/ . Embryol. exp. Morph. Vol. 25, 3, pp. 321-329, 1971
Printed in Great Britain
321
Tissue-specific mitotic inhibition
in the kidneys of embryonic grafts and partially
nephrectomized host Xenopus laevis
By D. P. CHOPRA 1 AND J. D. SIMNETT 1
From the Department of Pathology, University of Newcastle upon Tyne
SUMMARY
Immature Xenopus laevis were unilaterally nephrectomized and implanted subcutaneously
with stage 38 (prefeeding) larvae.
Rat kidney extract was injected 8 and 16 days after the operation and the mitotic incidence
(MI) in the host and implant tissues was measured by the colcemid metaphase arrest technique.
The MI in the kidney of nephrectomized hosts was higher than normal at 8 days but had
returned to the control level at 16 days. Injection of kidney extract inhibited mitosis in the
host kidney both at 8 and 16 days after partial nephrectomy.
The MI of implant pronephros was higher in nephrectomized than in control hosts both at
8 and 16 days. Injection of kidney extract into nephrectomized hosts inhibited mitosis in the
implant pronephros both at 8 and 16 days.
Neither host nephrectomy nor injection of kidney extract had any effect on the MI in the
epidermis of the implants.
Unoperated immature Xenopus were injected with rat kidney or liver extracts. Kidney
extract inhibited mitosis in the kidney but not in the liver, while liver extract inhibited mitosis
in the liver but not in the kidney.
The rate of mitosis in the kidney and liver of normal animals may be controlled by tissuespecific inhibitors of cell division.
INTRODUCTION
Partial nephrectomy in vertebrates is typically followed by compensatory
growth in the residual tissue (Goss, 1964; Malt, 1969). The compensatory response is characterized by a latent period followed by a sudden burst of mitotic
activity (Goss & Rankin, 1960; Williams, 1961). In the pronephros of Xenopus
larvae the maximum mitotic rate was observed 2 days after unilateral nephrectomy (Chopra & Simnett, 1969 a) while in metamorphosed Xenopus the maximum activity was recorded 6 days after operation. Partial nephrectomy in the
metamorphosed Xenopus produced an increased mitotic rate not only in the
remaining kidney tissue (mesonephros) but also in the pronephros of implanted
embryonic grafts (Chopra & Simnett, 19706), which suggests that the two tissues
have regulatory factors in common. Mitosis in cultures of pronephros is
inhibited by adding kidney extracts from adult Xenopus (Chopra & Simnett,
1
Authors'1 address: Department of Pathology, The Royal Victoria Infirmary, Newcastle
upon Tyne, NE1 4LP, England.
322
D. P. CHOPRA AND J. D. SIMNETT
19696; Simnett & Chopra, 1969) or rats (Chopra & Simnett, 1970 a) to the
culture medium. This indicates that under natural conditions the rate of cell
division in the kidney may be controlled by a tissue-specific inhibitor (Saetren,
1956) or chalone (Bullough, 1965) as has been described in a variety of other
tissues (Bullough & Laurence, 1964; Rytomaa & Kiviniemi, 1968; Simnett,
Fisher & Heppleston, 1969). This view is supported by the observation that an
antiserum against the antimitotic kidney extract stimulated mitosis specifically
in kidney cultures (Chopra & Simnett, 1970 a). Further evidence in support of
this theory has been obtained by showing that tissue-specific mitotic inhibition
in the kidney can be effected in vivo.
MATERIAL AND METHODS
Extirpation of hoist kidney and implantation of embryos. In earlier experiments
(Chopra & Simnett, 19706) embryos were implanted after a 2-day recovery
period following partial nephrectomy in the host and implants were removed 8
and 16 days after implantation (i.e. 10 and 18 days after partial nephrectomy).
In a number of experiments it has, however, been shown that the host can
withstand long periods of anaesthesia (D. P. Chopra, unpublished results)
which makes it possible to perform both operations in immediate succession.
This method was therefore adopted for the experiments presented in this paper.
