Pathophysiology of Congenital Diaphragmatic Hernia IV:

Renal Hyperplasia Is Associated With Pulmonary Hypoplasia

By Yataro Hosoda, Jon E. Rossman, and Philip L. Glick
Buffalo, New York

l The hypothesis of this article is that growth of the fetal

lung is stimulated by a pulmonary growth factor (PGF)
produced by the kidneys, which is modulated by a feedback
signal from the lungs, a pulmonary-derived renotropin (PDR).
In the fetus with pulmonary hypoplasia (PH), the lungs may
maximally stimulate this feedback loop to release more PDR,
resulting in continual stimulation of the kidneys and renal
enlargement. If such a schema plays a role in the pathophysi-
ology of PH. newborn infants with congenital diaphragmatic
hernia (CDH) or chronic amniotic fluid leak (CAFL) should
have enlarged kidneys. To investigate this hypothesis, we
created models of CDH in fetal lambs and CAFL in fetal
rabbits, and then analyzed lung (Lu) and kidney (K) growth.
When compared to controls, newborn CDH lambs had signif-
icantly smaller lungs and larger kidneys. The lungs were
hypoplastic as defined by either decreased lung weight/
body weight (LuW/BW), lung DNA/body weight (Lu DNA/
BW), or lung total protein/ body weight (LuTP/BW) (P c .Ol).
Renal hyperplasia was confirmed by KW/BW, K DNA/BW
(P < .Ol), and KTP/BW (P < .05). An inverse relationship
between lung size and kidney size could be described by the
equation KW/BW = 1.04 - 0.12 LuW/BW (r = -.75). The
CAFL model in newborn rabbits produced severe oligohy-
dramnios when compared with controls (P c .Ol). This re-
sulted in fetuses with smaller lungs and larger kidneys as Fig 1. Theoretical feedback modulation of normal in utero lung and
compared with those of controls. The lungs were signifi- kidney growth. The black arrows indicate normal modulation of the
cantly smaller and more hypoplastic than controls when afferent and efferent limbs of this loop.
compared by LuW (P < .Ol), LuW/BW (P c .Ol), Lu DNA/BW
(P < .05), and Lu TP/BW (P < .Ol). The kidneys were signifi-
cantly larger and more hyperplastic than controls as judged
by the similar criteria (P c .Ol). In addition, the earlier in unclear, but recently a unifying hypothesis explaining
gestation the operation was performed, the greater was the the association of polyhydramnios, PH, and renal
effect on the kidneys and lungs. These data support our enlargement in CDH has been proposed.’ In this
hypothesis and demonstrate that significant renal enlarge-
hypothesis, Glick et al suggest that in utero growth of
ment is associated with PH. The presumed mechanisms of
renal enlargement associated with PH and the relation to the lungs is stimulated by a pulmonary growth factor
amniotic fluid volume are discussed. If such a PGF and PDR (PGF) produced by the kidneys. It is further hypothe-
can be isolated, the diagnostic and therapeutic implication sized that the release of PGF is modulated by a
for fetuses with PH are considerable.
Copyright o 7993 by W.B. Saunders Company

INDEX WORDS: Congenital diaphragmatic hernia; pulmo-

nary hypoplasia; pulmonary growth factors; renal hyperpla- From The Buffalo Institute of Fetal Therapy, the Division of
sia. Pediatric Surgery, The Children’s Hospital of Buffalo, and the Depart-
ments of Surgery and Pediatrics, School of Medicine and Biomedical
Sciences, The University at Buffalo, State University of New York,

