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. Detectable 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.

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 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, Hågå & Halvorsen, 1979; Hågå & 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, Hågå & 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 ?

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 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 μCi, 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.

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 (Hågå & 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 μl/ml.

The Ep levels found are expressed as the number of CFUe/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.

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.

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

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
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
Table 2.

Haematocrit levels, reticulocyte counts, and weights (means ± S.D.) of newborn SD mice. The mothers were hypertransfused during the pregnancies. Numbers of animals investigated in parentheses

Haematocrit levels, reticulocyte counts, and weights (means ± S.D.) of newborn SD mice. The mothers were hypertransfused during the pregnancies. Numbers of animals investigated in parentheses
Haematocrit levels, reticulocyte counts, and weights (means ± S.D.) of newborn SD mice. The mothers were hypertransfused during the pregnancies. Numbers of animals investigated in parentheses

The Fe59-uptakes in liver and red blood cells of the anephric newborn, expressed as the percentage of the mean uptake of their normal littermates, are shown in Fig. 1. The Fe59-incorporations are not statistically different in the normal and anephric animals.

Fig. 1.

Fe59-uptakes in livers and RBC of 15 anephric newborn mice. The values are expressed as the percentage of the mean uptake of their littermates 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.

Fig. 1.

Fe59-uptakes in livers and RBC of 15 anephric newborn mice. The values are expressed as the percentage of the mean uptake of their littermates 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.

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 concentrations of Ep (P < 0 001) 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.

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-III 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 μl/ml. The numbers within the bars are the number of culture plates counted.

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-III 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 μl/ml. The numbers within the bars are the number of culture plates counted.

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 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 iron-deficiency 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.

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