/ . Embryol. exp. Morph. Vol. 58, pp. 157-175,1980
Printed in Great Britain © Company of Biologists Limited 1980
\ 57
Differentiation of glomerular filter
and tubular reabsorption apparatus during foetal
development of the rat kidney
By JEAN SCHAEVERBEKE AND MADELEINE CHEIGNON 1
From the Laboratoire de Biologie Cellulaire,
Universite Paris VII
SUMMARY
Differentiation of the glomerulus and the proximal tubule was studied in the rat foetus,
especially with regard to the development of the protein filtration-reabsorption apparatus.
Filtration starts several days before full differentiation of the glomerulus, when the glomerular
basement membrane consists of a thin lamina alongside the podocyte membrane. Endocytosis
is functional from this time, but fusion between endocytic vesicles and lysosome-like bodies
occurs 2 days later. Foetal urine electrophoresis shows the presence of many proteins,
including high molecular weight ones, this proteinuria seeming chiefly due to the immaturity
of the glomerular barrier.
INTRODUCTION
Differentiation of the nephron and particularly of the glomerulus has been
investigated both by light and by electron microscopy (Montaldo & Piso, 1970;
Leeson, 1961; Kasimierczak, 1971; Miyoshi, Fujita & Tokunaga, 1971; Crocker
& Easterbrook, 1972; Potter, 1972; Zimmermann & Boseck, 1972; Larsson,
1975; Kasimierczak, 1976) but hitherto, no study has been performed on the
correlations between the structural development of the protein retention system
(glomerular filtration barrier and tubular reabsorption apparatus) and the
onset of filtration and selective permeability properties.
Development of the metanephros begins during foetal life but is only
completed after birth. All the nephrons do not develop at the same time. The
first nephrogenic masses appear under the kidney capsule and are progressively
differentiated and connected to the collecting tubules; new nephron generations
are formed at the periphery of the kidney so that, at any time of foetal life,
the most differentiated ones will be the most deeply embedded. At birth, most
nephrons appear as mature formations but in certain species, particularly in
the rat, some nephrogenic masses can still be seen, their differentiation being
achieved several days or weeks following birth (Kasimierczak, 1971; Larsson
1975; Kasimierczak, 1976).
1
Authors' address; Laboratoire de Biologie Cellulaire, Universite Paris VII, Tour 23-33
ler etage, 2 place Jussieu, 75221 Paris Cedex 05, France.
11-2
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J. SCHAEVERBEKE AND M. CHEIGNON
The purpose of this paper is to examine ultrastructural differentiation
(especially with regard to the glomerular filtration barrier and proximal tubule
endocytosis apparatus) and protein filtration-reabsorption properties of
immature nephrons, at different stages of foetal life.
MATERIALS AND METHODS
Animals
Sherman rats were used in all experiments. For foetal studies, two-month-old
females weighing 200-300 g were mated overnight, the following day being
considered as day 0 of gestation.
Ultrastructural differentiation
Studies were performed on rat foetuses from day 15 to day 21 of gestation
and on newborn, 3-day-old, 25-day-old and adult animals. Ten animals were
used for each stage. Rats were anaesthetized intraperitoneally with sodium
pentobarbital (6 mg/100 g of body weight). In all cases fixation was performed
by vascular perfusion. It was essential to prevent disruption of blood perfusion
and vascular hypertension to avoid structural disturbances (Maunsbach, 1966).
Thus, the fixative perfusion rate was adapted to animal body weight and a
peripheral vessel was sectioned.
For foetal kidney studies the abdomen of pregnant females was opened
and the uterus exposed. The fixative (glutaraldehyde 1 % plus paraformaldehyde
0-8% in 0-12 M cacodylate or phosphate buffer with an admixture of 0-25%
sodium chloride, pH 7-3) was introduced through a fine hypodermic needle
into the vitelline vein at a flow rate of 10/4/min (15- to 18-day-old foetuses)
or 20 [A I mm (19- and 21-day-old foetuses) for 20 min.
