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Stimulation of cell division in ectopic kidney grafts following unilateral removal of the lung.

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Stimulation of Cell Division in Ectopic Kidney Grafts
Following Unilateral Removal of the Lung
JOHN SIMNETT, JOHN WALTON AND CHRISTINE OATES
Uniuersity of Newcastle upon Tyne, Department of Pathology, Royal Victoria
Infirmary, Newcastle upon Tyne, NEI 4LP, England
ABSTRACT
Small fragments of kidney tissue were grafted into the right lung
(Xenopus laevis) and the left lung was subsequently removed. This stimulated compensatory hyperplasia (increased mitotic rate) in alveolar tissue of the right lung
and in the kidney graft. This suggests that the stimulus to compensatory hyperplasia is location-specific rather than tissue- or organ-specific. After implantation into the lung the kidney grafts are revascdarized by the surrounding pulmonary supply. Damage or functional impairment of an organ usually produces a
localized increase in the rate of blood flow and we therefore propose that this may
lead to an increased rate of cell division in one of two ways: by permitting an increased functional capacity in the tissue concerned, or by causing a more rapid
local clearance of’ mitotic control factors.
In many organs of the body damage or
removal of living tissue is followed by an
increase in the rate of cell division so that
new replacement tissue is produced (Goss,
’64).Any hypothesis which attempts to explain this response must take account of
two important general observations. One is
that the response is tissue or organ specific
and the other is that compensatory hyperplasia is under organismal control such
that the response may occur in tissue situated at some distance from the original site
of injury. Most general hypotheses therefore make the following common assumptions (Simnett and Fisher, ’73): that the
rate of mitosis is regulated by chemical
factors, that each organ or possibly tissue
has its own chemically specific regulatory
factor and that, in the case of many organs,
these factors circulate in the bloodstream
forming a “systemic pool.” It is believed
that damage or loss of tissue causes a
change in the concentration of this systemic pool and that this, in turn, causes a
change in the rate of mitosis in the appropriate tissue. Hypotheses differ in the
nature of the proposed regulatory factors.
In the template/antitemplate model of
Weiss and Kavanau (’57) and the chalone
theory of Bullough and Laurence (‘64;
ANAT. REC., 1 8 7 : 273-280.
Bullough, ’73)the rate of cell division is envisaged as being under the control of tissue-specific mitotic inhibitors. Loss of tissue, it is proposed, causes a reduction in
the local or systemic concentration of the
regulatory factor and this permits an increase in the rate of mitosis in the homologous tissue. The “wound hormone” hypothesis (Abercrombie, ’57; Swann, ’581
proposes that in certain situations the rate
of mitosis is controlled by tissue-specific
stimulatory substances released from
damaged cells while in the “functional demand” hypothesis (Goss, ’64) it is assumed
that compensatory hyperplasia is the result
of an increased physiological load placed
upon the remaining tissues due to functional impairment of part of the organ. The
possible importance of these three different prerequisites, namely tissue loss, tissue
damage and increased physiological load,
were examined in a series of experiments
on the mammalian lung (Cohn, ’39; Fisher
and Simnett, ’73; Simnett, ’74; Simnett and
Fisher, ’761.Partial removal of the left lung
in rats caused an increased mitotic rate in
alveolar tissue of the right lung (Fisher and
Simnett, ’73).The experimental procedure
Received Mar. 15, ’76. Accepted July 20, ’76.
273
2 74
JOHN SIMNETT, JOHN WALTON AND CHRISTINE OATES
involved both damage and loss of tissue in
the left lung and an increased physiological
load on the right lung and the data were
therefore consistent with all of the three
general hypotheses described above. Simple collapse of the left lung, both in rats
(Fisher and Simnett, '73) and in mice (Simnett, '74) b y admitting air into the left part
of the thorax, also stimulated mitosis in the
right lung. In this case there was no
damage or loss of tissue and it would appear that the increased physiological load
placed on the right lung was the primary
stimulus. However, if the lung was collapsed and the residual space in the
thoracic cavity then filled with inert material the mitotic response (Fisher and Simnett, '73; Simnett, '74) and subsequent
growth (Cohn, '39)of the opposite lung was
inhibited. In this case the uncollapsed lung
failed to respond even though it had been
subjected to an increased physiological
load. These results cannot therefore he
fully explained by the template/antitemplate model, the chalone or wound hormone hypotheses or the concept of functional demand (Simnett and Fisher, '73).
