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Sites of renin production in fetal neonatal and postnatal syrian hamster kidneys.

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THE ANATOMICAL RECORD 235:144-150 (1993)
Sites of Renin Production in Fetal, Neonatal, and Postnatal Syrian
Hamster Kidneys
ALICE H. DODGE
Department Basic Sciences-Anatomical Science, California College of Podiatric Medicine,
S a n Francisco, California 941 15
ABSTRACT
Renin is first observed in the 14-dayfetal kidney. There is a
sharp increase in the number of renin positive cells in the 15-day fetal
kidney. Renin is located in the smooth muscle cells of arterioles, interlobular arteries, and branches of the renal artery. In the neonatal kidney, the
amount of renin appears to be equal to that observed in the 15-day fetal
kidney and is still located in the same blood vessels. In the 24-hour postnatal kidney, there is a sharp decrease in the total amount of renin. Renin
positive cells are now observed at the vascular pole. In the 48-hour postnatal kidney, there is a sharp increase in the total amount of renin. Most of
the renin positive cells are located at the vascular poles; however, a few
renin positive cells are seen in the interlobular arteries.
0 1993 Wiley-Liss, Inc.
Key words: Hamster, Kidney, Immunohistochemistry,Renin, Fetal, Neonatal, Postnatal
Renin is a small protolytic enzyme produced and
stored as prorenin by vascular smooth muscle. Renin is
produced primarily by the smooth muscle cells of the
juxtaglomerular complex of the adult kidney. Active
renin when released into the blood splits angiotensin
from renin substrate. Angiotensin displays a vasoconstrictor action on small blood vessels. The renin-angiotensin-vasoconstriction system is activated in a
matter of minutes (Guyton, 1991).
Renin production has been reported to begin in fetuses in several animal species (guinea pigs-Raimbach and Thomas, 1990; rats-Gomez et al., 1989;
sheep-Brace and Cheung, 1986; humans-Brar et al.,
1985, 1987; Symonds et al., 1985; Gomez et al., 1991).
In guinea pigs, the fetal plasma renin concentration is
higher near term than the circulating renin level in the
adult male guinea pig (Raimbach and Thomas, 1990).
In rats, renin mRNA levels are higher in fetal and
newborn kidneys than in adult kidneys. The fetal
mRNA is found in the vascular poles, afferent arterioles, interlobular arteries, and arcuate arteries (Gomez
et al., 1989). In pregnant women, circulating fetal renin levels have been found to be higher than maternal
levels in some cases of pregnancy-induced hypertension (Brar et al., 1987). In other humans, fetal kidney
renin activity is greater than adult kidney renin (Gomez et al., 1991).
A recent study demonstrated that circulating renin
levels were elevated when the host animal was treated
with the synthetic estrogen diethylstilbestrol (DES).
DES treatment of adult male hamsters resulted in the
production of renin by nonjuxtaglomerular renal vascular smooth muscle cells. Renin positive cells were
noted in renal artery branches, interlobular arteries,
and veins (Dodge et al., 1990).
0
1993 WILEY-LISS, INC
The purpose of the present study was to determine
when fetal hamster kidneys produce renin and what
cells produced it first. Another purpose was t o determine if renin production in fetal kidneys would parallel
renin production in DES-treated adult kidneys.
MATERIALS AND METHODS
The fetal, neonatal, and postnatal Syrian hamsters
used in this study were obtained from the Dodge hamster colony. The maintenance of this colony has been
described previously (Dodge et al., 1988). The day and
time of all hamster breedings were recorded. Fetal day
one was calculated to have begun 24 hours after the
hamsters were mated. Subsequent fetal days were calculated a t 24-hour intervals. The gestation period for
the hamsters in the Dodge colony ranged from 16 to 17
days. Fetal kidneys were collected from fetuses at the
beginning of 13, 14, and 15 days. Neonatal kidneys
were collected from newborns, and postnatal kidneys
were collected from 24-hour postnatal and 48-hour
postnatal hamsters. A total of 27 kidneys were obtained and investigated. For each age group, the kidneys were subjected to light microscopic and electron
microscopic procedures. There were six 13-day fetal
kidneys, six 14-day fetal kidneys, five 15-day fetal kidneys, four neonatal kidneys, four 24-hour postnatal
kidneys, and three 48-hour postnatal kidneys.
Collection of Fetal Kidneys
The hamster mothers were sacrificed by means of
intrathoracic doses of sodium pentobarbital. The uteri
Received January 24, 1992; accepted June 1, 1992
HAMSTER RENIN PRODUCTION
Fig. 1 , A 7-km section of a 13-day fetal kidney reacted with a n antibody to rat kidney renin. No renin
positive cells are observed, x 382.