In postnatal stages the fixative was back perfused into the abdominal aorta
proximal to its distal bifurcation, just after clamping the aorta above the renal
pedicles, at a flow rate of 20 /d/min (newborn and 3-day-old rats), 50 /tl/min
(25-day-old rats) or 100 /tl/min (adults) for 20 min. The kidneys were then
quickly removed, immediately immersed in the fixative and cut perpendicularly
to the renal capsule into pieces of 2-3 mm3 which were transferred into vials
containing fresh fixative at room temperature for 3 h. After a 10 min rinse in
0-12 M cacodylate or phosphate buffer containing 0-25% sodium chloride,
pH 7-3, tissues were stored overnight at 4 °C in the same solution and subsequently post-fixed with 2 % OsO4 in 0-1 M cacodylate or phosphate buffer,
pH 7-3 at 4 °C for 1 h. After washing successively with buffer (15 min) and
distilled water (three washes of 10 min each), tissues were dehydrated in graded
series of ethanol and embedded in Epon-Araldite (Mollenhauer & Totten,
1971). The blocks were cut with glass knives on a Porter-Blum MT2 ultramicrotome. Thick sections (1-5 /on) perpendicular to the kidney capsule were
stained in a mixture 1/1 (v/v) of 1 % methylene blue in saturated sodium
Differentiation in foetal rat kidney
159
borate and 1 % of Azure blue in water, and then examined by light microscopy
in order to localize the most differentiated nephron fields. In these selected
fields, ultra-thin sections were cut, sequentially stained in a mixture 1/1 (v/v)
of a saturated aqueous solution of uranyl acetate and pure acetone for 20 min
and in lead citrate (Reynolds, 1963) for 6 min. They were examined in a
Hitachi HS-8 electron microscope at 50 kV.
Qualitative analysis of urinary proteins
Urine samples were collected by transbladder aspiration. Proteins were
separated by electrophoresis in 4-26% polyacrylamide gel gradient slabs
(electrode buffer: Tris-borate-EDTA, pH 8-35, according to Kitchin (1965), at
220 V and 10 °C for 24 h). The slabs were stained with Coomassie blue (0-1 %
solution in methanol-acetic acid-water: 10/1/10, v/v).
Experiments with horseradish peroxidase
The protein tracer (horseradish peroxidase type II, Sigma Chemical Company,
molecular weight: 40000) was dissolved in physiological saline and perfused
in a small volume to prevent hemodynamical changes which are known to
affect the glomerular transport of macromolecules (Ryan & Karnovsky, 1976).
Thus, horseradish peroxidase, 100-200 /*g per g of body weight was infused
over 1 min into the vitelline vein of 17- to 21-day-old foetuses in volumes of
5-10/d according to their age. Animals were fixed by perfusion 1, 2, 4, 6, 8,
10, 15 or 20 min after horseradish peroxidase injection (three or four animals
for each interval) as described above. After additional fixation by immersion
in the glutaraldehyde-paraformaldehyde solution, tissues were rinsed at once
in 0-12 M cacodylate or phosphate buffer plus 0-25 % sodium chloride, pH 7-3,
overnight and then in 0-05 M Tris-HCl buffer, pH 7-6, for 30 min. Peroxidase
activity was revealed according to Graham & Karnovsky's method (1966a) on
small pieces excised with a razor blade (when we attempted to use frozen
40 /tin slices, the tissue was torn during successive manipulations since foetal
kidney is quite delicate). The kidneys from two animals injected with saline
solution were studied as controls. After incubation in test medium, tissues
were rinsed, post-fixed in osmium tetroxide, dehydrated and embedded in
Epon-Araldite. The sections were examined without additional staining.
RESULTS
Ultrastructural differentiation
Only the most differentiated, that is the deepest nephrons, will be described
at every stage of foetal development.
Day 15 of gestation. The first structures identifiable as nephrons appear.
They are still scattered in a tissue composed of sparse cells and are seen as
discrete masses in the immediate vicinity of the end of a collecting tubule.