T HE MORTALITY rate for congenital diaphrag- Buffalo, NY

matic hernia (CDH) is thought to depend on
both the severity of pulmonary hypoplasia (PH) and
the pulmonary hypertension present in the neonatal
period, and remains at approximately 50%.lm4Ironi-
cally, prenatal diagnosis and optimally managed pre-
natal, perinatal, and postnatal cases of CDH have
mortality rates as high as 8O%.5,6 Polyhydramnios
associated with CDH may further increase the mortal-
ity.5,6The etiology of the polyhydramnios in CDH is
Presented at the 23rd Annual Meeting of the American Pediatric
Surgical Association, Colorado Springs, Colorado, May 13-I 6, 1992.
Supported in part by The Women S and Children’s Health Research
Foundation of the Children S Hospital of Buffalo, The March of Dimes
Birth Defects Foundation Basic Research Grant (#l-1244), and The
United States Surgical Carp, Norwalk, CT.
Address reprint requests to Philip L. Glick, MD, The Buffalo
Institute of Fetal Therapy, The Children’s Hospital of Buffalo, 219
Bryant St, Buffalo, NY 14222.
Copyright o 1993 by W.B. Saunders Company
0022-3468/9312803-0031$03.00/O

464 JournalofPediafric Surgery, Vol28, No 3 (March), 1993: pp 464-470

RENAL HYPERPLASIA & PULMONARY HYPOPLASIA IN CDH 465

feedback signal from the lungs (to the kidneys) by a
pulmonary-derived renotropin (PDR) (Fig 1).
If this hypothesis is true, CDH and other causes of
PH, ie, chronic amniotic fluid leak (CAFL), should be
associated with renal enlargement. This theory re-
quires experimental verification. The purpose of the
present study is to document the effects of PH on
renal growth and to determine if the changes seen are
due to a change in the cell number (hyperplasia v
hypoplasia), cell size (hypertrophy v atrophy), or
both.
MATERIALS AND METHODS
CDH Model (Fetal Lambs)
At 80 days of gestation, diaphragmatic hernias were created
surgically in 24 fetuses by methods previously described.* We used
the GIA stapling device (US Surgical Corp, Norwalk, CT) to open
and the TA-90 stapler (US Surgical Corp) to close the uterus.9 At
139 to 14.5 days of gestation, the pregnant ewes underwent
cesarean section using the anesthetic technique previously de-
scribed.x The lambs were then killed by intravenous potassium
chloride injection as the cord was divided. Next, the lambs were
delivered without spilling the amniotic fluid, and amniotic fluid
volume was directly measured.
The newborn lamb body weight (BW), lung wet weight (LuW),
and kidney wet weight (KW) of all specimens were measured. In an
effort to standardize the size of the organs and to account for
differences in BW, ratios were calculated (LuW/BW. KW/BW).
The lung and kidney samples were homogenized using a tissue
homogenizer and an ultrasonicator (Braun-Sonic 1510; B. Braun
Biotech Inc, Allentown, PA), and DNA was isolated by phenol-
chloroform extraction.10 The extracted DNA content was deter-
mined by the Hoechst 33258 method with the TKO 100 Mini
Fluorometer (Hoefer Scientific Instruments, San Francisco,
CA).lt,l” The total lung and kidney DNA (Lu DNA, K DNA) were
estimated from the following equation: total DNA = DNA of
aliquot x organ weight/weight of aliquot. Ratios were then
calculated (Lu DNA/BW, K DNA/BW). The total DNA and the
DNA/BW were assumed to be directly proportional to total cell
population of the organ sampled; differences between experimen-
tal groups were thought to reflect hyperplastic or hypoplastic
changes in the organ’s development.‘3-15
The total protein (TP) content of lung and kidney tissues was
assayed (Lu TP, K TP) using the coomassie blue spectrophotomet-
ric analysisI and again corrected for differences in body weight (Lu

140 3

ae
120
C’
7
100

T OL
cm comro, CDH COnnd CD” conwe,
9
E Lung WVBW DNAIBW Total Proteln/BW
w
60

CDH Control CDH Control

A
Lung Weight Kidney Weight
0.06

CDH Control CDH Control

D
Lung Kidney

Fig 2. (A) The effect of the procedure on lung and kidney growth in the CDH fetal lamb model (**P < .Ol v controls). (6) The effect of the
procedure on lung weight/body weight, lung DNA/body weight, and lung total protein/body weight in the CDH fetal lamb model (**P < .Ol v
controls), indicating significant pulmonary hypoplasia. (C)The effect of the procedure on kidney weight/body weight, kidney DNA/ body weight,
and kidney total protein/body weight in the CDH fetal lamb model (‘P c .05 v controls, l*P < .Ol Y controls), indicating significant renal
hyperplasia. (D) Differences were not reflected in lung DNA/TP and kidney DNA/TP ratios, indicating that the cell size in the lungs and the kidneys
was unchanged, but that the cell population was decreased in lungs (hypoplasia but not atrophy) and was increased in kidneys (hyperplasia but
not hypertrophy) in the CDH fetal lamb model.
466 HOSODA, ROSSMAN, AND GLICK

