/. Embryo!, e.xp. Morph. Vol. 31, 3, pp. 655-665, 1974
Printed in Great Britain
655
Investigation of a substance
of renal origin which inhibits the growth of
renal cortex explant in vitro
By S. E. DICKER1 AND CHRISTINE A. MORRIS1
From the Department of Chemistry, University College London
SUMMARY
(1) Explants of renal cortex from 7-day mice or 5-day rats were reared on plasma clots,
in the presence or absence of chick embryo metanephros.
(2) The size of outgrowth, estimated after 48 h, was similar for renal cortex of both species.
When reared in chick embryo extract in the presence or absence of metanephros, the outgrowth estimated after 48 h was 2-08 ±0-092 greater than that of the initial explant.
(3) Addition of supernatant fluid from a homogenate of renal cortex of adult mouse, rat,
guinea-pig, rabbit or pig inhibited the outgrowth of renal cortex explant from neonate
mouse or rat. The supernatant fluid from a homogenate of adult renal medulla had no significant effect.
(4) The inhibitory activity of the supernatant fluid from a homogenate of renal cortex
from adult animals was destroyed by heating at 99 °C.
(5) The compound with growth-inhibitory activity appeared to be cortex-specific but not
species-specific. Extracts of liver, spleen, duodenum, and heart muscle of adult animals had
no significant effect on the outgrowth of renal explants of neonate animals; and extracts
from renal cortex of adult mammals had no significant effect on the growth of other tissues
from neonate mice or rats.
(6) The degree of growth inhibition was related to the amount of protein in the extract of
renal cortex. Maximum inhibition was observed with extracts of renal cortex containing
2-5 mg protein/0-5 ml.
(7) Immune serum samples were obtained from rabbits and guinea-pigs immunized against
extracts of renal cortex and medulla and liver from rats and mice. Only the serum from either
rabbit or guinea-pig immunized against extracts of renal cortex blocked the growth-inhibitory
action of renal cortex extracts.
INTRODUCTION
Weiss (1952) showed that by adding embryonic kidney extract to cultures of
chick metanephros, only 74 out of 1007 differentiated new tubules in the
growth zone, while in the absence of kidney extract 176 out of 1006 cultures
showed differentiation. Three years later, Andres (1955) found that the intravenous injection of a homogenate of mesonephros produced an increase of the
mitotic activity in the mesonephros of the host of 59% in 24 h and 41 %
after 48 h. He further showed that this stimulating effect upon proliferation of
1
Authors' address: The Christopher Ingold Laboratories, The Department of Chemistry,
University College London, 20 Gordon Street, London WC1 H0AJ.
41
EM B 31
656
S. E. DICKER AND C. A. MORRIS
the homologous organ was tissue-specific. Grobstein (1955), however, found
that the formation of tubules in metanephros reared in vitro depended on the
presence of an extrinsic factor normally supplied by either the ureteric bud, the
salivary epithelium or extracts from embryonic central nervous tissue, but not
by other extracts from either embryonic or adult tissues. While the stimulating
effect on the proliferation of mesonephros or metanephros in vitro appears to
be only partly specific, the inhibitor effect seems to be specific. This was confirmed by Simnett & Chopra (1969) who reared trunk regions from Xenopus
laevis larvae with their attached pronephroi: they observed that addition of
supernatant fluid from a homogenate of adult Xenopus kidney produced a
sharp fall in the mitotic incidence, whereas addition of supernatant from a
liver extract had no effect.
Similar evidence has come from in vivo experiments on unilaterally nephrectomized rats (Saetren, 1956, 1963; Roels, 1969; Dicker, 1972). In all instances,
the injection of either kidney homogenates or of the microsome fraction of a
kidney homogenate from an adult animal inhibited the compensatory growth
of the remaining kidney. Injections of liver extracts, however, had no effect.
From the results of these experiments it would appear that the control of renal
growth might be of humoral nature, involving both stimulating and inhibiting
compounds. The presence of growth-inhibiting substances in the epidermis
and the liver has been postulated by Bullough (1965) and Moolten & Bucher
(1967) respectively. Their presence in kidneys has still not been fully confirmed,
and doubts as to their existence and role will remain until they have been
isolated and identified. Attempts to achieve this are now in progress in this
laboratory, but success will depend partly on the availability of a suitable assay
system. The purpose of the present work was threefold: first, to show that the
culture of renal cortex from neonate animals might provide a suitable means
for estimating the inhibiting activity of tissue extracts; second, to establish
the specificity of the system; third, to explore the possibility of immunological
properties of the renal extracts and test the action of antibodies against the
growth-inhibiting compound.