J. SCHAEVERBEKE AND M. C H E I G N O N
Fig. 1. Glomerulus of 16-day-old foetus. The epithelial cell (Ep) has no foot
process. The endothelial cell {En) is thick, without fenestra. The space between
the epithelium and the endothelium is occupied by a thin basement lamina (B)
located next to the podocytes and by a sparse material alongside the endothelial
cells, x 28000.
Fig. 2. Proximal tubule of 16-day-old foetus. The cells are high and connected by
an intermediate junction. The tubular lumen (TL) is narrow. Very short microvilli
can be seen, x 6000.
Differentiation in foetal rat kidney
161
v>
IM
Fig. 3. Glomerulus of 17-day-old foetus. The epithelial cell (Ep) has no foot process
and presents some cytoplasmic invaginations near the urinary space (US). The
endothelial cell (En) is thick, without fenestra. B, Future glomerular basement
lamina, x 6500.
Fig. 4. Glomerulus of 17-day-old foetus. The epithelial cell (Ep) has only a few
shallow infoldings. The space between podocytes end endothelium is occupied by
a thin basement lamina (arrow) from which filamentous material extends up to the
endothelial cell (En), x 27000.
Fig. 5. Proximal tubule of 17-day-old foetus. Note the presence of short microvilli
(Mi) and apical tubular invaginations (at). Polysomes, lysosomes (Ly) and small
mitochondria are dispersed in the cytoplasm. The intercellular space is narrow,
without infolding, x 12000.
These nephrogenic buds develop a central lumen rapidly and become renal
vesicles. The most differentiated nephrons are S-shaped bodies resulting from
the elongation and curving of renal vesicles. These S-shaped bodies are completely surrounded by a very thin basement lamina. At this developmental
stage, intercellular spaces are locally irregular in width and all cells exhibit
almost the same ultrastructure. Each nucleus contains several large nucleoli.
162
J. SCHAEVERBEKE AND M. C H E I G N O N
1*
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Fig. 6. Glomerulus of 18-day-old foetus. The epithelial cells have an irregular
shape. In the endothelial cell (En), there are a few fenestrae irregularly arranged.
US, urinary space; B, glomerular basement lamina, x 8000.
Fig. 7. Glomerulus of 18-day-old foetus. The glomerular basement lamina is
composed of two layers: a continuous lamina close to the podocytes (large arrow)
and a discontinuous one next to the endothelium (En). The epithelial cell (Ep)
presents broad foot processes (Fp). Slit diaphragms can be seen bridging the
narrow gap between neighbouring foot processes (small arrow), x 36000.
Fig. 8. Proximal tubule of 18-day-old foetus. Mi, Microvilli; at, apical tubular invaginations; sav, small apical vesicles, x 30000.
Differentiation in foetal rat kidney
163
Ribosomes and polysomes are numerous but only a few long channels of
rough endoplasmic reticulum are seen. Golgi apparatus presents very short
saccules. Mitochondria are small and randomly dispersed. Microtubules,
parallel to the plasma membrane, are restricted to the basal half of the cell,
on both sides of the nucleus.
Day 16 of gestation. The lower limb of the S-shaped body increases in width
and forms a cup, the centre of which is invaded by a capillary loop. This cup
deepens and constitutes a double-walled hemisphere. The cells of the outer
wall soon begin to flatten and will become the Bowman's capsule; the cells
of the inner wall are the future podocytes. The two walls are separated by a
narrow space. The early podocytes have a cuboidal shape and their lateral
cell membranes are closely apposed. No foot process can be seen. The capillary
wall is thick and without fenestra; endothelial cells have irregular surfaces
with thin evaginations inside the capillary lumen and, facing the podocytes,
flat cytoplasmic expansions tangential to the capillary wall section. The space
between the epithelium and the endothelium is irregular in width (from 0-15
to 0-45 /.tin) and occupied by a thin basement lamina located next to the
podocytes (Fig. 1).
The middle limb of the S-shaped body will give rise to the proximal tubule
which is always seen close to the glomerulus in sections. The tubular lumen is
very narrow and the lining cells are connected by a long subapical intermediate
junction (Zonula adherens). The distribution of cellular organelles is the same
as previously (Fig. 2).