Fig, 2.A 7-km section of a 14-day fetal kidney reacted with an antibody to rat kidney renin. A few renin
positive cells are observed (open arrowhead), x 489.
145
146
A.H. DODGE
were removed and rinsed in phosphate-buffered saline
(PBS). The fetuses were dissected free and placed in
PBS. Each fetus was floated in PBS in the well of a
maximov slide; the kidneys were dissected free while
viewed with a dissecting microscope.
Collection of Neonatal and Postnatal Kidneys
Neonates, 24-hour postnatal, and 48-hour postnatal
hamsters were sacrificed by means of a quickly severed
spinal cord. The kidneys were dissected free and rinsed
in PBS.
Microscopic Procedures
The dissected kidneys were fixed in either Bouin’s
fixative, 4% paraformaldehyde/ Tris HCL pH 7.6 fixative, or formaldehyde-glutaraldehydecresol fixative
(FGC). The kidneys were used for electron microscopic
and immunohistochemical studies.
Electron Microscopy
All FGC fixed kidneys were postfixed in 1% OsO4,
stained in block with 10% uranyl acetate in methyl
alcohol, processed routinely, and embedded in Epon.
Pale gold to silver sections were double stained with
hot 2% uranyl acetate and sodium bismuth. The sections were viewed in a RCA EMU-3F microscope.
lmmunohistochemistry
Photomicroscopy
Immunohistochemical-stained sections were photographed with Kodak ektaphan film #4162. A #80A
Wratten gelatin filter was used; the filter enhanced the
contrast between the DAB-stained renin deposits and
the hematoxylin-stained nuclei.
RESULTS
In the developing hamster kidney, renin was observed in nonjuxtaglomerular vascular smooth muscle.
In the 13-day fetal kidney, tubular structures and nondifferentiated masses of cells were observed, but no renin positive cells were found (Fig. 1). In the 14-day
fetal kidney, a few renin positive cells were observed
located in the walls of tubularlike structures (Fig. 2).
In the 15-day fetal kidney, there was a sharp increase
in renin positive cells. Renin was located in the smooth
muscle cells of arterioles, interlobular arteries, and
branches of the renal artery (Figs. 3-5).
In the neonatal kidney, the total renin expression
was similar to that observed in the 15-day fetal kidney.
Renin positive cells were observed in the developing
arteries (Fig. 6).
In the 24-four postnatal kidney, the total renin expression was sharply reduced. Secretory granules were
observed in smooth muscle cells at the vascular pole
(Fig. 7). A small number of renin positive juxtaglomerular complexes were observed (Fig. 8). In the 48-hour
postnatal kidney, there were more renin positive cells
than had been observed in the 24-hour postnatal kidney. Renin positive juxtaglomerular complexes were
observed and a few renin positive cells were observed
in interlobular arteries (Fig. 9).
The 4% paraformaldehyde fixed kidneys were processed as previously described (Dodge et al., 1988). Sections (7 pm) were reacted with antibody to r a t kidney
renin supplied by Dr. T. Inagami (Department of BioDISCUSSION
chemistry, Vanderbilt University School of Medicine,
Renin, a proteolytic enzyme, is thought to be proNashville, TN). This antibody to rat kidney renin has
been proven effective in demonstrating hamster kidney duced principally in juxtaglomerular cells in the adult
animal. The results reported in this study support the
renin (Dodge et al., 1988).
The kidney sections were processed using Vector’s hypothesis that nonjuxtaglomerular vascular smooth
biotinylated secondary antibody performed avidin DH- muscle of the hamster kidney can produce renin. In the
biotinylated peroxidase procedure (Vector Elite ABC developing hamster kidney, vascular smooth muscle
PK-6101 Rabbit IgG immunoperoxidase kit, Vector cells located in the developing arterioles, interlobular
Laboratories, Burlingame, CA). The kidney sections arteries, and branches of the renal artery were renin
were reacted with a 1:1,000 dilution of the antibody to positive. These vascular smooth muscle cells continued
rat kidney renin in 1%NaCl PBS. Dilution of the pri- to produce renin in the neonatal kidney and then
mary antibody in this NaCl concentration resulted in abruptly reduced renin production by 24 hours postnalittle to no nonspecific staining. The peroxidase-stained tal. Renin production increased in the 48-hour kidney
components in the sections were demonstrated by and was produced primarily by the juxtaglomerular
means of Vector’s peroxidase substrate DAB kit cells.