METHODS
Preparation of culture medium
and plasma
Extracts of whole chick embryo or of embryos from which the kidneys had
been removed, prepared as described by Paul (1972), were added to an equal
volume of Earle's balanced salt solution (Earle, 1943); this was centrifuged for
lOmin at 2000 g and the supernatant was stored at - 2 0 °C. Before use, the
supernatant fluid was adjusted to pH 7-0 by gassing with CO2, and was added
to medium 199 (Morton, Morgan & Parker, 1950) and inactivated horse serum
(No. 5, Wellcome Reagents) in the proportions 1:2:2, respectively.
Desiccated chick plasma (T.C. chicken plasma, Difco B 354) was mixed
Characteristics of a renal growth inhibitor
657
with 5 ml tissue culture reconstituting fluid (Difco B 352), and its pH adjusted
to 7-2 by gently agitating the plasma in the tube in the presence of carbon
dioxide, using phenol red as indicator.
Preparation of explants and cultures
Animals were killed by decapitation. Organs were removed and immersed
in medium 199. Explants of about 1 mm diameter or less were cut, using a
cataract knife. Tissue explants used were either from 6- to 7-day mice or 5-day
rats.
To one drop of chick plasma was added one drop of the culture medium, in
which was incorporated a small piece of tissue explant. When the plasma had
clotted, it was covered with 0-5 ml of culture medium and 0-5 ml Earle's
solution or experimental tissue extract. The preparation was gassed with a
mixture of 95 % O2 and 5 % CO2 for 20 min and kept in a humidifier as described by Trowell (1959). Each plate contained four explants in plasma clots,
and up to ten plates could be housed in the humidifier. The humidifier with the
tissue cultures was kept in a dark room at 37 °C. The medium was renewed
after 24 h, and the preparation was gassed again; the cultures were kept usually
for 48 h, though sometimes up to 96 h.
Preparation of tissue extracts
A known weight of tissue was homogenized in 10 volumes of Earle's solution
and spun for 20 min at 2000 g. The supernatant fluid was kept cool until it
was used. Unless otherwise stated, the volume of supernatant fluid from tissue
homogenates added to tissue explants was 0-5 ml. Tissues used were kidney,
liver, duodenum, spleen and heart muscle, obtained from neonate and adult
mice and rats, adult guinea-pigs, rabbits and pigs.
Estimation of growth
For measurement of the outgrowth the explants were examined under an
inverted microscope, the image of the preparation being projected on a white
background and the contours of the explants and outgrowth being drawn out
carefully. The 'growth' was estimated as the ratio between the surface occupied
by the explant and its outgrowth (E+G) and the surface of the explant (E),
and expressed as (E+G)/E. Thus the absence of growth was represented by 1.
All measurements were made after 48 h.
Production of immune sera
Six rabbits (2-3 kg body wt.) and 14 guinea-pigs (Hartley strain; 600-700 g
body wt.) were used. The renal cortex of adult rats or mice was dissected out
from the medulla, and both were homogenized separately in buffered saline
solution in the usual way. Rabbits were injected subcutaneously once a week
41-2
658
S. E. DICKER AND C. A. MORRIS
for up to 6 weeks with 0-2 ml of the supernatant fluid from tissue homogenates
containing approximately 1 mg protein, emulsified in an equal volume of incomplete Freund's adjuvant. Blood was taken from the marginal ear vein
before each injection. For guinea-pigs, the supernatant fluids from either renal
cortex or medulla emulsified in complete Freund's adjuvant were used. The
guinea-pigs were injected according to the following schedule: on day 1,
injections of 0-6 mg protein in the pad of two paws; followed on days 15, 22
and 29 by intradermal injections (Benacerraf, Ovary, Bloch & Franklin, 1963).
Each injection contained approximately 0-6 mg protein. A week after the last
injection, the animals were killed and blood collected. Similar experiments
were done with samples of livers from adult rats. A volume of unheated serum
from rabbits or guinea-pigs was then mixed with an equal volume of the tissue
extract to be tested; this was allowed to stay at 37 °C for 1 h before being added
to the tissue explants. Experiments using unheated serum samples alone were
carried out as controls.