The partial nephrectomy exerted its maximum effect on the pronephros of
embryonic implants during the period of increased mitotic rate in the host
kidney, which for juvenile metamorphosed Xenopus laevis occurred between 5
and 10 days after the operation (Chopra & Simnett, 19706). Subsequently the
mitotic rate decreased and had at 16 days returned to normal. In the present
series of experiments implants were therefore removed 8 and 16 days after partial
nephrectomy or implantation.
A small incision was made in the ventral abdominal cavity of anaesthetized
juvenile metamorphosed Xenopus laevis and the kidney was partially extirpated
by diathermic cauterization. The total amount of renal tissue thus removed was
about 40%. The incision was closed with two sutures of fine silk. Control hosts
were subjected to a similar procedure although no renal tissue was removed
(sham operation).
In a previous study (Simnett, 1966) a comparison between embryos of different developmental stages examined at varying intervals after implantation
had demonstrated that implants of prefeeding larvae (stage 38, Nieuwkoop &
Faber, 1967) showed the best histological differentiation. Consequently
embryos of this stage were used for implantation in the present investigation.
After anaesthetizing the animals the kidney operation was performed and subsequently a small incision was made into the dorsal lymph sac of each host for
the implantation of two embryos. Since the incision was small it healed quickly
without any suturing. After recovery from anaesthesia, the animals were kept
in standing tapwater at a temperature of 16-17° C.
Tissue-specific mitotic inhibition
323
Method of preparing tissue extract. Since the inhibitory effect of kidney
extract is not species-specific (Chopra & Simnett, 1970 a) extracts prepared from
adult rats were used in the present experiments. The rats were killed by dislocation of the neck and the kidneys were perfused with warm normal saline
solution supplemented with sodium citrate (1-9 mg/ml). Subsequently the
kidneys were removed, finely minced and homogenized with 3-5 times their
volume of 0-3 % cold saline solution in a glass homogenizer for 7-10 min. After
spinning in a high-speed centrifuge at 25000 rev./min (100000 g) the supernatant was collected, freeze-dried and stored at - 7 0 ° C until used. Rat liver
extract was prepared in the same manner. Under these conditions the extract
remained active for at least 16 weeks. Before use the freeze-dried extract was
dissolved in glass-distilled water and individual animals were injected with a
volume of extract equivalent to 10 mg dry weight of the original kidney or liver
tissue.
Measurement of mitotic incidence (MI). Mitotic activity in the host kidney,
liver and implant pronephros and epidermis was measured by the metaphase
arrest technique (Chopra & Simnett, 1969 a). At various intervals after partial
nephrectomy both operated and control hosts were injected with colcemid
(0-03 ml of a 100 mg % solution). In animals injected with tissue extracts the
two injections - extract and colcemid - were given at the same time. Four hours
after injection the animals were killed and, according to the requirements of the
experiment the kidneys, implants or liver were removed and fixed in Worcester's fluid for 20 h. Serial sections of implant (7 fi) and specimen sections (5 fi)
of host kidney or liver were cut and stained in Harris's hematoxylin and Weigert's
hematoxylin respectively.
Mitotic incidence (MI) in the kidney tubules and liver of the host was calculated by counting the number of arrested metaphases in two samples each of
4000-8000 nuclei in both tissues. The MI for the pronephric tubule and epidermis
of implants was calculated from one sample of 1000 nuclei of both types from
each implant using the same method. In all cases the MI was expressed as the
number of arrested metaphases per 105 nuclei for each sample, which represents
the proportion of cells entering mitosis during the 4 h period following colcemid
injection.
EXPERIMENTS AND RESULTS
The effect of kidney extract injected into implant-bearing hosts
Twenty-four hosts, divided into four groups of six animals each, were partially
nephrectomized and two embryos implanted into the dorsal lymph sac of each
of them. Ten sham-operated control hosts, divided into two groups of five
animals each, also received two implants each. Altogether one control and six
nephrectomized hosts died during the course of the experiment, leaving between
three and five animals in each group. Two groups of nephrectomized and one
group of control animals were sacrificed at 8 days and the same number of
324
D. P. CHOPRA AND J. D. SIMNETT
groups at 16 days after the operation. Four hours before sacrifice all hosts were
injected with colcemid and two of the nephrectomized groups (one at 8 and
one at 16 days) also received injections of kidney extract (10 mg/host). The
embryos were thus subjected to three different types of treatment: implantation
into non-nephrectomized hosts and implantation into partially nephrectomized
hosts with or without subsequent injection of kidney extract.