TPIBW, K TP/BW). Ratios between DNA and TP were calculated
(Lu DNA/TP, K DNA/TP, Lu TP/DNA, and K TP/DNA) as a
measure of cell size, and differences between experimental groups
were thought to reflect hypertrophic or atrophic changes in organ
development.i7 Serving as controls, 15 nonoperated litter mates
underwent cesarean section at 139 to 145 days of gestation and the
lungs and the kidneys were analyzed in a similar manner.
CAFL Model (Fetal Rabbits)
New Zealand white rabbits of known gestational age underwent
operations to simulate CAFL to produce oligohydramnios and
PH.‘s The operations were performed on day 17, 20, or 23 of
gestation in the pseudoglandular period of lung development.
Hysterotomy and amniotomy were carried out on 3 or 4 sacs of one
uterine horn. An S-mm segment of Silastic tubing was placed into
the amniotic sac and secured by a purse-string suture. This tubing
assured continued drainage of amniotic fluid into the maternal
peritoneal cavity. Six fetuses on day 17 of pregnancy, 24 fetuses on
day 20, and 20 fetuses on day 23 underwent this procedure.
Gestation was then allowed to continue until day 30 of pregnancy,
when all fetuses underwent cesarean delivery. The newborn rabbits
were checked for survival and then killed.
By methods similar to the iamb study, BW, LuW, KW, LuW/
BW, KWIBW, Lu DNA, K DNA, Lu DNA/BW, K DNA/BW, Lu
TP, K TP, Lu TPIBW, K TPIBW, Lu DNAITP, K DNA/TP, Lu
TP/DNA and K TP/DNA were determined. The liver wet weight
(LiW), LiW/BW, liver DNA (LiDNA), Li DNA/BW, liver TP (Li
TP), Li TP/BW, Li DNAITP, and Li TPiDNA was used as a
control organ to determine the effect of the procedure.la These
data were compared to that of corresponding nonoperated litter
mates for each group. In another study, 8 operated fetuses of each
group underwent reexploration at 27 to 28 days of pregnancy, then
each amniotic sac was aspirated of all fluid using a 20-gauge needle
and syringe, and the volume was recorded.18.19 These were com-
pared with control fetuses to assess the changes in amniotic fluid
volume due to the procedure.
phy) in the CDH model. Comparison of LuW/BW
and KW/BW showed that there was an inverse
relationship between lung size and kidney size in the
fetal lamb that could be described by the equation
KW/BW = 1.04 - 0.12 LuW/BW (r = -.75) (Fig 3).
The mean amniotic fluid volume of the CDH model
was larger than that of controls (848 + 382 v 704 5 227
mL) but difference between groups was not statisti-
cally significant.
CAFL Model
All six fetuses that were operated on day 17 of
pregnancy died in utero for presumed technical
causes, and this arm of protocol was stopped. Eigh-
teen of 24 fetuses that were operated on at 20 days
and all of 20 that were operated on at 23 days survived
until term.
This procedure produced severe oligohydramnios
when compared to unoperated controls (P < .Ol, Fig
4A). The oligohydramnios from the CAFL resulted in
fetuses with smaller lungs and larger kidneys as
compared with those of controls (Fig 4B). The lungs
were significantly smaller and more hypoplastic when
compared by LuW (P < .Ol), LuW/BW (P < .Ol),
Lu DNA/BW (P < .05), and Lu TP/BW (P < .Ol,
Fig 4C). The kidneys were significantly larger and
more hyperplastic than controls as judged by the
similar criteria (P < .Ol, Fig 4D). In addition, the
2.0 -

