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Effects of maternal bilateral ureteral ligation on the differentiation of glomerular anionic sites in fetal rat kidney.

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THE ANATOMICAL RECORD 230:267-272 (1991)
Effects of Maternal Bilateral Ureteral Ligation on
the Differentiation of Glomerular Anionic Sites in
Fetal Rat Kidney
TOSHIYA OKADA AND YOSHIO MORIKAWA
Department of Veterinary Anatomy, College of Agriculture, University of Osaka Prefecture,
Mozuumemachi 4-804, Sakai, Osaka 591, Japan
ABSTRACT
Effects of maternal bilateral ureteral ligation on the differentiation of glomerular anionic sites in fetal kidney were studied with the colloidal iron
method. The ligation was performed on days 17, 19, and 21 of pregnancy, and the
animal were euthanized 1 day after each ligation. On fetal day 20, in the fetuses
from ligated mothers, colloidal iron was concentrated in laminae rarae interna and
externa of the glomerular basement membrane. In the fetuses from sham-ligated
mothers, however, a different pattern of iron distribution was noted. In most areas
of the glomerular basement membrane, colloidal iron was randomly distributed as
in both fetuses of fetal day 18. In the remaining areas, the same pattern of colloidal
iron distribution as was noted in the fetuses from ligated mothers of fetal day 20
was observed. On fetal day 22, colloidal iron was densely accumulated in laminae
rarae interna and externa of the glomerular basement membrane in both groups.
However, the iron-free zone was slightly thicker in the fetuses from the ligated
mothers. These findings suggest that maternal bilateral ureteral ligation accelerates the differentiation of glomerular anionic sites in the kidney of fetal rats.
The glomerular capillary wall, especially the basement membrane, functions as a size- and charge-selective barrier to macromolecules (Reeves et al., 1980).
Effective filtration starts with morphological differentiation of filtering membrane consisting of the epithelial slit, glomerular basement membrane, and endothelial fenestrae (Friis, 1980). Furthermore, anionic sites
in renal glomerulus, which are observed on free surfaces of the epithelial and endothelial cells and in glomerular basement membrane, are involved in effective
filtration (Kanwar and Farquhar, 1979a).
In fetal rats, waste products are metabolized largely
in the placenta (not in the fetal kidney) and are transported into the maternal circulation throughout gestation (Liggins, 1972).Matsuo et al. (1986) have reported
that maternal bilateral ureteral ligation for 1 day
causes a remarkable increase in blood urea nitrogen
(BUN) level, which in late gestation passes through the
placenta into the fetal circulation to stimulate fetal
kidney urine production. We have reported that maternal bilateral ureteral ligation at the time of active
glomerular maturation results in an increased number
of mature-type glomeruli in fetal rats (Okada and
Morikawa, 1988). These findings led us to wonder
whether some unexplained change might be taking
place in the glomerular filtration property a t the anionic sites in fetal kidney. Moreover, there is very little
information available on this subject.
Therefore, this study was undertaken to clarify the
events surrounding the differentiation of glomerular
anionic sites of fetal kidney when the maternal kidneys are rendered dysfunctional by ureteral ligation.
0 1991 WILEY-LISS, INC.
MATERIALS AND METHODS
Animals
Wistar rats were given water and a commercial diet
(NMF; Oriental Yeast Co., Japan), both ad libitum.
The day after an overnight mating was considered as
day 1 of gestation. A total of 18 pregnant rats were
divided into two groups, “ligated” and “sham-ligated.’’
Bilateral ureteral ligation was performed on days 17,
19, and 21 of gestation by the method of Matsuo et al.
(1986). Under ether anesthesia, seven fetuses were obtained from each rat, in the six groups of three animals
each, 1 day after the operation was performed. The
fetuses from ligated mothers were designated E fetus
and those from sham-ligated mothers C fetus.