Gomez et al. (1991) reported that under the approSK-4100. Renin positive cells stained a browdblack
priate
physiological stimulus adult renal vasculature
color. The sections were counterstained with GillBaker-Mayer hematoxylin solution, routinely pro- could revert to a fetal pattern of renin distribution. The
cessed, and mounted with coverslips. In order to make
sure that each immunohistochemical staining reaction
was positive, rat kidney sections were processed with
the hamster sections. Renin positive juxtaglomerular
Fig. 3. A 7-km section of a 15-day fetal kidney reacted with an
sites in the rat kidneys indicated a positive staining antibody to rat kidney renin. Renin positive cells are observed in the
reaction. Control kidney sections were reacted with wall of a branch of the renal artery (open arrowhead), x 443.
Dako’s normal rabbit serum (NRS) #902 diluted 1:
Fig. 4. A 7 pm section as a 15-day fetal kidney reacted with an
1,000 with 1% NaCl PBS. The control sections were antibody to rat kidney renin. Renin positive cells are observed in the
processed similarly to the antibody to RKR-treated sec- wall of an interlobular artery (open arrowhead), x 550.
tions. No renin positive cells were observed. Substrate
Fig. 5. A 7-pm section of a 15-day fetal kidney reacted with an
only controls and secondary antibody plus Vector’s antibody
to rat kidney renin. There are numerous renin positive cells
ABC plus substrate controls were negative.
in the wall of several interlobular arteries (open arrowhead), x 458.
Figs. 3-5.
Fig. 6.A 7-(rm section of a neonatal kidney reacted with a n antibody to rat kidney renin. There are
numerous renin positive cells in a branching interlobular artery (open arrowhead), x 367.
Fig. 7. A 24-hour postnatal kidney section in which is seen an afferent arteriole. The vascular smooth
muscle contains secretory granules (S.G.), x 15,000.
Fig. 8. A 7-pm section of a 24-hour postnatal kidney reacted with a n antibody to rat kidney renin. A
small amount of renin positive material is observed in the wall of the afferent arteriole at the vascular
pole (open arrowhead), x 489.
Fig. 9. A 7-pm section of a 48-hour postnatal kidney reacted with an antibody to rat kidney renin.
Renin positive cells (open arrowheads) are observed in the walls of afferent arterioles and in the wall of
the interlobular artery (*I, x 475.
150
A.H. DODGE
DES-induced renin distribution pattern in the adult
hamster kidney reported by Dodge et al. (1990) is similar to the developing renal renin pattern described in
this report. For example, renin positive cells were demonstrated in branches of the renal artery i n the DEStreated adult kidneys and the 15-day fetal kidneys. It
can be concluded that the adult renal renin pattern can
revert to the developing pattern. The hamster DES animal model provides a system that can be used to study
reversion to a fetal pattern.
LITERATURE CITED
Brace, R.A., and C.Y. Cheung 1986 Fetal cardiovascular and endocrine responses to prolonged fetal hemorrhage. Am. J. Physiol.
251 (2 PtL):R417-424.
Brar, H.S., S.L. Kjos, W. Dougherty, Y.S. Do, H.B. Tam, and W.A.
Hsuch 1987 Increased fetal olacental active renin Droduction in
pregnancy-induced hypertension. Am. J . Obstit. Gynecol.,
157(2):363-367.
Brar, H.S., Y.S. Do, H.B. Tam, G.J. Valenzula, R.D. Murray, L.D.
Longo, M.L. Yonekura, and W.A. Hsuch 1985 Uteroplacental unit
as a source of elevated circulating prorenin levels in normal pregnancy. Am. J. Obstet. Gynecol., 155(6):1223-1226.
Dodge, A.H., LA. Reid, and T. Inagami 1990 DES-induced renin production by hamster renal vascular smooth muscle. Thirtieth Annual Meeting of the American Society for Cell Biology, p. 345a.
Dodge, A.H., M. Brownfield, I.A. Reid, and T. Inagami, 1988 Immunohistochemical renin study of DES-induced renal tumor in the
Syrian hamster. Am. J . Anat., 182:347-352.
Gomez, R.A., C. Pupilli, and A.D. Everett 1991 Molecular and cellular
aspects of renin during kidney ontogeny. Pediat. Nephrology,
5(1):80-87.
Gomez, R.A., K.R. Lynch, B.C. Sturgill, R.L. Chevalier, R.M. Carey,
and M.J. Peach 1989 Distribution of renin mRNA and its protein
in the developing kidney. Am. J. Physiol. 257:F850-858.
Guyton, A.C. 1991 Blood pressure control-special role of the kidneys
and body fluids. Science, 252:1813-1816.
Raimbach, S.J., and A.L. Thomas, 1990 Renin and angiotensin converting enzyme concentrations in the fetal and neonatal guinea
pig. J . Physiol., 423:441-451.
Symonds, E.M., D.J. Craven, and C.H. Redeck 1985 Fetal plasma
renin and renin substrate in midtrimester pregnancy. Br. J . Obstet. Gynaecol., 92(6):618-621.
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