Statistical Analysis
The data were analyzed on a PC-286LE computer (EPSON
Corp, Tokyo, Japan) using l-2-3 (Lotus Development Corp,
Cambridge, MA) and Statview II (Abacus Concepts Inc, Berkeley, 1.5 -
2
n
CA). Statistical determinations were performed using the Mann-
E
Whitney U test, with P < .05 considered significant. 9

5
RESULTS

CDH Model
Fifteen of 24 lambs that underwent fetal surgery
survived until term. The operated lambs had signifi-
cantly smaller lungs, and larger kidneys when com-
pared with controls (Fig 2A). The lungs were signifi-
cantly hypoplastic as defined by either decreased
LuW/BW, Lu DNA/BW or Lu TP/BW (P < .Ol, Fig
2B). Renal hyperplasia was confirmed by KW/BW, K
DNA/BW (P < .Ol) and K TP/BW (P < .05, Fig
2C). However, these differences were not reflected in 0.0 ’ I , ! I ,

0 1 2 3 4 5
Lu DNA/TP and K DNA/TP ratios (Fig 2D), indicat-
ing that the cell size in the lungs and the kidneys was Lung Wt I Body Wt (gm%)
unchanged, but that the cell population was de-
Fig 3. The relationship between LuW/BW and KW/BW can be
creased in lungs (hypoplasia but not atrophy) and was described by the regression equation KW/BW = 1.04 - 0.12 LuW/
increased in kidneys (hyperplasia but not hypertro- BW,r = -.75 (P c .OOl). Squares = controls. Triangles = CDH.
RENAL HYPERPLASIA & PULMONARY HYPOPLASIA IN CDH 467

Rabbits : n=8

1
Kidney WVBW DNlVBW Total ProteinlBW

0.8

0.6

0.4

0.2

A CAFL C CAFL C
20 days 23 days
n
“CAFL C CAFL C “CAFL C CAFL C
E 20 d 23 d 20 d 23 d