Tissue Processing
Four fetuses were studied for localization of colloidal
iron in the glomeruli. Fetuses were decapitated and the
left kidneys were quickly removed. The kidneys were
then cut into small blocks and fixed in a mixture of
1.0% glutaraldehyde and 0.8% paraformaldehyde in
0.1 M cacodylate buffer (pH 7.4) for 3 hr a t room temperature. After washing overnight in the same buffer
a t 4”C, the blocks were cut into 40 pm sections with a
Sorvall TC-2 tissue sectioner. For detection of anionic
sites in the glomerulus, sections were floated for 3 hr in
colloidal iron at pH 1.8, prepared according to the
method of Nicolson (1972), a t room temperature. After
washing overnight in the buffer, the sections were
Received August 3, 1990; accepted October 31, 1990
268
T. OKADA AND Y. MORIKAWA
postfixed with 1.0% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) for 2 hr. The sections were dehydrated in a graded series of alcohol, embedded in EponAraldite, cut for electron microscopy, and examined
under a Hitachi H-600 electron microscope a t 75 kV,
without counterstaining. The kidneys from the remaining three fetuses of the ligated and the sham-ligated
mothers were processed for standard electron microscopy. Since three glomeruli per animal had sufficed in
a similar study by Avasthi and Koshy (1988), a minimum of three glomeruli per fetus, which were the most
differentiated, were studied in the present study.
had foot processes and slits. The endothelial cells were
fenestrated. The epithelial and endothelial cells shared
a common trilaminar basement membrane, in which
laminae rarae interna and externa as well a s lamina
densa were clearly visible (Figs. 9, 10). Colloidal iron
was concentrated in laminae rarae externa and interna, between which a n iron-free area, the lamina
densa, was interposed. The lamina densa in the E fetus
was slightly thicker (40-50 nm) than in the C fetus
(30-40 nm) (Figs. 11, 12).
DISCUSSION
The present results indicate that differentiation of
RESULTS
the glomerular basement membrane in fetal rat kidney
All observations described here refer to the most dif- is accelerated by maternal bilateral ureteral ligation
ferentiated glomeruli, located in the deepest portion of performed after day 19 of pregnancy. On fetal day 18,
the renal cortex. On fetal day 18, neither epithelial there was no remarkable difference between E and C
slits nor endothelial fenestrae were seen in C fetus, fetuses in anionic sites, as demonstrated by the agwhereas endothelial fenestrae were often observed in E gregation of colloidal iron. On fetal days 20 and 22,
fetus. Neither fetus showed any significant glomerular however, the glomerular basement membrane in E febasement membrane development. There was a large tus was more developed than t h a t in C fetus. In E fetus,
space between the epithelial and endothelial cells, on fetal day 20, colloidal iron was preferentially conwhere mature basement membrane would later ap- centrated in laminae interna and externa, between
pear. Thin lamina, from which fine fibrils were ori- which the lamina densa was recognized as a n iron-free
ented to the endothelium, was observed running as a zone. On the other hand, in C fetus of the same day,
continuous line along the basal surface of the epithelial different patterns in iron distribution were observed in
cells (Figs. 1, 2). Colloidal iron was randomly distrib- the glomerular basement membrane. These findings
uted in the space between the epithelial and endothe- indicate that the anionic sites were more differentiated
lial cells. Accumulation of colloidal iron was also ob- in E fetus than in C fetus on fetal day 20. The fact that
served on the free surfaces of the epithelial and the width of the lamina densa in E fetus was larger
endothelial cells. The iron was more concentrated on than that in C fetus on fetal day 22 also suggests that
the surface of the epithelial cells than on the endothe- differentiation of the glomerular basement membrane
lial cells, and the same degree of accumulation was is further accelerated by maternal ureteral ligation.
noted in all subsequent fetuses. No difference in iron This notion is supported by the following reports: diflocalization on glomerular anionic sites was observed ferentiation of the glomerular basement membrane folat this point (Figs. 3, 4).