RESULTS
'Growth' of postembryonic or adult organs, reared on plasma clots, is
characterized essentially by an outgrowth of fibroblast-like cells (Trowell,
1959; Paul, 1972). In relation to the size of the explant the extent to which
these cells grew varied according to the type of organ cultured, the length of
time of the culture and the age of the animals. Explants from renal cortex
'grew' better than those of the medulla, and explants of newborn animals
'grew' more than those of adults. Explants from the renal cortex of 6- to 7day mice or 5-day'rats were used as standard preparations. Their 'growth'
was represented by a mixed population of narrow fibroblast-like or
epitheloid type cells, the former outweighing the latter in frequency. Estimated after 48 h, the extent of their 'outgrowth' was similar. Whether the
culture medium used was prepared from whole chick embryos or from embryos
from which the metanephros had been removed, the 'growth' of explants of
renal cortex from neonate mice or rats was 2-08 ± 0-092 (140 explants). This
figure was adopted as the standard 'growth' of control preparations, after 48 h.
Effects of renal extracts of adult animals on the 'growth' of renal cortex
explants from neonate mice and rats. In preliminary experiments the supernatant
fluid of homogenates of whole kidneys of adult mice or rats was added to
renal cortex explants of neonate mouse or rat. This clearly inhibited their
growth. If, however, after a contact of 24 h, or even 48 h, the supernatant fluid
was removed and replaced by Earle's solution, outgrowth of fibroblast-like
cells started again, indicating that the explant was still alive. In later experiments,
supernatant fluid of homogenates from the renal cortex or medulla were used
separately. This showed that the inhibitory activity was confined essentially to
the cortex, while the medulla contained little, if any. Comparing the effect of
cortical extracts from adult and neonate mice or rats, much less inhibitory
Characteristics of a renal growth inhibitor
659
(140)
200
1-90
ISO
"
" 1-70
•" 1-60
%
f
1-50
o
•_
c
1 40
1-30
1-20
MO
100
0
0-5
10
1-5
20
2-5
Amount of protein (nig 0-5 ml)
Fig. 1. Relation between the amounts of protein (mg/g wet wt.) content of a renal
cortical extract of adult mouse and its inhibitory effect on the' growth' of an explant
of renal cortex of a neonate mouse. Ordinate: 'growth' of the explant as measured
after 48 h. Vertical bars: ±S.E. In parentheses, number of estimations.
activity for the same weight of tissue was found in the extracts from newborn
animals.
The inhibitory activity of cortical extracts from adult mouse or rat was not
significantly altered by heating at 60 °C, but was destroyed by heating at
99 °C for 6-8 min.
Assay of the inhibitory activity from the renal cortex. Figure 1 shows the graded
inhibitory effect of various amounts of protein contents of supernatant fluids
from homogenates of adult mouse cortex on the 'growth' of 7-day mouse
cortical explants. About 90 % inhibition of outgrowth was observed with
extracts containing 2-5 mg protein/0-5 ml. Similar degrees of inhibition were
660
S. E. DICKER AND C. A. MORRIS
Table 1. Protein content of kidneys of adult rats, mice and pigs
Cortex
Medulla
Rat kidney
(mg/g wet wt.)
Mouse kidney
(mg/g wet wt.)
Pig kidney
(mg/g wet wt.)
161-7 ±0-52 (6)
101-2 ±0-37 (6)
183-7 ±0-49 (6)
108-9 ±0-75 (5)
237-8(1)
132-2(1)
Number of estimations in parentheses.
observed with extracts from the renal cortex of adult rats and pigs, though in
the case of the former somewhat larger amounts (30-3-5 mg) of protein were
needed. This difference in amounts of protein required for maximum inhibition
may have been due to the difference in concentration of proteins in the renal
cortex of these species (Table 1).
Animal specificity versus tissue specificity. Supernatant fluids from homogenates of renal cortex of adult mouse, rat, guinea-pig and pig all inhibited
to some extent the 'growth' of renal explants of neonate mice or rats. Supernatant fluids from homogenates of liver, duodenum, spleen, or heart muscle of
adult animals had no significant effect on the standard 'growth' of renal
cortex explants of neonate animals. Conversely, extracts from the renal cortex
of adult animals had no effect on the outgrowth of liver, spleen and heart
muscle explants from young mice or rats. Finally, addition of supernatant fluids
from homogenates of liver, spleen or heart muscle of adult mice, rats or guineapigs had no effect on the outgrowth of explants of homologous organs of young
mice or rats.