Day 17 of gestation. The S-shaped body is connected to the collecting tubule.
The podocytes appear still as cuboidal cells and are joined by several junctions
near their capillary side. Elsewhere, they are separated from each other by
wide intercellular spaces. A few broad and short processes begin to develop
on the cell surface adjacent to the capillary; they probably represent the
first-order podocyte branches. Endothelial cells have a thick and non-fenestrated
cytoplasm. As before, a thin basement lamina from which filamentous material
extends up to the endothelial cells, is seen close to the podocyte membrane
(Figs. 3, 4).
Some of the proximal tubule sections are located next to the glomerulus
while others are apart, suggesting that these tubules are longer than in the
preceding stage. Adjacent cells show several kinds of junctions: one short
tight junction near the tubular lumen, one junction just beneath, and several
gap junctions up to the base of the cells. A few short microvilli first appear
at this stage. In the underlying cytoplasm there are tubular invaginations, small
vesicles and, towards the base, a few lysosomes and autophagic vacuoles. In
the lower part of the cell, scanty peroxisomes and lipid droplets can be seen.
The other cytoplasmic organelles have the same structure and distribution as
before (Fig. 5).
Day 18 of gestation. The podocytes have an irregular shape and come apart
164
J. SCHAEVERBEKE AND M. CHEIGNON
Differentiation in foetal rat kidney
165
from each other except near the [basement membrane where they are fastened
together by a few junctions. The urinary space is about 4 fim wide. Broad foot
processes take shape facing the endothelial cells, the slits between them being
bridged by a diaphragm; elsewhere, cell membranes exhibit scarce and short cytoplasmic extensions. The capillary wall is generally thick but occasionally thin
and even fenestrated, pores being obstructed by electron-dense material. Besides
the podocyte basement lamina, a thin discontinuous lamina is seen next to
the endothelial cells. These two laminae are joined by fine filaments (Figs. 6,7).
In the proximal tubule, the microvilli are more numerous and longer than
in the previous stage. The cell membranes present a few basal and lateral
infoldings. Most of the apical endocytic vesicles are small (0-4 /im in diameter)
and a few middle-sized (between 0-4 and 0-8 /im in diameter), both bearing a
thick internal coat (Fig. 8). Mitochondria remain small and scattered. Golgi
apparatus consists of short saccules and is located on both sides of the nucleus
in a direction parallel to the lateral plasma membrane.
Day 19 of gestation. The foot processes are longer than in the previous stage.
In the endothelial cells a few pores obstructed by a fibrillar material are
observed. The basement lamina of these cells is continuous and separated from
that of the epithelial cells by a thin filamentous zone of low electron density
(Fig. 9, 10).
The proximal tubule microvilli increase in length. There are only a few
plasma membrane invaginations on the basal and lateral sides of the cells. The
apical-coated vesicles are numerous and uncoated or thinly coated large
vesicles (1 fim in diameter) appear just beneath. The microtubules, either alone
or in bundles, are located not only alongside the nucleus but also in the apical
cytoplasm of the cells, especially between the endocytic vesicles. The mitochondria, similar in size as in the preceding developmental stage, are found
alongside channels of rough endoplasmic reticulum (Fig. 11).
Day 20 of gestation. The podocytes present many long and thin foot processes
and are tightly packed so that, in sections, their monolayer disposition is no
longer obvious. They enlarge and tend to fill the urinary space which is consequently reduced to interpodocyte gaps, as in the mature glomerulus. The
F I G U R E S 9-11
Fig. 9. Glomerulus of 19-day-old foetus. The glomerular basement membrane is
composed of two continuous layers (arrows) sparsely joined by a filamentous
material, x 37000.
Fig. 10. Glomerulus of 19-day-old foetus. Ep, Epithelial cell; Fp, foot process;
En, endothelial cell; B, glomerular basement membrane; US, urinary space,
x 23 000.