Removal or collapse of the left lung
causes the heart and mediastinum to be
displaced towards the left which provides
the right lung with a greater potential for
expansion and air intake while mediastinal
displacement and the consequent hyperinflation of the right lung is prevented
by implantation of inert material into the
thorax (Fisher and Simnett, '73). In the experiments described above increased cell
division occurred only when the lung was
free to hyperinflate and we suggest that
this may be a causal connection. Assuming
that hyperinflation constitutes a stimulus,
direct or indirect, to cell division we wish
to know whether this stimulus is specific to
lung tissue. This question is examined in
the present investigation by implanting ectopic grafts of kidney tissue into one lung
which is then stimulated by removal of the
contralateral lung.
proved impracticable so the present work
was performed on South African Clawed
Frogs (Xenopw laevis laevis). These were
bred and reared in the laboratory and used
when they had reached a weight of 5-7 gm
(trunk length 35-40 mml. Surgical procedures were carried out under anaesthesia, achieved by total immersion in 0.05%
MS-222, and incisions in the skin and body
were closed with fine silk sutures. The animals were maintained postoperatively at
23°C. Mitotic counts were made b y first injecting the animals intra-abdominally with
a solution of Colcemid (2 pg per gm of body
weight). Tissues were fixed four hours later
in Carnoy's fluid, embedded in paraffin
wax and sectioned serially at 6 p. Sections
were stained in haematoxylin and eosin
and the numbers of arrested metaphases
were counted, under an oil immersion lens
at a magnification of x 1,000,in samples of
at least 50,000 cells for kidney graft or
100,000cells for alveolar tissue of the lung.
Before counting all slides were "shuffled"
into random order and each slide assigned
an arbitrary code number. The mitotic incidence (MI) was calculated separately for
lung and graft tissue in each animal and
was expressed as the proportion of arrested metaphases per lo3 cells. At the
concentration used Colcemid arrests all
mitotic cells at metaphase without affecting the rate at which cells enter mitosis.
The full effect of a single injection persists
for at least six hours and, as calculated, the
MI is therefore a measure of the relative
proportions of cells entering mitosis in the
4-hour period between injection of Colcemid and fixation of the tissue. In the
present case the MI is an aggregate value
covering a number of cell types since, due
to sampling difficulties, we did not attempt
to make separate estimates for the different types of cells within the kidney or lung
alveolar tissue.
Stimulation of mitosis in lung
alveolar tissue
The object ofthis first experiment was to
DESCRIPTION OF EXPERIMENTS
confirm that unilateral pneumonectomy in
Ectopic grafting on the mammalian lung Xenopus leads to compensatory hyper-
275
STIMULATION OF MITOSIS IN GRAFTS OF KIDNEY
1ABLE 1
Effect of unilateral pneumonectomy on mitotic incidence in the lung alceolar tissue
Interval between
removal of left
and right lungs
Mitotic incidence
Animal no.
1
0 (control)
2 days
4 days
6 days
2
3
4
5
6
7
8
9
10
11
12
Right
Left
0.04
0.14
0.16
0.06
0.05
0.16
0.05
0.04
0.05
0.04
0.08
0.44
0.09
0.07
LIR
ratio
::?:
I
0.96
0.08
0.5
0.8
1.01
0.64
2.9
0.66
0.78
0.66
0.79
0.67
i16.0
::
7::;
9.75
1.5
8.8
9.6
1
1
I
16.7
28.1
6.6
1. The right lung was removed and the left lung fixed at different intervals thereafter.
2. Mitotic incidence (MI) is the proportion ofarrcsted inetaphase nuclei per 103 cells four hours after Colcemid
injection.