lows thickening of the lamina densa without any
On fetal day 20, no morphological difference in the change in anionic sites after establishment of trilamiepithelial cells was observed between E and C fetuses. nar structure (laminae rarae interna and externa and
The cells had foot processes and slits. The space be- lamina densa) of the glomerular basement membrane
tween the endothelial and epithelial cells was nar- (Cheignon et al., 1981; Bakala et al., 1988).
rower than t h a t in both fetuses of fetal day 18. HowAnionic sites in the glomerular basement membrane
ever, a morphologically different developmental are closely related to the presence of proteoglycans
pattern in the basement membrane was noted. In C (Farquhar et al., 1982; Kanwar and Farquhar, 1979b),
fetus, two distinct thin laminae were observed in most which, owing to their strongly negative charge, play
areas; one was running along the basal surface of the critical roles in permeselective seiving properties of the
epithelial cells (epithelial basement lamina) and the glomerular basement membrane that establish size
other along the basal surface of the endothelial cells and charge barrier to serum proteins (Kanwar, 1984;
(endothelial basement lamina). These laminae some- Rennke e t al., 1975). The present study showed that, in
times fused to form a dense line, the lamina densa (Fig. E fetus on fetal days 20 and 22, the anionic sites in the
5). In E fetus, on the other hand, the epithelial and glomerular basement membrane were restricted to
endothelial laminae fused as lamina densa in all areas both margins of the membrane, showing a similar
observed (Fig. 6). A different pattern in colloidal iron structure of the glomerular basement membrane as is
distribution was observed in C fetus. In most areas of seen in adult rats. On the other hand, in C fetus on day
the space, the iron was randomly distributed, or the 20, in most areas of the glomerular basement memepithelial basement lamina was locally free of iron. In
other areas, the iron was accumulated in both margins
of the space in which a clear iron-free zone was interposed (Fig. 7). In E fetus, colloidal iron was heavily
Abbreviations:
accumulated in both margins of the space in which a n En
endothelium
iron-free zone was noted as a thick clear line in all EP
epithelium
areas observed (Fig. 8).
f
fenestra
foot process
On fetal day 22, in both fetuses, the epithelial and FP
lamina densa
endothelial cells as well as glomerular basement mem- LD
LRI
lamina rara interna
brane were well developed, showing morphological fea- LRE
lamina rara externa
tures similar to those of adult rats. The epithelial cells S
epithelial slit
EFFECTS OF MATERNAL URETERAL LIGATION
Fig. 1. Glomerulus of an 18-day-old C fetus stained with uranyl
acetate and lead citrate. The epithelial cell has no foot process. The
endothelial cell is thick and is not fenestrated. A thin basement lamina, from which filamentous materials extend up to the endothelial
cell, is seen in the space between the epithelial and endothelial cells.
x 38,000.
Fig. 2. Glomerulus of a n 18-day-old E fetus stained with uranyl
acetate and lead citrate. The endothelial cell is fenestrated and becomes thinner than that of age-matched C fetus in Figure 1. The
epithelial cell and basement membrane show the same structure as in
the age-matched C fetus. x 38,000.
brane, the anionic sites were randomly distributed, indicating the immaturity of the glomerular basement
membrane; in the other areas, the sites were restricted
to both margins of the membrane, indicating its matu-
269
Fig. 3. Glomerulus of a n 18-day-old C fetus stained with colloidal
iron. Free surface of the epithelium is heavily stained. Colloidal iron
appears randomly distributed in the glomerular basement membrane.
X 52,000.
Fig. 4. Glomerulus of a n 18-day-old E fetus stained with colloidal
iron. The iron labelling is similar to that of age-matched C fetus in
Figure 3. X52,OOO.
rity. These observations are in accordance with our
previous reports that maternal bilateral ureteral ligation, performed after day 19 of pregnancy, accelerates
maturation of both the glomerulus (Okada and
270
T. OKADA AND Y. MORIKAWA
Fig. 5. Glomerulus of a 20-day-old C fetus stained with uranyl acetate and lead citrate. The epithelial cell has foot processes, and the
endothelial cell is fenestrated. The glomerular basement membrane is
partly composed of two continuous layers (epithelial basement lamina
and endothelial basement lamina) (arrowheads) and partly composed
of the lamina densa (arrow) bordered on both sides by two laminae
rarae. x 22,500.