Effects of immune serum on the inhibitory activity of extracts
from the renal cortex of adult animals
Immune sera against renal cortex, renal medulla and liver from adult mice
and rats were obtained from 6 rabbits and 14 guinea-pigs. They were mixed
with the supernatant fluid of homogenates of renal cortex from adult mice or
rats in the ratio 1:1, and after incubation at 37 °C they were tested on the
'growth' of renal cortical explants from neonate mice. Each experiment was
planned so that the inhibitory effect of the renal cortex from either adult mouse
or rat could be compared with the effect of (a) the cortical extract mixed
with serum of a control animal, (b) the immune serum alone and (c) the
immune serum mixed with the extract of the renal cortex. Table 2 represents
a typical experiment with serum from rabbits. It was not possible to detect
an effect of the immune sera against liver or renal medulla after they had been
incubated with extracts of liver or medulla from adult animals, which was to
be expected since extracts from neither liver nor renal medulla affected the
outgrowth of kidney explants. Nor did the immune samples alone,whether against
liver, renal cortex or medulla, have an inhibitory effect on the growth of
(3)
(2)
(1)
(4)
Effect of serum
of rabbit F5
on explant
(5)
Effect of serum
of rabbit F5
+ extr. of cortex
on explant
(6)
Effect of serum
of rabbit E5
+ extr. of cortex
on explant
^
>«:
5
ON
200 ±0100 (4)
206 + 0071 (4)
100 ±0030 (4)
200 ±0102 (4)
1-01 ±0-020 (4)
1-89 ±0-118 (4)
105 ±0010 (4)
2-65 ± 0-225 (4)
1-03 ±0-010 (4)
206±0105 (4)
2-95±0079 (4)
3-32 ±0-292 (4)
1 10 ±0041 (4)
1 02 ±0010 (4)
210±0099 (4)
1-90 ±0-061 (4)
1 92±0099 (4)
2-14 ±0173 (4)
2-27±0079 (4)
2-82 ±0-241 (4)
1-92 ±0-095 (4)
1 02 ±0010(4)
1-80 ±0081 (4)
108±0026 (4)
Mean
2-21 ±0-129 (16)
102±0012 (16)
1 06+ 0022 (16)
219 + 0111 (16)
2-33 ±0-152 (16)
2-22 ±0148 (16)
Rabbit F5 had been immunized against the supernatant fluid from a homogenate of adult rat renal medulla; rabbit E5 had been S'
immunized against the supernatant fluid from a homogenate of adult rat renal cortex (see text). Each experiment consisted of four plates for ^
each series (l)-(6). Note that the growth was similar for columns (1), (3), (4) and (6), and markedly different from that in columns (2) and (5). ^-
Effect of serum
of rabbit E5
on expiant
Effect of extract
of renal cortex
on explant
Control
explants in
Earle's
solution
Table 2. Comparison of the effects of antisera on extracts from adult rat renal cortex, as estimated by the
rate of outgrowth after 48 h, of renal cortex explants of 7-day mice
662
S. E. DICKER AND C. A. MORRIS
neonate mice cortical explants. But when samples of immune serum against
renal cortex were mixed with renal cortical extracts of adult mice or rats, the
inhibitory activity of the latter disappeared, the 'growth' of the explants being
similar to that of controls. Finally, when samples of immune serum against
renal medulla or liver were incubated with extracts of the renal cortex of adult
mouse or rat, the inhibitory effect of the latter was not affected.
Histology of kidneys of rabbits and guinea-pigs injected
with extracts from the cortex and medulla
It is well known that animals injected with extracts of organs show destructive
inflammatory lesions specific to the organ used for immunization. Kidneys of
animals injected with extracts of either cortex or medulla showed inflammatory
and haemorrhagic lesions, with swollen glomeruli and cloudy swelling of cells
of the proximal tubules. In animals injected with extracts of renal cortex,
lesions appeared to be more marked in the outer cortex, where numerous
haemorrhagic areas could be seen, together with cellular proliferation in the
lining of distal tubules and cloudy swelling of cells in the proximal convoluted tubules; in rabbits immunized against extracts of renal medulla,
alterations were more pronounced in the medulla and the cortico-medullary
junction.
DISCUSSION
It would appear from the present results that kidneys of adult animals
contain a substance which inhibits the 'growth' of a renal cortical explant of
neonate mouse or rat. The inhibitory substance appears to be highly tissuespecific. Addition to the cultures of extracts from liver, spleen, duodenum, or
heart muscle from adult animals did not affect the growth of the explants.
Conversely, kidney extracts from adult mouse or rat had no effect on the outgrowth of liver, spleen, duodenum and heart muscle of newborn mice or rats.
The inhibitory activity, however, was not species-specific since renal extracts
from adult rat, guinea-pig, rabbit and pig had an inhibiting action similar to
that of adult mouse. Finally, the renal inhibiting factor appeared to exist predominantly in the cortex of adult animals; extracts from the medulla of adult
animals or from the cortex and the medulla of neonates had little effect.
