/. Embryol. exp. Morph. Vol. 61, pp. 165-173, 1981
Printed in Great Britain © Company cf Biologists Limited 1981
165
Role of the kidney in foetal erythropoiesis:
Erythropoiesis and erythropoietin levels in newborn
mice with renal agenesis
By PER HAGA 1 AND STEIN KRISTIANSEN 2
From the Paediatric Research Institute, National Hospital of Norway, and
the Departments of Paediatrics and Pathology, Ullevdl Hospital, Oslo
SUMMARY
The role of the kidney in foetal erythropoiesis was studied in newborn SD mice on the day
of birth. Some of the homozygotes and heterozygotes of this strain are born anephric. Red cell
production was evaluated by haematocrit levels, reticulocyte counts, and Fe59-uptake in liver
and RBC, the isotope given to the mothers during pregnancy.
Erythropoiesis of the newborn with renal agenesis was not different from that of animals
with intact kidneys. When the mothers were exposed to hypoxia during pregnancy, significantly higher haematocrit- and reticulocyte levels were observed, and there was no difference
in erythropoiesis of anephric newborn compared with newborn with intact kidneys. Red cell
production was also similar in those with and without kidneys when the mothers were
hypertransfused.
Plasma erythropoietin levels in the offspring of normal pregnancies were determined.
Delectable concentrations of the hormone were found, and the levels were the same in
anephric and normal newborn. Exposure to hypoxia (0-5 atm for 6 h) significantly increased
plasma erythropoietin levels. This increase was of the same magnitude in animals with and
without kidneys.
This study indicates that murine foetal erythropoiesis is regulated by erythropoietin in the
same way as later in life. Since abolition of the erythropoiesis of the mothers through hypertransfusion, did not influence the red cell production of the foetuses, Ep seems not to cross
placenta. Erythropoietin is, therefore, produced extrarenally during this period.
INTRODUCTION
Several studies indicate that erythropoietin (Ep) is the main regulator of
erythropoiesis during mammalian foetal life (Finne, 1964; Zanjani, Poster,
Mann & Wasserman 1977). The most conclusive evidence has been the works
of Zanjani et al. (1977) on sheep and goats during the last third of pregnancy.
In these mammals, and also in man, erythropoiesis takes place in the bone
marrow during the last part of pregnancy, while mice and rats are more immature in this respect, since at the time of birth, their red cell production is still
1
Author's address (for reprints): Department of Paediatrics, Ulleval Hospital, Oslo 1,
Norway.
2
Author's address: Department of Microbiology, National Hospital of Norway, Oslo 1,
Norway.
166
p. HXGX AND S. KRISTIANSEN
predominatly hepatic. Foetal erythropoiesis in mice has been shown to be
independent of maternally produced Ep (Jacobsen, Marks & Gaston, 1959), and
increased rates of erythropoiesis in foetuses of rats subjected to hypoxia or
bleeding have been observed (Matoth & Zaizov, 1971). In vitro culture of cells
from yolk sac (Bateman & Cole, 1971) and foetal liver (Cole & Paul, 1966;
Stephenson, Axelrad, McLeod & Shreeve, 1971) from the mouse has shown
these cells capable of haem production and differentiation when stimulated by
Ep. Recently, we have in our laboratory been able to detect significant concentrations of Ep in plasma from rat foetuses and newborn mice on the day of
birth (Meberg, Haga & Halvorsen, 1979; Haga & Falkanger, 1979). These
observations indicate a regulatory role for Ep during rodent foetal life.
The kidney is well established as the primary erythropoietin producing organ
in the mammalian adult, while the ability to produce Ep externally varies from
species to species. Chance observations in human newborn with renal agenesis
show that foetal erythropoiesis may proceed normally despite the absence of
nephric tissue (Mauer, Dobrin & Vernier, 1974; Halvorsen, Haga & Halvorsen,
1975), while nephrectomized foetal sheep and goats produce Ep as well as
foetuses with intact kidneys (Zanjani et al. 1977). This suggests that sites, other
than the kidney, are the major production sites for Ep during foetal life in these
species.
Nephrectomy of neonatal rats has been found to have little effect on erythropoiesis (Lucarelli, Howard & Stohlman, 1964), or on the Ep response to
hypoxia (Carmena, Howard & Stohlman, 1968; Gruber et al. 1977), and suggests
that, also in rodents, foetal erythropoiesis may be independent of renally
produced Ep.