Fig. 7. Glomerulus of a 20-day-old C fetus stained with colloidal
iron. Three areas showing different distributional patterns of iron in
the space between the epithelial and endothelial cells are seen: the
area in which the iron distribution is restricted to the laminae rarae
interna and externa (large arrow), the area in which the epithelial
basement lamina is locally free of iron (small arrows), and the area in
which the iron is randomly distributed (arrowheads). x 26,500.
Fig. 6. Glomerulus of a 20-day-old E fetus stained with uranyl acetate and lead citrate. The glomerular basement membrane is composed of the lamina densa bordered on both sides by two laminae
rarae. x 22,500.
Fig. 8. Glomerulus of a 20-day-old E fetus stained with colloidal
iron. The distribution of colloidal iron is restricted to the laminae
rarae interna and externa. x 26,500.
Morikawa, 1988) and the proximal tubules with relation to the glomerular functional development of kidney in fetal rats (Okada and Morikawa, 1990). Furthermore, the present results are supported by several lines
of evidence concerning the onset of fetal kidney function in rats. The kidney is functional in urine production 2 days before birth (Wells, 1946). Glomerular filtration occurs on fetal day 18 (Schaeverbeke and
EFFECTS O F MATERNAL URETERAL LIGATION
271
Fig. 9.Glomerulus of a 22-day-old C fetus stained with uranyl acetate and lead citrate. The glomerular basement membrane is composed of a lamina densa bordered on both sides by two laminae rarae.
The endothelium is fenestrated. x 38,000.
Fig. 11. Glomerulus of a 22-day-old C fetus stained with colloidal
iron. The distribution of colloidal iron is restricted to the laminae
rarae interna and externa. x 63,000.
Fig. 10. Glomerulus of a 22-day-old E fetus stained with uranyl
Fig. 12.Glomerulus of a 22-day-old E fetus stained with colloidal
iron. The iron-free zone, lamina densa, is slightly thicker than that in
the age-matched C fetus in Figure 11. ~ 6 3 , 0 0 0 .
acetate and lead citrate. The lamina densa is thicker than that in
age-matched C fetus in Figure 9. x 38,000.
Cheignon, 1980), and effective ultrafiltration starts on
fetal day 20 (Bakala et al., 1985). The days as described
by these authors are equivalent to fetal days 19 and 21
in the present study. In addition, Matsuo et al. (1986)
reported that, when maternal ureters were ligated on
day 21 of pregnancy to cause uremia, there is a highly
significant inverse relationship between maternal
blood urea nitrogen (BUN) level and the number of
fetuses on day 22 of pregnancy. They suggested that
the function of the fetal kidney is stimulated by the
elevated BUN level on late fetal days. In conclusion,
the present study revealed that, when maternal bilateral ureteral ligation was performed on day 19 or 21 of
pregnancy, the days when the rat fetal kidney is func-
272
T. OKADA AND Y. MORIKAWA
tional, the differentiation of the glomerular basement
membrane of fetal kidney was accelerated.
ACKNOWLEDGMENTS
This work was supported in part by Grant-in-Aid
02760178 from the Ministry of Education, Science and
Culture of Japan.
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1988 Basement membrane proteoglycans and anionic sites in the
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Okada, T., and Y. Morikawa 1990 Effects of maternal bilateral ureteral ligation on the development of the proximal tubule of kidney in fetal rats: Morphometry and electron microscopic study.
Anat. Rec., 228t456-460.
Reeves, W.H., Y.S. Kanwar, and M.G. Farquhar 1980 Assembly of the
glomerular filtration surface. Differentiation of anionic sites in
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735-753.
Rennke, H.G., R.S. Cotran, and M.A. Venkatachalam 1975 Role of
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site, glomerular, ureteral, effect, ligation, differentiation, anionic, kidney, rat, bilateral, maternal, fetal
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