Fig. 11. Proximal tubule of 19-day-old foetus. At the base of the microvilli, endocytic vesicles in formation can be seen (arrows). Membrane of the small apical
vesicles (sav) is covered by a thick internal coat, x 34000.
166
J. SCHAEVERBEKE AND M. C H E I G N O N
Fig. 12. Glomerulus of 20-day-old foetus. The glomerular basement membrane
(B) is composed of a lamina densa bordered on both sides by two laminae rarae.
The endothelium (En) is fenestrated CO- Slit diaphragm, arrow; Ep, epithelial cell;
Fp, foot process, x 30000.
Fig. 13. Proximal tubule of 20-day-old foetus, sav, small apical vesicles; lav, large
apical vesicle; Ly, lysosome; bi, basal infolding, x 11000.
capillary loops are twisted and intermixed with epithelial and mesangial cells.
A very thin layer of endothelium extends around the capillary and shows many
open pores. The epithelial and endothelial cells draw close to one another and
then share a common basement membrane resulting probably from the coalescence of previous thin laminae. On both sides of this preliminary lamina
densa, one sees a loose network which will become the laminae rarae (Fig. 12).
Differentiation in foetal rat kidney
167
miLM^L-i£M:'i!t;
a
Fig. 14. Glomerulus of 21-day-old foetus. The structure is the same as in the
previous stage except a thickening of the lamina densa. Ep, Epithelial cell; Fp, foot
process; slit diaphragm, arrow; B, glomerular basement membrane; En, endothelialcell;/, fenestra. x 30000.
Fig. 15. Proximal tubule of 21-day-old foetus. The small (sav) and large (lav) apical
endocytic vesicles are numerous. Mi, microvilli; Ly, lysosome. x 21000.
The proximal tubule cells present even longer microvilli. The main feature
of this developmental stage is the presence of profuse large endocytic vesicles
sometimes seen close by lysosome-like bodies. Golgi apparatus contains
saccules longer than in the previous stage (about 2/*m). The mitochondria
enlarge, come together at the basal half of the cells and show a tendency to
be oriented perpendicularly to the tubule axis. They are bordered by channels
of rough endoplasmic reticulum (Fig. 13).
Day 21 of gestation. The former features of glomerulus differentiation are
168
J. SCHAEVERBEKE AND M. C H E I G N O N
Fig. 16. Proximal tubule of 3-day-old animal. The small apical vesicles (sav) are
less numerous than previously. The large vesicles (lav) are seen close to lysosome
(Ly), these two organelles being likely in process of fusion. Mi, microvilli. x 13000.
Differentiation in foetal rat kidney
Start
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169
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Fig. 17. Electrophoresis of rat urine in a 4-26% polyacrylamide gel gradient.
A, 21-day-old foetus; B, 3-day-old animal; C, adult. 1, prealbumins; 2, albumin;
3, a-foetoprotein; 4, a2-macroglobulin.
strongly marked. This stage is more particularly characterized by a thickening
of the glomerular basement membrane to about 0-1 /tm in width (Fig. 14)
The proximal tubule cells have extensively interdigitating processes which
contain long mitochondria arranged perpendicular to the tubule axis, close
to the plasma membrane. The endocytic vesicles are very numerous in the
apical part of the cells, the large ones being almost contiguous to each other.
Some large vesicles are seen apparently in the process of fusing with lysosomes
(Fig. 15).
Newborns. The structure of the most differentiated nephrons is almost the
same as in the last foetal stage, except that the number and the size of the
large endocytic vesicles are increased.
3-day-old animals. The process of intermingling of the epithelial and
endothelial cells becomes more accentuated than previously and the glomerular
basement membrane is as thick as in adult stage (0-15 /on).
In the proximal tubule, there are a few small vesicles, while the large ones
remain numerous, most of them being probably engaged in fusion with
170
J. SCHAEVERBEKE AND M. CHEIGNON
Differentiation in foetal rat kidney
111
lysosomes. Voluminous droplets are observed at the base of the cells (Fig. 16).
25-day-old animals. The nephrons have the same structure as in the adult.