3. Significance of difkrence between right and left lung MI (2,4 and 6 days combined);p
test).
plasia in the remaining lung and to find
out the time at which the maximum mitotic
response occurs. One group of three animals was injected with Colcemid and
sacrificed four hours later for fixation of
left and right lungs. A further nine animals
were injected with Colcemid and after
four hours, under anaesthesia, the right
lung was ligatured at its base and fixed.
The animals were allowed to recover and
were then sacrificed in groups of three following Colcemid treatment after 2,4 and 6
days for fixation of the left lung. The experiment provides data (table 1)for MI in
the left and right lungs of untreated animals, for the right lung at pneumonectomy
and for the left lung at different intervals
after right pneumonectomy. In the control
animals (simultaneous fixation of both
lungs) the L/R ratio (MI in the left over LI
in the right lung) was 0.96:l.O-close to
the expected 1:1 ratio. In animals with the
right lung removed there was a large increase in the L/R ratio, the maximum (28:11
being observed four days after operation.
It should be noted that in the pneumonectomized animals the left lung received two
exposures to Colcemid, one four hours
before fixation and another 2, 4 or 6 days
previously. In a separate experiment we
<
0.01 (Student’st.
showed that injections of Colcemid SO
spaced do not increase the MI above the
values found after the 4-hour single Colcemid treatement. We therefore conclude
that the increased L/R ratio is a measure of
the degree of compensatory hyperplasia
due to unilateral pneumonectomy and that
the maximum response occurs after approximately four days.
Cell division in ectopic
kidney grafts
A right dorso-lateral incision was made
through the skin and body wall and a strip
approximately 1 x 3 mm was cut from the
edge of the exposed kidney (mesonephrosl.
This was transferred to a pool ofphysiological saline solution, taken up in a glass
pipette (internal diameter 1 mml with a
sharp bevel tip and injected through an incision into the right lung. Animals were
then maintained for three weeks to allow
the skin and body wall incisions to heal and
the right lung to reinflate. In half the animals the left lung was then removed. After
a further four days all animals were injected with Colcernid four hours before
sacrifice and fixation of the lungs. Mortality
over the whole period of the experiment
was approximately 20% leaving a total of
276
JOHN SIMNE?T, JOHN WALTON AND CHRISTINE OATES
TABLE 2
Effect of unilateral pneumonectomy
on mitotic incidence in the lung
and in kidney grafts
Mitotic incidence with
standard deviation
Mitotic stimulation following unilateral
removal of the lung was not restricted to
alveolar tissue since a highly significant increase in MI (2.7-fold) was also observed in
the ectopic kidney grafts.
DlSCUSSION
Right lung
Kidney grnft
~
Control group
Group with left
pneumonectomy
Increase in
pneumonectomized
animals
0.65? 0.38
(18)
C181
1.12r0.51
(17)
C171
x 1.72
p < 0.005
2.09k 1.55
(18)
1531
5.60* 3.15
(16)
C411
x 2.68
p < 0.001
1. The left lung was removed three weeks after the implantation of kidney tissue in the right lung. The right lung
and kidney graft were fixed four days d t e r removal ofthe left
lung.
2. For definition ofmitotic incidence see table 1.Figures in
round brackets denote the number of animals per group,
figures in square brackets the number of sample mitotic
counts.
3. The significance of the differencc between control and
unilaterally pneumonectomked groups was calculated by
Studmt's t. test, in which N was derived from the number of
sample mitotic counts.
35 survivors in 34 of which kidney grafts
were subsequently identified in the right
lung (figs. 1, 2). Examination of tubules and
glomeruli (fig.3) suggested that the grafted
tissue was actively revascularized.
In the lung the MI was estimated from
one sample (1 serial section) from each animal (total animals 35, total samples 35)
while for the kidney grafts two, or more
usually three samples were counted for
each animal (total animals 34, total samples
94).
Unilateral removal of the lung was again
shown to stimulate mitosis in the remaining
lung (table 2) although, because of the
higher values in the control group, the
magnitude of the response(a 72% increase)
was less than in the previous experiment
(table 1).Such natural variation between
individuals and between experiments in respect of mitotic rate is common in Xenopus
which, being a poikilothermic species is
sensitive to small temperature differences
and also to other factors such as regularity
of feeding and the age and size of individuals.