Lung Kidney

0.4

0.2

”
“CAFL C CAFL C CAFL C CAFL C Fig 4. (A) The fetal rabbit CAFL model produced severe oligohy-
B 20d 23 d 20 d 23 d dramnios by decreasing amniotic fluid volume to less than that of
Lung Weight Kidney Weight controls (**f c .Ol). (B) The effect of the procedure on lung and
kidney growth in the rabbf chronic amniotic fluid leak model (**P e .Ol
Y controls). (C) The effect of the procedure on lung weight/body
weight, lung DNA/ body weight, and lung total protein/ body weight
in the rabbit chronic amniotic fluid leak model (*p < .lXi v controls,
l*p c .Ol v controls), indicating significant pulmonary hypoplasia. (D)
The effect of the procedure on kidney weight/body weight, kidney
DNA/body weight, and kidney total protein/body weight in the
chronic amniotic fluid leak model (**P < .Ol v controls), indicating
significant renal hyperplasia. (E) Differences were not reflected in lung
DNA/TP and kidney DNA/TP ratios, indicating that the cell size in the
lungs and the kidneys was unchanged, but that the cell population
was decreased in lungs (hypoplasia but not atrophy) and was in-
creased in kidneys (hyperplasia but not hypertrophy) in the rabbit
CAFL model.
468
earIier in gestation the operation was performed, the
greater was the effect on the kidneys and lungs.
Similar to the lamb study, DNA/TP and TP/DNA
ratios of lungs and kidneys did not differ significantly
between groups and thus cell atrophy or hypertrophy
are not involved in the changes in organ size which
were observed (Fig 4E). The liver did not vary
significantly between groups, and its growth appeared
to be unaffected by the procedure.
DISCUSSION
These results in the fetal lamb support our hypoth-
esis that PH in CDH is associated with significant
renal enlargement. The renal enlargement is hyper-
plastic and not hypertrophic growth. This experimen-
tal verification confirms our previous clinical observa-
tion.’
According to our hypothesis, in the fetus with
CDH, the space-occupying nature of the herniated
viscera hinders normal pulmonary growth and devel-
opment and results in PH. The lungs may “sense” this
inadequate pulmonary growth and development and
secrete excess PDR. The kidneys would respond to
this afferent signal, enlarge by hyperplastic growth,
and secrete increased amounts of PGF.
We had previously speculated that, if our theory
were true and broadly applicable to other instances of
PH, then PH associated with oligohydramnios, ie,
CAFL, should also be associated with renal enlarge-
ment. Our data now confirm this and the effect in
rabbits seems to be proportional to the length of
gestation in which the stimulus exists. Presumably in
1. Anderson KD: Congenital diaphragmatic hernia, in Welch
KJ, Randolph JG, Ravitch MM, et al (eds): Pediatric Surgery (ed
1). Chicago, IL, Year Book, 1986, pp 589-601
2. Reynolds M, Luck SR, Lappen R: The “critical” neonate with
diaphragmatic hernia: A 21-year perspective. J Pediatr
19:364-369,1984
3. Touloukian RJ, Markowitz RI: A preoperative x-ray scoring
system for risk assessment of newborns with congenital diaphrag-
matic hernia. J Pediatr Surg 19:252-257, 1984
4. Simson JNL, Eckstein HB: Congenital diaphragmatic
A 20 year experience. Br J Surg 72:733-736, 1985
5. Adzick NS, Harrison MR, Glick PL, et al: Diaphragmatic
hernia in the fetus: Prenatal diagnosis and outcome in 94 cases. J
Pediatr Surg 20:357-361,1985
6. Steinhorn RH, Kriesmer PJ, Green PP, et al: Natural history
of congenital diaphragmatic hernia in Minnesota: Impact of in
utero diagnosis on survival. Pediatr Res 29:236A, 1991 (abstr)
7. Glick PL, Siebert JR, Benjamin DR: Pathophysiology
congenital diaphragmatic hernia I: Renal enlargement
feedback modulation by pulmonary derived renotropins-A
ing hypothesis to explain pulmonary hypoplasia, polyhydramnios,
and renal enlargement in the fetus/newborn with congenital
diaphragmatic hernia. J Pediatr Surg 25:492-495,199O
HOSODA, ROSSMAN, AND GLICK
CAFL, as in CDH when the lungs “sense” inadequate
growth, they also produce increased amounts of
PDR. The kidneys become hyperplastic and produce
more PGF, but the CAFL and oligohydramnios
persists and PH results.
The precise etiologic role of amniotic fluid in the
process of lung growth is not known. Several possible
etiologies for PH with oligohydramnios have been
described.i8-20 Oligohydramnios may cause fetal tho-
racic compression by the amniotic membranes and
the uterine wall and limit intrathoracic space and
fetal breathing movement.2’-23 However, attempts at
intraamniotic saline infusions to reconstitute amni-
otic fluid volume have not been successful in prevent-
ing PH. 24Therefore, we believe the quality of amni-
otic fluid rather than just the quantity may be
important. PGF may be an important component of