In particular, the proximal tubules show many long and slender microvilli and
only a few endocytic vesicles. Basal and lateral infoldings are very deep,
setting up high septa between which are seen very long mitochondria fringed
with rough endoplasmic reticulum channels.
Development of protein filtration-reabsorption properties
Qualitative analysis of urine proteins. Foetal electrophoregrams show the
presence of most of the plasma proteins, including high-molecular-weight ones,
such as a-2-macroglobulin (molecular weight: 800000). In the urine of young
animals (3-day-old) the same components are found except for the proteins
of molecular weight above 150000-200000 which are absent. In the urine of
adults only a few traces of light proteins are present (Fig. 17).
Location of horseradish peroxidase after intravenous injection. Before day 18
of gestation, peroxidase reaction product cannot be detected either in the
glomerulus or in the proximal tubule. At day 18, enzyme activity is found
after 4 min of intravenous injection of peroxidase in the glomerular basement
membrane, in the urinary space (Fig. 18), on the proximal tubule microvilli, in
the tubular invaginations and in the small apical vesicles (Fig. 19). At day 19
of gestation, peroxidase filtration occurs as early as 2 min after perfusion is
stopped and reaction product is observed in the apical tubules of the proximal
tubule cells. Two minutes later all the apical vesicles contain reaction product
at their periphery. At days 20 and 21 filtration is still faster, starting from 1 min
following peroxidase injection (Fig. 20). After 2 min, all the small apical
vesicles contain peroxidase and the reaction product is usually located on the
inside of the vesicle membrane (Fig. 21); on the contrary, large vesicles which
FIGURES
18-21
Fig. 18. Glomerulus of 18-day-old foetus fixed 4 min after horseradish peroxidase
injection. The reaction product (arrow) is seen in the glomerular basement membrane (B) between epithelial (Ep) and endothelial (En) cells, especially alongside
the podocyte. Unstained, x 34000.
Fig. 19. Proximal tubule of 18-day-old foetus fixed 4 min after horseradish peroxidase
injection. The reaction product is present on the brush border, in the apical tubular
invaginations and in the small vesicles farrows). Unstained, x 16000.
Fig. 20. Glomerulus of 21-day-old foetus fixed 1 min after horseradish peroxidase
injection. The reaction product (arrows) is present in the capillary lumen, in the
glomerular basement membrane (B), on the podocyte membrane and in the urinary
space (US). Ep, Epithelial cell; Fp, foot process; En, endothelial cell. Unstained.
x 30000.
Fig. 21. Proximal tubule of 21 -day-old foetus fixed 2 min after horseradish peroxidase
injection. The tracer (arrows), present on the brush border membrane, is picked
up into small vesicles (sav). Unstained, x 31000.
12
EMB 58
172
J. SCHAEVERBEKE AND M. C H E I G N O N
Fig. 22. Apical cytoplasm of a proximal tubule cell of 21-day-old foetus fixed
4 min after horseradish peroxidase injection. The tracer (arrow) is present on the
microvilli, in the apical tubular invaginations and in the large apical vesicles (lav)
in fusing process with a lysosome (Ly); Unstained, x 18000.
Fig. 23. Proximal tubule of 21-day-old foetus fixed 10 minutes after horseradish
peroxidase injection. The microvilli (Mi) and the small apical vesicles (sav) lacking
reaction product. The large apical vesicles (lav) contain the reaction product
(arrow). Ly, lysosome. Unstained, x 13000.
fuse with lysosomes are filled with scattered precipitates which soon become
less and less contrasted (Figs. 22, 23). In addition, from day 18 onwards, some
peroxidase activity is observed in intercellular spaces, but this is probably
a result of enzyme diffusion from peritubular capillaries.