All these experiments have, however, been performed with such a mutilating
procedure as nephrectomy, and the results must be evaluated in this context. An
almost ideal model to study the role of the foetal kidney in the regulation of
erythropoiesis would be animals with renal agenesis. The SD strain of mice
offers such a model. Some of the homozygotes and heterozygotes of this strain
are anephric (Gluecksohn-Schoenheimer, 1943). The present study was undertaken to answer the following questions: 1. Does erythropoiesis proceed at
a normal rate in anephric foetuses during the hepatic stage of erythropoiesis ?
2. Is Ep involved in the regulation of this erythropoiesis ?
MATERIAL AND METHODS
Adult heterozygous SD mice were kindly supplied by MRC-Berks, England,
and mated in our laboratory. Newborn animals less than 24 h were examined.
Heterozygotes and homozygotes of the SD strain have, in addition to urogenital
malformations, skeletal malformations of which a short tail or no tail is the
most conspicuous. To rule out the presence of nephric tissue, the following
procedure was followed: After blood was obtained, the animals that did not
Role of the kidney in mouse foetal erythropoiesis
167
void any urine during the blood sampling, were dissected under a stereo microscope. Serial transverse sections of the posterior abdominal walls were made of
those animals in which stereo microscopy failed to locate any kidney(s).
These slides were examined microscopically, and the newborn with no
demonstrable kidney tissue were judged anephric. The number of animals that
were anephric or had ectopic kidneys varied considerably from litter to litter,
with some litters containing none. Table 2 shows all newborn of 13 litters,
except for four dead foetuses, and it is seen that approximately a quarter of the
newborn had these major urogential malformations.
Normal erythropoiesis
Weights of the newborn mice were recorded, and blood was obtained by
severing the cervical vessels. Haematocrit levels were measured by a standard
technique in micro-haematocrit tubes filled to a calibration mark (Red-Tip no.
8889-302009, Sherwood, St Louis, Mo., U.S.A.). The reticulocytes were counted
after staining with brilliant cresyl blue.
Fe59, in a dose of approximately 1 /^Ci, was given subcutaneously to the
mothers towards the end of the pregnancy on two consecutive days. In our
hands it was difficult to make this strain reproduce. For this reason, a female
and a male were caged together for 3 days, and the exact date of conception was
therefore unknown. Thus all the litters did not receive the iron on exactly the
same days of pregnancy, and some mothers received only one injection. Because
of this, the Fe59-uptake of individuals within each litter has been compared, the
littermates having kidneys serving as controls. The radioiron uptake of liver and
red blood cells (RBC) was calculated as counts per gram liver weight or blood
volume respectively, divided by total body count per gram bodyweight. The
blood volumes of the newborn mice were assumed to be 10 % of the bodyweights.
Exposure to hypoxia during pregnancy
One female was caged with one male for 3 days, then removed from the
partner, and from 4 days later until the termination of the pregnancy she was
exposed to intermittent hypoxia (0-5 atm, 8 h a day). In this way, of the ten
litters examined, five were exposed to hypoxia for 14 days, three for 15 days, and
two for 16 days. The procedures were otherwise as described for normal erythropoiesis.
Hypertransfusion during pregnancy
After a mating period of 3 days, the mothers received an intraperitoneal injection of 1-0 ml of packed homologous red blood cells on two consecutive days.
Transfusions (0-5 ml of packed RBC) were thereafter given every other day
until the end of pregnancy. The RBC transfused were collected into heparin, the
plasma removed and the cells washed twice in 0-9 % NaCl before being injected.
168
p. HAGA AND S. KRISTIANSEN
Table 1. Haematocrit levels, reticulocyte counts, and weights (means ±S.D.) of
newborn SD mice, born after normal pregnancies and to mothers exposed to
intermittent hypoxia during the pregnancies. Numbers of animals investigated in
parentheses
Normal pregnancies
Anephric Kidney(s) +
Hypoxic pregnancies
Ectopic Anephric Kidney(s) +
kidney
Ectopic
kidney
Haematocrit (%)
43-2±4-9 44-2±5-2 42-9±4-8 43-4± 12-3 46-6±7-9 49-2±6-3
(20)
(85)
(10)
(7)
(45)
(6)
Reticulocytes (%) 55-2±5-2 54-2±7-9 55-1 ±3-5 701 ±10-2 670±7-6 610±4-9
(17)
(44)
(8)
(7)
(40)
(6)
Weight (g)
l-36±0-14 1-43 + 0-17 l-32±015 1-21 ±013 1-25±0-13 106±010
(20)
(83)
(9)
(8)
(51)
(7)
At the time of birth these mothers had tail haematocrits of 69 + 4 % (n = 9)
and were reticulocyte-free.