It is generally accepted that the mitotic
response in compensatory hyperplasia is
strictly organ or tissue specific. Our data
suggest that it is the Eocation which is
specific but that, within this location -the
lung in the present experiments -a tissue
may respond even if it is of a different type
to that which has been damaged or removed.
From experiments on the mammalian
lung already described we concluded that
hyperinflation of the pulmonary tissue was
a causal factor in inducing compensatory
hyperplasia. From the present experiments
on ectopic grafts it would appear that the
causal role of hyperinflation is indirect
since the kidney tissue, which because of
its solid structure has no capacity for immediate physical expansion, nevertheless
responds. Evidently there must be some
more direct causal factor which is common
to the lung and to the kidney graft.
Removal of one lung, which necessitates
the ligature of the pulmonary artery, or
collapse of the lung, which increases its resistance to blood flow (Szidon and Fishman,
'69) causes blood to be diverted to the remaining lung. According to the state of expansion of the lung this increased vascular
flow can either pass through the alveolar
capillaries or be diverted from the alveoli
through bypass systems in the capillaries,
through precapillary anastomoses between
the pulmonary and bronchial circulatory
systems or through arteriovenous shunts in
the pulmonary circulation (Daly and Hebb,
'66). Regulation of blood flow at this level
appears to be under the control of the
autonomic nervous system (Szidon and
Fishman, '69). In the experiments on the
mammalian lung (Cohn, '39; Fisher and
Simnett, '73; Simnett, '74) we can therefore assume that hyperinflation of alveolar
tissue and the increased mitotic rate or
STIMULATION OF MITOSIS IN GHAETS OF KIDNEY
growth in the alveolar tissue also correlate
with an increased blood perfusion rate.
Following implantation into the lung the
kidney graft is revascularized from the surrounding alveolar circulation and we
therefore propose that the increased rate
of capillary blood flow to the tissue, both
for alveolar and kidney cells, may be the
more immediate stimulus to compensatory
hyperplasia. A definite correlation between blood flow and cell division rate has
also been noted in the liver following partial hepatectomy (Brauer, '63) and it has
been suggested (Mann, '44) that the restoration of liver tissue is dependent on the
rate of portal blood flow through the organ.
This hypothesis has largely been discounted (Brauer, '63; Bucher, '63) on the
basis of experiments in which compensatory hyperplasia was observed to occur even
when the normal portal blood supply was
replaced b y blood from the inferior vena
cava or from the arterial system. These experiments tacitly assume that if the liver
vasculature has any causal function it must
be in supplying some systemically circulating ("humoral") regulatory factor. However, there are strong theoretical (Weiss
and Kavanau, '57;Iversen, '65)and experimental (Bullough, '73)reasons for believing
that cell division is regulated through the
negative feedback system involving mitotic
inhibitors synthesized locally within the
tissue. It has been suggested that an increased rate of blood flow through a tissue
will cause a local reduction in the tissue
concentration of the mitotic inhibitor and
hence permit an increase in the rate of cell
division (Simnett and Fisher, '73).
Increased blood flow following tissue
damage is under organismal control (usually uia the autonomic nervous system) and
it is organ specific (Cameron, '67).If this increased rate of flow does play any part in
the regulation of compensatory hyperplasia there is no essential theoretical
reason to suppose, as in other hypotheses
already described, that the chemical factors controlling cell division are either
organ specific or that they circulate systemically. Arguments against the concept
277
of a systemic pool have already been
presented on the basis of evidence from
the cell division response to localized tissue damage (Abercrombie, '57; Simnett
and Fisher, '76) but there is good experimental evidence to support the existence
of specific chemical factors in a number of
organs (Bullough, '73) including the lung
(Simnett et al., '69) and the kidney (Chopra
and Simnett, '70).
The results of earlier experiments (Simnett, '74) are not consistent with the idea
(Goss, '64) that cell division is stimulated
by an increased functional demand in the
organ concerned, but rather that it may be
related to an increased capacity for functional response. An increased rate of blood
flow could promote functional activity and
this might be an alternative explanation for
the data on cell division rate obtained in
the present experiments.