DISCUSSION
Foetal urine analysis shows the presence of many plasma proteins as compared
with the urine of adult animals. This proteinuria seems to be largely due to
immaturity of the glomerular filtration barrier since even high-molecular-weight
proteins, which do not permeate the glomerular filter in the differentiated
nephron (Graham & Karnovsky, 19666), are found in foetal urine. Two kinds
of observations are in agreement with this assumption. Firstly, at the start of
glomerular filtration, indicated by the first detection of horseradish peroxidase
Differentiation
in foetal rat kidney
173
in the urinary space (namely at day 18), the glomerular basement membrane
is merely composed of a thin lamina probably much more permeable than the
thick three-layered lamina of the mature glomerulus. Although we are unaware
of the composition of the glomerular filtrate at the different stages of nephron
differentiation, it is likely that selective permeability appears gradually with
successive biochemical deposits onto the glomerular barrier. Secondly, the
endocytic apparatus, which comprises the apical vesicles and the secondary
lysosomes resulting from coalescence between the large endocytic vesicles and
the primary lysosomes, is clearly more profuse at foetal stages than in the
mature tubule. This may be due to a relatively high concentration of plasma
proteins in the glomerular filtrate. Indeed, it has been suggested that the
number of endocytic vesicles increases either in proximal tubule (Bergelin &
Karlsson, 1975; Larsson & Maunsbach, 1975) or in other cells (Cohn &
Fedorko, 1969) when they are exposed to increasing concentrations of macromolecules.
Foetal proteinuria may also be related to immaturity of the tubular reabsorption system. In the differentiated proximal tubule, nearly all the filtered
proteins are picked up into endocytic vesicles via the apical tubular invaginations and then digested into secondary lysosomes (Larsson & Maunsbach,
1975). As indicated by tracer studies, the whole endocytic process occurs
within a few minutes either in the mature nephron or at the late stages of its
differentiation (days 20 and 21 of gestation). On the contrary, in the early
stages (days 18 and 19 of gestation) the formation of secondary lysosomes
takes place only 1 or 2 days after the onset of endocytosis, though the primary
lysosomes are present from the first steps of tubule development. The congestion of apical vesicles, possibly consequent on the lack of formation of
secondary lysosomes, may be the cause of a slower endocytosis; this, in addition
to the abundance of plasma proteins in the glomerular filtrate, may account
for foetal proteinuria. However, that cannot explain the presence of large
protein molecules in foetal urine since protein reabsorption in the proximal
tubule is a non-specific phenomenon: therefore, any qualitative changes in the
composition of urine, i.e. the presence or the absence of large protein molecules,
is likely to be a consequence of glomerular permeability.
The gradual differentiation of the protein reabsorption mechanisms is
perhaps in close relation to the cellular distribution of microtubules. So, it is
noteworthy that the apical ordering of microtubules takes place just before
the first appearance of secondary lysosomes. Previously, they were located
along the lateral cell membranes to the exclusion of apical cytoplasm. Since
microtubules are involved in intracellular movement (Silverblatt, Tyson &
Bulger, 1974) it is possible that the delayed appearance of secondary lysosomes
in immature proximal tubules results from the lack of apical microtubules as
endocytosis commences.
In other respects, as shown by urine electrophoresis, proteinuria is more
174
J. SCHAEVERBEKE AND M. CHEIGNON
selective in the newborn than in the foetus, in spite of the presence of immature
nephrons for several days after birth. It is possible that the difference between
prenatal and postnatal selective permeability is at least partially due to an
earlier differentiation of the glomerular basement membrane in postnatal than
in foetal developing nephrons. Thus, in the postnatal kidney, Kazimierczak
(1971) and Larsson & Maunsbach (1975) have reported that the epithelial
and endothelial basement lamina occasionally merge as early as the S stage
and that a three-layered basement membrane is observed when the endothelial
cells have only a few pores, whereas, in the foetal kidney, we observe a thin
epithelial lamina at the S-shaped-body stage and a typical basement membrane
only when the capillary wall is largely fenestrated.
In conclusion, the present study shows that the transient proteinuria observed
during foetal life is chiefly due to the immaturity of the glomerular barrier.
This investigation was supported by Institut National de la Sante et de la Recherche
Medicale (ATP 62-78-94).
REFERENCES
I. S. S. & KARLSSON, B. W. (1975). Functional structure of the glomerular
filtration barrier and the proximal tubuli in the developing foetal and neonatal pig kidney.