The procedures were otherwise as described under normal erythropoiesis,
except that radioiron incorporation was not evaluated.
Erythropoietin concentrations in plasma
A recently developed cell culture method (Haga & Falkanger, 1979) for
plasma erythropoietin determinations was used. Blood was collected into
micro-haematocrit tubes from newborn at the day of birth; as a rule the animals
were only a few hours old. The plasmas from the possibly anephric animals were
not pooled until they were proven anephric, while the other plasmas were
pooled after collection. Other litters were together with their mothers, exposed
to hypoxia (0-5 atm for 6 h), blood being obtained from the newborn immediately afterwards. Plasma samples from 18 anephric animals were pooled
for the determination of the normal Ep level. Similarly, plasmas from 18
anephric mice were pooled to determine the concentration after hypoxic
exposure. The Ep levels were compared with the levels in newborn mice with
intact kidney(s), as well as with the concentrations of Ep in newborn from
another strain (WLO). The plasma concentrations used in the cultures were
50/d/ml.
The Ep levels found are expressed as the number of CFU e /cell number
plated in the culture dishes. A Connaught step-III preparation of erythropoietin
was used as standard.
Student's t-test was used for the statistical evaluation.
Role of the kidney in mouse foetal erythropoiesis
169
•
150
•
• •
:
§ 100
a>
• •
- •
•
• •
• •
*•
•
• •
•
••*
•
50
•
RBC
Liver
59
Fig. 1. Fe -uptakes in livers and RBC of 15 anephric newborn mice. The values
are expressed as the percentage of the mean uptake of their Httermates with
kidneys. The horizontal lines show the average! 2 S.D. of the 8 litters with kidneys
examined. The isotope was given to the mothers at the end of the pregnancies.
RESULTS
The microscopic examinations of the serial sections of the posterior abdominal
walls, revealed that quite a few of the animals thought to be anephric has a small
kidney placed in the midline behind the bladder. Data from this group is also
presented, and termed 'ectopic kidney'.
Normal erythropoiesis
Table 1 shows the haematocrit levels, reticulocyte counts, and weights oi
anephric and normal newborn as well as those with ectopic kidney. There is no
difference in haematocrit levels nor in reticulocyte counts between the newborn
without kidney tissue and those with nephric tissue. Although the weights of
those with normally located kidney(s) tended to be a little higher, the differences were not statistically significant.
The Fe59-uptakes in liver and red blood cells of the anephric newborn,
expressed as the percentage of the mean uptake of their normal Httermates, are
shown in Fig. 1. The Fe59-incorporations are not statistically different in the
normal and anephric animals.
170
p. H A G A A N D S. K R I S T I A N S E N
Table 2. Haematocrit levels, reticulocyte counts, and weights {means ± S.D.) of
newborn SD mice. The mothers were hyper transfused during the pregnancies.
Numbers of animals investigated in parentheses
Anephric
Haematocrit (%)
Reticulocytes (%)
Weight (g)
41-8 + 50
(12)
59-4 ±7-0
(12)
116 + 016
(12)
Kidney(s) +
43-8 ±5-5
(59)
62-8 ±7-8
(32)
1-20 + 015
(61)
Ectopic kidney
38-3 ±6-3
(6)
65 6± 15 1
(6)
1-24 + 0-18
(6)
Exposure to hypoxia during pregnancy
Exposing the mothers to intermittent hypoxia during the last 2 weeks of
pregnancy produced no differences in haematocrit levels or reticulocyte counts
between anephric and normal newborn (Table 1). The weights of the anephric
and normal newborn were similar, while those with ectopic kidney had significantly lower weights (Table 1) (P < 0-001). Compared with the offspring of
normal non-hypoxic pregnancies, all three groups of newborn weighed less.
When all the animals born after hypoxic exposure were compared with the
offspring of normal pregnancies, hypoxia was shown to cause a small but
significant increase in haematocrit levels (P < 0-02) and a pronounced increase
in reticulocyte counts (P < 0-001).
The Fe59-uptakes in liver and RBC of six anephric newborn of mothers
exposed to hypoxia were not different from their litter-mates with kidneys.
Hypertransfusion during pregnancy
As shown in Table 2 hypertransfusion of the mothers during the pregnancies
caused no difference in haematocrit levels or reticulocyte counts between
anephric newborn mice and their counterparts having kidneys, nor did their
weights differ. They did, however, weigh significantly less than the offspring of
normal pregnancies.