The concept of vascular control of cell
division is consistent with the observation
that administration of autonomic nervous
system blockers or surgical transection of
the autonomic nervous supply to damaged
organs inhibits compensatory hyperplasia
(Cohn, '39; Goss, '64).
ACKNOWLEDGMENTS
This work was supported by grants from
the Northern Council of the Cancer Research Campaign (John Simnett and John
Walton) and from the Medical Research
Council (Christine Oates).
LITERATURE CITED
Abercrombie, 34. 1957 Localized formation of new
tissue in an adult animal. Symp. Soc. Exp. Biol., 11:
235-254.
Brauer. R. W. 1963 Liver circulation and function,
Physiol. Rev., 43: 115-213.
Bucher, N. L. H. 1963 Regeneration of mammalian
liver. Int. Rev. Cytol., 15: 245-300.
Bullough, W. S. 1973 The chalones: a review. Nat.
Cancer Inst. Monograph, 38: 5-16.
Bullough, W. S., and E. B. Laurence 1964 Mitotic
control by internal secretion. Exp. Cell Res., 33:
176-194.
Cameron, R. C. 1967 Inflammation and repair. In:
Patholcgy. S . L. Robbins, ed. W. B. Saunders, Philadelphia and London, pp. 31-73.
Cbopra, D. P., and J. D. Simnett 1969 Demonstration
278
JOHN SIMNETT, JOHN WALTON AND CHRISTINE OATES
of an organ-specific mitotic inhibitor in the amphihian kidney. Exp. Cell Res., 58: 319-322.
Cohn, R. 1939 Factors affecting the postnatal
growth of the lung. Anat. Rec., 75: 195-205.
Daly, I. d e B., and C. Hebb 1966 Pulmonary and
Bronchial Vascular Systems. Arnold, London.
Fisher, J. M., and J. D. Simnett 1973 hlorphogenetic
and proliferative changes in the regenerating lung
of the rat. h a t . Rec., 176: 389-396.
G o s s , R. J. 1964 Adaptive Growth. Logos Press,
London.
Iversen, 0. H. 1965 Cybernetic aspects of the
cancer problem. Prog. Biocybern., 2: 76-110.
Mann, F. C. 1944 Restoration and pathologic reactions of the liver. J. Mount Sinai Hosp., 11: 65-74.
Simnett, J. D. 1974 Stimulation of' cell division following unilateral collapse of the lung. h a t . Hec.,
180: 681-686.
Simnett, J. D., and J. M. Fisher 1973 Description of
growth phenomena and the formulation of growth
control models. Nat. Cancer Inst. Monograph, 38:
29-36.
1976 Cell division and tissue repair following localized damage to the lung. J. Morph., 148:
177-184.
Simnett, J. D., J. M. Fisher and A. G. Heppleston 1969
Tissue-specific inhibition of lung alveolar cell
mitosis in organ culture. Nature (London), 223:
944-946.
Swann, M. M. 1958 The control of cell division: a
review. Cancer Res., 18: 1118-1160.
Szidon,J. P., and A. P. Fishman 1969 Antonomic control of the pulmonary circulation. In: The Pulmonary Circulation and Interstitial Spaces. A. P. Fishman and H. H. Hecht, eds. University Press,
Chicago, pp. 239-265.
Weiss, P., and J. L. Kavanau 1957 A model of' growth
and growth control in mathematical terms. J. Gen.
Physiol., 41; 1-47.
-
PLATE 1
EXPLANATION OF FIGURES
1 Section of a frog lung showing the solid kidney graft (K) surrounded by the
spongy lung alveolar tissue (A).
2 High power photomicrograph ofpart ofa kidney graft showing a mitotic cell
(.MI in the epithelium lining one of'the kidney tubules (T).
3 Photomicrograph of' part of' a graft showing a kidney glomerulus. The presence of numbers of red blood cells (marked by arrow) suggests that the graft
has an active blood supply.
STIMULATION OF hlITOSlS IN GRAFTS OF KIDNEY
John Simnett, John Walton and Christine O t t r s
PLATE 1
279
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