Mitotic incidence in the host kidney. Eight days after the operation the mitotic
activity in the nephrectomized animals was significantly (P < 0-001) higher
than in the control animals (Table 1). At this time the values of MI recorded in
the kidney of nephrectomized and control hosts were 323 ± 121 and 124 ±34
respectively. After 16 days the MI in the kidney of partially nephrectomized
hosts (306 ±171) appeared to be higher than in control animals (188 ±65),
although the difference was not significant.
Table 1. The effects of partial nephrectomy and injection of kidney extract
on mitotic incidence {MI)* in the kidney (Xenopus laevis)
Time after
partial nephrectomy
(days)
16
Control
124 + 34
(10)
188 + 65
(8)
Partially
nephrectomized
323 ±121$
(10)
306±171§
(8)
Partially
nephrectomized with
kidney extractf
39±16||
(10)
87 ±531|
(6)
Figures in parentheses represent the number of samples analysed per group.
* MI is the number of arrested metaphases per 105 cells after 4 h of colcemid treatment.
t Each animal injected with 10 mg of extract.
% Stimulation of mitosis significant (P < 0001) as compared with the corresponding
controls.
§ Stimulation of mitosis not significant as compared with the corresponding controls.
|| Depression significant (P < 0001) as compared with the corresponding partially
nephrectomized animals.
Injection of kidney extract into partially nephrectomized animals resulted in a
specific and significant inhibition of cell proliferation both at 8 and 16 days after
the operation. At 8 days the MI in the kidney of these nephrectomized animals
(39 ± 16) was significantly lower (P < 0-001) than that in uninjected, partially
nephrectomized (323 ± 121) and control (124 ± 34) animals. Similarly, at 16 days
after the operation the MI in the kidney of nephrectomized hosts injected with
kidney extract (87 ± 53) was significantly lower (P < 0-01) than that in uninjected
partially nephrectomized (306 ±171) and control (188 ± 65) hosts.
Mitotic incidence in implant pronephros and epidermis. In implants removed
from control hosts the MI in pronephros was 740 ±259 and 901 ±282 and in
epidermis 2880 ±489 and 3121 ±707 at 8 and 16 days respectively (Table 2).
There was no significant difference between the values at 8 and 16 days.
Tissue-specific mitotic inhibition
325
Partial nephrectomy in the host caused a significant increase in cell proliferation in the pronephros but not in the epidermis of the implant. At 8 days the MI
in the pronephros of implants from nephrectomized hosts reached a value of
2088 ± 629, which is significantly higher (P < 0-001) than in that from control
hosts (740 ± 259). Even 16 days after partial nephrectomy, when the MI in the
regenerating host kidney had returned to near normal values (Table 1), the MI
in the implant pronephros removed from the nephrectomized hosts (1474 ± 463)
remained significantly higher (P < 002) than in that from control hosts (901 ±
282). At no time after partial nephrectomy was there any significant difference
between the MI in the epidermis of nephrectomized and control implants. The MI
in epidermis of implants removed from nephrectomized hosts 8 and 16 days after
implantation was 3130 ± 308 and 3206 ±516 respectively (Table 2).
Table 2. Mitotic incidence in the pronephros and epidermis
of implanted embryos (Xenopus laevis)
Treatment of host
Implant
tissue
Pronephros
Days after
implantation
8
16
Epidermis
8
16
Control
Partially
nephrectomized
740 ±259
2088 ±629*
(8)
(9)
901+ 282
1474 +463 f
Partially
nephrectomized
with kidney
extract
468 +151 f
(9)
242±139J
(8)
(8)
(6)
2880 ±489
313O±3O8§
3324±780§
(5)
(5)
(5)
3121 ±707
(5)
3206±516§
(5)
2670 ±493§
(5)
Figures in parentheses represent the number of implants analysed.
* Stimulation of mitosis significant (P < 0-001) as compared with the corresponding
controls.
f Stimulation of mitosis significant (P < 002) as compared with the corresponding
controls.