When all animals born to hypertransfused mothers were compared with newborn of normal pregnancies, it was shown that hypertransfusion of the mothers
did not alter the haematocrit levels of the newborn, but produced a significant
increase in reticulocyte counts (P < 0-001).
Plasma erythropoietin concentrations
Figure 2 depicts the plasma erythropoietin levels of anephric newborn on the
day of birth, compared with the levels in their normal counterparts, as well as
their response to a period of hypoxia (0-5 atm for 6 h). The levels of newborn
of another strain of mice are also shown. Newborn normal mice have detectable
Role of the kidney in mouse foetal erythropoiesis
171
3800 i-
3400 -
3000
1200 -
800 -
400 -
SD
kidney +
WLO
Fig. 2. Erythropoietin concentrations in plasma on the day of birth (open bars) in
anephric mice (SD —) and in newborn with intact kidneys (SD + ). Ep levels in
newborn of the normal mouse strain WLO are also shown. The plasma concentrations of Ep in response to hypoxia (0-5 atm for 6 h) after birth are shown as crossed
bars. Connaught step-HT erythropoietin (50 mU/ml) was used as standard. Ep concentrations are expressed as the number of CFUe formed in the culture plates. The
plasmas were added to the cultures in a concentration of 50/<l/ml. The numbers
within the bars are the number of culture plates counted.
concentrations of Ep (P < 0001) at birth, and the level is the same in anephric
newborn. After hypoxia, a substantial increase in Ep concentrations occur
(P < 0-001), and the levels are the same whether the animals have nephric tissue
or not.
DISCUSSION
Zanjani et al. (1977) found that foetal erythropoiesis in sheep and goats
during the last third of pregnancy was regulated by Ep both under normal and
hypoxic conditions in a manner similar to that later in life. The Ep was produced
by the foetus itself, however, not in the kidney, but in the liver. The present
study shows clearly that foetal erythropoiesis in mice may also proceed independently of the presence of nephric tissue, both under normal and hypoxic
conditions. Hypoxia during pregnancy caused increased erythropoiesis in the
foetuses, as expected if the regulatory mechanisms are the same in this period
as later in life. In concordance with earlier data (Jacobsen et al. 1959), red cell
production of the foetuses was not reduced after hypertransfusion of the
172
p. H&GA AND S. KRISTIANSEN
mothers. Transfer of maternally produced Ep to any significant degree was thus
unlikely. The newborn mice, both with and without kidneys, had detectable
plasma concentrations of Ep at birth, and responded with increased levels after
exposure to hypoxia (Fig. 2). The present findings thus indicate that murine
erythropoiesis in the foetal period is regulated by Ep in a similar way as later in
life. In the foetus, however, Ep is produced extrarenally. The results of this study,
are in agreement with the findings of Zanjani et al. (1977), but extend them to
the hepatic stage of foetal erythropoiesis. Jacobsen et al. (1959) found increased
haematocrit levels in the offspring of hypertransfused mothers compared with
the foetuses born after normal pregnancies, and attributed this to irondeficiency anaemia in the laboratory animals studied. In the present study, the
haematocrit levels were similar in the newborn of hypertransfused and normal
mothers. The reticulocyte counts, however, were significantly higher in the offspring of hypertransfused mothers. This increase is difficult to explain, but may
suggest a hypoxic condition for the foetus due to decreased blood flow in the
placenta because of increased viscosity.
The newborn were exposed to hypoxia together with their mothers so as to
survive in good condition. Some authors have reported evidence for the transmittance of Ep through maternal milk (Grant, 1955; Carmichael, Gordon &
LoBue, 1978) while we and others have been unable to confirm this (Lucarelli
et al. 1964; Meberg et al. 1980). That transfer of Ep through the milk should be
able to raise the Ep levels of the newborn during a 6 h period to such an extent
as found (Fig. 2), seems at any rate unlikely, but cannot be ruled out entirely.
This investigation does not clarify which organ produces Ep during foetal
life. Most of the evidence points to the liver as the primary site of extrarenally
produced Ep both during this period and later in life (Zanjani et al. 1977;
Gruber et al. 1977; Fisher, 1979). However, there is also data that indicates the
submandibular glands as important extrarenal sites (Zangheri et al. 1977). We
have previously reported polycythemia and increased Ep values both in plasma
and cystic fluid of a newborn with bilateral renal cysts (Halvorsen et al. 1975).
This suggests that, although the foetal kidney is not the primary production site
for Ep, it is capable of Ep production.
This study was supported by a grant from the Norwegian Research Council for Science and
the Humanities.