Anat. Embryol. 148, 223-234.
COHN, Z. A. & FEDORKO, M. E. (1969). The formation and fate of lysosomes, in Lysosomes
in Biology and Pathology, vol. 1 (ed. by J. T. Dingle and H. B. Fell), pp. 43-61. London:
North-Holland.
CROCKER, J. F. S. & EASTERBROOK, K. (1972). Differentiation of the human glomerulus.
Proc. 5th Int. Congr. Nephrol., Mexico, vol. 1, pp. 2-5.
GRAHAM, R. C. & KARNOVSKY, M. J. (1966a). The early stages of absorption of injected
horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. / . Histochem. Cytochem. 14, 291-302.
GRAHAM, R. C. & KARNOVSKY, M. J. (19666). Glomerular permeability: ultrastructural
cytochemical studies using peroxidases as protein tracers. / . exp. Med. 124, 1123-1134.
KASIMIERCZAK, J. (1971). Development of the renal corpuscle and the juxta-glomerular
apparatus: a light and electron microscopic study. Acta Pathol. Microbial. Scand., Suppl.
218.
KASIMIERCZAK, J. (1976). Developpement du glomerule renal en microscopie electronique
a transmission et a balayage. Bull. Assoc. Anat. (Nancy) 60, 137-143.
KITCHIN, F. D. (1965). Demonstration of the inherited serum group specific protein by
acrylamide electrophoresis. Proc. Soc. exp. Biol. Med. 119, 1153-1155.
LARSSON, L. (1975). The ultrastructure of the developing proximal tubule in the rat kidney.
/. Ultrastruct. Res. 51,119-139.
LARSSON, L. & MAUNSBACH, A. B. (1975). Differentiation of the vacuolar apparatus in
cells of the developing proximal tubule in the rat kidney. / . Ultrastruct. Res. 53, 254270.
LEESON, T. S. (1961). An electron microscope study of the postnatal development of the
hamster kidney. Lab. Invest. 10, 466-480.
MAUNSBACH, A. B. (1966). The influence of different fixatives and fixation methods on the
ultrastructure of rat kidney proximal tubule cells. I. Comparison of different perfusion
fixation methods and of glutaraldehyde, formaldehyde and osmium tetroxide fixatives.
/. Ultrastruct. Res. 15, 242-282.
MIYOSHI, M., FUJITA, T. & TOKUNAGA, J. (1971). The differentiation of renal podocytes: a
combined scanning and electron microscope study in rats. Archs Histol. Jap. 33, 161—
178.
BERGELIN,
Differentiation in foetal rat kidney
175
H. H. & TOTTEN, C. (1971). Studies on seeds. I. Fixation of seeds. /. Cell
Biol. 48, 387-394.
MONTALDO, G. & Piso, A. (1970). Observations au microscope electronique sur la morphogenese de la membrane basale du glomerule renal. Septieme Congres International de
Microscopie Electronique, Grenoble, pp. 603-604.
POTTER, E. L. (1972). Normal and Abnormal Development of the Kidney. Year Book Medical
Publ. Inc. Chicago.
REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in
electron microscopy. /. Cell Biol. 17, 208-212.
RYAN, G. B. & KARNOVSKY, M. J. (1976). Distribution of endogenous albumin in the rat
glomerulus: role of hemodynamic factors in glomerular barrier function. Kidney Int. 9,
36-45.
SILVERBLATT, F. J., TYSON, G. E. & BULGER, R. E. (1974). Effects of vinblastine on the
phagolysosome system of proximal tubule cells of rat kidney: administration of horseradish peroxidase. Lab. Invest. 31, 170-180.
ZIMMERMANN, H. D. & BOSECK, S. (1972). Muofilamentare Strukturen in Glomerulus und
Tubulusepithelien in der friihfetalen Nachniere des Menschen. Virchows Arch. path.
Anat. Physiol. A 357, 53-66.
MOLLENHAUER,
(Received 30 October 1979, revised 9 February 1980)