X Depression of mitosis significant as compared with the controls or partially nephrectomized hosts (P < 0001).
§ No significant depression as compared with the controls.
Injection of kidney extract into partially nephrectomized hosts inhibited cell
division in the implant pronephros. The MI in pronephros of implants removed
at 8 days from nephrectomized hosts injected with kidney extract (468 ± 151)
was significantly lower than that in the implant pronephros from uninjected
nephrectomized (2088 ± 629; P < 0-001) and control hosts (740 ± 259) (Table 2).
A similar reduction was also recorded 16 days after implantation, when the
MI in pronephros from nephrectomized hosts injected with kidney extract
22
E M B 25
326
D. P. CHOPRA AND J. D. SIMNETT
(242 ±139) was significantly lower (P < 0-001) than in that from uninjected
nephrectomized (1474 ±463) and control hosts (901 ±282) (Table 2).
Injection of the kidney extract into partially nephrectomized hosts had no
significant effect on the epidermis of the implants. At 8 and 16 days after implantation the MI in epidermis of these implants was 3324 ± 780 and 2670 ± 493
respectively (Table 2).
The effect of kidney and liver extracts injected into unoperated animals
The results suggest that a tissue extract exerts an antimitotic effect only upon
its organ of origin, since kidney extract inhibited host kidney and implant pronephros but not the implant epidermis. This conclusion is supported by the
following experiment.
One group of six non-nephrectomized unimplanted animals was injected with
colcemid and kidney extract and a second group with colcemid and liver extract.
As described earlier, the animals were killed 4 h later and their kidneys and livers
removed for histological examination.
Table 3. The effects of liver or kidney extracts on the MI in the kidney
and liver of unoperated animals (Xenopus laevis)
(Each figure of MI is the mean of twelve values unless otherwise indicated.)
Control
Kidney
Liver
Kidney extract
303 ±108
(30)
305 ± 28
69 ±27*
291 ±48t
Liver extract
289±95f
88 ±25*
* Depression of mitosis significant (P < 001) as compared with the corresponding control.
t Depression not significant as compared with the corresponding control.
Injection of kidney extract reduced the MI significantly in the kidney (from
303 ± 108 to 69 ± 27; P < 0-01) but had no effect on the MI of the liver. Conversely, the injection of liver extract produced a significant reduction of the MI
in the liver (from 305 ± 28 to 88 ± 25; P < 0-01) but had no effect on the MI in
the kidney (Table 3). Thus, the antimitotic effect of kidney extract is specific
for the kidney since it does not affect the liver. The present experiment also
shows that the liver contains a factor with antimitotic action on the liver but
not on the kidney.
DISCUSSION
A supernatant fraction of homogenized ultracentrifuged Xenopus laevis or
rat kidney inhibited mitosis in the pronephros of cultured explants of Xenopus
embryos but had no effect upon the epidermis, while similar preparations of lung
and liver had no antimitotic effect on either the pronephros or the epidermis
(Simnett & Chopra, 1969; Chopra & Simnett, 19696). The present work shows
Tissue-specific mitotic inhibition
327
that the same tissue-specific effect can be obtained in vivo not only in the pronephros of implanted embryos but also in the mesonephric kidney of the host.
In culture experiments the full effect of the kidney extract is only obtained when
the stress hormones, adrenalin and hydrocortisone are added to the culture
medium (Chopra & Simnett, 19696; Simnett & Chopra, 1969). In the present
in vivo experiments injection of kidney extract alone reduced the mitotic rate
by as much as 79 % (implant pronephros) and 88 % (host kidney), possibly due
to the potentiating action of circulating stress hormones.
In experiments on whole unimplanted embryos maintained at 16-17 °C,
labelled mitosis did not appear until 8 h after administration of tritiated thymidine, which shows that the minimum length of G 2 at this temperature is 8 h
(authors' unpublished results). The fact that mitotic inhibition was obtained in
the 4 h period following administration of the extract suggests that, at least in
these experiments, the inhibitory factor acts by slowing the rate of movement
from G2 into M rather than by modifying the rate of DNA synthesis.
