoxia in the EPO producing
tissues.1 Tissue EPO mRNA levels appear to reflect
the contribution of the respective tissue to overall EPO
production.
From organ ablation studies in animals, evidence can be derived that
the liver is the primary site of EPO production in fetal life.6,12-14 Several experimental studies in different
mammalian species (mouse, rat, sheep, cat, dog, monkey, and pig) in
which EPO mRNA levels were determined in kidneys and the liver at
different stages of gestation have supported this
notion.11,17,19,27-32 For humans, circumstantial evidence
of hepatic EPO production during fetal life was provided by the finding
that fetuses/neonates suffering from bilateral renal agenesis had
normal or even elevated EPO serum concentrations.8,9 To our
knowledge, only scarce data are available on EPO gene expression in the
human fetus. In three preliminary reports, EPO gene expression was
detected in liver, kidneys, and brain of human fetuses with a
gestational age between 11 to 24 weeks.33-35 Calhoun et
al,36 who focussed on the contribution of the spleen in
erythropoiesis, found no EPO mRNA in the spleen. To eludicate possible
sites of EPO synthesis and their contribution to overall EPO
production, we systematically screened liver, kidneys, spleen, and bone
marrow in a considerable number of samples by means of competitive PCR.
We were able to detect and quantitate EPO gene expression not only in
liver and kidneys, but also in spleen and bone marrow of human fetuses
and neonates.
Erythropoietin gene expression in human fetal and neonatal liver.
EPO transcripts were expressed in 97% of liver samples from fetuses
between weeks 16 and 40 of gestation, from neonates, and from two
children who died at 7 and 18 months of age. EPO mRNA expression per
gram tissue in the liver was the highest of all organs examined.
Considering the size of this organ, it clearly determines the liver as
the dominant site for EPO expression in fetal and also in neonatal
life. Hepatic EPO gene expression in the mouse was localized in
hepatocytes surrounding central veins and in fibroblast-like Ito
cells.37,38 Both types of cells are functionally developed
as early as the eighth week of gestation in human
embryogenesis.39 Therefore, the histological/morphological development of the human liver makes it conceivable that expression of
EPO after 16 weeks of gestation may be localized in the same cells in
humans as well.
Our samples have been derived from postmortem examinations. Because
fetal circulation differences in the perfusion of the left and the
right liver lobe have been described,40 we quantitatively compared EPO mRNA expression in the left and right liver lobe. We did
not find significantly different EPO mRNA levels between the two lobes.
Therefore, in case the perfusion of the two liver lobes was different,
this had no impact on EPO gene expression. This finding is in agreement
with data from animal studies in which EPO mRNA was present in all
lobes of the liver to a similar degree.41 One may conclude
that in humans, as well, EPO mRNA is uniformly expressed in the lobes
of the liver.
Erythropoietin expression in human fetal and neonatal kidneys.
We found the EPO gene expressed in human fetal kidney tissue as early
as 17 weeks of gestation. Using PCR, EPO mRNA has been detected very
early in gestation in fetal rat and sheep, at a time when renal EPO
production was not expected considering the results of organ ablation
studies.6,14,32 In the present study, human EPO mRNA
expression per g renal tissue increased significantly beyond 30 weeks
of gestation. Although in fetuses and neonates the amount of EPO mRNA
per gram of renal tissue is less than one tenth of that in the liver at
that age, the steep increase in renal EPO expression after 30 weeks is
of note. EPO gene expression in the kidney of mouse, sheep, and humans
has been localized in the interstitial cells between the proximal tubulus.28,29,42-44 At 17 weeks of gestation, the earliest
time at which we have quantitated EPO mRNA in this study, the
metanephros develops. This process begins around the eleventh week of
gestation.45 Therefore, comparable to fetal
sheep,32 the human fetus is able to express EPO mRNA in the
metanephros. In humans, morphogenesis of the kidney with respect to
formation of nephrons and the ampulla is completed around 32 weeks of
gestation. Thereafter, enlargement of the existing structures and the
interstitial tissue is dominant.45 One may assume that
according to the morphologic and histologic development, the increase
in EPO mRNA expression (amol per gram tissue) after 30 weeks of gestation results from the increasing number of cells that
express the EPO gene.43
Erythropoietin expression in human fetal and neonatal spleen and bone
marrow.
Together with liver and kidney, EPO mRNA was detected in
fetal spleen and bone marrow throughout gestation. So far, the
expression of the EPO gene in the spleen has not been reported for
humans or sheep, but for adult rat19 and
mice.46 Calhoun et al36 did not detect any EPO
mRNA in spleen tissue from six human fetuses with a gestational age of
14 to 22 weeks. Because our earliest samples were from gestational week
16, it is unlikely that gestational age accounts for the different
results. Our results from bone marrow samples strengthen the notion
that the EPO gene is in fact expressed in cells in this tissue. Mouse
macrophages taken from the bone marrow have been reported to produce
EPO47 and might be responsible for the EPO mRNA content in
this organ. In addition, a very recent report indicates that
CD34+ early erythroid progenitors themselves may express
the EPO gene.48 Whether EPO expression plays an important
role as an autocrine growth factor in the human bone marrow, as it has
been suggested by Hermine et al49 remains to be studied.
Characteristics of the "switch" of the primary EPO production
site.
From animal studies, it is known that although the liver is the primary
site of EPO production during fetal life, this function is gained by
the kidneys in the adult. However, the time at which the switch occurs
varies from species to species.14,17,29 To characterize the
switch from the liver to the kidneys in humans, it is mandatory to know
the fractional contribution of each organ to the total body EPO mRNA
content. For the first time, this was realized by means of the
competitive RT-PCR for EPO mRNA. In all fetuses and neonates of this
study, over 80% of total EPO mRNA was localized in the liver. The 95%
confidence interval for the contribution of the kidneys until week 30 of gestation to total EPO mRNA did not exceed 9%. Thereafter, the
kidneys contributed up to 27% of total EPO gene
expression. This was mainly due to the increase in EPO mRNA per gram of
kidney tissue, while the liver only showed a very moderate decrease in
EPO expression. At birth, the dominant organ for EPO expression in
humans therefore appears to still be the liver. Assuming that the
changes in EPO gene expression continue in a similar way, the switch in
humans should be completed not before several months after birth.
Finally, it is of note that in fetuses with bilateral renal agenesis,
no increase in hepatic EPO mRNA content was observed. Expression in the
liver appears to be sufficient and no need to compensate for the lack
of renal EPO production arises. The liver clearly is the primary EPO
production site during human gestation, whereas in adults, EPO
production is located in the kidneys. Therefore, in humans as in other
species, EPO expression switches from the liver to the kidneys.
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FOOTNOTES |
Submitted February 17, 1998;
accepted June 24, 1998.
Address reprint requests to Christof Dame, MD, Department of
Neonatology, University of Bonn, Adenauerallee 119, D-53113 Bonn, Germany.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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Abstract
Introduction
Abstract