The skilful animal caretaking of Borghild Hansen, Laila Holmsen, and Mai Monsen is
gratefully acknowledged.
REFERENCES
A. E. & COLE, R. J. (1971). Stimulation of haem synthesis by erythropoietin in
mouse yolk-sac-stage embryonic cells. / . Embryol. exp. Morph. 26, 475-480.
CARMENA, A. O., HOWARD, D. & STOHLMAN, F. JR. (1968). Regulation of erythropoiesis.
XXII. Erythropoietin production in the newborn animal. Blood 32, 376-382.
CARMICHAEL, R. D., GORDON, A. S. & LOBUE, J. (1978). The effects of maternal phlebotomy
and orally-administered erythropoietin (Ep) on erythropoiesis in the suckling rat. Bio).
Neonate 33, 119-131.
BATEMAN,
Role of the kidney in mouse foetal erythropoiesis
173
R. J. & PAUL, J. (1966). The effects of erythropoietin on haem synthesis in mouse yolk
sac and cultured foetal liver cells. J. Embryo), exp. Morph. 15, 245-260.
FINNE, P. H. (1964). Erythropoietin levels in the amniotic fluid, particularly in Rh-immunized
pregnancies. Acta Paediat., Uppsala 53, 269-281.
FISHER, J. W. (1979). Extrarenal erythropoietin production. /. Lab. din. Med. 93, 695-699.
GLUECKSOHN-SCHOENHEIMER, S. (1943). The morphological manifestations of a dominant
mutation in mice affecting tail and urogential system. Genetics 28, 341-348.
GRANT, W. C. (1955). The influence of anoxia of lactating rats and mice on blood of their
normal offspring. Blood 10, 334-340.
GRUBER, D. F., ZUCALI, J. R., WLEKLINSKY, J., LARUSSA, V. & MIRAND, E. A. (1977).
Temporal transition in the site of rat erythropoietin production. Expl Hemat. 5, 399-407.
HALVORSEN, K., HAGA, P. & HALVORSEN, S. (1975). Regulation of erythropoiesis in the foetus
and neonate. In Erythropoiesis (ed. K. Nakao, J. W. Fisher & F. Takaku), pp. 349-355.
University of Tokyo Press.
HAGA, P. & FALKANGER, B. (1979). In vitro assay for erythropoietin: Erythroid colony
formation in methyl cellulose used for the measurement of erythropoietin in plasma.
Blood 53, 1172-1181.
JACOBSEN, L. O., MARKS, E. K. & GASTON, E. O. (1959). Studies on erythropoiesis. XII. The
effect of transfusion-induced polycythemia in the mother on the fetus. Blood 14, 644-653
LUCARELLI, G., HOWARD, D. & STOHLMAN, F. JR. (1964). Regulation of erythropoiesis. XV
Neonatal erythropoiesis and the effect of nephrectomy. /. din. Invest. 43, 2195-2203.
MATOTH, Y. & ZAIZOV, R. (1971). Regulation of erythropoiesis in the fetal rat. IsraelJ. med.
Sci. 7, 839-843.
MAUER, S. M., DOBRIN, R. S. & VERNIER, R. L. (1974). Unilateral and bilateral renal agenesis
in monoamniotic twins. /. Pediat. 84, 236-238.
MEBERG, A., HAGA, P. & JOHANSEN, M. (1980). Plasma erythropoietin levels in mice during
the growth period. Br. J. Haemat. 45, 569-574.
MEBERG, A., HAGA, P. & HALVORSEN, S. (1979). Pre- and postnatal serum erythropoietin
(ESF) levels in mice and rats. Response to hypoxia. Pediat. Res. 13, 957.
STEPHENSON, J. R., AXELRAD, A. A., MCLEOD, D. L. & SHREEVE, M. M. (1971). Induction
of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proc. natn. Acad.
Sci., U.S.A. 68, 1542-1546.
COLE,
ZANGHERI, E. O., LOPEZ, O. I., HONORATO, L. E., PUSCAMA, L. O., RODRIGUEZ, M. E. &
RETA, E. (1977). The role of the submandibular glands in extrarenal erythropoietin
production in mice. Expl Hemat. 5, 237-240.
E. D., POSTER, J., MANN, L. I. & WASSERMAN, L. R. (1977). Regulation of erythropoiesis in the fetus. In Kidney Hormones vol. II, (ed. J. W. Fisher), pp. 463-493. Academic
Press, Inc.
ZANJANI,
(Received 2 April 1980, revised 7 August 1980)