The mitotic rate in regenerating rat liver or kidney can be inhibited by injection of macerate prepared from the homologous organ (Saetren, 1956), and
a similar organ-specific inhibitory effect was shown in the present experiments
on Xenopus. It has, however, been demonstrated that the action of tissue extracts
may depend upon the method of preparation since, although a nuclear fraction
prepared from liver inhibited cell division in embryonic chick liver, a stimulatory
effect was obtained with the cytoplasmic fraction (Tumanishvili & Salamatina,
1968). In the present study the prolonged period of homogenization (7-10 min)
is likely to have ruptured the nuclei, and therefore the supernatant extract
probably contains both soluble nuclear and cytoplasmic components. If, as
suggested by Tumanishvili & Salamatina (1968), the cytoplasmic fraction is
stimulatory, it would appear that its effect must be counteracted by a very
potent inhibitor present in the nucleus. Most work on the antimitotic effects of
tissue extract employs crude methods of homogenization and the question of
intracellular localization merits further investigation.
The active inhibitory factor present in the kidney appears to be antigenic in
nature since an antiserum prepared against kidney extract stimulates mitosis
specifically in the pronephros of Xenopus (Chopra & Simnett, 1970a). This is
probably due to the tissue-specific antibodies neutralizing the endogenous
mitotic inhibitor present in the pronephros. Immunoelectrophoretic preparations
in which inhibitory kidney extracts are run against the stimulatory antiserum
show that the extract contains at least two tissue-specific antigens, though the
effect of these on cell division is yet to be studied (D. P. Chopra & J. D. Simnett,
unpublished results).
Partial ablation of epidermis and kidney by mechanical means led to an increased rate of cell division in the remaining tissue, but when the tissues were
destroyed by invading tumour no compensatory increase in mitotic rate was
observed (Argyris, 1968). Thus it was concluded that although the mass of
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D. P. CHOPRA AND J. D. SIMNETT
tissue removed may control the degree of cell proliferation, the direct stimulus
to cell division may not be the loss of tissue per se, but some other factor which
depends upon an abrupt change in tissue mass, possibly a stimulatory product
released by sublethally damaged cells. However, a comparison between the
present experiments and a previous study (Chopra & Simnett, 1970/?) shows that
the compensatory response in implant pronephros is the same whether implantation is done at the same time as partial nephrectomy of the host or whether it is
delayed by 2 days. The fact that compensation occurred even when the implant
was not present at the time of host kidney ablation suggests that in this case it is
the continued deficiency rather than the initial damage to the host kidney which
enhances the mitotic rate in the implant pronephros.
The rates of DNA synthesis and mitosis in a compensating tissue start to
decrease even before the mass of lost tissue is restored, and overcompensation is
thereby avoided. This observation has led to the suggestion (Lajtha, Oliver &
Gurney, 1962) that after tissue damage a cohort of cells, the number of which is
proportional to the extent of the tissue deficiency, enters the mitotic cycle, after
which the signal for deficiency is cancelled. In our earlier experiments (Chopra
& Simnett, 1970/?) embryos were implanted 2 days after host partial nephrectomy and vascularization of the implants has been shown to require a further
4 days. It therefore took 6 days before the embryonic pronephros was exposed to
the host circulation, and the fact that it still responded by an increased mitotic
rate, as in the present series of experiments, suggests that the message for host
kidney deficiency may persist for at least 6 days after partial nephrectomy. An
alternative hypothesis to that of Lajtha et al. (1962) is that once sufficient cells
have entered the mitotic cycle the tissue becomes unresponsive to the deficiency
signal - reception of the signal may be blocked even though transmission of the
signal persists. This idea could be tested by studying the rates of mitosis in the
pronephros of embryos implanted at different intervals after host partial
nephrectomy.
This work was supported by a grant from the North of England Council of the British
Empire Cancer Campaign for Research. Thanks are due to Professor A. G. Heppleston for
kindly providing research facilities in his Department, to Mr and Mrs H. Elliot for skilled
technical assistance, and to Mrs M. Jackson, our Editor of Research Publications, for
valuable editorial help.
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(Manuscript received 8 June 1970, revised 9 October 1970)