In contrast with foetal renal explants, which when reared in vitro differentiate
new tubules in the growth zone (Weiss, 1952), the 'growth' of postembryonic
or mature renal tissue in synthetic medium is characterized essentially by a
proliferation and/or migration of fibroblast-like cells mixed with epitheliocytes.
Such outgrowth is not specific to renal tissue; it appears to be a common
feature to cultures of postembryonic and adult tissues (Trowell, 1959; Paul,
1972). This raises the question as to whether such outgrowth can be used to
assess the 'growth' of explants. There are two ways of measuring the rate of
growth of tissue explants in vitro; one is by measuring the increase in the area
Characteristics of a renal growth inhibitor
663
of the culture or in the width of the zone of migration of fibroblasts, the other
is by evaluating the rate of cell divisions, expressed in terms of the mitotic
index. Neither of these criteria is entirely satisfactory (Cunningham & Kirk,
1942; Romanoff, 1960). For practical reasons, it was decided to use the first
method; i.e. to estimate the 'growth' by measuring the area occupied by the
outgrowth, as observed after 48 h of culture. This showed that the 'growth'
of explants of renal cortex from young mice and rats were similar. It was of
interest to see that the extent to which epitheliocytes and fibroblast-like cells
from renal explants 'grow' could be reduced by addition to the culture medium
of extracts of the cortex of adult kidneys. The fact that this reduction of outgrowth from renal explants occurred only in the presence of kidney extracts
of adult animals, irrespective of their species, and not with extracts of any
other tissue, is crucial to the interpretation of the present results.
Since the outgrowth of renal explants consisted of a mixture of fibroblast
and epithelioid type cells, it is possible that they may be controlled separately.
This is suggested by the observation that when inhibition of the outgrowth
started, the epithelioid type cells disappeared first. If this interpretation is correct,
the renal cortex extract may prove to contain at least two controlling substances.
However, until this point has been clarified further and the culture-assay made
more precise, it may be wise to refer to the presence in the cortex of adult
kidneys of a cortex-specific rather than a tissue-specific compound.
The inhibitory activity present in the supernatant fluid of a homogenate
from renal cortex of adult animals was destroyed at 99 °C. This does not tell
much as to its nature, beyond the fact that the active compound may possibly
be a protein of high molecular weight. Investigation as to its nature is in
progress.
One of the results of work carried out between 1920 and 1935 by Fleisher &
Arnstein (1921) but mainly by Landsteiner (1936) was the concept of speciesspecificity and organ-specificity. This led to the recognition of the antigenic
complexity of tissue extracts and the similarity of antigenic properties between
species. In spite of the obvious antigenic complexity of renal extracts, the
results obtained were surprisingly clear: while immune sera prepared by immunizing rabbits and guinea-pigs against renal medulla or liver from adult
animals did not interfere with the inhibitory activity of extracts of the renal
cortex, immune sera obtained by immunization of rabbits and guinea-pigs
against the renal cortex of adult animals blocked the inhibitory effect of the
latter, with the result that cultures of explants of cortex from neonate mice or
rats showed proliferation and/or migration of epitheliocytes and fibroblasts
similar to that observed in control cultures.
When animals have been injected with extracts of organs, autoantibodies
and destructive inflammatory lesions specific to the organ used for immunization occur (Roitt, 1972). This explains the lesions observed in the kidneys of
rabbits injected with extracts of either renal cortex or medulla. Though both
664
S. E. DICKER AND C. A. MORRIS
extracts produced damage, it is of interest that in the animals injected with
extracts of the cortex the lesions were much more marked in the cortex and
outer-cortex than in those injected with extracts of renal medulla.
Thus, from direct and indirect evidence, it would appear that the renal
cortex of adult animals contains a substance which inhibits the outgrowth of
renal cortex explants of neonate mice and rats in vitro and has antigenic properties in vivo. Though not species-specific, this substance appears to be highly
cortex-specific. While it would be tempting to assume that such a substance
may play a role in the control of kidney growth, it must be realized that extrapolation from tissue culture to a kidney in vivo is a very long one and that
until more work is done, the present results can be no more than suggestive.
One of us (S.E.D.) would like to most sincerely thank Professor C. A. Vernon for
generous hospitality in his laboratory, helpful advice and continuous interest in the work.
We would also like to thank Dr Marguerite Blake (Chelsea College) for her help in the
histological investigation of kidneys.
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