the amniotic fluid to promote proper lung growth and
development.
The present data encourage continued investiga-
tion to identify, characterize, and synthesize PGF and
PDR. These may be unique growth factors specific to
the lungs and kidneys, or ubiquitous growth factors
being modulated in a unique fashion under the
physiologic circumstances. In the future, PGF might
be used therapeutically for fetuses and newborns with
PH. Accelerating pulmonary growth and develop-
ment in newborns with CDH or PH due to other
causes may hasten weaning from mechanical ventila-
tion or extracorporeal membrane oxygenation and
decrease their mortality and morbidity.
REFERENCES
8. Glick PL, Stannard VA, Leach CL, et al: Pathophysiology of
Congenital diaphragmatic hernia II: The fetal lamb CDH model is
surfactant deficient. J Pediatr Surg 27:382-388, 1992
9. Adzick NS, Harrison MR, Flake AW, et al: Automatic
Surg uterine stapling device in fetal surgery. Surgical Forum 36:479-481,
1985
10. Strauss WM: Preparation of genomic DNA from mamma-
lian tissue, in Ausubel FM, Brent R, Kingston RE, et al (eds):
Current Protocols in Molecular Biology 1. New York, NY, Wiley,
hernia: 1988, Sections 2.2.1-2.2.3
11. Brunk CF, Jones KC, James TW: Assay for nanogram
quantities of DNA in cellular homogenates. Anal Biochem 92:497-
500,1979
12. Labarca C, Paigen K: A simple, rapid, and sensitive DNA
assay procedure. Anal Biochem 102:344-352,198O
13. Wigglesworth JS, Desai R: Use of DNA estimation for
growth assessment in normal and hypoplastic fetal lungs. Arch Dis
of Child 56:601-605,198l
suggests 14. Wigglesworth JS, Desai R, Guerrini P: Fetal lung hypopla-
unify- sia: biochemical and structural variations and their possible signifi-
cance. Arch Dis Child 56:606-615, 1981
15. Wigglesworth JS, Desai R: Is fetal respiratory functional a
major determinant of perinatal survival? Lancet 1:264-267, 1982
RENAL HYPERPLASIA & PULMONARY HYPOPLASIA IN CDH
16. Bradford MM: A rapid and sensitive method for the quanti-
fication of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 72:248-254, 1976
17. Winick M, Noble A: Quantitative change in DNA, RNA and
protein during prenatal and postnatal growth in the rat. Dev Biol
12:451-466, 1965
18. Nakayama DK, Glick PL, Harrison MR, et al: Experimental
pulmonary hypoplasia due to oligohydramnios and its reversal by
relieving thoracic compression. J Pediatr Surg 18:347-353,1983
1Y. Adzick NS. Harrison MR, Glick PL, et al: Experimental
pulmonary hypoplasia and oligohydramnios: Relative contribu-
tions of lung fluid and fetal breathing movements. J Pediatr Surg
19:658-665,1984
20. Perlman M. Williams J, Hirsch M: Neonatal pulmonary
Discussion
T.F. Tracy (St Louis, MO): This excellent presenta-
tion by Dr Hosado and mentored by Dr Glick has
really given us two distinct experiments that give us
some insight into the long sought after pulmonary-
renal axis in fetal development. Throughout the
1970s Dr William Blanc of Columbia University,
struggled with this problem and the problem of
pulmonary hypoplasia that resulted from oligohydram-
nios. In a rat model with amniotic fluid leak in late
gestation, he not only succeeded in inducing pulmo-
nary hypoplasia but also succeeded in inducing global
intrauterine growth retardation. Hence he had the
wrong model.
In the 1980s many studies by Dr Harrison and his
colleagues at the University of San Francisco took up
the banner with a successful larger animal model and
were able to demonstrate that oligohydramnias in-
deed did induce pulmonary hypoplasia. However,
with the removal of the thoracic compression, the
hypoplasia was also reversed. Unfortunately, no obser-
vations throughout these several studies noted any
renal hypertrophy or hyperplasia in any of these
experiments. In the 1990s this search for this Holy
Grail of lung development continues in Buffalo. In
these elegant studies their hypothesis is that fetal
lung growth is directed by fetal renal growth. Also
there is a second arm to their hypothesis that there is
feedback loop from the lung to the kidney. In this
paper today, it is clear that renal hyperplasia follows
two sequential modifications that induce pulmonary
hypoplasia. This confirms the second arm of their
hypothesis in that a small lung equals a large kidney.
One of the most interesting figures was the regression
analysis for those animals with congenital diaphrag-
matic hernia. Significant regression for small lungs
leading to large kidneys as achieved by combining
469
hypoplasia after prolonged leakage of amniotic Huid. Arch Dis
Child 51:349-353,1976
21. Blott M, Greenough A, Nicolaides KH, et al: Fetal breathing
movements as predictor of favorable pregnancy outcome after
oligohydramnios due to membrane rupture in second trimester.
Lancet 2:129-131, 1987
22. Collins MH, Moessinger AC, Kleinerman J. et al: Morphom-
etry of hypoplastic fetal guinea pig lungs following amniotic fluid
leak. Pediatr Res 20:955-960, 1986
23. Harrison MR, Jester JA, Ross NA: Correction of congenital
diaphragmatic hernia in utero. I. The model: lntrathoracic balloon
produced fetal pulmonary hypoplasia. Surgery 88:174-182,198O
24. Glick PL, Harrison MR, Adzick NS, et al: Management of
fetus with congenital hydronephrosis II: Prognostic criteria and
selection for treatment. J Pediatr Surg 20:376-387. 1985
control animals as well as experimental animals. My
first question is, does this same regression coefficient
apply in normal controls as well as those diaphrag-
matic controls? In other words, is there a graded
hypoplasia of the lung that results in a graded
hyperplasia within the kidney? If your hypothesis is
correct, the same regression coefficient should be
found in controls and experimental animals, yet both
groups appear flat. The second question pertains to
the problems with amniotic fluid leak. Your attempts
at early induction of amniotic fluid leak led to
increased fetal loss. Was Dr Blanc right in that
amniotic fluid leak is capable of inducing global
growth retardation and only late gestational changes
in amniotic fluid volume will result in the changes that
you found in your kidneys? One minor point with
your DNA body weight data in this chronic amniotic
fluid model was that your kidney weight loss was
significant with increases in your margin of .07 to .04.
You had some interesting liver data that showed a
similar increase in DNA synthesis at the same time.
Could we not be seeing a global regenerative re-
sponse as a stimulus for proliferation?
J. Lunger (St Louis, MO): If your hypothesis is
correct, I assume there is a single cell type in the
kidney that is producing this pulmonary growth fac-
tor. The hypertrophy in the kidney would be caused
by hypertrophy of that single cell type. Have you done
any histological studies to determine what cell type
that is?
.I. Wilson (Boston, MA): We looked at your data
very carefully and even compared your kidney weights
with those of our own control animals in our fetal
studies and there is no question in my mind that your
kidneys are hyperplastic. I think this is fascinating but
I am not sure I agree with your hypothesis. In our own
470
studies of lung growth in fetal lambs, we have been
able to correlate pulmonary hypoplasia with bilateral
nephrectomy which is certainly in keeping with your
model. However, by performing a tracheal ligation on
these anephric animals, we have obtained lungs 3
times the size of normal lungs and 5 times the size of
the lungs with nephrectomy alone. By volume, weight,
alveolar count, and protein/DNA ratios, these are
hyperplastic lungs despite the fact that no kidneys
were present from either 70 or 90 days’ on. We have
repeated this model utilizing experimental diaphrag-
matic hernia rather than nephrectomy as the pro-
moter of pulmonary hypoplasia with similar results. I
believe your description of renal hyperplasia is real
but can you reconcile your model of feedback employ-
ing a renally previous pulmonary growth factor with
our data in anephric fetal sheep?
RL. Glick (response): Thank you very much for
your comments. I would like to begin with Dr Wil-
son’s comments. We had originally speculated that
PDR and PGF were unique growth factors produced
by the lungs and the kidneys, respectively. In work we
have not yet published from our laboratory, we have
shown that fetal serum, amniotic fluid, and homoge-
HOSODA, ROSSMAN, AND GLICK
nates of lungs from the diaphragmatic hernia model
stimulate kidney organ cell cultures. And serum,
amniotic fluid and kidney homogenates in organ lung
cultures stimulate the kidneys to grow. However, in
light of Dr Wilson’s data, we have to rethink our
hypothesis. We think that PDR and PGF may be
unique growth factors specific to the lungs and
kidneys. However, they may be ubiquitous growth
factors being modulated in an unique fashion under
these physiologic circumstances. We have not actually
tried to find these growth factors as much as to prove
that they are there. It may turn out that they are
existing growth factors being modulated uniquely in
this situation. So I don’t think that Dr Wilson’s data
are incompatible with our original hypothesis. The
PGF may just be coming from another organ. As far
as Dr Tracy’s comments go, if you look at the
regression curve, the control animals also fit right
along that regression curve in that the control animals
that have smaller lungs have larger kidneys. The liver
data are interesting and that may go along with Dr
Wilson’s thought that this is a ubiquitous growth
factor.